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Sample records for cytopathology image cytometry

  1. Digital Imaging in Cytopathology

    PubMed Central

    Khalbuss, Walid E.; Pantanowitz, Liron; Parwani, Anil V.

    2011-01-01

    Rapid advances are occurring in the field of cytopathology, particularly in the field of digital imaging. Today, digital images are used in a variety of settings including education (E-education), as a substitute to multiheaded sessions, multisite conferences, publications, cytopathology web pages, cytology proficiency testing, telecytology, consultation through telecytology, and automated screening of Pap test slides. The accessibility provided by digital imaging in cytopathology can improve the quality and efficiency of cytopathology services, primarily by getting the expert cytopathologist to remotely look at the slide. This improved accessibility saves time and alleviates the need to ship slides, wait for glass slides, or transport pathologists. Whole slide imaging (WSI) is a digital imaging modality that uses computerized technology to scan and convert pathology and cytology glass slides into digital images (digital slides) that can be viewed remotely on a workstation using viewing software. In spite of the many advances, challenges remain such as the expensive initial set-up costs, workflow interruption, length of time to scan whole slides, large storage size for WSI, bandwidth restrictions, undefined legal implications, professional reluctance, and lack of standardization in the imaging process. PMID:21785680

  2. The impact of digital imaging in the field of cytopathology

    PubMed Central

    Hornish, Maryanne; Goulart, Robert A.

    2009-01-01

    With the introduction of digital imaging, pathology is undergoing a digital transformation. In the field of cytology, digital images are being used for telecytology, automated screening of Pap test slides, training and education (e.g. online digital atlases), and proficiency testing. To date, there has been no systematic review on the impact of digital imaging on the practice of cytopathology. This article critically addresses the emerging role of computer-assisted screening and the application of digital imaging to the field of cytology, including telecytology, virtual microscopy, and the impact of online cytology resources. The role of novel diagnostic techniques like image cytometry is also reviewed. PMID:19495408

  3. Imaging Mass Cytometry.

    PubMed

    Chang, Qing; Ornatsky, Olga I; Siddiqui, Iram; Loboda, Alexander; Baranov, Vladimir I; Hedley, David W

    2017-02-01

    Imaging Mass Cytometry (IMC) is an expansion of mass cytometry, but rather than analyzing single cells in suspension, it uses laser ablation to generate plumes of particles that are carried to the mass cytometer by a stream of inert gas. Images reconstructed from tissue sections scanned by IMC have a resolution comparable to light microscopy, with the high content of mass cytometry enabled through the use of isotopically labeled probes and ICP-MS detection. Importantly, IMC can be performed on paraffin-embedded tissue sections, so can be applied to the retrospective analysis of patient cohorts whose outcome is known, and eventually to personalized medicine. Since the original description in 2014, IMC has evolved rapidly into a commercial instrument of unprecedented power for the analysis of histological sections. In this Review, we discuss the underlying principles of this new technology, and outline emerging applications of IMC in the analysis of normal and pathological tissues. © 2017 International Society for Advancement of Cytometry.

  4. Cytopathological image analysis using deep-learning networks in microfluidic microscopy.

    PubMed

    Gopakumar, G; Hari Babu, K; Mishra, Deepak; Gorthi, Sai Siva; Sai Subrahmanyam, Gorthi R K

    2017-01-01

    Cytopathologic testing is one of the most critical steps in the diagnosis of diseases, including cancer. However, the task is laborious and demands skill. Associated high cost and low throughput drew considerable interest in automating the testing process. Several neural network architectures were designed to provide human expertise to machines. In this paper, we explore and propose the feasibility of using deep-learning networks for cytopathologic analysis by performing the classification of three important unlabeled, unstained leukemia cell lines (K562, MOLT, and HL60). The cell images used in the classification are captured using a low-cost, high-throughput cell imaging technique: microfluidics-based imaging flow cytometry. We demonstrate that without any conventional fine segmentation followed by explicit feature extraction, the proposed deep-learning algorithms effectively classify the coarsely localized cell lines. We show that the designed deep belief network as well as the deeply pretrained convolutional neural network outperform the conventionally used decision systems and are important in the medical domain, where the availability of labeled data is limited for training. We hope that our work enables the development of a clinically significant high-throughput microfluidic microscopy-based tool for disease screening/triaging, especially in resource-limited settings.

  5. Masks in imaging flow cytometry.

    PubMed

    Dominical, Venina; Samsel, Leigh; McCoy, J Philip

    2017-01-01

    Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis.

  6. Cytopathology whole slide images and virtual microscopy adaptive tutorials: A software pilot

    PubMed Central

    Van Es, Simone L.; Pryor, Wendy M.; Belinson, Zack; Salisbury, Elizabeth L.; Velan, Gary M.

    2015-01-01

    Background: The constant growth in the body of knowledge in medicine requires pathologists and pathology trainees to engage in continuing education. Providing them with equitable access to efficient and effective forms of education in pathology (especially in remote and rural settings) is important, but challenging. Methods: We developed three pilot cytopathology virtual microscopy adaptive tutorials (VMATs) to explore a novel adaptive E-learning platform (AeLP) which can incorporate whole slide images for pathology education. We collected user feedback to further develop this educational material and to subsequently deploy randomized trials in both pathology specialist trainee and also medical student cohorts. Cytopathology whole slide images were first acquired then novel VMATs teaching cytopathology were created using the AeLP, an intelligent tutoring system developed by Smart Sparrow. The pilot was run for Australian pathologists and trainees through the education section of Royal College of Pathologists of Australasia website over a period of 9 months. Feedback on the usability, impact on learning and any technical issues was obtained using 5-point Likert scale items and open-ended feedback in online questionnaires. Results: A total of 181 pathologists and pathology trainees anonymously attempted the three adaptive tutorials, a smaller proportion of whom went on to provide feedback at the end of each tutorial. VMATs were perceived as effective and efficient E-learning tools for pathology education. User feedback was positive. There were no significant technical issues. Conclusion: During this pilot, the user feedback on the educational content and interface and the lack of technical issues were helpful. Large scale trials of similar online cytopathology adaptive tutorials were planned for the future. PMID:26605119

  7. Imaging flow cytometry for phytoplankton analysis.

    PubMed

    Dashkova, Veronika; Malashenkov, Dmitry; Poulton, Nicole; Vorobjev, Ivan; Barteneva, Natasha S

    2017-01-01

    This review highlights the concepts and instrumentation of imaging flow cytometry technology and in particular its use for phytoplankton analysis. Imaging flow cytometry, a hybrid technology combining speed and statistical capabilities of flow cytometry with imaging features of microscopy, is rapidly advancing as a cell imaging platform that overcomes many of the limitations of current techniques and contributed significantly to the advancement of phytoplankton analysis in recent years. This review presents the various instrumentation relevant to the field and currently used for assessment of complex phytoplankton communities' composition and abundance, size structure determination, biovolume estimation, detection of harmful algal bloom species, evaluation of viability and metabolic activity and other applications. Also we present our data on viability and metabolic assessment of Aphanizomenon sp. cyanobacteria using Imagestream X Mark II imaging cytometer. Herein, we highlight the immense potential of imaging flow cytometry for microalgal research, but also discuss limitations and future developments.

  8. Dictionary-enhanced imaging cytometry

    PubMed Central

    Orth, Antony; Schaak, Diane; Schonbrun, Ethan

    2017-01-01

    State-of-the-art high-throughput microscopes are now capable of recording image data at a phenomenal rate, imaging entire microscope slides in minutes. In this paper we investigate how a large image set can be used to perform automated cell classification and denoising. To this end, we acquire an image library consisting of over one quarter-million white blood cell (WBC) nuclei together with CD15/CD16 protein expression for each cell. We show that the WBC nucleus images alone can be used to replicate CD expression-based gating, even in the presence of significant imaging noise. We also demonstrate that accurate estimates of white blood cell images can be recovered from extremely noisy images by comparing with a reference dictionary. This has implications for dose-limited imaging when samples belong to a highly restricted class such as a well-studied cell type. Furthermore, large image libraries may endow microscopes with capabilities beyond their hardware specifications in terms of sensitivity and resolution. We call for researchers to crowd source large image libraries of common cell lines to explore this possibility. PMID:28225061

  9. Dictionary-enhanced imaging cytometry

    NASA Astrophysics Data System (ADS)

    Orth, Antony; Schaak, Diane; Schonbrun, Ethan

    2017-02-01

    State-of-the-art high-throughput microscopes are now capable of recording image data at a phenomenal rate, imaging entire microscope slides in minutes. In this paper we investigate how a large image set can be used to perform automated cell classification and denoising. To this end, we acquire an image library consisting of over one quarter-million white blood cell (WBC) nuclei together with CD15/CD16 protein expression for each cell. We show that the WBC nucleus images alone can be used to replicate CD expression-based gating, even in the presence of significant imaging noise. We also demonstrate that accurate estimates of white blood cell images can be recovered from extremely noisy images by comparing with a reference dictionary. This has implications for dose-limited imaging when samples belong to a highly restricted class such as a well-studied cell type. Furthermore, large image libraries may endow microscopes with capabilities beyond their hardware specifications in terms of sensitivity and resolution. We call for researchers to crowd source large image libraries of common cell lines to explore this possibility.

  10. Probing bacterial cell biology using image cytometry.

    PubMed

    Cass, Julie A; Stylianidou, Stella; Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2017-03-01

    Advances in automated fluorescence microscopy have made snapshot and time-lapse imaging of bacterial cells commonplace, yet fundamental challenges remain in analysis. The vast quantity of data collected in high-throughput experiments requires a fast and reliable automated method to analyze fluorescence intensity and localization, cell morphology and proliferation as well as other descriptors. Inspired by effective yet tractable methods of population-level analysis using flow cytometry, we have developed a framework and tools for facilitating analogous analyses in image cytometry. These tools can both visualize and gate (generate subpopulations) more than 70 cell descriptors, including cell size, age and fluorescence. The method is well suited to multi-well imaging, analysis of bacterial cultures with high cell density (thousands of cells per frame) and complete cell cycle imaging. We give a brief description of the analysis of four distinct applications to emphasize the broad applicability of the tool.

  11. Resources for flow and image cytometry

    SciTech Connect

    Cassidy, M.

    1990-01-01

    This paper describes resources available to the flow and image cytometry community. I have been asked to limit the discussion to resources available in the United States, so reference to resources exclusively available in Japan, Europe, or Australia are not included. It is not the intention of this paper to include each and every resource available, rather, to describe the types available and give some examples. Included in this manuscript are listings of some of the examples of resources which readers may find useful. Addresses of commercial companies are not included in the interest of space. Most of the examples listed advertise on a regular basis in journals publishing in cytometry fields. The resources to be described are divided into five categories: instrument resources, computer and software resources, standards, physical or user'' resources, and instructional resources. Each of these resources will be discussed separately. 4 tabs.

  12. Cellular Image Analysis and Imaging by Flow Cytometry

    PubMed Central

    Basiji, David A.; Ortyn, William E.; Liang, Luchuan; Venkatachalam, Vidya; Morrissey, Philip

    2007-01-01

    Synopsis Imaging flow cytometry combines the statistical power and fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. The technique is a good fit for clinical applications by providing a convenient means for imaging and analyzing cells directly in bodily fluids. Examples are provided of the discrimination of cancerous from normal mammary epithelial cells and the high throughput quantitation of FISH probes in human peripheral blood mononuclear cells. The FISH application will be further enhanced by the integration of extended depth of field imaging technology with the current optical system. PMID:17658411

  13. Cytopathology fellowship milestones.

    PubMed

    Naritoku, Wesley Y; Black-Schaffer, W Stephen

    2014-12-01

    The American Society of Cytopathology has provided guidelines for goals and objectives for cytopathology fellows. There are 90 Accreditation Council for Graduate Medical Education-accredited cytopathology fellowship training programs in the United States, each with its own unique curriculum designed to achieve these goals and objectives. The Accreditation Council for Graduate Medical Education cytopathology fellowship milestones were developed to ensure some uniformity in the outcomes of the various skill sets and competencies expected of a graduating cytopathology fellow. The rationale, development, and details of the cytopathology fellowship milestones are described herein.

  14. Flow cytometry what you see matters: Enhanced clinical detection using image-based flow cytometry.

    PubMed

    McFarlin, Brian K; Gary, Melody A

    2017-01-01

    Image-based flow cytometry combines the throughput of traditional flow cytometry with the ability to visually confirm findings and collect novel data that would not be possible otherwise. Since image-based flow cytometry borrows measurement parameters and analysis techniques from microscopy, it is possible to collect unique measures (i.e. nuclear translocation, co-localization, cellular synapse, cellular endocytosis, etc.) that would not be possible with traditional flow cytometry. The ability to collect unique outcomes has led many researchers to develop novel assays for the monitoring and detection of a variety of clinical conditions and diseases. In many cases, investigators have innovated and expanded classical assays to provide new insight regarding clinical conditions and chronic disease. Beyond human clinical applications, image-based flow cytometry has been used to monitor marine biology changes, nano-particles for solar cell production, and particle quality in pharmaceuticals. This review article summarizes work from the major scientists working in the field of image-based flow cytometry.

  15. Extracting information from imaging cytometry: a review.

    PubMed

    Gokhale, P J

    2016-11-01

    The extraction of statistically meaningful quantitative information from microscopy images is increasingly important for modern biological research. Obtaining accurate, quantitative information from biological specimens, however, is a complex process that requires optimization of several parameters. One must consider the number of probes, fluorescent channels required, type of plate to be used, number of fields to be acquired and optimal resolution for image acquisition. The extraction of information from images is dependent on and can be aided greatly by careful consideration of the factors involved in the image acquisition process. I summarize here the general principles behind the imaging and software technology that is used to quantify images and highlight particular issues of concern for critically applying image quantitation techniques for research.

  16. Comparison study of five different display modalities for whole slide images in surgical pathology and cytopathology in Europe

    NASA Astrophysics Data System (ADS)

    D'Haene, Nicky; Maris, Calliope; Rorive, Sandrine; Moles Lopez, Xavier; Rostang, Johan; Marchessoux, Cédric; Pantanowitz, Liron; Parwani, Anil V.; Salmon, Isabelle

    2013-03-01

    User experience with viewing images in pathology is crucial for accurate interpretation and diagnosis. With digital pathology, images are being read on a display system, and this poses new types of questions: such as what is the difference in terms of pixelation, refresh lag or obscured features compared to an optical microscope. Is there a resultant change in user performance in terms of speed of slide review, perception of adequacy and quality or in diagnostic confidence? A prior psychophysical study was carried out comparing various display modalities on whole slide imaging (WSI) in pathology at the University of Pittsburgh Medical Center (UPMC) in the USA. This prior study compared professional and non-professional grade display modalities and highlighted the importance of using a medical grade display to view pathological digital images. This study was duplicated in Europe at the Department of Pathology in Erasme Hospital (Université Libre de Bruxelles (ULB)) in an attempt to corroborate these findings. Digital WSI with corresponding glass slides of 58 cases including surgical pathology and cytopathology slides of varying difficulty were employed. Similar non-professional and professional grade display modalities were compared to an optical microscope (Olympus BX51). Displays ranged from a laptop (DELL Latitude D620), to a consumer grade display (DELL E248WFPb), to two professional grade monitors (Eizo CG245W and Barco MDCC-6130). Three pathologists were selected from the Department of Pathology in Erasme Hospital (ULB) in Belgium to view and interpret the pathological images on these different displays. The results show that non-professional grade displays (laptop and consumer) have inferior user experience compared to professional grade monitors and the optical microscope.

  17. Photothermal image cytometry of human neutrophils

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitry

    2001-07-01

    Photothermal imaging, when being applied to the study of living cells, provides morpho-functional information about the cell populations. In technical terms, the method is complementary to optical microscopy. The photothermal method was used for cell imaging and quantitative studies. Preliminary results of the studies on living human neutrophils are presented. Differences between normal and pathological neutrophil populations from blood of healthy donors and patients with saracoidosis and pleuritis are demonstrated.

  18. Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy

    PubMed Central

    Wu, Cheng-Ham; Wang, Tzung-Dau; Hsieh, Chia-Hung; Huang, Shih-Hung; Lin, Jong-Wei; Hsu, Szu-Chun; Wu, Hau-Tieng; Wu, Yao-Ming; Liu, Tzu-Ming

    2016-01-01

    Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood. PMID:27845443

  19. Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Cheng-Ham; Wang, Tzung-Dau; Hsieh, Chia-Hung; Huang, Shih-Hung; Lin, Jong-Wei; Hsu, Szu-Chun; Wu, Hau-Tieng; Wu, Yao-Ming; Liu, Tzu-Ming

    2016-11-01

    Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood.

  20. Analysis of chromosome damage for biodosimetry using imaging flow cytometry.

    PubMed

    Beaton, L A; Ferrarotto, C; Kutzner, B C; McNamee, J P; Bellier, P V; Wilkins, R C

    2013-08-30

    The dicentric chromosome assay (DCA), which involves counting the frequency of dicentric chromosomes in mitotic lymphocytes and converting it to a dose-estimation for ionizing radiation exposure, is considered to be the gold standard for radiation biodosimetry. Furthermore, for emergency response, the DCA has been adapted for triage by simplifying the scoring method [1]. With the development of new technologies such as the imaging flow cytometer, it may now be possible to adapt this microscope-based method to an automated cytometry method. This technology allows the sensitivity of microscopy to be maintained while adding the increased throughput of flow cytometry. A new protocol is being developed to adapt the DCA to the imaging cytometer in order to further increase the rapid determination of a biological dose. Peripheral blood mononuclear cells (PBMC) were isolated from ex vivo irradiated whole blood samples using a density gradient separation method and cultured with PHA and Colcemid. After 48h incubation, the chromosomes were isolated, stained for DNA content with propidium iodide (PI) and labelled with a centromere marker. Stained chromosomes were then analyzed on the ImageStream(×) (EMD-Millipore, Billerica, MA). Preliminary results indicate that individual chromosomes can be identified and mono- and dicentric chromosomes can be differentiated by imaging cytometry. A dose response curve was generated using this technology. The details of the method and the dose response curve are presented and compared to traditional microscope scoring. Imaging cytometry is a new technology which enables the rapid, automated analysis of fluorescently labelled chromosomes. Adapting the dicentric assay to this technology has the potential for high throughput analysis for mass casualty events.

  1. Lensfree holographic imaging for on-chip cytometry and diagnostics.

    PubMed

    Seo, Sungkyu; Su, Ting-Wei; Tseng, Derek K; Erlinger, Anthony; Ozcan, Aydogan

    2009-03-21

    We experimentally illustrate a lensfree holographic imaging platform to perform on-chip cytometry. By controlling the spatial coherence of the illumination source, we record a 2D holographic diffraction pattern of each cell or micro-particle on a chip using a high resolution sensor array that has approximately 2 microm pixel size. The recorded holographic image is then processed by using a custom developed decision algorithm for matching the detected hologram texture to existing library images for on-chip characterization and counting of a heterogeneous solution of interest. The holographic diffraction signature of any microscopic object is significantly different from the classical diffraction pattern of the same object. It improves the signal to noise ratio and the signature uniformity of the cell patterns; and also exhibits much better sensitivity for on-chip imaging of weakly scattering phase objects such as small bacteria or cells. We verify significantly improved performance of this holographic on-chip cytometry approach by automatically characterizing heterogeneous solutions of red blood cells, yeast cells, E. coli and various sized micro-particles without the use of any lenses or microscope objectives. This lensless on-chip holography platform will especially be useful for point-of-care cytometry and diagnostics applications involving e.g., infectious diseases such as HIV or malaria.

  2. Optofluidic fluorescent imaging cytometry on a cell phone.

    PubMed

    Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

    2011-09-01

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in

  3. Cell streak imaging cytometry for rare cell detection.

    PubMed

    Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

    2015-02-15

    Detection of rare cells, such as circulating tumor cells, have many clinical applications. To measure rare cells with increased sensitivity and improved data managements, we developed an imaging flow cytometer with a streak imaging mode capability. The new streak mode imaging mode utilizes low speed video to capture moving fluorescently labeled cells in a flow cell. Each moving cell is imaged on multiple pixels on each frame, where the cell path is marked as a streak line proportional to the length of the exposure. Finding rare cells (e.g., <1 cell/mL) requires measuring larger sample volumes to achieve higher sensitivity, therefore we combined streak mode imaging with a "wide" high throughput flow cell (e.g. flow rates set to 10 mL/min) in contrast to the conventional "narrow" hydrodynamic focusing cells typically used in cytometry that are inherently limited to low flow rates. The new flow cell is capable of analyzing 20 mL/min of fluorescently labeled cells. To further increase sensitivity, the signal to noise ratio of the images was also enhanced by combining three imaging methods: (1) background subtraction, (2) pixel binning, and (3) CMOS color channel selection. The streaking mode cytometer has been used for the analysis of SYTO-9 labeled THP-1 human monocytes in buffer and in blood. Samples of cells at 1 cell/mL and 0.1 cell/mL were analyzed in 30 mL with flow rates set to 10 mL/min and frame rates of 4 fps (frame per second). For the target of 1 cell/mL, an average concentration of 0.91 cell/mL was measured by cytometry, with a standard error of 0.03 (C(95) = 0.85-0.97). For the target of 0.1 cell/mL, an average concentration of 0.083 cell/mL was measured, with a standard error of 0.01 (C(95) = 0.065-0.102). Whole blood was also spiked with SYTO-9 labeled cells to a concentration of 10 cell/mL, and the average flow cytometry measurement was 8.7 cells/mL (i.e. 0.87 cells/mL in diluted blood) with a 95% CL of 8.1-9.2 cells/mL. This demonstrated the ability

  4. Label-free high-throughput imaging flow cytometry

    NASA Astrophysics Data System (ADS)

    Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

    2014-03-01

    Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

  5. Ultrafast quantitative time-stretch imaging flow cytometry of phytoplankton

    NASA Astrophysics Data System (ADS)

    Lai, Queenie T. K.; Lau, Andy K. S.; Tang, Anson H. L.; Wong, Kenneth K. Y.; Tsia, Kevin K.

    2016-03-01

    Comprehensive quantification of phytoplankton abundance, sizes and other parameters, e.g. biomasses, has been an important, yet daunting task in aquatic sciences and biofuel research. It is primarily because of the lack of effective tool to image and thus accurately profile individual microalgae in a large population. The phytoplankton species are highly diversified and heterogeneous in terms of their sizes and the richness in morphological complexity. This fact makes time-stretch imaging, a new ultrafast real-time optical imaging technology, particularly suitable for ultralarge-scale taxonomic classification of phytoplankton together with quantitative image recognition and analysis. We here demonstrate quantitative imaging flow cytometry of single phytoplankton based on quantitative asymmetric-detection time-stretch optical microscopy (Q-ATOM) - a new time-stretch imaging modality for label-free quantitative phase imaging without interferometric implementations. Sharing the similar concept of Schlieren imaging, Q-ATOM accesses multiple phase-gradient contrasts of each single phytoplankton, from which the quantitative phase profile is computed. We employ such system to capture, at an imaging line-scan rate of 11.6 MHz, high-resolution images of two phytoplankton populations (scenedesmus and chlamydomonas) in ultrafast microfluidic flow (3 m/s). We further perform quantitative taxonomic screening analysis enabled by this technique. More importantly, the system can also generate quantitative phase images of single phytoplankton. This is especially useful for label-free quantification of biomasses (e.g. lipid droplets) of the particular species of interest - an important task adopted in biofuel applications. Combining machine learning for automated classification, Q-ATOM could be an attractive platform for continuous and real-time ultralarge-scale single-phytoplankton analysis.

  6. Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

    PubMed

    Schimenti, K J; Lacerna, K; Wamble, A; Maston, L; Iaffaldano, C; Straight, M; Rabinovitch, A; Lazarus, H M; Jacobberger, J W

    1992-01-01

    Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis > manual counting.

  7. High content image cytometry in the context of subnuclear organization.

    PubMed

    De Vos, W H; Van Neste, L; Dieriks, B; Joss, G H; Van Oostveldt, P

    2010-01-01

    The organization of proteins in space and time is essential to their function. To accurately quantify subcellular protein characteristics in a population of cells with regard for the stochasticity of events in a natural context, there is a fast-growing need for image-based cytometry. Simultaneously, the massive amount of data that is generated by image-cytometric analyses, calls for tools that enable pattern recognition and automated classification. In this article, we present a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear spots using automated fluorescence mosaic microscopy, optimized image processing tools, and supervised classification. We demonstrate the efficiency of our analysis by determination of differential DNA damage repair patterns in response to genotoxic stress and radiation, and we show the potential of data mining in pinpointing specific phenotypes after transient transfection. The presented approach allowed for systematic analysis of subnuclear features in large image data sets and accurate classification of phenotypes at the level of the single cell. Consequently, this type of nuclear fingerprinting shows potential for high-throughput applications, such as functional protein assays or drug compound screening.

  8. Image and flow cytometry: companion techniques for adherent and non-adherent cell analysis and sorting.

    PubMed

    Métézeau, P

    1993-01-01

    Flow cytometry (FMC) is an analytical and preparative technique whereas image analysis is only applied to cell analysis. Recently, image analysis has been adapted as a preparative method using a new technique: image cytometry for analysis and sorting (ICAS). FCM and ICAS are complementary. Flow cytometry allows rapid, quantitative and precise study of fluorescence and light scattering in a large number of cells in suspension, while ICAS analyses fewer cells (adherent cells or tissue) on the basis of fluorescence, morphology and size. ICAS can use these criteria to destroy unwanted cells and hence sort selected cells. ICAS can also be used for confocal microscopy and laser surgery.

  9. Applications of imaging flow cytometry in the diagnostic assessment of acute leukaemia.

    PubMed

    Grimwade, Lizz F; Fuller, Kathryn A; Erber, Wendy N

    2017-01-01

    Automated imaging flow cytometry integrates flow cytometry with digital microscopy to produce high-resolution digital imaging with quantitative analysis. This enables cell identification based on morphology (cell size, shape), antigen expression, quantification of fluorescence signal intensity and localisation of detected signals (i.e. surface, cytoplasm, nuclear). We describe applications of imaging flow cytometry for the diagnostic assessment of acute leukaemia. These bone marrow malignancies are traditionally diagnosed and classified by cell morphology, phenotype and cytogenetic abnormalities. Traditionally morphology is assessed by light microscopy, phenotyping by conventional flow cytometry and genetics by karyotype and fluorescence in situ hybridisation (FISH) on interphase nuclei/metaphase spreads of cells on slides. Imaging flow cytometry adds a new dimension to the diagnostic assessment of these neoplasms. We describe three specific applications: From this we conclude that imaging flow cytometry offers benefits over conventional diagnostic methods. Specifically the ability to visualise the cells of interest, the pattern and localisation of expressed antigens and assess cytogenetic abnormalities in one integrated automated high-throughput test. Imaging flow cytometry presents a new paradigm for the diagnostic assessment of leukaemia.

  10. Visualization of pulmonary clearance mechanisms via noninvasive optical imaging validated by near-infrared flow cytometry.

    PubMed

    Zhou, Haiying; Gunsten, Sean P; Zhegalova, Natalia G; Bloch, Sharon; Achilefu, Samuel; Christopher Holley, J; Schweppe, Daniel; Akers, Walter; Brody, Steven L; Eades, William C; Berezin, Mikhail Y

    2015-05-01

    In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as 4-h post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies.

  11. Imaging Flow Cytometry for the Study of Erythroid Cell Biology and Pathology

    PubMed Central

    Samsel, Leigh; McCoy, J Philip

    2015-01-01

    Erythroid cell maturation and diseases affecting erythrocytes are frequently accompanied by morphologic and immunophenotypic changes to these cells. In the past, these changes have been assessed primarily through the use of manual microscopy, which substantially limits the statistical rigor, throughput, and objectivity of these studies. Imaging flow cytometry provides a technology to examine both the morphology of cells as well as to quantify the staining intensity and signal distribution of numerous fluorescent markers on a cell-by-cell basis with high throughput in a statistically robust manner, and thus is ideally suited to studying erythroid cell biology. To date imaging flow cytometry has been used to study erythrocytes in three areas: 1) erythroid cell maturation, 2) sickle cell disease, and 3) infectious diseases such as malaria. In the maturation studies, imaging flow cytometry can closely recapitulate known stages of maturation and has led to the identification of a new population of erythroid cell precursors. In sickle cell disease, imaging flow cytometry provides a robust method to quantify sickled erythrocytes and to identify cellular aggregates linked to morbidities, and in malaria, imaging flow cytometry has been used to screen for new chemotherapeutic agents. These studies have demonstrated the value of imaging flow cytometry for investigations of erythrocyte biology and pathology. PMID:25858229

  12. Quantifying autophagy: Measuring LC3 puncta and autolysosome formation in cells using multispectral imaging flow cytometry.

    PubMed

    Pugsley, Haley R

    2017-01-01

    The use of multispectral imaging flow cytometry has been gaining popularity due to its quantitative power, high throughput capabilities, multiplexing potential and its ability to acquire images of every cell. Autophagy is a process in which dysfunctional organelles and cellular components that accumulate during growth and differentiation are degraded via the lysosome and recycled. During autophagy, cytoplasmic LC3 is processed and recruited to the autophagosomal membranes; the autophagosome then fuses with the lysosome to form the autolysosome. Therefore, cells undergoing autophagy can be identified by visualizing fluorescently labeled LC3 puncta and/or the co-localization of fluorescently labeled LC3 and lysosomal markers. Multispectral imaging flow cytometry is able to collect imagery of large numbers of cells and assess autophagy in an objective, quantitative, and statistically robust manner. This review will examine the four predominant methods that have been used to measure autophagy via multispectral imaging flow cytometry.

  13. DNA tetraploidy in Feulgen-stained bladder washings assessed by image cytometry.

    PubMed

    Kline, M J; Wilkinson, E J; Askeland, R; Given, R W; Stephen, C; Hendricks, J B

    1995-04-01

    The prognostic utility of DNA cytometry has been demonstrated for irrigation specimens from bladder neoplasms. While the traditional method of measuring the DNA content of cells recovered by bladder irrigation is flow cytometry, image analysis has been applied increasingly, with successful results. In some cases, image analysis has been shown to detect DNA aneuploid populations missed by flow cytometry. The DNA aneuploid population most frequently missed by flow cytometry is in the DNA tetraploid range. The purpose of the present study was to review image cytometry data on bladder washings analyzed at the University of Florida Diagnostic Referral Laboratories during a one-year period, with special emphasis on the subset with DNA tetraploid histograms. Of the 205 cases reviewed, 127 (62%) were DNA diploid, 36 (18%) DNA aneuploid and 42 (20%) DNA tetraploid. Corresponding cytology was negative in 113/127 (89%) of DNA diploid, 3/36 (8%) of DNA aneuploid and 29/42 (69%) of DNA tetraploid cases. Within the DNA tetraploid group, 45% of cases had no clinical (cystoscopic) or pathologic (cytologic and histologic) evidence of neoplasia. None of these patients developed tumors during follow-up. The presence of DNA tetraploidy in cytologically negative cases should be interpreted cautiously.

  14. Quantitative analysis of centromeric FISH spots during the cell cycle by image cytometry.

    PubMed

    Amakawa, Genta; Ikemoto, Kenzo; Ito, Hideaki; Furuya, Tomoko; Sasaki, Kohsuke

    2013-10-01

    Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G2 phase was larger than that in the G0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G2 phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.

  15. Visualization of Pulmonary Clearance Mechanisms via Noninvasive Optical Imaging Validated by Near-Infrared Flow Cytometry

    PubMed Central

    Zhou, Haiying; Gunsten, Sean P.; Zhegalova, Natalia G.; Bloch, Sharon; Achilefu, Samuel; Holley, J. Christopher; Schweppe, Daniel; Akers, Walter; Brody, Steven L.; Eades, William; Berezin, Mikhail Y.

    2016-01-01

    In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as four-hours post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies. PMID:25808737

  16. Intercellular carbon nanotube translocation assessed by flow cytometry imaging.

    PubMed

    Marangon, Iris; Boggetto, Nicole; Ménard-Moyon, Cécilia; Venturelli, Enrica; Béoutis, Marie-Lys; Péchoux, Christine; Luciani, Nathalie; Wilhelm, Claire; Bianco, Alberto; Gazeau, Florence

    2012-09-12

    The fate of carbon nanotubes in the organism is still controversial. Here, we propose a statistical high-throughput imaging method to localize and quantify functionalized multiwalled carbon nanotubes in cells. We give the first experimental evidence of an intercellular translocation of carbon nanotubes. This stress-induced longitudinal transfer of nanomaterials is mediated by cell-released microvesicles known as vectors for intercellular communication. This finding raises new critical issues for nanotoxicology, since carbon nanotubes could be disseminated by circulating extracellular cell-released vesicles and visiting several cells in the course of their passage into the organism.

  17. A deep semantic mobile application for thyroid cytopathology

    NASA Astrophysics Data System (ADS)

    Kim, Edward; Corte-Real, Miguel; Baloch, Zubair

    2016-03-01

    Cytopathology is the study of disease at the cellular level and often used as a screening tool for cancer. Thyroid cytopathology is a branch of pathology that studies the diagnosis of thyroid lesions and diseases. A pathologist views cell images that may have high visual variance due to different anatomical structures and pathological characteristics. To assist the physician with identifying and searching through images, we propose a deep semantic mobile application. Our work augments recent advances in the digitization of pathology and machine learning techniques, where there are transformative opportunities for computers to assist pathologists. Our system uses a custom thyroid ontology that can be augmented with multimedia metadata extracted from images using deep machine learning techniques. We describe the utilization of a particular methodology, deep convolutional neural networks, to the application of cytopathology classification. Our method is able to leverage networks that have been trained on millions of generic images, to medical scenarios where only hundreds or thousands of images exist. We demonstrate the benefits of our framework through both quantitative and qualitative results.

  18. Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

  19. Assessment of Granulocyte Subset Activation: New Information from Image-Based Flow Cytometry.

    PubMed

    McFarlin, Brian K; Venable, Adam S; Henning, Andrea L; Williams, Randall R; Prado, Eric A

    2016-01-01

    Analysis of granulocyte function can provide important information about the state of the body's innate immune system. Existing flow cytometry methods that lack image-based analysis capabilities fail to fully evaluate granulocyte function. In the present method, we combine simultaneous detection of phagocytosis and oxidative burst in granulocytes to identify unique subsets of activated granulocytes. This analysis method provides novel information about granulocytes that allows our lab and others to evaluate the effectiveness of nutritional and lifestyle countermeasures, designed to improve immunity.

  20. A telepathology based Virtual Reference and Certification Centre for DNA image cytometry.

    PubMed

    Haroske, G; Giroud, F; Kunze, K D; Meyer, W

    2000-01-01

    An increasing need for flexible consultation between pathologists, including the application of fast evolving supplementary technologies, has been identified during the last years. Although pathology is already one of the most advanced application of telemedicine there is more to come from the fast evolution towards computerized microscope image analysis: A reproducible quantification of measurable descriptors of the lesions in cells and tissues (so-called biological markers) is an indispensable adjunct to routine diagnostic application. Among such quantitative methods DNA image cytometry is increasingly applied by pathologists for assistance in diagnostics. As for other pathological issues, too, a reference center for the clinical application of DNA image cytometry might be therefore of utmost value for pathologists using that method. Based on advanced telematic technologies, a Virtual Reference and Certification Center (VRCC) could be installed for certifying the cytometry hardware and software, the analytical procedures, and the basic interpretation of the results. It will be designed to be operated as a non-attended service, based on quantification servers accessible via Internet round the clock. The VRCC will supply appropriate standardization and normalization materials and run a GroupWare platform for consensus making by experts.

  1. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    NASA Astrophysics Data System (ADS)

    Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J.

    2015-09-01

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min-1 with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels-1.

  2. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    SciTech Connect

    Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J.

    2015-09-15

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min{sup −1} with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels{sup −1}.

  3. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization.

    PubMed

    Hutcheson, Joshua A; Majid, Aneeka A; Powless, Amy J; Muldoon, Timothy J

    2015-09-01

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min(-1) with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels(-1).

  4. Cytopathologic diagnosis on joint lavage fluid for patients with temporomandibular joint disorders.

    PubMed

    Mikami, Toshinari; Kumagai, Akiko; Aomura, Tomoyuki; Javed, Fawad; Sugiyama, Yoshiki; Mizuki, Harumi; Takeda, Yasunori

    2014-01-01

    Temporomandibular joint (TMJ) disorders (TMD) are usually diagnosed based on the patient's clinical findings and the results of image investigations; however, understanding of the inflammatory process in TMJ is difficult. In addition, many of the TMJ disease types share common principal symptoms. Therefore, TMJ diseases in the early stage can be misdiagnosed with TMD. It is hypothesized that cytopathologic examination of the joint lavage fluids is useful in interpreting the TMD-associated inflammatory process from a cellular aspect. The aim of this study was to assess the TMJ lavage fluid cytopathologically in TMD patients. Thirty-nine patients, clinically diagnosed as TMD, were included in the present study. Clinical symptoms of the patients were recorded. Forty-four samples of TMJ lavage fluid were collected and paraffin-embedded cell sections were made by cell block tissue array method. Cytologic conditions in upper articular cavity of TMJ were cytopathologically diagnosed and were compared with the clinical symptoms of each patient. Cell components were detected in 22 of the 44 analyzed joint lavage fluids. There was a correlation between cytopathologic findings and clinical symptoms. Variety of cytopathology and inflammatory conditions in patients with similar clinical symptoms were also found. The results suggested that cytopathologic examination of the joint lavage fluids from TMD patients is helpful for gaining an understanding of the inner local conditions of TMJ at the cellular level.

  5. A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.

    PubMed

    Somanchi, Srinivas S; McCulley, Kelsey J; Somanchi, Anitha; Chan, Leo L; Lee, Dean A

    2015-01-01

    Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the "gold standard" for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.

  6. Triggering of leukocytes by phase contrast in imaging cytometry with scanning fluorescence microscope (SFM)

    NASA Astrophysics Data System (ADS)

    Bocsi, József; Pierzchalski, Arkadiusz; Marecka, Monika; Malkusch, Wolf; Tárnok, Attila

    2009-02-01

    Slide-based cytometry (SBC) leads to breakthrough in cytometry of cells in tissues, culture and suspension. Carl Zeiss Imaging Solutions' new automated SFM combines imaging with cytometry. A critical step in image analysis is selection of appropriate triggering signal to detect all objects. Without correct target cell definition analysis is hampered. DNA-staining is among the most common triggering signals. However, the majority of DNA-dyes yield massive spillover into other fluorescence channels limiting their application. By microscopy objects of >5μm diameter can be easily detected by phase-contrast signal (PCS) without any staining. Aim was to establish PCS - triggering for cell identification. Axio Imager.Z1 motorized SFM was used (high-resolution digital camera, AxioCam MRm; AxioVision software: automatic multi-channel scanning, analysis). Leukocytes were stained with FITC (CD4, CD8) and APC (CD3) labelled antibodies in combinations using whole blood method. Samples were scanned in three channels (PCS/FITC/APC). Exposition-times for PCS were set as low as possible; the detection efficiency was verified by fluorescence. CD45-stained leukocytes were counted and compared to the number of PCS detected events. Leukocyte subtyping was compared with other cytometers. In focus the PCS of cells showed ring-form that was not optimal for cell definition. Out of focus PCS allows more effective qualitative and quantitative cell analyses. PCS was an accurate triggering signal for leukocytes enabling cell counting and discrimination of leukocytes from platelets. Leukocyte subpopulation frequencies were comparable to those obtained by other cytometers. In conclusion PCS is a suitable trigger-signal not interfering with fluorescence detection.

  7. Quantitative morphometric measurements using site selective image cytometry of intact tissue.

    PubMed

    Kwon, Hyuk-Sang; Nam, Yoon Sung; Wiktor-Brown, Dominika M; Engelward, Bevin P; So, Peter T C

    2009-02-06

    Site selective two-photon tissue image cytometry has previously been successfully applied to measure the number of rare cells in three-dimensional tissue specimens up to cubic millimetres in size. However, the extension of this approach for high-throughput quantification of cellular morphological states has not been demonstrated. In this paper, we report the use of site-selective tissue image cytometry for the study of homologous recombination (HR) events during cell division in the pancreas of transgenic mice. Since HRs are rare events, recombinant cells distribute sparsely inside the organ. A detailed measurement throughout the whole tissue is thus not practical. Instead, the site selective two-photon tissue cytometer incorporates a low magnification, wide field, one-photon imaging subsystem that rapidly identifies regions of interest containing recombinant cell clusters. Subsequently, high-resolution three-dimensional assays based on two-photon microscopy can be performed only in these regions of interest. We further show that three-dimensional morphology extraction algorithms can be used to analyse the resultant high-resolution two-photon image stacks providing information not only on the frequency and the distribution of these recombinant cell clusters and their constituent cells, but also on their morphology.

  8. Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics†

    PubMed Central

    de la Zerda, Adam; Kim, Jin-Woo; Galanzha, Ekaterina I.; Gambhir, Sanjiv S.; Zharov, Vladimir P.

    2013-01-01

    Various nanoparticles have raised significant interest over the past decades for their unique physical and optical properties and biological utilities. Here we summarize the vast applications of advanced nanoparticles with a focus on carbon nanotube (CNT)-based or CNT-catalyzed contrast agents for photoacoustic (PA) imaging, cytometry and theranostics applications based on the photothermal (PT) effect. We briefly review the safety and potential toxicity of the PA/PT contrast nanoagents, while showing how the physical properties as well as multiple biological coatings change their toxicity profiles and contrasts. We provide general guidelines needed for the validation of a new molecular imaging agent in living subjects, and exemplify these guidelines with single-walled CNTs targeted to αvβ3, an integrin associated with tumor angiogenesis, and golden carbon nanotubes targeted to LYVE-1, endothelial lymphatic receptors. An extensive review of the potential applications of advanced contrast agents is provided, including imaging of static targets such as tumor angiogenesis receptors, in vivo cytometry of dynamic targets such as circulating tumor cells and nanoparticles in blood, lymph, bones and plants, methods to enhance the PA and PT effects with transient and stationary bubble conjugates, PT/PA Raman imaging and multispectral histology. Finally, theranostic applications are reviewed, including the nanophotothermolysis of individual tumor cells and bacteria with clustered nanoparticles, nanothrombolysis of blood clots, detection and purging metastasis in sentinel lymph nodes, spectral hole burning and multiplex therapy with ultrasharp rainbow nanoparticles. PMID:22025336

  9. An open-source solution for advanced imaging flow cytometry data analysis using machine learning.

    PubMed

    Hennig, Holger; Rees, Paul; Blasi, Thomas; Kamentsky, Lee; Hung, Jane; Dao, David; Carpenter, Anne E; Filby, Andrew

    2017-01-01

    Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using "user-friendly" platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data sets. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery.

  10. Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method.

    PubMed

    Laverty, Daniel J; Kury, Alexandria L; Kuksin, Dmitry; Pirani, Alnoor; Flanagan, Kevin; Chan, Leo Li-Ying

    2013-06-01

    The measurements of concentration, viability, and budding percentages of Saccharomyces cerevisiae are performed on a routine basis in the brewing and biofuel industries. Generation of these parameters is of great importance in a manufacturing setting, where they can aid in the estimation of product quality, quantity, and fermentation time of the manufacturing process. Specifically, budding percentages can be used to estimate the reproduction rate of yeast populations, which directly correlates with metabolism of polysaccharides and bioethanol production, and can be monitored to maximize production of bioethanol during fermentation. The traditional method involves manual counting using a hemacytometer, but this is time-consuming and prone to human error. In this study, we developed a novel automated method for the quantification of yeast budding percentages using Cellometer image cytometry. The automated method utilizes a dual-fluorescent nucleic acid dye to specifically stain live cells for imaging analysis of unique morphological characteristics of budding yeast. In addition, cell cycle analysis is performed as an alternative method for budding analysis. We were able to show comparable yeast budding percentages between manual and automated counting, as well as cell cycle analysis. The automated image cytometry method is used to analyze and characterize corn mash samples directly from fermenters during standard fermentation. Since concentration, viability, and budding percentages can be obtained simultaneously, the automated method can be integrated into the fermentation quality assurance protocol, which may improve the quality and efficiency of beer and bioethanol production processes.

  11. Single point vs. mapping approach for spectral cytopathology (SCP).

    PubMed

    Schubert, Jennifer M; Mazur, Antonella I; Bird, Benjamin; Miljković, Milos; Diem, Max

    2010-08-01

    In this paper we describe the advantages of collecting infrared microspectral data in imaging mode opposed to point mode. Imaging data are processed using the PapMap algorithm, which co-adds pixel spectra that have been scrutinized for R-Mie scattering effects as well as other constraints. The signal-to-noise quality of PapMap spectra will be compared to point spectra for oral mucosa cells deposited onto low-e slides. Also the effects of software atmospheric correction will be discussed. Combined with the PapMap algorithm, data collection in imaging mode proves to be a superior method for spectral cytopathology.

  12. Analysis of Individual Molecular Events of DNA Damage Response by Flow and Image Assisted Cytometry

    PubMed Central

    Darzynkiewicz, Zbigniew; Traganos, Frank; Zhao, Hong; Halicka, H. Dorota; Skommer, Joanna; Wlodkowic, Donald

    2010-01-01

    This chapter describes molecular mechanisms of DNA damage response (DDR) and presents flow- and image-assisted cytometric approaches to assess these mechanisms and measure the extent of DDR in individual cells. DNA damage was induced by cell treatment with oxidizing agents, UV light, DNA topoisomerase I or II inhibitors, cisplatin, tobacco smoke, and by exogenous and endogenous oxidants. Chromatin relaxation (decondensation) is an early event of DDR chromatin that involves modification of high mobility group proteins (HMGs) and histone H1 and was detected by cytometry by analysis of the susceptibility of DNA in situ to denaturation using the metachromatic fluorochrome acridine orange. Translocation of the MRN complex consisting of Meiotic Recombination 11 Homolog A (Mre11), Rad50 homolog and Nijmegen Breakage Syndrome 1 (NMR1) into DNA damage sites was assessed by laser scanning cytometry as the increase in the intensity of maximal pixel as well as integral value of Mre11 immunofluorescence. Examples of cytometric detection of activation of Ataxia telangiectasia mutated (ATM), and Check 2 (Chk2) protein kinases using phospho-specific Abs targeting Ser1981 and Thr68 of these proteins, respectively are also presented. We also discuss approaches to correlate activation of ATM and Chk2 with phosphorylation of p53 on Ser15 and histone H2AX on Ser139 as well as with cell cycle position and DNA replication. The capability of laser scanning cytometry to quantify individual foci of phosphorylated H2AX and/or ATM that provides more dependable assessment of the presence of DNA double-strand breaks is outlined. The new microfluidic Lab-on-a-Chip platforms for interrogation of individual cells offer a novel approach for DDR cytometric analysis. PMID:21722802

  13. High-throughput label-free image cytometry and image-based classification of live Euglena gracilis.

    PubMed

    Lei, Cheng; Ito, Takuro; Ugawa, Masashi; Nozawa, Taisuke; Iwata, Osamu; Maki, Masanori; Okada, Genki; Kobayashi, Hirofumi; Sun, Xinlei; Tiamsak, Pimsiri; Tsumura, Norimichi; Suzuki, Kengo; Di Carlo, Dino; Ozeki, Yasuyuki; Goda, Keisuke

    2016-07-01

    We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-Hz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner.

  14. High-throughput label-free image cytometry and image-based classification of live Euglena gracilis

    PubMed Central

    Lei, Cheng; Ito, Takuro; Ugawa, Masashi; Nozawa, Taisuke; Iwata, Osamu; Maki, Masanori; Okada, Genki; Kobayashi, Hirofumi; Sun, Xinlei; Tiamsak, Pimsiri; Tsumura, Norimichi; Suzuki, Kengo; Di Carlo, Dino; Ozeki, Yasuyuki; Goda, Keisuke

    2016-01-01

    We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-Hz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner. PMID:27446699

  15. Using multispectral imaging flow cytometry to assess an in vitro intracellular Burkholderia thailandensis infection model.

    PubMed

    Jenner, Dominic; Ducker, Catherine; Clark, Graeme; Prior, Jo; Rowland, Caroline A

    2016-04-01

    The use of in vitro models to understand the interaction of bacteria with host cells is well established. In vitro bacterial infection models are often used to quantify intracellular bacterial load by lysing cell populations and subsequently enumerating the bacteria. Modern established techniques employ the use of fluorescence technologies such as flow cytometry, fluorescent microscopy, and/or confocal microscopy. However, these techniques often lack either the quantification of large data sets (microscopy) or use of gross fluorescence signal which lacks the visual confirmation that can provide additional confidence in data sets. Multispectral imaging flow cytometry (MIFC) is a novel emerging field of technology. This technology captures a bright field and fluorescence image of cells in a flow using a charged coupled device camera. It allows the analysis of tens of thousands of single cell images, making it an extremely powerful technology. Here MIFC was used as an alternative method of analyzing intracellular bacterial infection using Burkholderia thailandensis E555 as a model organism. It has been demonstrated that the data produced using traditional enumeration is comparable to data analyzed using MIFC. It has also been shown that by using MIFC it is possible to generate other data on the dynamics of the infection model rather than viable counts alone. It has been demonstrated that it is possible to inhibit the uptake of bacteria into mammalian cells and identify differences between treated and untreated cell populations. The authors believe this to be the first use of MIFC to analyze a Burkholderia bacterial species during intracellular infection. © 2016 Crown copyright. Published by Wiley Periodicals Inc. on behalf of ISAC.

  16. Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry.

    PubMed

    Beaton-Green, Lindsay A; Rodrigues, Matthew A; Lachapelle, Sylvie; Wilkins, Ruth C

    2017-01-01

    Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®(X) (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

  17. Computer-aided cytological cancer diagnosis: cell type classification as a step towards fully automatic cancer diagnostics on cytopathological specimens of serous effusions

    NASA Astrophysics Data System (ADS)

    Schneider, Timna E.; Bell, André A.; Meyer-Ebrecht, Dietrich; Böcking, Alfred; Aach, Til

    2007-03-01

    Compared to histopathological methods cancer can be detected earlier, specimens can be obtained easier and with less discomfort for the patient by cytopathological methods. Their downside is the time needed by an expert to find and select the cells to be analyzed on a specimen. To increase the use of cytopathological diagnostics, the cytopathologist has to be supported in this task. DNA image cytometry (DNA-ICM) is one important cytopathological method that measures the DNA content of cells based on the absorption of light within Feulgen stained cells. The decision whether or not the patient has cancer is based on the histogram of the DNA values. To support the cytopathologist it is desirable to replace manual screening of the specimens by an automatic selection of relevant cells for DNA-ICM. This includes automated acquisition and segmentation of focused cells, a recognition of cell types, and a selection of cells to be measured. As a step towards automated cell type detection we show the discrimination of cell types in serous effusions on a selection of about 3, 100 manually classified cells. We present a set of 112 features and the results of feature selection with ranking and a floating-search method combined with different objective functions. The validation of the best feature sets with a k-nearest neighbor and a fuzzy k-nearest neighbor classifier on a disjoint set of cells resulted in classification rates of 96% for lymphocytes and 96.8% for the diagnostically relevant cells (mesothelial+ cells), which includes benign and malign mesothelial cells and metastatic cancer cells.

  18. Analysis of DNA-guided self-assembly of microspheres using imaging flow cytometry.

    PubMed

    Tang, Hao; Deschner, Ryan; Allen, Peter; Cho, Younjin; Sermas, Patrick; Maurer, Alejandro; Ellington, Andrew D; Willson, C Grant

    2012-09-19

    Imaging flow cytometry was used to analyze the self-assembly of DNA-conjugated polystyrene microspheres. This technique enables quantitative analysis of the assembly process and thereby enables detailed analysis of the effect of structural and process variables on the assembly yield. In a demonstration of the potential of this technique, the influence of DNA strand base pair (bp) length was examined, and it was found that 50 bp was sufficient to drive the assembly of microspheres efficiently, forming not only dimers but also chainlike structures. The effect of stoichiometry on the yield was also examined. The analysis demonstrated that self-assembly of 50 bp microspheres can be driven nearly to completion by stoichiometric excess in a manner similar to Le Chatelier's principle in common chemical equilibrium.

  19. Characterization of functional variables in epididymal alpaca (Vicugna pacos) sperm using imaging flow cytometry.

    PubMed

    Santiani, Alexei; Ugarelli, Alejandra; Evangelista-Vargas, Shirley

    2016-10-01

    Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model.

  20. Study of low speed flow cytometry for diffraction imaging with different chamber and nozzle designs.

    PubMed

    Sa, Yu; Feng, Yuanming; Jacobs, Kenneth M; Yang, Jun; Pan, Ran; Gkigkitzis, Ioannis; Lu, Jun Q; Hu, Xin-Hua

    2013-11-01

    Achieving effective hydrodynamic focusing and flow stability at low speed presents a challenging design task in flow cytometry for studying phenomena such as cell adhesion and diffraction imaging of cells with low-cost cameras. We have developed different designs of flow chamber and sheath nozzle to accomplish the above goal. A 3D computational model of the chambers has been established to simulate the fluid dynamics in different chamber designs and measurements have been performed to determine the velocity and size distributions of the core fluid from the nozzle. Comparison of the simulation data with experimental results shows good agreement. With the computational model significant insights were gained for optimization of the chamber design and improvement of the cell positioning accuracy for study of slow moving cells. The benefit of low flow speed has been demonstrated also by reduced blurring in the diffraction images of single cells. Based on these results, we concluded that the new designs of chamber and sheath nozzle produce stable hydrodynamic focusing of the core fluid at low speed and allow detailed study of cellular morphology under various rheological conditions using the diffraction imaging method.

  1. Photothermal Multispectral Image Cytometry for Quantitative Histology of Nanoparticles and Micrometastasis in Intact, Stained and Laser Burned Tissues

    PubMed Central

    Nedosekin, Dmitry A.; Shashkov, Evgeny V.; Galanzha, Ekaterina I.; Hennings, Leah; Zharov, Vladimir P.

    2012-01-01

    There is a rapidly growing interest in the advanced analysis of histological data and the development of appropriate detection technologies, including mapping of nanoparticle distributions in tissue in nanomedicine applications. We evaluated photothermal (PT) scanning cytometry for color-coded imaging, spectral identification, and quantitative detection of individual nanoparticles and abnormal cells in histological samples with and without staining. Using this tool, individual carbon nanotubes, gold nanorods, and melanoma cells with intrinsic melanin markers were identified in unstained (e.g. sentinel lymph nodes) and conventionally-stained tissues. In addition, we introduced a spectral burning technique for histology through selective laser bleaching areas with nondesired absorption background and nanobubble-based PT signal amplification. The obtained data demonstrated the promise of PT cytometry in the analysis of low-absorption samples and mapping of various individual nanoparticles' distribution that would be impossible with existing assays. Comparison of PT cytometry and photoacoustic (PA) cytometry previously, developed by us, revealed that these methods supplement each other with a sensitivity advantage (up to 10-fold) of contactless PT technique in assessment of thin (≤100 μm) histological samples, while PA imaging provides characterization of thicker samples which, however, requires an acoustic contact with transducers. A potential of high-speed integrated PT–PA cytometry for rapid examination of both intact and stained heterogeneous tissues with high sensitivity at the zepromolar concentration level is further highlighted. PMID:20949577

  2. Romanowsky staining in cytopathology: history, advantages and limitations.

    PubMed

    Krafts, K P; Pambuccian, S E

    2011-04-01

    If the entire discipline of diagnostic cytopathology could be distilled into a single theme, it would be the Papanicolaou stain. Yet it was the Romanowsky stain upon which the discipline of cytopathology was founded. Both stains are used today in the cytopathology laboratory, each for a different and complementary purpose. We trace the history of cytopathological stains and discuss the advantages and limitations of Romanowsky-type stains for cytological evaluation. We also provide suggestions for the advantageous use of Romanowsky-type stains in cytopathology.

  3. Morphologic changes in rat urothelial cells during carcinogenesis. II. Image cytometry

    SciTech Connect

    Young, I.T.; Vanderlaan, M.; Kromhout, L.; Jensen, R.; Grover, A.; King, E.

    1984-01-01

    Improved early detection of neoplasia by screening of urothelial cells requires an understanding of the features distinguishing normal and neoplastic cell populations. The authors have begun a program of study based upon a rate model system for the controlled observation of early-stage lesions produced by the carcinogen N-butyl-N-(4-hydroxybutyl)- nitrosamine. Cells dissociated directly from normal and malignant urothelium were characterized by conventional cytopathology techniques and by quantitative microscopy (for nuclear texture and nuclear and cytoplasmic size, shape, and stain content) to derive a comprehensive picture of bladder tumor development. By following the changes that occur in the dissociated urothelial cells the authors have found that the nuclear area, total nuclear stain, nuclear shape, and the nuclear chromatin change significantly over a 48-wk interval as the lesions progress toward malignancy. 24 references, 10 figures, 1 table.

  4. CMOS based image cytometry for detection of phytoplankton in ballast water

    PubMed Central

    Pérez, J. M.; Jofre, M.; Martínez, P.; Yáñez, M. A.; Catalan, V.; Parker, A.; Veldhuis, M.; Pruneri, V.

    2017-01-01

    We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results. PMID:28271014

  5. CMOS based image cytometry for detection of phytoplankton in ballast water.

    PubMed

    Pérez, J M; Jofre, M; Martínez, P; Yáñez, M A; Catalan, V; Parker, A; Veldhuis, M; Pruneri, V

    2017-02-01

    We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results.

  6. Direct concentration and viability measurement of yeast in corn mash using a novel imaging cytometry method.

    PubMed

    Chan, Leo L; Lyettefi, Emily J; Pirani, Alnoor; Smith, Tim; Qiu, Jean; Lin, Bo

    2011-08-01

    Worldwide awareness of fossil-fuel depletion and global warming has been increasing over the last 30 years. Numerous countries, including the USA and Brazil, have introduced large-scale industrial fermentation facilities for bioethanol, biobutanol, or biodiesel production. Most of these biofuel facilities perform fermentation using standard baker's yeasts that ferment sugar present in corn mash, sugar cane, or other glucose media. In research and development in the biofuel industry, selection of yeast strains (for higher ethanol tolerance) and fermentation conditions (yeast concentration, temperature, pH, nutrients, etc.) can be studied to optimize fermentation performance. Yeast viability measurement is needed to identify higher ethanol-tolerant yeast strains, which may prolong the fermentation cycle and increase biofuel output. In addition, yeast concentration may be optimized to improve fermentation performance. Therefore, it is important to develop a simple method for concentration and viability measurement of fermenting yeast. In this work, we demonstrate an imaging cytometry method for concentration and viability measurements of yeast in corn mash directly from operating fermenters. It employs an automated cell counter, a dilution buffer, and staining solution from Nexcelom Bioscience to perform enumeration. The proposed method enables specific fluorescence detection of viable and nonviable yeasts, which can generate precise results for concentration and viability of yeast in corn mash. This method can provide an essential tool for research and development in the biofuel industry and may be incorporated into manufacturing to monitor yeast concentration and viability efficiently during the fermentation process.

  7. Circulating tumor cell detection in hepatocellular carcinoma based on karyoplasmic ratios using imaging flow cytometry

    PubMed Central

    Liu, Zixin; Guo, Weixing; Zhang, Dandan; Pang, Yanan; Shi, Jie; Wan, Siqin; Cheng, Kai; Wang, Jiaqi; Cheng, Shuqun

    2016-01-01

    Circulating tumor cells (CTCs) originate from tumor tissues and are associated with cancer prognosis. However, existing technologies for CTC detection are limited owing to a lack of specific or accurate biomarkers. Here, we developed a new method for CTC detection based on the karyoplasmic ratio, without biomarkers. Consecutive patients with liver cancer or non-cancer liver diseases were recruited. CTCs in blood samples were analyzed by imaging flow cytometry based on the karyoplasmic ratio as well as EpCAM and CD45. Microvascular invasion (MVI), tumor recurrence, and survival were recorded for all patients. A total of 56.2 ± 23.8/100,000 cells with high karyoplasmic ratios (HKR cells) were detected in cancer patients, which was higher than the number of HKR cells in the non-cancer group (7.6 ± 2.2/100,000). There was also a difference in HKR cells between liver cancer patients with and without MVI. Based on a receiver operating characteristic curve analysis, the threshold was 21.8 HKR cells per 100,000 peripheral blood mononuclear cells, and the area under the curve was higher than those of traditional methods (e.g., CD45 and EpCAM staining). These results indicate that the new CTC detection method was more sensitive and reliable than existing methods. Accordingly, it may improve clinical CTC detection. PMID:28009002

  8. Rapid Patterning of 1-D Collagenous Topography as an ECM Protein Fibril Platform for Image Cytometry

    PubMed Central

    Xue, Niannan; Li, Xia; Bertulli, Cristina; Li, Zhaoying; Patharagulpong, Atipat; Sadok, Amine; Huang, Yan Yan Shery

    2014-01-01

    Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among

  9. Biodistribution of cisplatin revealed by imaging mass cytometry identifies extensive collagen binding in tumor and normal tissues

    PubMed Central

    Chang, Qing; Ornatsky, Olga I.; Siddiqui, Iram; Straus, Rita; Baranov, Vladimir I.; Hedley, David W.

    2016-01-01

    Imaging mass cytometry was used for direct visualization of platinum localization in tissue sections from tumor and normal tissues of cisplatin-treated mice bearing pancreas cancer patient-derived xenografts. This recently-developed technology enabled simultaneous detection of multiple markers to define cell lineage, DNA damage response, cell proliferation and functional state, providing a highly detailed view of drug incorporation in tumor and normal tissues at the cellular level. A striking and unanticipated finding was the extensive binding of platinum to collagen fibers in both tumor and normal mouse tissues. Time course experiments indicated the slow release of stroma-bound platinum, although it is currently unclear if released platinum retains biological activity. Imaging mass cytometry offers a unique window into the in vivo effects of platinum compounds, and it is likely that this can be extended into the clinic in order to optimize the use of this important class of agent. PMID:27812005

  10. In vivo imaging flow cytometry based on laser scanning two-photon microscopy at kHz cross-sectional frame rate

    NASA Astrophysics Data System (ADS)

    Kong, Lingjie; Tang, Jianyong; Cui, Meng

    2016-03-01

    In vivo flow cytometry has found numerous applications in biology and pharmacology. However, conventional cytometry does not provide the detailed morphological information that is needed to fully determine the phenotype of individual circulating cells. Imaging cytometry, capable of visualizing the morphology and dynamics of the circulating cells at high spatiotemporal resolution, is highly desired. Current wide-field based image cytometers are limited in the imaging depth and provide only two-dimensional resolution. For deep tissue imaging, laser scanning two-photon fluorescence microscopy (TPM) is widely adopted. However, for applications in flow cytometry, the axial scanning speed of current TPMs is inadequate to provide high-speed cross-sectional imaging of vasculature. We have integrated an optical phase-locked ultrasound lens into a standard TPM and achieved microsecond-scale axial scanning. With a galvo scanner for transverse scanning, we achieved kHz cross-sectional frame rate. Here we report its applications for in vivo deformability cytometry and in vivo imaging flow cytometry, and demonstrate the capability of imaging dynamical morphologies of flowing cells, distinguishing cells and cellular clusters, and simultaneously quantifying different cell populations based on their fluorescent labels.

  11. Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.

    PubMed

    van Beers, Eduard J; Samsel, Leigh; Mendelsohn, Laurel; Saiyed, Rehan; Fertrin, Kleber Y; Brantner, Christine A; Daniels, Mathew P; Nichols, James; McCoy, J Philip; Kato, Gregory J

    2014-06-01

    In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = -0.558, P = 0.027), negatively with pH (R = -0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = -0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.

  12. Cytometry standards continuum

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.; Spidlen, Josef; Brinkman, Ryan R.

    2008-02-01

    Introduction: The International Society for Analytical Cytology, ISAC, is developing a new combined flow and image Analytical Cytometry Standard (ACS). This standard needs to serve both the research and clinical communities. The clinical medicine and clinical research communities have a need to exchange information with hospital and other clinical information systems. Methods: 1) Prototype the standard by creating CytometryML and a RAW format for binary data. 2) Join the ISAC Data Standards Task Force. 3) Create essential project documentation. 4) Cooperate with other groups by assisting in the preparation of the DICOM Supplement 122: Specimen Module and Pathology Service-Object Pair Classes. Results: CytometryML has been created and serves as a prototype and source of experience for the following: the Analytical Cytometry Standard (ACS) 1.0, the ACS container, Minimum Information about a Flow Cytometry Experiment (MIFlowCyt), and Requirements for a Data File Standard Format to Describe Flow Cytometry and Related Analytical Cytology Data. These requirements provide a means to judge the appropriateness of design elements and to develop tests for the final ACS. The requirements include providing the information required for understanding and reproducing a cytometry experiment or clinical measurement, and for a single standard for both flow and digital microscopic cytometry. Schemas proposed by other members of the ISAC Data Standards Task Force (e.g, Gating-ML) have been independently validated and have been integrated with CytometryML. The use of netCDF as an element of the ACS container has been proposed by others and a suggested method of its use is proposed.

  13. A quantitative method for measurement of HL-60 cell apoptosis based on diffraction imaging flow cytometry technique.

    PubMed

    Yang, Xu; Feng, Yuanming; Liu, Yahui; Zhang, Ning; Lin, Wang; Sa, Yu; Hu, Xin-Hua

    2014-07-01

    A quantitative method for measurement of apoptosis in HL-60 cells based on polarization diffraction imaging flow cytometry technique is presented in this paper. Through comparative study with existing methods and the analysis of diffraction images by a gray level co-occurrence matrix algorithm (GLCM), we found 4 GLCM parameters of contrast (CON), cluster shade (CLS), correlation (COR) and dissimilarity (DIS) exhibit high sensitivities as the apoptotic rates. It was further demonstrated that the CLS parameter correlates significantly (R(2) = 0.899) with the degree of nuclear fragmentation and other three parameters showed a very good correlations (R(2) ranges from 0.69 to 0.90). These results demonstrated that the new method has the capability for rapid and accurate extraction of morphological features to quantify cellular apoptosis without the need for cell staining.

  14. A quantitative method for measurement of HL-60 cell apoptosis based on diffraction imaging flow cytometry technique

    PubMed Central

    Yang, Xu; Feng, Yuanming; Liu, Yahui; Zhang, Ning; Lin, Wang; Sa, Yu; Hu, Xin-Hua

    2014-01-01

    A quantitative method for measurement of apoptosis in HL-60 cells based on polarization diffraction imaging flow cytometry technique is presented in this paper. Through comparative study with existing methods and the analysis of diffraction images by a gray level co-occurrence matrix algorithm (GLCM), we found 4 GLCM parameters of contrast (CON), cluster shade (CLS), correlation (COR) and dissimilarity (DIS) exhibit high sensitivities as the apoptotic rates. It was further demonstrated that the CLS parameter correlates significantly (R2 = 0.899) with the degree of nuclear fragmentation and other three parameters showed a very good correlations (R2 ranges from 0.69 to 0.90). These results demonstrated that the new method has the capability for rapid and accurate extraction of morphological features to quantify cellular apoptosis without the need for cell staining. PMID:25071957

  15. Cytometry metadata in XML

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.; Leif, Stephanie H.

    2016-04-01

    Introduction: The International Society for Advancement of Cytometry (ISAC) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). CytometryML will serve as a common metadata standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). The datatypes are primarily based on the Flow Cytometry and the Digital Imaging and Communication (DICOM) standards. A small section of the code was formatted with standard HTML formatting elements (p, h1, h2, etc.). Results:1) The part of MIFlowCyt that describes the Experimental Overview including the specimen and substantial parts of several other major elements has been implemented as CytometryML XML schemas (www.cytometryml.org). 2) The feasibility of using MIFlowCyt to provide the combination of an overview, table of contents, and/or an index of a scientific paper or a report has been demonstrated. Previously, a sample electronic publication, EPUB, was created that could contain both MIFlowCyt metadata as well as the binary data. Conclusions: The use of CytometryML technology together with XHTML5 and CSS permits the metadata to be directly formatted and together with the binary data to be stored in an EPUB container. This will facilitate: formatting, data- mining, presentation, data verification, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate a publication's adherence to the MIFlowCyt standard, promote interoperability and should also result in the textual and numeric data being published using web technology without any change in composition.

  16. Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical image analysis and flow cytometry

    PubMed Central

    Blackbeard, J.; O’Dea, K.P.; Wallace, V.C.J.; Segerdahl, A.; Pheby, T.; Takata, M.; Field, M.J.; Rice, A.S.C.

    2007-01-01

    Microgliosis is implicated in the pathophysiology of several neurological disorders, including neuropathic pain. Consequently, perturbation of microgliosis is a mechanistic and drug development target in neuropathic pain, which highlights the requirement for specific, sensitive and reproducible methods of microgliosis measurement. In this study, we used the spinal microgliosis associated with L5 spinal nerve transection and minocycline-induced attenuation thereof to: (1) evaluate novel software based semi-quantitative image analysis paradigms for the assessment of immunohistochemical images. Microgliosis was revealed by immunoreactivity to OX42. Several image analysis paradigms were assessed and compared to a previously validated subjective categorical rating scale. This comparison revealed that grey scale measurement of the proportion of a defined area of spinal cord occupied by OX42 immunoreactive cells is a robust image analysis paradigm. (2) Develop and validate a flow cytometric approach for quantification of spinal microgliosis. The flow cytometric technique reliably quantified microgliosis in spinal cord cell suspensions, using OX42 and ED9 immunoreactivity to identify microglia. The results suggest that image analysis of immunohistochemical revelation of microgliosis reliably detects the spinal microgliosis in response to peripheral nerve injury and pharmacological attenuation thereof. In addition, flow cytometry provides an alternative approach for quantitative analysis of spinal microgliosis elicited by nerve injury. PMID:17553569

  17. Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical image analysis and flow cytometry.

    PubMed

    Blackbeard, J; O'Dea, K P; Wallace, V C J; Segerdahl, A; Pheby, T; Takata, M; Field, M J; Rice, A S C

    2007-08-30

    Microgliosis is implicated in the pathophysiology of several neurological disorders, including neuropathic pain. Consequently, perturbation of microgliosis is a mechanistic and drug development target in neuropathic pain, which highlights the requirement for specific, sensitive and reproducible methods of microgliosis measurement. In this study, we used the spinal microgliosis associated with L5 spinal nerve transection and minocycline-induced attenuation thereof to: (1) evaluate novel software based semi-quantitative image analysis paradigms for the assessment of immunohistochemical images. Microgliosis was revealed by immunoreactivity to OX42. Several image analysis paradigms were assessed and compared to a previously validated subjective categorical rating scale. This comparison revealed that grey scale measurement of the proportion of a defined area of spinal cord occupied by OX42 immunoreactive cells is a robust image analysis paradigm. (2) Develop and validate a flow cytometric approach for quantification of spinal microgliosis. The flow cytometric technique reliably quantified microgliosis in spinal cord cell suspensions, using OX42 and ED9 immunoreactivity to identify microglia. The results suggest that image analysis of immunohistochemical revelation of microgliosis reliably detects the spinal microgliosis in response to peripheral nerve injury and pharmacological attenuation thereof. In addition, flow cytometry provides an alternative approach for quantitative analysis of spinal microgliosis elicited by nerve injury.

  18. Cytopathologic diagnosis of spontaneous infarction of fibroadenoma of the breast.

    PubMed

    Wadhwa, Neelam; Joshi, Richa; Mangal, Nidhi; Khan, Nirupma Panikar; Joshi, Mohit

    2014-01-01

    Infarction is an uncommon event in a fibroadenoma, which is the commonest benign tumor of the breast. Most often it occurs in pregnancy, lactation or is secondary to fine needle aspiration. Spontaneous infarction of a fibroadenoma in the absence of a predisposing condition is very rare. The cytopathologic features of infarction are necrosis and worrisome nuclear features, which are often misinterpreted as either inflammation or malignancy. We detail a report of accurate cytopathologic diagnosis of spontaneous infarction of fibroadenoma in a 17-year-old adolescent non pregnant girl. Careful attention to the cytopathologic clues like uniform thickness of the necrotic epithelial fragments, branching pattern reminiscent of the staghorn pattern despite atypical nuclear features and clinical details like young age of the patient and recent onset pain in a pre-existing lump helped arrive at the correct diagnosis and spared the patient of a radical excision. To the best of our knowledge, there are no earlier reports of correct cytopathologic diagnosis.

  19. Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

    PubMed Central

    Schaub, Franz X; Reza, Md Shamim; Flaveny, Colin A; Li, Weimin; Musicant, Adele M; Hoxha, Sany; Guo, Min; Cleveland, John L; Amelio, Antonio L

    2015-01-01

    Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer (BRET) that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bi-functional nature of these LumiFluor reporters enables greatly improved spatio-temporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, non-invasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes. PMID:26424696

  20. Subnuclear foci quantification using high-throughput 3D image cytometry

    NASA Astrophysics Data System (ADS)

    Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

    2015-07-01

    Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

  1. Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system

    NASA Astrophysics Data System (ADS)

    Futia, Gregory L.; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A.

    2016-04-01

    Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.

  2. Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry.

    PubMed

    Maguire, Orla; O'Loughlin, Kieran; Minderman, Hans

    2015-08-01

    Aberrant activity of Nuclear Factor-kappaB (NF-κB) is associated with many diseases and is therapeutically targeted. Post-translational modifications, particularly phosphorylation of the RELA/p65 sub-unit, are essential for cytoplasmic to nuclear localization of NF-κB/p65 and initiation of transcription of downstream target genes. Immunoblot and phospho-flow cytometry have been used to study the relationship between phosphorylation motifs and NF-κB activation and microscopic analysis of nuclear localization of p65 is also used as a parameter for activation. The labor intensive nature of these approaches commonly limits the number of sampling points or replicates. Recent insights into the relationship between p65 phosphorylation motifs and their nuclear localization indicate that these parameters have different significances and should not be used interchangeably. In this study, we demonstrate feasibility and reproducibility of studying the relationship between p65 phosphorylation and nuclear translocation using imaging flow cytometry (IFC). TNFα- or PMA/Ionomycin-induced phosphorylation of p65 at serine 529 in cell line models and healthy donor lymphocytes served as the experimental model. IFC analysis demonstrated that expression of phosphorylated serine 529 (P-p65(s529)) increased rapidly following stimulation and that nuclear localization of P-p65(s529) followed the nuclear localization pattern of total p65. However, in the presence of tacrolimus, P-p65(s529) expression was inhibited without affecting nuclear localization of total p65. The data demonstrate the application of IFC to simultaneously assess phosphorylation of p65 and its cellular localization and the results obtained by this analysis corroborate current insights regarding the specific effect of tacrolimus on serine 529 phosphorylation.

  3. Interfacing Lab-on-a-Chip Embryo Technology with High-Definition Imaging Cytometry.

    PubMed

    Zhu, Feng; Hall, Christopher J; Crosier, Philip S; Wlodkowic, Donald

    2015-08-01

    To spearhead deployment of zebrafish embryo biotests in large-scale drug discovery studies, automated platforms are needed to integrate embryo in-test positioning and immobilization (suitable for high-content imaging) with fluidic modules for continuous drug and medium delivery under microperfusion to developing embryos. In this work, we present an innovative design of a high-throughput three-dimensional (3D) microfluidic chip-based device for automated immobilization and culture and time-lapse imaging of developing zebrafish embryos under continuous microperfusion. The 3D Lab-on-a-Chip array was fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining, while the off-chip interfaces were fabricated using additive manufacturing processes (fused deposition modelling and stereolithography). The system's design facilitated rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It was conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. Compared with the conventional Petri dish assays, the chip-based bioassay was much more convenient and efficient as only small amounts of drug solutions were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility.

  4. A light sheet confocal microscope for image cytometry with a variable linear slit detector

    NASA Astrophysics Data System (ADS)

    Hutcheson, Joshua A.; Khan, Foysal Z.; Powless, Amy J.; Benson, Devin; Hunter, Courtney; Fritsch, Ingrid; Muldoon, Timothy J.

    2016-03-01

    We present a light sheet confocal microscope (LSCM) capable of high-resolution imaging of cell suspensions in a microfluidic environment. In lieu of conventional pressure-driven flow or mechanical translation of the samples, we have employed a novel method of fluid transport, redox-magnetohydrodynamics (redox-MHD). This method achieves fluid motion by inducing a small current into the suspension in the presence of a magnetic field via electrodes patterned onto a silicon chip. This on-chip transportation requires no moving parts, and is coupled to the remainder of the imaging system. The microscopy system comprises a 450 nm diode 20 mW laser coupled to a single mode fiber and a cylindrical lens that converges the light sheet into the back aperture of a 10x, 0.3 NA objective lens in an epi-illumination configuration. The emission pathway contains a 150 mm tube lens that focuses the light onto the linear sensor at the conjugate image plane. The linear sensor (ELiiXA+ 8k/4k) has three lateral binning modes which enables variable detection aperture widths between 5, 10, or 20 μm, which can be used to vary axial resolution. We have demonstrated redox-MHD-enabled light sheet microscopy in suspension of fluorescent polystyrene beads. This approach has potential as a high-throughput image cytometer with myriad cellular diagnostic applications.

  5. Phaedra, a protocol-driven system for analysis and validation of high-content imaging and flow cytometry.

    PubMed

    Cornelissen, Frans; Cik, Miroslav; Gustin, Emmanuel

    2012-04-01

    High-content screening has brought new dimensions to cellular assays by generating rich data sets that characterize cell populations in great detail and detect subtle phenotypes. To derive relevant, reliable conclusions from these complex data, it is crucial to have informatics tools supporting quality control, data reduction, and data mining. These tools must reconcile the complexity of advanced analysis methods with the user-friendliness demanded by the user community. After review of existing applications, we realized the possibility of adding innovative new analysis options. Phaedra was developed to support workflows for drug screening and target discovery, interact with several laboratory information management systems, and process data generated by a range of techniques including high-content imaging, multicolor flow cytometry, and traditional high-throughput screening assays. The application is modular and flexible, with an interface that can be tuned to specific user roles. It offers user-friendly data visualization and reduction tools for HCS but also integrates Matlab for custom image analysis and the Konstanz Information Miner (KNIME) framework for data mining. Phaedra features efficient JPEG2000 compression and full drill-down functionality from dose-response curves down to individual cells, with exclusion and annotation options, cell classification, statistical quality controls, and reporting.

  6. The Application of Imaging Flow Cytometry to High-Throughput Biodosimetry

    PubMed Central

    Wilkins, Ruth C.; Rodrigues, Matthew A.; Beaton-Green, Lindsay A.

    2017-01-01

    Biodosimetry methods, including the dicentric chromosome assay, the cytokinesis-block micronucleus assay and the γH2AX marker of DNA damage are used to determine the dose of ionizing radiation. These techniques are particularly useful when physical dosimetry is absent or questioned. While these assays can be very sensitive and specific, the standard methods need to be adapted to increase sample throughput in the case of a large-scale radiological/nuclear event. Recent modifications to the microscope-based assays have resulted in some increased throughput, and a number of biodosimetry networks have been, and continue to be, established and strengthened. As the imaging flow cytometer (IFC) is a technology that can automatically image and analyze processed blood samples for markers of radiation damage, the microscope-based biodosimetry techniques can be modified for the IFC for high-throughput biological dosimetry. Furthermore, the analysis templates can be easily shared between networked biodosimetry laboratories for increased capacity and improved standardization. This review describes recent advances in IFC methodology and their application to biodosimetry. PMID:28250914

  7. An Imaging Flow Cytometry-based approach to analyse the fission yeast cell cycle in fixed cells.

    PubMed

    Patterson, James O; Swaffer, Matthew; Filby, Andrew

    2015-07-01

    Fission yeast (Schizosaccharomyces pombe) is an excellent model organism for studying eukaryotic cell division because many of the underlying principles and key regulators of cell cycle biology are conserved from yeast to humans. As such it can be employed as tool for understanding complex human diseases that arise from dis-regulation in cell cycle controls, including cancers. Conventional Flow Cytometry (CFC) is a high-throughput, multi-parameter, fluorescence-based single cell analysis technology. It is widely used for studying the mammalian cell cycle both in the context of the normal and disease states by measuring changes in DNA content during the transition through G1, S and G2/M using fluorescent DNA-binding dyes. Unfortunately analysis of the fission yeast cell cycle by CFC is not straightforward because, unlike mammalian cells, cytokinesis occurs after S-phase meaning that bi-nucleated G1 cells have the same DNA content as mono-nucleated G2 cells and cannot be distinguished using total integrated fluorescence (pulse area). It has been elegantly shown that the width of the DNA pulse can be used to distinguish G2 cells with a single 2C foci versus G1 cells with two 1C foci, however the accuracy of this measurement is dependent on the orientation of the cell as it traverses the laser beam. To this end we sought to improve the accuracy of the fission yeast cell cycle analysis and have developed an Imaging Flow Cytometry (IFC)-based method that is able to preserve the high throughput, objective analysis afforded by CFC in combination with the spatial and morphometric information provide by microscopy. We have been able to derive an analysis framework for subdividing the yeast cell cycle that is based on intensiometric and morphometric measurements and is thus robust against orientation-based miss-classification. In addition we can employ image-based metrics to define populations of septated/bi-nucleated cells and measure cellular dimensions. To our knowledge

  8. An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells.

    PubMed

    Cerveira, Joana; Begum, Julfa; Di Marco Barros, Rafael; van der Veen, Annemarthe G; Filby, Andrew

    2015-08-01

    Calcium ions (Ca(2+)) are a ubiquitous transducer of cellular signals controlling key processes such as proliferation, differentiation, secretion and metabolism. In the context of T cells, stimulation through the T cell receptor has been shown to induce the release of Ca(2+) from intracellular stores. This sudden elevation within the cytoplasm triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca(2+) that in turn activates key molecules such as calcineurin. This cascade ultimately results in gene transcription and changes in the cellular state. Traditional methods for measuring Ca(2+) include spectrophotometry, conventional flow cytometry (CFC) and live cell imaging techniques. While each method has strengths and weaknesses, none can offer a detailed picture of Ca(2+) mobilisation in response to various agonists. Here we report an Imaging Flow Cytometry (IFC)-based method that combines the throughput and statistical rigour of CFC with the spatial information of a microscope. By co-staining cells with Ca(2+) indicators and organelle-specific dyes we can address the spatiotemporal patterns of Ca(2+) flux in Jurkat cells after stimulation with anti-CD3. The multispectral, high-throughput nature of IFC means that the organelle co-staining functions to direct the measurement of Ca(2+) indicator fluorescence to either the endoplasmic reticulum (ER) or the mitochondrial compartments without the need to treat cells with detergents such as digitonin to eliminate cytoplasmic background. We have used this system to look at the cellular localisation of Ca(2+) after stimulating cells with CD3, thapsigargin or ionomycin in the presence or absence of extracellular Ca(2+). Our data suggest that there is a dynamic interplay between the ER and mitochondrial compartments and that mitochondria act as a sink for both intracellular and extracellular derived Ca(2+). Moreover, by generating an NFAT-GFP expressing Jurkat line, we were able to

  9. HUMN project initiative and review of validation, quality control and prospects for further development of automated micronucleus assays using image cytometry systems.

    PubMed

    Fenech, Michael; Kirsch-Volders, Micheline; Rossnerova, Andrea; Sram, Radim; Romm, Horst; Bolognesi, Claudia; Ramakumar, Adarsh; Soussaline, Francoise; Schunck, Christian; Elhajouji, Azeddine; Anwar, Wagida; Bonassi, Stefano

    2013-08-01

    The use of micronucleus (MN) assays in in vitro genetic toxicology testing, radiation biodosimetry and population biomonitoring to study the genotoxic impacts of environment gene-interactions has steadily increased over the past two decades. As a consequence there has been a strong interest in developing automated systems to score micronuclei, a biomarker of chromosome breakage or loss, in mammalian and human cells. This paper summarises the outcomes of a workshop on this topic, organised by the HUMN project, at the 6th International Conference on Environmental Mutagenesis in Human Populations at Doha, Qatar, 2012. The aim of this paper is to summarise the outcomes of the workshop with respect to the set objectives which were: (i) Review current developments in automation of micronucleus assays by image cytometry; (ii) define the performance characteristics of automated MN scoring using image cytometry and methods of assessment for instrument validation and quality control and (iii) discuss the design of inter-laboratory comparisons and standardisation of micronucleus assays using automated image cytometry systems. It is evident that automated scoring of micronuclei by automated image cytometry using different commercially available platforms [e.g. Metafer (MetaSystems), Pathfinder™ (IMSTAR), iCyte(®) (Compucyte)], particularly for lymphocytes, is at a mature stage of development with good agreement between visual and automated scoring across systems (correlation factors ranging from 0.58 to 0.99). However, a standardised system of validation and calibration is required to enable more reliable comparison of data across laboratories and across platforms. This review identifies recent progress, important limitations and steps that need to be taken into account to enable the successful universal implementation of automated micronucleus assays by image cytometry.

  10. Validation of nanobody and antibody based in vivo tumor xenograft NIRF-imaging experiments in mice using ex vivo flow cytometry and microscopy.

    PubMed

    Bannas, Peter; Lenz, Alexander; Kunick, Valentin; Fumey, William; Rissiek, Björn; Schmid, Joanna; Haag, Friedrich; Leingärtner, Axel; Trepel, Martin; Adam, Gerhard; Koch-Nolte, Friedrich

    2015-04-06

    This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies. First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results. The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

  11. The 1990s--interface of cytopathology and new technology.

    PubMed

    Linder, J

    1992-01-01

    In physical chemistry, the most unique and dramatic reactions occur at the interface between different phases of matter. An analogy can be drawn between this observation and the interface that currently exists between the traditional practice of cytopathology and the technologies discussed in this editorial. It is natural to be excited about new technologies. They offer the potential to improve our diagnostic ability, to save time, and to expand the range of cytopathology services. Our enthusiasm for new technology should be tempered by the inherent appeal of cytopathology--its relative simplicity. Cytologic diagnoses are possible with a glass slide, extracts of tree bark, and a well-trained observer. This can be rapid and tremendously cost effective, not only identifying the type of abnormality, but often providing prognostic information. New technology, while offering additional information, may not be cost effective, or may not offer more information than is available by traditional methods. Whether or not to accept new technologies is the choice of cytotechnologists and cytopathologists. It is the goal of the Editorial Board of Diagnostic Cytopathology that this be a well-informed choice. The current and coming issues of Diagnostic Cytopathology will describe technological advances in the Focus on Technology section of the journal. We trust that you will find this information useful in your evaluation of new technology.

  12. Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry.

    PubMed

    Fendl, Birgit; Weiss, René; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

    2016-09-09

    Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells.

  13. Oscillatory Dynamics of Cell Cycle Proteins in Single Yeast Cells Analyzed by Imaging Cytometry

    PubMed Central

    Ball, David A.; Marchand, Julie; Poulet, Magaly; Baumann, William T.; Chen, Katherine C.; Tyson, John J.; Peccoud, Jean

    2011-01-01

    Progression through the cell division cycle is orchestrated by a complex network of interacting genes and proteins. Some of these proteins are known to fluctuate periodically during the cell cycle, but a systematic study of the fluctuations of a broad sample of cell-cycle proteins has not been made until now. Using time-lapse fluorescence microscopy, we profiled 16 strains of budding yeast, each containing GFP fused to a single gene involved in cell cycle regulation. The dynamics of protein abundance and localization were characterized by extracting the amplitude, period, and other indicators from a series of images. Oscillations of protein abundance could clearly be identified for Cdc15, Clb2, Cln1, Cln2, Mcm1, Net1, Sic1, and Whi5. The period of oscillation of the fluorescently tagged proteins is generally in good agreement with the inter-bud time. The very strong oscillations of Net1 and Mcm1 expression are remarkable since little is known about the temporal expression of these genes. By collecting data from large samples of single cells, we quantified some aspects of cell-to-cell variability due presumably to intrinsic and extrinsic noise affecting the cell cycle. PMID:22046265

  14. Nanobarcoded superparamagnetic iron oxide nanoparticles for nanomedicine: Quantitative studies of cell-nanoparticle interactions by scanning image cytometry.

    PubMed

    Eustaquio, Trisha; Leary, James F

    2016-02-01

    Oligonucleotide-functionalized nanoparticles (NPs) are promising agents for nanomedicine, but the potential in vitro nanotoxicity that may arise from such conjugates has yet to be evaluated in a dose response manner. Since nanomedicine functions on the single-cell level, measurements of nanotoxicity should also be performed as such. In vitro single-cell nanotoxicity assays based on scanning image cytometry are used to study a specific type of oligo-functionalized NP, "nanobarcoded" superparamagnetic iron oxide NPs (NB-SPIONs). The selected panel of single-cell assays measures well-known modes of nanotoxicity--apoptosis, necrosis, generation of reactive oxygen species (ROS), and cell number. Using these assays, the cytotoxicity of two sizes of NB-SPIONs (10 nm and 30 nm core size) was compared to the parent NP, carboxylated SPIONs (COOH-SPIONs). The results suggest that the conjugated NB confers a biocompatible coating that protects against cytotoxicity at very high SPION doses, but both NB- and COOH-SPIONs of either size generally have low in vitro cytotoxicity at physiologically relevant doses.

  15. Using image-based flow cytometry to measure monocyte oxidized LDL phagocytosis: A potential risk factor for CVD?

    PubMed

    Henning, Andrea L; Venable, Adam S; Prado, Eric A; Best Sampson, Jill N; McFarlin, Brian K

    2015-08-01

    Obesity and cardiovascular disease is a worldwide health concern that has been a major focus in research for several decades. Among these diseases, atherosclerosis is one of the leading causes of death and disability nationwide. Circulating monocytes are believed to be primary cells responsible for foam cell formation. The present report describes a novel method for measuring monocyte oxLDL phagocytosis capacity using image-based flow cytometry. Human venous blood monocytes were incubated with different concentrations of oxLDL for different lengths of time to optimize the assay. High (post-meal) and low (pre-meal) responder samples were generated by feeding human subjects a high-fat (~85% of daily fat allowance), high-calorie (~65% of daily calorie needs) meal. This is a relevant model with respect to obesity and risk of developing atherogenesis. After the functional assay, classic (CD14+/CD16-) and pro-inflammatory (CD14+/CD16+) monocytes were assessed for oxLDL uptake, adhesion molecule expression (CD11b and CD18), and scavenger receptor expression (CD36) using an image-based flow cytometer (FlowSight). The present method represents a novel advance in methods available to detect the propensity of circulating monocytes to become intima foam cells. We found the assay to be most effective at separating high from low responder samples when using a fixed oxLDL concentration (120 μL/mL) and incubation length (1-h). In a clinical application, this method demonstrated that consuming a single high-fat meal causes an increase in the proportion of monocyte oxLDL phagocytosis and their adhesion capacity, suggesting a higher propensity to become foam cells.

  16. Optimized automated data analysis for the cytokinesis‐block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry

    PubMed Central

    Rodrigues, M. A.; Probst, C. E.; Beaton‐Green, L. A.

    2016-01-01

    Abstract The cytokinesis‐block micronucleus (CBMN) assay is a well‐established technique that can be employed in triage radiation biodosimetry to estimate whole body doses of radiation to potentially exposed individuals through quantitation of the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using traditional microscope‐based methods and most recently has been modified for application on the ImageStreamX (ISX) imaging flow cytometer. This modification has allowed for a similar number of BNCs to be automatically scored as compared to traditional microscopy in a much shorter time period. However, the MN frequency measured was much lower than both manual and automated slide‐based methods of performing the assay. This work describes the optimized analysis template which implements newly developed functions in the IDEAS® data analysis software for the ISX that enhances specificity for BNCs and increases the frequency of scored MN. A new dose response calibration curve is presented in which the average rate of MN per BNC is of similar magnitude to those presented in the literature using automated CBMN slide scoring methods. In addition, dose estimates were generated for nine irradiated, blinded samples and were found to be within ±0.5 Gy of the delivered dose. Results demonstrate that the improved identification accuracy for MN and BNCs in the ISX‐based version of the CBMN assay will translate to increased accuracy when estimating unknown radiation doses received by exposed individuals following large‐scale radiological or nuclear emergencies. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC PMID:27272602

  17. Image-based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst.

    PubMed

    McFarlin, Brian K; Williams, Randall R; Venable, Adam S; Dwyer, Karen C; Haviland, David L

    2013-08-01

    Granulocytes play a key role in innate immunity and the most common functional assays are phagocytosis and oxidative burst. The purpose of this technical note is to use image-based flow cytometry to divide activated granulocytes into unique subsets based on their degree of phagocytosis and oxidative burst in response to different experimental incubations. Prior to the experiments, all reagents were titered to determine the lowest dose that resulted in an acceptable signal to noise ratio. Heparinized, whole blood (100 µl) was mixed with one of two bioparticles (E. coli and S. aureus) and DHE (10 µg/ml) and incubated for 5, 10, 20, 40, 60, 80, 100, 120, and 140 min in a 37°C water bath. An additional tube kept on ice was used as a negative control. All subsequent processing steps were completed on ice in the dark to minimize additional activation of cells. After the 37°C incubation, N-ethylmaleimide (15 mM) was added to halt phagocytosis, preventing the uptake of additional microparticles. Suspensions were labeled with CD66b-APC and CD45-APCeFluor780 for 60 min and a fix/lyse solution was added. Prior to acquisition, 7AAD was added to stain nuclear DNA. A minimum of 5,000 granulocyte (CD66b+) events were acquired using a Millipore-Amnis FlowSight equipped with blue (488 nm, 60 mW), red (642 nm, 100 mW), and side scatter (785 nm, 12 mW) lasers. Samples were compensated and analyzed using Amnis IDEAS software (v.5.0.983.0). Image-based analysis allowed us to divide activated granulocytes into three distinct subsets, whose relative abundance changed as a function of both bioparticle type and incubation length. The method described in this technical note represents a potential novel adaptation to common methods of assessing granulocyte function. More research is needed to test and validate our image-based method in clinical conditions that impair granulocyte function.

  18. Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols.

    PubMed

    Figueroa, Gloria; Parira, Tiyash; Laverde, Alejandra; Casteleiro, Gianna; El-Mabhouh, Amal; Nair, Madhavan; Agudelo, Marisela

    2016-10-18

    Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

  19. Image cytometry determination of ploidy level, proliferative activity, and nuclear size in a series of 314 transitional bladder cell carcinomas.

    PubMed

    van Velthoven, R; Petein, M; Oosterlinck, W J; Zandona, C; Zlotta, A; Van der Meijden, A P; Pasteels, J L; Roels, H; Schulman, C; Kiss, R

    1995-01-01

    Image cytometry was carried out on 281 superficial (Ta and T1) and 33 invasive (T2 to T4) bladder cancers. The parameters used to characterize these bladder tumors were: (1) histopathological grading, (2) clinical staging, (3) tumor size, (4) deoxyribonucleic acid (DNA) index (DI), (5) DNA histogram type (DHT), (6) percentage of euploid (diploid plus tetraploid) cells, (7) percentage of polyploid cells (> 5C DNA content), (8) proliferative activity (S phase fraction value), and (9) nuclear area (NA). The proliferative activity of the tumors was not related to either histopathological grade or to clinical stage, but it was related to the DHT parameter, which made it possible to identify diploid, hyperdiploid, triploid, hypertriploid, tetraploid, and polymorphic tumors. The hypertriploid tumors exhibited a significantly lower proliferative activity than the nonhypertriploid ones. Although both the DI and the NA values correlated significantly with histopathological grading, only the NA values correlated significantly with clinical staging. We further observed that some grade III bladder tumors were definitely diploid, whereas some grade I tumors were highly aneuploid. We thus hypothesize that the ploidy level of a given tumor reflects its age directly and its aggressiveness only very indirectly. In our opinion aneuploidy is only an indirect marker of aggressiveness because it reflects the fact that a malignant tumor is old, ie, has been present in a patient over a long period of time and has had ample time to express its malignancy at the clinical level. A significant relationship was accordingly obtained between tumor size and ploidy level with the highest proportion of aneuploid tumors and the highest percentage of polyploid cell nuclei being observed among the largest bladder tumors.

  20. New tools in cytometry.

    PubMed

    Depince-Berger, A-E; Aanei, C; Iobagiu, C; Jeraiby, M; Lambert, C

    2016-12-01

    Cytometry aims to analyze cells, of any type, using dedicated instruments. The quantitative aspect makes flow cytometry (FCM) a good complementary tool for morphology. Most of the identification tools are based on immunostaining of cell structure details and more and more tools are available in terms of specificities and labels. FCM is under exponential development thanks to technical, immunological and data analysis progresses. Actual generations are now routinely using 6 to 10 simultaneous immuno-labeling on 20 to 100,000 cells, at high speed and short sample preparation and can easily detect rare events at frequency below 10(-4) cells. Data interpretation is complex and requires expertise. Mathematical tools are available to support analysis and classification of cells based. Cells from tissues can also be analyzed by FCM after mechanical and or enzymatic separation, but in situ cells can also be analyzed with the help of cytometry. Very new instruments bring spectral analysis, image in flow and mass spectrometry. Medical applications are very broad, notably in hemopathies, immunology, solid tumors, but also microbiology, toxicology, drug discovery, food and environmental industry. But, the limit of FCM is its dependence on operator from sample preparation, instrument settings up to data analysis and a strong effort is now under progress for standardization and constitution of international data bank for references and education.

  1. Microfluidic devices and methods for integrated flow cytometry

    DOEpatents

    Srivastava, Nimisha; Singh, Anup K.

    2011-08-16

    Microfluidic devices and methods for flow cytometry are described. In described examples, various sample handling and preparation steps may be carried out within a same microfluidic device as flow cytometry steps. A combination of imaging and flow cytometry is described. In some examples, spiral microchannels serve as incubation chambers. Examples of automated sample handling and flow cytometry are described.

  2. Diagnostic accuracy assessment of cytopathological examination of feline sporotrichosis.

    PubMed

    Jessica, N; Sonia, R L; Rodrigo, C; Isabella, D F; Tânia, M P; Jeferson, C; Anna, B F; Sandro, A

    2015-11-01

    Sporotrichosis is an implantation mycosis caused by pathogenic species of Sporothrix schenckii complex that affects humans and animals, especially cats. Its main forms of zoonotic transmission include scratching, biting and/or contact with the exudate from lesions of sick cats. In Brazil, epidemic involving humans, dogs and cats has occurred since 1998. The definitive diagnosis of sporotrichosis is obtained by the isolation of the fungus in culture; however, the result can take up to four weeks, which may delay the beginning of antifungal treatment in some cases. Cytopathological examination is often used in feline sporotrichosis diagnosis, but accuracy parameters have not been established yet. The aim of this study was to evaluate the accuracy and reliability of cytopathological examination in the diagnosis of feline sporotrichosis. The present study included 244 cats from the metropolitan region of Rio de Janeiro, mostly males in reproductive age with three or more lesions in non-adjacent anatomical places. To evaluate the inter-observer reliability, two different observers performed the microscopic examination of the slides blindly. Test sensitivity was 84.9%. The values of positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and accuracy were 86.0, 24.4, 2.02, 0.26 and 82.8%, respectively. The reliability between the two observers was considered substantial. We conclude that the cytopathological examination is a sensitive, rapid and practical method to be used in feline sporotrichosis diagnosis in outbreaks of this mycosis.

  3. Advancing cytometry for immunology.

    PubMed

    Cossarizza, Andrea; Nolan, John; Radbruch, Andreas; Tárnok, Attila

    2012-12-01

    Cytometry is a key technology for immunology. It allows researchers to scrutinize the cells of the immune system in molecular detail, and to assess phenotype and function at the level of individual cells, no matter how rare these cells may be. The International Society for the Advancement of Cytometry, ISAC, by way of its meetings, online resources and publications (e.g. Cytometry Part A and Current Protocols in Cytometry, which are all published by Wiley) track the ever advancing developments regarding cytometry instrumentation and reagents, and the analysis of complex data sets. In June this year in Leipzig, Germany, ISAC held its annual conference "CYTO 2012", a marketplace of innovation in cytometry.

  4. [Pseudo-parasites in histology and cytopathology].

    PubMed

    Pierre, C; Carloz, E; Marlier-Civatte, M; Branquet, D; Gros, P

    1995-01-01

    When interpreting smears and specimens, histologist and cytopathologists can be misled by images mimicking micro-organisms especially parasites such as protozoa, mycotic agents or helminths. Although some of these pitfalls are well-known, others can be problematic especially if nature of the contaminant is the same as that of the parasite that it mimics. False protozoa parasites can correspond either to exogenous agents such as spores, remnants of human cells, or inert exogenous particles. Pseudo-yeast images can be due to pollen, starch or soot but especially to cells such as macrophages, spermatozoids, and neurons or to various inert bodies such as pigments or calcifications. Pseudomycotic filaments can result from vegetable silk, asbestos bodies, radiate granules or fibrin. Curschmann's spirals and vegetable fibers can be confused with helminths and bacterial particles or pollen with helminth eggs.

  5. Proposal for a novel management of indeterminate thyroid nodules on the basis of cytopathological subclasses.

    PubMed

    Rossi, Martina; Lupo, Sabrina; Rossi, Roberta; Franceschetti, Paola; Trasforini, Giorgio; Bruni, Stefania; Tagliati, Federico; Buratto, Mattia; Lanza, Giovanni; Damiani, Luca; Degli Uberti, Ettore; Zatelli, Maria Chiara

    2016-09-13

    Indeterminate thyroid nodules include heterogeneous lesions that could benefit from a differential management. Our aim is to better define the management of the Bethesda System for Reporting Thyroid Cytopathology class III and IV nodules, by identifying cytological subcategories among Bethesda System for Reporting Thyroid Cytopathology class III associated with different clinical risk, by means of ultrasound, repeated FNAB, and BRAFV600E molecular analysis. We also evaluated the outcome of nodules not operated, over a 5-year follow-up. Out of 460 nodules (269 Bethesda System for Reporting Thyroid Cytopathology class III and 191 Bethesda System for Reporting Thyroid Cytopathology class IV), 344 were operated on surgical group and 116 followed-up conservatively (follow-up group). Bethesda System for Reporting Thyroid Cytopathology class III was divided into four subcategories on the basis of cytomorphological features (III-1, III-2, III-3, III-4). Clinical risk was defined on the basis of histological, cytological, and ultrasound data. Malignancy was higher in Bethesda System for Reporting Thyroid Cytopathology class III vs. Bethesda System for Reporting Thyroid Cytopathology class IV (34.4 vs. 26.2 %; p < 0.01). Papillary thyroid carcinoma was the most frequent cancer in each Bethesda System for Reporting Thyroid Cytopathology class (35 %). BRAFV600E diagnostic accuracy was 87 %. Repeated FNAB reclassified as benign nearly 40 % of nodules, selecting patients where surgery could be spared. Significant nodule growth occurred in 13.7 % of nodules, belonging mostly to Bethesda System for Reporting Thyroid Cytopathology class III-2 and Bethesda System for Reporting Thyroid Cytopathology class IV. Overall clinical risk was higher in Bethesda System for Reporting Thyroid Cytopathology III-1, III-4, and IV classes. We propose a differential management of Bethesda System for Reporting Thyroid Cytopathology III and IV classes and related subcategories

  6. Cytopathologic characteristics and differential diagnostic considerations of osteolytic myxopapillary ependymoma.

    PubMed

    Hayashi, Toshitetsu; Haba, Reiji; Kushida, Yoshio; Kadota, Kyuichi; Katsuki, Naomi; Bando, Kenji; Shibuya, Shinsuke; Matsunaga, Toru

    2014-09-01

    Myxopapillary ependymoma (MPE) is a rare variant of conventional ependymoma found predominantly in the sacrococcygeal region in young adults and characterized by its distinct epithelial and stromal components (WHO grade I designation). MPE with extensive osteolysis is extremely uncommon and only up to 40 cases have been documented. A case is presented here in which imprint smears of a sacral tumor in an 18-year-old man revealed complex papillary structures, small loose clusters, or cord-like structures of bland tumor cells embedded in a myxoid or mucinous background. The tumor cells possessed uniformly round nuclei with a smooth nuclear outline, fine granular chromatin, and small nucleoli. Slender cytoplasmic fibrillary processes and occasional intracytoplasmic vacuoles were observed. A cytologic diagnosis of a MPE was suggested and histochemical and immunohistochemical studies were conducted on formalin-fixed, paraffin-embedded material. Immunohistochemically, the tumor cells showed diffuse and strong membranous and cytoplasmic staining for cytokeratin AE1/AE3, glial fibrillary protein, and S-100 protein, but negative for epithelial membrane antigen, pan-neuroendocrine markers (i.e., NSE, chromogranin A, synaptophysin), or brachyury. The proliferative index with MIB-1 was around 10%. The diagnosis of osteolytic MPE was confirmed based on cytopathologic, histopathological, immunohistochemical results, radiologic findings, and the location of the tumor. We demonstrated here the cytopathological features of osteolytic MPE with emphasis on differential diagnostic considerations.

  7. Cytopathologic diagnosis of fine needle aspiration biopsies of thyroid nodules

    PubMed Central

    Misiakos, Evangelos P; Margari, Niki; Meristoudis, Christos; Machairas, Nickolas; Schizas, Dimitrios; Petropoulos, Konstantinos; Spathis, Aris; Karakitsos, Petros; Machairas, Anastasios

    2016-01-01

    Fine-needle aspiration (FNA) cytology is an important diagnostic tool in patients with thyroid lesions. Several systems have been proposed for the cyropathologic diagnosis of the thyroid nodules. However cases with indeterminate cytological findings still remain a matter of debate. In this review we analyze all literature regarding Thyroid Cytopathology Reporting systems trying to identify the most suitable methodology to use in clinical practice for the preoperative diagnosis of thyroid nodules. A review of the English literature was conducted, and data were analyzed and summarized and integrated from the authors’ perspective. The main purpose of thyroid FNA is to identify patients with higher risk for malignancy, and to prevent unnecessary surgeries for benign conditions. The Bethesda System for Reporting Thyroid Cytopathology is the most widely used system for the diagnosis of thyroid FNA specimens. This system also contains guidelines for the diagnosis and treatment of indeterminate or suspicious for malignancy cases. In conclusion, patients who require repeated FNAs for indeterminate diagnoses will be resolved by repeat FNA in a percentage of 72%-80%. PMID:26881190

  8. The use of the decision tree technique and image cytometry to characterize aggressiveness in World Health Organization (WHO) grade II superficial transitional cell carcinomas of the bladder.

    PubMed

    Decaestecker, C; van Velthoven, R; Petein, M; Janssen, T; Salmon, I; Pasteels, J L; van Ham, P; Schulman, C; Kiss, R

    1996-03-01

    The aggressiveness of human bladder tumours can be assessed by means of various classification systems, including the one proposed by the World Health Organization (WHO). According to the WHO classification, three levels of malignancy are identified as grades I (low), II (intermediate), and III (high). This classification system operates satisfactorily for two of the three grades in forecasting clinical progression, most grade I tumours being associated with good prognoses and most grade III with bad. In contrast, the grade II group is very heterogeneous in terms of their clinical behaviour. The present study used two computer-assisted methods to investigate whether it is possible to sub-classify grade II tumours: computer-assisted microscope analysis (image cytometry) of Feulgen-stained nuclei and the Decision Tree Technique. This latter technique belongs to the Supervised Learning Algorithm and enables an objective assessment to be made of the diagnostic value associated with a given parameter. The combined use of these two methods in a series of 292 superficial transitional cell carcinomas shows that it is possible to identify one subgroup of grade II tumours which behave clinically like grade I tumours and a second subgroup which behaves clinically like grade III tumours. Of the nine ploidy-related parameters computed by means of image cytometry [the DNA index (DI), DNA histogram type (DHT), and the percentages of diploid, hyperdiploid, triploid, hypertriploid, tetraploid, hypertetraploid, and polyploid cell nuclei], it was the percentage of hyperdiploid and hypertetraploid cell nuclei which enabled identification, rather than conventional parameters such as the DI or the DHT.

  9. Detection of pathogenic E. coli O157:H7 by a hybrid microfluidic SPR and molecular imaging cytometry device.

    PubMed

    Zordan, Michael D; Grafton, Meggie M G; Acharya, Ghanashyam; Reece, Lisa M; Cooper, Christy L; Aronson, Arthur I; Park, Kinam; Leary, James F

    2009-02-01

    Current methods to screen for bacterial contamination involve using costly reagents such as antibodies or PCR reagents or time-costly growth in cultures. There is need for portable, real-time, multiplex pathogen detection technology that can predict the safety of food. Surface plasmon resonance (SPR) imaging is a sensitive, label-free method that can detect the binding of an analyte to a surface by the changes in refractive index that occur upon binding. We have designed a hybrid microfluidic biochip to perform multiplexed detection of single-celled pathogens using a combination of SPR and fluorescence imaging. The device consists of an array of gold spots, each functionalized with a capture biomolecule targeting a specific pathogen. This biosensor array is enclosed by a polydimethylsiloxane microfluidic flow chamber that delivers a magnetically concentrated sample to be tested. The sample is imaged by SPR on the bottom of the biochip and epi-fluorescence on the top. The prototype instrument was successfully able to image antibody-captured E. coli O157:H7 bacteria by SPR and fluorescence imaging. The efficiency of capture of these bacteria by the magnetic particles was determined using spectrophotometric ferric oxide absorbance measurements. The binding of the E. coli to each spot was quantified by measuring the percent of the gold spot area upon which the bacteria was bound and analyzed using NIH ImageJ software. This hybrid imaging approach of pathogenic E. coli detection coupled with an estimate of relative infectivity is shown to be a working example of a testing device for potential foodborne pathogens.

  10. Lasers in flow cytometry.

    PubMed

    Telford, William G

    2011-01-01

    Laser technology has advanced tremendously since the first gas lasers were incorporated into early flow cytometers. Gas lasers have been largely replaced by solid-state laser technology, making virtually any desirable visible light wavelength available for flow cytometry. Multiwavelength, white light, and wavelength tunable lasers are poised to enhance our analytical capabilities even further. In this chapter, I summarize the role that lasers play in cytometry, and the practical characteristics that make a laser appropriate for flow cytometry. I then review the latest single wavelength lasers available for flow cytometry, and how they can be used to excite the ever-expanding array of available fluorochromes. Finally, I review the contribution and potential of the latest tunable laser technology to flow cytometry, and show several examples of these novel sources integrated into production instruments. Technical details and critical parameters for successful application of these lasers for biomedical analysis are covered in depth.

  11. A shared standard for cytometry and pathology

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.; Leif, Stephanie H.

    2013-02-01

    Introduction: The development of cytometry standards is complicated by their being relevant to pathology and biological science, which already have standards. CytometryML, the cytometry markup language, is an XML standard for flow and image cytometry, which includes both objects and their relationships, and is based upon existing standards: the International Society for Advancement of Cytometry ( ISAC) FCS, Digital Imaging and Communication in Medicine ( DICOM), and International Digital Publishing Forum (EPUB). Methods: The CytometryML schemas are written in XML Schema Definition (XSD1.1). Object-oriented methodology was employed to create the CytometryML schemas, which were tested by translating specific XSD elements into XML and filling in the values. The attribute based syntax description of relationships in the Resource Description Framework (RDF) has been replaced by an XSD element based implementation. The ISAC Archival Cytometry Standard (ACS) concept of a zipped data container file was further refined to be a EPUB file. Since Table of Contents information is present in an EPUB container, it was minimized in the Relations schema, which replaced the ToC schema of the ACS and includes a modified and extended version of the ToC RDF capabilities. Results: An XML based system that includes the DICOM specified separation of series and instances and includes relationships has been created. Conclusions: CytometryML and EPUB could be used for the transmission of research and medical data and be extension some of the pathology part of DICOM. The CytometryML version of RDF in XSD could be extended to provide XSD with full RDF capabilities.

  12. High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device.

    PubMed

    Kim, Min Jung; Lee, Su Chul; Pal, Sukdeb; Han, Eunyoung; Song, Joon Myong

    2011-01-07

    Drug-induced cardiotoxicity or cytotoxicity followed by cell death in cardiac muscle is one of the major concerns in drug development. Herein, we report a high-content quantitative multicolor single cell imaging tool for automatic screening of drug-induced cardiotoxicity in an intact cell. A tunable multicolor imaging system coupled with a miniaturized sample platform was destined to elucidate drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability and apoptosis/necrosis in H9c2(2-1) cell line. Cells were treated with cisapride (a human ether-à-go-go-related gene (hERG) channel blocker), digoxin (Na(+)/K(+)-pump blocker), camptothecin (anticancer agent) and a newly synthesized anti-cancer drug candidate (SH-03). Decrease in potassium channel permeability in cisapride-treated cells indicated that it can also inhibit the trafficking of the hERG channel. Digoxin treatment resulted in an increase of intracellular [Na(+)]. However, it did not affect potassium channel permeability. Camptothecin and SH-03 did not show any cytotoxic effect at normal use (≤300 nM and 10 μM, respectively). This result clearly indicates the potential of SH-03 as a new anticancer drug candidate. The developed method was also used to correlate the cell death pathway with alterations in intracellular [Na(+)]. The developed protocol can directly depict and quantitate targeted cellular responses, subsequently enabling an automated, easy to operate tool that is applicable to drug-induced cytotoxicity monitoring with special reference to next generation drug discovery screening. This multicolor imaging based system has great potential as a complementary system to the conventional patch clamp technique and flow cytometric measurement for the screening of drug cardiotoxicity.

  13. Cytopathology laboratory accreditation, with special reference to the American Society of Cytology programs.

    PubMed

    Gupta, P K; Erozan, Y S

    1989-01-01

    The features of the Laboratory Accreditation Program of the American Society of Cytology that pertain to quality assurance in cytopathology are reviewed. The areas considered include: (1) specimen procurement and cytopreparation, (2) the role of the cytotechnologist in cytoscreening, evaluation and reporting, (3) the role of the cytopathologist and (4) quality control measures. Attention to the issues raised in these areas is essential to achieving the best possible cytopathology practice in the most efficient and economical manner.

  14. High-Throughput 3D Tumor Spheroid Screening Method for Cancer Drug Discovery Using Celigo Image Cytometry.

    PubMed

    Kessel, Sarah; Cribbes, Scott; Déry, Olivier; Kuksin, Dmitry; Sincoff, Eric; Qiu, Jean; Chan, Leo Li-Ying

    2016-06-01

    Oncologists have investigated the effect of protein or chemical-based compounds on cancer cells to identify potential drug candidates. Traditionally, the growth inhibitory and cytotoxic effects of the drugs are first measured in 2D in vitro models, and then further tested in 3D xenograft in vivo models. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed under a 3D environment. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids using the Celigo image cytometer. First, optimal seeding density for tumor spheroid formation was determined by investigating the cell seeding density of U87MG, a human glioblastoma cell line. Next, the dose-response effects of 17-AAG with respect to spheroid size and viability were measured to determine the IC50 value. Finally, the developed high-throughput method was used to measure the dose response of four drugs (17-AAG, paclitaxel, TMZ, and doxorubicin) with respect to the spheroid size and viability. Each experiment was performed simultaneously in the 2D model for comparison. This detection method allowed for a more efficient process to identify highly qualified drug candidates, which may reduce the overall time required to bring a drug to clinical trial.

  15. High Throughput Measurement of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-derived Cardiomyocytes by Kinetic Image Cytometry

    PubMed Central

    Cerignoli, Fabio; Charlot, David; Whittaker, Ross; Ingermanson, Randy; Gehalot, Piyush; Savtchenko, Alex; Gallacher, David J.; Towart, Rob; Price, Jeffrey H.; McDonough, Patrick M.; Mercola, Mark

    2013-01-01

    Current methods to measure physiological properties of cardiomyocytes and predict fatal arrhythmias that can cause sudden death, such as Torsade de Pointes, lack either the automation and throughput needed for early-stage drug discovery and/or have poor predictive value. To increase throughput and predictive power of in vitro assays, we developed kinetic imaging cytometry (KIC) for automated cell-by-cell analyses via intracellular fluorescence Ca2+ indicators. The KIC instrument simultaneously records and analyzes intracellular calcium concentration [Ca2+]i at 30-ms resolution from hundreds of individual cells/well of 96-well plates in seconds, providing kinetic details not previously possible with well averaging technologies such as plate readers. Analyses of human embryonic stem cell and induced pluripotent stem cell-derived cardiomyocytes revealed effects of known cardiotoxic and arrhythmogenic drugs on kinetic parameters of Ca2+ dynamics, suggesting that KIC will aid in the assessment of cardiotoxic risk and in the elucidation of pathogenic mechanisms of heart disease associated with drugs treatment and/or genetic background. PMID:22926323

  16. A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.

    PubMed

    Hazin, John; Moldenhauer, Gerhard; Altevogt, Peter; Brady, Nathan R

    2015-08-01

    Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity.

  17. Spectral Cytopathology of Cervical Samples: Detecting Cellular Abnormalities in Cytologically Normal Cells

    PubMed Central

    Schubert, Jennifer M.; Bird, Benjamin; Papamarkakis, Kostas; Miljković, Miloš; Bedrossian, Kristi; Laver, Nora; Diem, Max

    2010-01-01

    Aim Spectral Cytopathology (SCP) is a novel spectroscopic method for objective and unsupervised classification of individual exfoliated cells. The limitations of conventional cytopathology are well-recognized within the pathology community. In SCP, cellular differentiation is made by observing molecular changes in the nucleus and the cytoplasm, which may or may not produce morphological changes detectable by conventional cytopathology. This proof of concept study demonstrates SCP’s potential as an enhancing tool for cytopathologists by aiding in the accurate and reproducible diagnosis of cells in all states of disease. Method Infrared spectra are collected from cervical cells deposited onto reflectively coated glass slides. Each cell has a corresponding infrared spectrum that describes its unique biochemical composition. Spectral data are processed and analyzed by an unsupervised chemometric algorithm, Principal Component Analysis (PCA). Results In this blind study, cervical samples are classified by analyzing the spectra of morphologically normal looking squamous cells from normal samples and samples diagnosed by conventional cytopathology with low grade squamous intraepithelial lesions (LSIL). SCP discriminated cytopathological diagnoses amongst twelve different cervical samples with a high degree of specificity and sensitivity. SCP also correlated two samples with abnormal spectral changes: these samples had a normal cytopathological diagnosis but had a history of abnormal cervical cytology. The spectral changes observed in the morphologically normal looking cells are most likely due to an infection with human papillomavirus, HPV. HPV DNA testing was conducted on five additional samples, and SCP accurately differentiated these samples by their HPV status. Conclusions SCP tracks biochemical variations in cells that are consistent with the onset of disease. HPV has been implicated as the cause of these changes detected spectroscopically. SCP does not depend on

  18. Structured illumination-based super-resolution optical microscopy for hemato- and cyto-pathology applications.

    PubMed

    Zhang, Tieqiao; Osborn, Samantha; Brandow, Chloe; Dwyre, Denis; Green, Ralph; Lane, Stephen; Wachsmann-Hogiu, Sebastian

    2013-01-01

    Structured illumination fluorescence microscopy utilizes interfering light and the moiré effect to enhance spatial resolution to about a half of that of conventional light microscopy, i.e. approximately 90 nm. In addition to the enhancement in the x and y directions, it also allows enhancement of resolution in the z- direction by the same factor of two (to approximately 220 nm), making it a powerful tool for 3-D morphology studies of fluorescently labeled cells or thin tissue sections. In this report, we applied this technique to several types of blood cells that are commonly seen in hematopathology. Compared with standard brightfield and ordinary fluorescence microscopy images, the 3-D morphology results clearly reveal the morphological features of different types of normal blood cells. We have also used this technique to evaluate morphologies of abnormal erythrocytes and compare them with those recorded on normal cells. The results give a very intuitive presentation of morphological structures of erythrocytes with great details. This research illustrates the potential of this technique to be used in hematology and cyto-pathology studies aimed at identifying nanometer-sized features that cannot be distinguished otherwise with conventional optical microscopy.

  19. Clinical and cytopathological aspects in phyllodes tumors of the breast.

    PubMed

    Pătraşcu, Anca; Popescu, Carmen Florina; Pleşea, I E; Bădulescu, Adriana; Tănase, Florentina; Mateescu, Garofiţa

    2009-01-01

    The frequency of mesenchymal breast tumors is very low, being represented mostly by tumors with biphasic proliferation (phyllodes tumors) and less by other types of non-epithelial tumors. From clinical point of view, phyllodes tumors (PT) can mimic a breast carcinoma. Therefore, the preoperative diagnosis by cytological examination on material obtained by fine needle aspiration (FNA) is very important for adequate treatment of these tumors. In current study, we assessed clinical aspects of 79 phyllodes tumors regarding patient's age and localization of the tumors. In 17 out of 79 cases, it has been performed FNA within the tumors with further cytological examination on the smears obtained. The median age of the patients was 46.07-year-old, being progressively higher with grade of the tumors with significant values between benign and borderline tumors (p=0.04954) and between benign and malignant ones (p=0.02890). The distinguish on the smears of stromal fragments and naked stromal nuclei with variable grade of atypia regarding the tumoral type, in detriment of epithelial elements have been conclusive for fibroepithelial lesion as cytopathological diagnosis. The preoperative differentiation between a breast phyllodes tumor and a breast carcinoma is extremely important for avoiding of a useless radical surgery for the patient. If the fine needle aspiration was correctly performed, the accuracy of the cytodiagnosis has been 82% in current study.

  20. Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism.

    PubMed

    Fujiki, Yutaka; Tao, Kai; Bianchi, Diana W; Giel-Moloney, Maryann; Leiter, Andrew B; Johnson, Kirby L

    2008-02-01

    Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P < 0.0001). By PCR, more transgene from CAG mice was detected compared to ROSA26-EGFP mice (P = 0.04). By FCM, ROSA26-EGFP cell fluorescence was more uniform than CAG cells. A greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs, but CAG mice had a greater proportion of cells with the brightest fluorescent intensity. Each transgenic strain possesses characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for

  1. Flow cytometry of sperm

    SciTech Connect

    Gledhill, B.L.

    1987-09-21

    This brief paper summarizes automated flow cytometric determination of sperm morphology and flow cytometry/sorting of sperm with application to sex preselection. In the latter context, mention is made of results of karyotypic determination of sex chromosome ratios in albumin-processed human sperm. 23 refs., 1 fig., 1 tab.

  2. Lettuce infectious yellows virus-encoded P26 induces plasmalemma deposit cytopathology

    SciTech Connect

    Stewart, Lucy R.; Medina, Vicente; Sudarshana, Mysore R.; Falk, Bryce W.

    2009-05-25

    Lettuce infectious yellows virus (LIYV) encodes a 26 kDa protein (P26) previously shown to associate with plasmalemma deposits (PLDs), unique LIYV-induced cytopathologies located at the plasmalemma over plasmodesmata pit fields in companion cells and phloem parenchyma. To further characterize the relationship of P26 and PLDs, we assessed localization and cytopathology induction of P26 expressed from either LIYV or a heterologous Tobacco mosaic virus (TMV) vector using green fluorescent protein (GFP) fusions, immunofluorescence microscopy, biochemical fractionation, and transmission electron microscopy (TEM). TEM analyses demonstrated that P26 not only associated with, but induced formation of PLDs in the absence of other LIYV proteins. Interestingly, PLDs induced by P26-expressing TMV were no longer confined to phloem cells. Putative P26 orthologs from two other members of the genus Crinivirus which do not induce conspicuous PLDs exhibited fractionation properties similar to LIYV P26 but were not associated with any PLD-like cytopathology.

  3. Artificial Neural Networks as Decision Support Tools in Cytopathology: Past, Present, and Future

    PubMed Central

    Pouliakis, Abraham; Karakitsou, Efrossyni; Margari, Niki; Bountris, Panagiotis; Haritou, Maria; Panayiotides, John; Koutsouris, Dimitrios; Karakitsos, Petros

    2016-01-01

    OBJECTIVE This study aims to analyze the role of artificial neural networks (ANNs) in cytopathology. More specifically, it aims to highlight the importance of employing ANNs in existing and future applications and in identifying unexplored or poorly explored research topics. STUDY DESIGN A systematic search was conducted in scientific databases for articles related to cytopathology and ANNs with respect to anatomical places of the human body where cytopathology is performed. For each anatomic system/organ, the major outcomes described in the scientific literature are presented and the most important aspects are highlighted. RESULTS The vast majority of ANN applications are related to cervical cytopathology, specifically for the ANN-based, semiautomated commercial diagnostic system PAPNET. For cervical cytopathology, there is a plethora of studies relevant to the diagnostic accuracy; in addition, there are also efforts evaluating cost-effectiveness and applications on primary, secondary, or hybrid screening. For the rest of the anatomical sites, such as the gastrointestinal system, thyroid gland, urinary tract, and breast, there are significantly less efforts relevant to the application of ANNs. Additionally, there are still anatomical systems for which ANNs have never been applied on their cytological material. CONCLUSIONS Cytopathology is an ideal discipline to apply ANNs. In general, diagnosis is performed by experts via the light microscope. However, this approach introduces subjectivity, because this is not a universal and objective measurement process. This has resulted in the existence of a gray zone between normal and pathological cases. From the analysis of related articles, it is obvious that there is a need to perform more thorough analyses, using extensive number of cases and particularly for the nonexplored organs. Efforts to apply such systems within the laboratory test environment are required for their future uptake. PMID:26917984

  4. Photoacoustic flow cytometry

    PubMed Central

    Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2016-01-01

    Conventional flow cytometry using scattering and fluorescent detection methods has been a fundamental tool of biological discoveries for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents the long-term study of cells in their native environment. Here, we summarize recent advances of new generation flow cytometry for in vivo noninvasive label-free or targeted detection of cells in blood, lymph, bone, cerebral and plant vasculatures using photoacoustic (PA) detection techniques, multispectral high-pulse-repetition-rate lasers, tunable ultrasharp (up to 0.8 nm) rainbow plasmonic nanoprobes, positive and negative PA contrasts, in vivo magnetic enrichment, time-of-flight cell velocity measurement, PA spectral analysis, and integration of PA, photothermal (PT), fluorescent, and Raman methods. Unique applications of this tool are reviewed with a focus on ultrasensitive detection of normal blood cells at different functional states (e.g., apoptotic and necrotic) and rare abnormal cells including circulating tumor cells (CTCs), cancer stem cells, pathogens, clots, sickle cells as well as pharmokinetics of nanoparticles, dyes, microbubbles and drug nanocarriers. Using this tool we discovered that palpation, biopsy, or surgery can enhance CTC release from primary tumors, increasing the risk of metastasis. The novel fluctuation flow cytometry provided the opportunity for the dynamic study of blood rheology including red blood cell aggregation and clot formation in different medical conditions (e.g., blood disorders, cancer, or surgery). Theranostics, as a combination of PA diagnosis and PT nanobubble-amplified multiplex therapy, was used for eradication of CTCs, purging of infected blood, and thrombolysis of clots using PA guidance to control therapy efficiency. In vivo flow cytometry using a portable fiber-based devices can provide a breakthrough platform for early diagnosis of cancer, infection and

  5. Documentation of immunocytochemistry controls in the cytopathologic literature: a meta-analysis of 100 journal articles.

    PubMed

    Colasacco, Carol; Mount, Sharon; Leiman, Gladwyn

    2011-04-01

    Although a detailed description of the procedure and tissue used as controls is considered a necessary component in surgical pathology articles in which immunohistochemistry is utilized, such documentation seems less stringent in the cytopathologic literature. A comprehensive literature search was done for articles published in English within the last 15 years on nine of the most widely used antibodies in cytopathology. Individual case reports were excluded. Of the 100 articles reviewed, 13 articles were review articles or commentaries and hence not included in the analysis. Only 11 (13%) of the remaining 87 articles described positive and negative controls run on identically prepared samples. Forty-seven articles (54%) either did not mention controls or did not run controls as separate specimens. Sixteen articles (18%) included a vague statement about controls. Twelve (14%) commented only on the negative control, included only histology tissue controls, or included cell block controls, but the study also included other types of preparations, such as cytospins. One article (1%) did not include controls because of insufficient material. The College of American Pathologists recognizes the impracticality of maintaining separate positive control samples for every possible combination of fixation, processing, and specimen type. However, more stringent documentation of procedure and use of controls in the cytopathologic literature will ensure that immunocytochemistry results in diagnostic cytopathology as well as in research are valid and reproducible.

  6. Plankton Analysis by Automated Submersible Imaging Flow Cytometry: Transforming a Specialized Research Instrument into a Broadly Accessible Tool and Extending its Target Size Range

    DTIC Science & Technology

    2012-09-30

    the particles suspended in seawater is crucial to an understanding of the biology , optics, and geochemistry of the oceans. The composition and size...been interested in marine applications of flow cytometry, and had sold several slightly-modified instruments (called Influx Marina ) to oceanographic...our WHOI Biology Department colleague Don Anderson, who was funded by NSF to purchase several Environmental Sample Processors (ESP), to be

  7. Uses of flow cytometry in virology.

    PubMed Central

    McSharry, J J

    1994-01-01

    This article reviews some of the published applications of flow cytometry for in vitro and in vivo detection and enumeration of virus-infected cells. Sample preparation, fixation, and permeabilization techniques for a number of virus-cell systems are evaluated. The use of flow cytometry for multiparameter analysis of virus-cell interactions for simian virus 40, herpes simplex viruses, human cytomegalovirus, and human immunodeficiency virus and its use for determining the effect of antiviral compounds on these virus-infected cells are reviewed. This is followed by a brief description of the use of flow cytometry for the analysis of several virus-infected cell systems, including blue tongue virus, hepatitis C virus, avian reticuloendotheliosis virus, African swine fever virus, woodchuck hepatitis virus, bovine viral diarrhea virus, feline leukemia virus, Epstein-Barr virus, Autographa californica nuclear polyhedrosis virus, and Friend murine leukemia virus. Finally, the use of flow cytometry for the rapid diagnosis of human cytomegalovirus and human immunodeficiency virus in peripheral blood cells of acutely infected patients and the use of this technology to monitor patients on antiviral therapy are reviewed. Future prospects for the rapid diagnosis of in vivo viral and bacterial infections by flow cytometry are discussed. Images PMID:7530594

  8. CytometryML binary data standards

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.

    2005-03-01

    CytometryML is a proposed new Analytical Cytology (Cytomics) data standard, which is based on a common set of XML schemas for encoding flow cytometry and digital microscopy text based data types (metadata). CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. The separation of the large binary data objects (list mode and image data) from the XML description of the metadata permits the metadata to be directly displayed, analyzed, and reported with standard commercial software packages; the direct use of XML languages; and direct interfacing with clinical information systems. The separation of the binary data into its own files simplifies parsing because all extraneous header data has been eliminated. The storage of images as two-dimensional arrays without any extraneous data, such as in the Adobe Photoshop RAW format, facilitates the development by scientists of their own analysis and visualization software. Adobe Photoshop provided the display infrastructure and the translation facility to interconvert between the image data from commercial formats and RAW format. Similarly, the storage and parsing of list mode binary data type with a group of parameters that are specified at compilation time is straight forward. However when the user is permitted at run-time to select a subset of the parameters and/or specify results of mathematical manipulations, the development of special software was required. The use of CytometryML will permit investigators to be able to create their own interoperable data analysis software and to employ commercially available software to disseminate their data.

  9. Inertial microfluidics for flow cytometry

    NASA Astrophysics Data System (ADS)

    Di Carlo, Dino

    2010-08-01

    Inertial components of the Navier-Stokes equations are usually not considered in microfluidic flows but have recently been shown to be of great practical use for continuous manipulation of particles and cells. After introducing the physical basis of the counter-intuitive self focusing of particles in a single inlet flow, I will discuss our current best focusing systems, and I will present results on using inertial focusing to create an extreme throughput flow cytometer for blood analysis. This system is an imaging cytometer implementation that can image 1 million focused blood cells per second, with the capability to increase to 20 million cells per second with appropriate wide-field of view imaging systems. The microfluidic device consists of 256 parallel high-aspect ratio microchannels in each of which two streams of focused cells assemble. These cells also form regular trains in the direction of flow such that cell coincidence is a rare occurrence, far below Poisson statistics suggest. Controlled inertially focused streams of particles are poised to provide next-generation filter-less filters and simplified flow cytometry instruments which ultimately may aid in water treatment environmental cleanup and cost-effective medical diagnostics.

  10. Observations on the application of the Papanicolaou Society of Cytopathology standardised terminology and nomenclature for pancreaticobiliary cytology.

    PubMed

    McKinley, Madeleine; Newman, Marsali

    2016-06-01

    In 2014 the Papanicolaou Society of Cytopathology (PSC) published a system of standardised terminology and nomenclature for pancreaticobiliary cytology (STNPC). In the present study, 232 previously reported pancreaticobiliary cytology specimens were categorised according to this set of guidelines in order to identify potential challenges to implementation of the PSC system into routine practice. Overall, 207 (89%) of the cases were found to comply with the PSC scheme in their original form. Twenty-five cases (11%) demonstrated that the application of the PSC system would result in a change of category. In the majority of these cases, the change was related to the method of categorising low grade and premalignant neoplasms, using the categories of 'Neoplastic: other' (a new category unique to STNPC classification scheme) and 'Atypical', for specimens deemed to be diagnostic of or suspicious for these lesions, respectively. The study also highlighted the emphasis on the inclusion of imaging context and cyst fluid analysis in the interpretation of endoscopic ultrasound guided fine needle aspiration specimens in the guidelines. The STNPC offers an approach to pancreaticobiliary cytology that reflects the considerable variation in the nature and treatment of the entities that may be encountered in these specimens. Challenges in utilisation of the scheme include awareness of the unique approach to the categorisation of premalignant and low grade neoplasms, and the amount and quality of available clinical and imaging information.

  11. Spectral cytopathology: new aspects of data collection, manipulation and confounding effects.

    PubMed

    Miljković, Miloš; Bird, Benjamin; Lenau, Kathleen; Mazur, Antonella I; Diem, Max

    2013-07-21

    This paper presents a short review on the improvements in data processing for spectral cytopathology, the diagnostic method developed for large scale diagnostic analysis of spectral data of individual dried and fixed cells. This review is followed by the analysis of the confounding effects introduced by utilizing reflecting "low-emissivity" (low-e) slides as sample substrates in infrared micro-spectroscopy of biological samples such as individual dried cells or tissue sections. The artifact introduced by these substrates, referred to as the "standing electromagnetic wave" artifact, indeed, distorts the spectra noticeably, as postulated recently by several research groups. An analysis of the standing wave effect reveals that careful data pre-processing can reduce the spurious effects to a level where they are not creating a major problem for spectral cytopathology and spectral histopathology.

  12. Cytometry of mammalian sperm

    SciTech Connect

    Gledhill, B.L.

    1983-10-11

    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. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples.

  13. Application of scanning cytometry and confocal-microscopy-based image analysis for investigation the role of cytoskeletal elements during equine herpesvirus type 1 (EHV-1) infection of primary murine neurons.

    PubMed

    Słońska, A; Cymerys, J; Godlewski, M M; Bańbura, M W

    2016-11-01

    Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the β-tubulin fibres within the neurites of infected cells. Alterations in β-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other.

  14. Near infrared lasers in flow cytometry.

    PubMed

    Telford, William G

    2015-07-01

    Technology development in flow cytometry has closely tracked laser technology, the light source that flow cytometers almost exclusively use to excite fluorescent probes. The original flow cytometers from the 1970s and 1980s used large water-cooled lasers to produce only one or two laser lines at a time. Modern cytometers can take advantage of the revolution in solid state laser technology to use almost any laser wavelength ranging from the ultraviolet to the near infrared. Commercial cytometers can now be equipped with many small solid state lasers, providing almost any wavelength needed for cellular analysis. Flow cytometers are now equipped to analyze 20 or more fluorescent probes simultaneously, requiring multiple laser wavelengths. Instrument developers are now trying to increase this number by designing fluorescent probes that can be excited by laser wavelength at the "edges" of the visible light range, in the near ultraviolet and near-infrared region. A variety of fluorescent probes have been developed that excite with violet and long wavelength ultraviolet light; however, the near-infrared range (660-800 nm) has yet seen only exploitation in flow cytometry. Fortunately, near-infrared laser diodes and other solid state laser technologies appropriate for flow cytometry have been in existence for some time, and can be readily incorporated into flow cytometers to accelerate fluorescent probe development. The near infrared region represents one of the last "frontiers" to maximize the number of fluorescent probes that can be analyzed by flow cytometry. In addition, near infrared fluorescent probes used in biomedical tracking and imaging could also be employed for flow cytometry with the correct laser wavelengths. This review describes the available technology, including lasers, fluorescent probes and detector technology optimal for near infrared signal detection.

  15. Multiplex immunoassay for persistent organic pollutants in tilapia: Comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays required a flow cytometer with sophisticated fluidics and optics. The new imaging superparamagnetic SEMs-based platform transports SEMs with considerably ...

  16. Two-Photon Flow Cytometry

    NASA Technical Reports Server (NTRS)

    Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

    2004-01-01

    Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

  17. The application of histo-cytopathological biomarkers in marine pollution monitoring: a review.

    PubMed

    Au, D W T

    2004-05-01

    During the past two decades, a variety of histopathological alterations in fish and bivalves have been developed and used as biomarkers in pollution monitoring. Some of these have been successfully adopted in major national monitoring programmes, while others, although show promise, are still in the experimental stage. This paper critically reviews the scientific basis, cause and effect relationship, reliability, advantages and limitations of 14 histo-cytopathological biomarkers. The usefulness and practical application of each biomarker have been evaluated against a number of objective criteria including: ecological relevance, sensitivity, specificity, dose-response relationship, confounding factors, technical difficulties and cost-effectiveness.

  18. Cytopathology and coagulopathy associated with viral erythrocytic necrosis in chum salmon

    USGS Publications Warehouse

    MacMillian, John R.; Mulcahy, D.; Landolt, M.L.

    1989-01-01

    The 8-month cytopathologic progression of viral erythrocytic necrosis (VEN) disease in chum salmon Oncorhynchus keta is described. Single to multiple acidophilic, cytoplasmic viral inclusion bodies developed first in mature erythrocytes and then, within 1–2 months, all morphologically identifiable hemopoietic cell types contained VEN inclusions. Cytologic analysis indicated that multinucleate giant erythroblasts, ineffective erythropoiesis, and abnormal erythroid cell maturation occurred. A significant increase in blood coagulation time occurred concomitantly. This severe and chronic blood dyscrasia accounts for some of the pathophysiologic sequelae previously observed.

  19. In Vivo Flow Cytometry: A Horizon of Opportunities

    PubMed Central

    Tuchin, Valery V.; Tárnok, Attila; Zharov, Vladimir P.

    2012-01-01

    Flow cytometry has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo flow cytometry which provides detection and imaging of circulating normal and abnormal cells directlyin blood or lymph flow. The goal of this mini-review is to provide a brief history, features and challenges of this new generation of flow cytometry methods and instruments. Spectrum of possibilities of in vivo flow cytometry in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, cardiovascular disorder) including integrated photoacoustic-photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform. PMID:21915991

  20. Analyzing the Tumor Microenvironment by Flow Cytometry.

    PubMed

    Young, Yoon Kow; Bolt, Alicia M; Ahn, Ryuhjin; Mann, Koren K

    2016-01-01

    Flow cytometry is an essential tool for studying the tumor microenvironment. It allows us to quickly quantify and identify multiple cell types in a heterogeneous sample. A brief overview of flow cytometry instrumentation and the appropriate considerations and steps in building a good flow cytometry staining panel are discussed. In addition, a lymphoid tissue and solid tumor leukocyte infiltrate flow cytometry staining protocol and an example of flow cytometry data analysis are presented.

  1. Big data from small samples: Informatics of next-generation sequencing in cytopathology.

    PubMed

    Roy-Chowdhuri, Sinchita; Roy, Somak; Monaco, Sara E; Routbort, Mark J; Pantanowitz, Liron

    2016-12-05

    The rapid adoption of next-generation sequencing (NGS) in clinical molecular laboratories has redefined the practice of cytopathology. Instead of simply being used as a diagnostic tool, cytopathology has evolved into a practice providing important genomic information that guides clinical management. The recent emphasis on maximizing limited-volume cytology samples for ancillary molecular studies, including NGS, requires cytopathologists not only to be more involved in specimen collection and processing techniques but also to be aware of downstream testing and informatics issues. For the integration of molecular informatics into the clinical workflow, it is important to understand the computational components of the NGS workflow by which raw sequence data are transformed into clinically actionable genomic information and to address the challenges of having a robust and sustainable informatics infrastructure for NGS-based testing in a clinical environment. Adapting to needs ranging from specimen procurement to report delivery is crucial for the optimal utilization of cytology specimens to accommodate requests from clinicians to improve patient care. This review presents a broad overview of the various aspects of informatics in the context of NGS-based testing of cytology specimens. Cancer Cytopathol 2016. © 2016 American Cancer Society.

  2. Rise of the Micromachines: Microfluidics and the Future of Cytometry

    PubMed Central

    Wlodkowic, Donald; Darzynkiewicz, Zbigniew

    2011-01-01

    The past decade has brought many innovations to the field of flow and image-based cytometry. These advancements can be seen in the current miniaturization trends and simplification of analytical components found in the conventional flow cytometers. On the other hand, the maturation of multispectral imaging cytometry in flow imaging and the slide-based laser scanning cytometers offers great hopes for improved data quality and throughput while proving new vistas for the multiparameter, real-time analysis of cells and tissues. Importantly, however, cytometry remains a viable and very dynamic field of modern engineering. Technological milestones and innovations made over the last couple of years are bringing the next generation of cytometers out of centralized core facilities while making it much more affordable and user friendly. In this context, the development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry. LOC devices promise new functionalities that can overcome current limitations while at the same time promise greatly reduced costs, increased sensitivity, and ultra high throughputs. We can expect that the current pace in the development of novel microfabricated cytometric systems will open up groundbreaking vistas for the field of cytometry, lead to the renaissance of cytometric techniques and most importantly greatly support the wider availability of these enabling bioanalytical technologies. PMID:21704837

  3. Complexities of bloom dynamics in the toxic dinoflagellate Alexandrium fundyense revealed through DNA measurements by imaging flow cytometry coupled with species-specific rRNA probes

    NASA Astrophysics Data System (ADS)

    Brosnahan, Michael L.; Farzan, Shahla; Keafer, Bruce A.; Sosik, Heidi M.; Olson, Robert J.; Anderson, Donald M.

    2014-05-01

    Measurements of the DNA content of different protist populations can shed light on a variety of processes, including cell division, sex, prey ingestion, and parasite invasion. Here, we modified an Imaging FlowCytobot (IFCB), a custom-built flow cytometer that records images of microplankton, to measure the DNA content of large dinoflagellates and other high-DNA content species. The IFCB was also configured to measure fluorescence from Cy3-labeled rRNA probes, aiding the identification of Alexandrium fundyense (syn. A. tamarense Group I), a photosynthetic dinoflagellate that causes paralytic shellfish poisoning (PSP). The modified IFCB was used to analyze samples from the development, peak and termination phases of an inshore A. fundyense bloom (Salt Pond, Eastham, MA, USA), and from a rare A. fundyense ‘red tide’ that occurred in the western Gulf of Maine, offshore of Portsmouth, NH (USA). Diploid or G2 phase (‘2C’) A. fundyense cells were frequently enriched at the near-surface, suggesting an important role for aggregation at the air-sea interface during sexual events. Also, our analysis showed that large proportions of A. fundyense cells in both the Salt Pond and red tide blooms were planozygotes during bloom decline, highlighting the importance of sexual fusion to bloom termination. At Salt Pond, bloom decline also coincided with a dramatic rise in infections by the parasite genus Amoebophrya. The samples that were most heavily infected contained many large cells with higher DNA-associated fluorescence than 2C vegetative cells, but these cells' nuclei were also frequently consumed by Amoebophrya trophonts. Neither large cell size nor increased DNA-associated fluorescence could be replicated by infecting an A. fundyense culture of vegetative cells. Therefore, we attribute these characteristics of the large Salt Pond cells to planozygote maturation rather than Amoebophrya infection, though an interaction between infection and planozygote maturation may

  4. Cytopathology laboratory improvement programs of the College of American Pathologists: Laboratory Accreditation Program (CAP LAP) and Performance Improvement Program in Cervicovaginal Cytology (CAP PAP).

    PubMed

    Nielsen, M L

    1997-03-01

    Major programs of the College of American Pathologists (CAP) are directed toward improvement of laboratory practices through peer review, interlaboratory comparison, education, and development of practice standards and guidelines. Two programs provided to cytopathology laboratories, the Laboratory Accreditation Program and the Interlaboratory Comparison Program in Cervicovaginal Cytology, are dedicated to these laboratory improvement principles. In 1996, each of these programs served over 2100 laboratories that provide cytopathology services. This paper reviews the peer development, structure, and administration of the Laboratory Accreditation Program and the Interlaboratory Comparison Program in Cervicovaginal Cytology, focusing on recent and ongoing initiatives to enhance their contribution to continued improvement of gynecologic cytopathology laboratory practices.

  5. Teaching phagocytosis using flow cytometry.

    PubMed

    Boothby, John T; Kibler, Ruthann; Rech, Sabine; Hicks, Robert

    2004-05-01

    Investigative microbiology on protists in a basic teaching laboratory environment is limited by student skill level, ease of microbial culture and manipulation, instrumentation, and time. The flow cytometer is gaining use as a mainstream instrument in research and clinical laboratories, but has had minimal application in teaching laboratories. Although the cost of a flow cytometer is currently prohibitive for many microbiology teaching environments and the number of trained instructors and teaching materials is limited, in many ways the flow cytometer is an ideal instrument for teaching basic microbiology. We report here on a laboratory module to study phagocytosis in Tetrahymena sp. using flow cytometry in a basic microbiology teaching laboratory. Students and instructors found the flow cytometry data analysis program, Paint-AGate(PRO-TM), to be very intuitive and easy to learn within a short period of time. Assessment of student learning about Tetrahymena sp., phagocytosis, flow cytometry, and investigative microbiology using an inquiry-based format demonstrated an overall positive response from students.

  6. Adaptive eLearning modules for cytopathology education: A review and approach.

    PubMed

    Samulski, T Danielle; La, Teresa; Wu, Roseann I

    2016-11-01

    Clinical training imposes time and resource constraints on educators and learners, making it difficult to provide and absorb meaningful instruction. Additionally, innovative and personalized education has become an expectation of adult learners. Fortunately, the development of web-based educational tools provides a possible solution to these challenges. Within this review, we introduce the utility of adaptive eLearning platforms in pathology education. In addition to a review of the current literature, we provide the reader with a suggested approach for module creation, as well as a critical assessment of an available platform, based on our experience in creating adaptive eLearning modules for teaching basic concepts in gynecologic cytopathology. Diagn. Cytopathol. 2016;44:944-951. © 2016 Wiley Periodicals, Inc.

  7. Standardized terminology and nomenclature for pancreatobiliary cytology: The Papanicolaou Society of Cytopathology Guidelines.

    PubMed

    Pitman, Martha B; Centeno, Barbara A; Ali, Syed Z; Genevay, Muriel; Stelow, Ed; Mino-Kenudson, Mari; Castillo, Carlos Fernandez-Del; Schmidt, C Max; Brugge, William R; Layfield, Lester J

    2014-01-01

    The Papanicolaou Society of Cytopathology has developed a set of guidelines for pancreatobiliary cytology including indications for endoscopic ultrasound (EUS) guided fine-needle aspiration (FNA) biopsy, techniques of EUS-FNA, terminology and nomenclature of pancreatobiliary disease, ancillary testing and post-biopsy treatment and management. All documents are based on the expertise of the authors, a review of the literature, discussion of the draft document at several national and international meetings over an 18 month period and synthesis of online comments of the draft document on the Papanicolaou Society of Cytopathology web site [www.papsociety.org]. This document selectively presents the results of these discussions and focuses on a proposed standardized terminology scheme for pancreatobiliary specimens that correlate cytological diagnosis with biological behavior and increasingly conservative patient management of surveillance only. The proposed terminology scheme recommends a six-tiered system: Non-diagnostic, negative, atypical, neoplastic [benign or other], suspicious and positive. Unique to this scheme is the "neoplastic" category separated into "benign" (serous cystadenoma) or "other" (premalignant mucinous cysts, neuroendocrine tumors and solid-pseudopapillary neoplasms (SPNs)). The positive or malignant category is reserved for high-grade, aggressive malignancies including ductal adenocarcinoma, acinar cell carcinoma, poorly differentiated neuroendocrine carcinomas, pancreatoblastoma, lymphoma and metastases. Interpretation categories do not have to be used. Some pathology laboratory information systems require an interpretation category, which places the cytological diagnosis into a general category. This proposed scheme provides terminology that standardizes the category of the various diseases of the pancreas, some of which are difficult to diagnose specifically by cytology. In addition, this terminology scheme attempts to provide maximum

  8. Effects of dietary boron on cervical cytopathology and on micronucleus frequency in exfoliated buccal cells.

    PubMed

    Korkmaz, Mehmet; Uzgören, Engin; Bakirdere, Sezgin; Aydin, Firat; Ataman, O Yavuz

    2007-02-01

    Recent evidence indicates that boron and borates may have anticarcinogenic properties. In this study, we have investigated the incidence of adverse cytological findings in cervical smears and the micronucleus (MN) frequency in women living in boron-rich and boron-poor regions. Cervical smears were prepared from 1059 women with low socioeconomic status; 472 of the women lived in relatively boron-rich rural areas, while 587 lived in relatively boron-poor regions. The average and standard deviation values for the age of the women screened with the cervical Pap smear test were 41.55 +/- 8.38. The mean dietary intake of boron was 8.41 mg/day for women from the boron-rich regions, and 1.26 mg/day for women living in the boron-poor regions (P < 0.0001). Women from the boron-rich regions had no cytopathological indications of cervical cancer, while there were cytopathological findings for 15 women from the boron-poor areas (chi(2) = 10.473, P < 0.05). Sixty women, 30 from each region, were chosen for evaluating MN frequencies in exfoliated buccal cells. MN frequencies for women from the boron-rich and boron-poor regions were not significantly different (t = -0.294, P > 0.05). Also, there were no significant correlations between age and MN frequency for women from both the boron-rich (r = 0.133, P = 0.48, P > 0.05) and boron-poor (r = -0.033, P = 0.861, P > 0.05) regions. The results suggest that ingestion of boron in the drinking water decreases the incidence of cervical cancer-related histopathological findings. There was no correlation between the pathological findings from the cervical smears and buccal cell MN frequency suggesting that the two study populations were exposed equally to gentotoxic agents. Nonetheless, cervical cancer-related histopathological findings should be validated by other researchers.

  9. Standardized terminology and nomenclature for pancreatobiliary cytology: the Papanicolaou Society of Cytopathology guidelines.

    PubMed

    Pitman, Martha B; Centeno, Barbara A; Ali, Syed Z; Genevay, Muriel; Stelow, Ed; Mino-Kenudson, Mari; Fernandez-del Castillo, Carlos; Max Schmidt, C; Brugge, William; Layfield, Lester

    2014-04-01

    The Papanicolaou Society of Cytopathology has developed a set of guidelines for pancreatobiliary cytology including indications for endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) biopsy, techniques of EUS-FNA, terminology and nomenclature of pancreatobiliary disease, ancillary testing, and postbiopsy treatment and management. All documents are based on the expertise of the authors, a review of the literature, discussions of the draft document at several national and international meetings over an 18-month period and synthesis of online comments of the draft document on the Papanicolaou Society of Cytopathology web site (www.papsociety.org). This document selectively presents the results of these discussions and focuses on a proposed standardized terminology scheme for pancreatobiliary specimens that correlate cytological diagnosis with biological behavior and increasingly conservative patient management of surveillance only. The proposed terminology scheme recommends a six-tiered system: Nondiagnostic, Negative, Atypical, Neoplastic (benign or other), Suspicious and Positive. Unique to this scheme is the "Neoplastic" category separated into "benign" (serous cystadenoma), or "Other" (premalignant mucinous cysts, neuroendocrine tumors, and solid-pseudopapillary neoplasms). The positive or malignant category is reserved for high-grade, aggressive malignancies including ductal adenocarcinoma, acinar cell carcinoma, poorly differentiated neuroendocrine carcinomas, pancreatoblastoma, lymphoma, and metastases. Interpretation categories do not have to be used. Some pathology laboratory information systems require an interpretation category, which places the cytological diagnosis into a general category. This proposed scheme provides terminology that standardizes the category of the various diseases of the pancreas, some of which are difficult to diagnose specifically by cytology. In addition, this terminology scheme attempts to provide maximum flexibility

  10. Diagnosis of blastomycosis in surgical pathology and cytopathology: correlation with microbiologic culture.

    PubMed

    Patel, Ajay Jitendra; Gattuso, Paolo; Reddy, Vijaya B

    2010-02-01

    Blastomycosis, a worldwide disease caused by the inhalation of Blastomyces dermatitidis spores, can be diagnosed by microbiologic culture or morphologic identification in tissue or cytologic material. A retrospective review of cases diagnosed as blastomycosis in surgical pathology and cytopathology was undertaken at a University Medical Center to assess the diagnostic value of morphologic methods and their correlation with microbiologic cultures. Surgical pathology/cytology records were reviewed for the period between January 1998 and April 2007 and 53 cases diagnosed as blastomycosis were retrieved: 38 males, 15 females; age 14 to 77 years, median 48. Twenty-nine cases (54.7%) involved lung, 14 (26.4%) soft tissue/bone, 5 (9.4%) skin, 3 (5.6%) other sites, and 2 (3.7%) involved both lung and skin. Forty-six of the 53 patients (87%) had concomitant cultures: 31 (67.4%) were positive for blastomycosis, 11 (23.9%) negative and 4 (8.7%) showed other fungal organisms. A review of microbiology laboratory results for the same period identified a total of 39 patients who were diagnosed with blastomycosis based on isolation of B. dermatitidis. These included 31 cases (79.5%) that were also diagnosed on histology/cytology specimens, 4 (10.25%) that were not submitted to surgical pathology and 4 (10.25%) cases in which pathologic examination failed to identify Blastomyces. This study shows that blastomycosis encountered in surgical/cytopathology can be reliably diagnosed by morphologic examination allowing for prompt treatment. However, microbiologic cultures still play a major role in clinical management of patients suspected of infection because 10.25% were false negative on morphology in our study.

  11. Standardized terminology and nomenclature for pancreatobiliary cytology: The Papanicolaou Society of Cytopathology Guidelines

    PubMed Central

    Pitman, Martha B.; Centeno, Barbara A.; Ali, Syed Z.; Genevay, Muriel; Stelow, Ed; Mino-Kenudson, Mari; Castillo, Carlos Fernandez-del; Schmidt, C. Max; Brugge, William R.; Layfield, Lester J.

    2014-01-01

    The Papanicolaou Society of Cytopathology has developed a set of guidelines for pancreatobiliary cytology including indications for endoscopic ultrasound (EUS) guided fine-needle aspiration (FNA) biopsy, techniques of EUS-FNA, terminology and nomenclature of pancreatobiliary disease, ancillary testing and post-biopsy treatment and management. All documents are based on the expertise of the authors, a review of the literature, discussion of the draft document at several national and international meetings over an 18 month period and synthesis of online comments of the draft document on the Papanicolaou Society of Cytopathology web site [www.papsociety.org]. This document selectively presents the results of these discussions and focuses on a proposed standardized terminology scheme for pancreatobiliary specimens that correlate cytological diagnosis with biological behavior and increasingly conservative patient management of surveillance only. The proposed terminology scheme recommends a six-tiered system: Non-diagnostic, negative, atypical, neoplastic [benign or other], suspicious and positive. Unique to this scheme is the “neoplastic” category separated into “benign” (serous cystadenoma) or “other” (premalignant mucinous cysts, neuroendocrine tumors and solid-pseudopapillary neoplasms (SPNs)). The positive or malignant category is reserved for high-grade, aggressive malignancies including ductal adenocarcinoma, acinar cell carcinoma, poorly differentiated neuroendocrine carcinomas, pancreatoblastoma, lymphoma and metastases. Interpretation categories do not have to be used. Some pathology laboratory information systems require an interpretation category, which places the cytological diagnosis into a general category. This proposed scheme provides terminology that standardizes the category of the various diseases of the pancreas, some of which are difficult to diagnose specifically by cytology. In addition, this terminology scheme attempts to provide

  12. Computational analysis of high-throughput flow cytometry data

    PubMed Central

    Robinson, J Paul; Rajwa, Bartek; Patsekin, Valery; Davisson, Vincent Jo

    2015-01-01

    Introduction Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology. Areas covered Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension. Expert opinion There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible. PMID:22708834

  13. Bacteria detection by flow cytometry.

    PubMed

    Karo, Oliver; Wahl, Alexandra; Nicol, Sven-Boris; Brachert, Julia; Lambrecht, Bernd; Spengler, Hans-Peter; Nauwelaers, Frans; Schmidt, Michael; Schneider, Christian K; Müller, Thomas H; Montag, Thomas

    2008-01-01

    Since bacterial infection of the recipient has become the most frequent infection risk in transfusion medicine, suitable methods for bacteria detection in blood components are of great interest. Platelet concentrates are currently the focus of attention, as they are stored under temperature conditions, which enable the multiplication of most bacteria species contaminating blood donations. Rapid methods for bacteria detection allow testing immediately before transfusion in a bed-side like manner. This approach would overcome the sampling error observed in early sampling combined with culturing of bacteria and would, at least, prevent the transfusion of highly contaminated blood components leading to acute septic shock or even death of the patient. Flow cytometry has been demonstrated to be a rapid and feasible approach for detection of bacteria in platelet concentrates. The general aim of the current study was to develop protocols for the application of this technique under routine conditions. The effect of improved test reagents on practicability and sensitivity of the method is evaluated. Furthermore, the implementation of fluorescent absolute count beads as an internal standard is demonstrated. A simplified pre-incubation procedure has been undertaken to diminish the detection limit in a pragmatic manner. Additionally, the application of bacteria detection by flow cytometry as a culture method is shown, i.e., transfer of samples from platelet concentrates into a satellite bag, incubation of the latter at 37 degrees C, and measuring the contaminating bacteria in a flow cytometer.

  14. Abstracts for the 59th Annual Scientific Meeting (November 2011) by American Society of Cytopathology (ASC) at Baltimore, MD, USA

    PubMed Central

    2011-01-01

    These are peer-reviewed poster-platform submissions finalized by the Scientific Program Committee. A total of 153 abstracts (14 Platforms [PP1 through PP14] & 139 Posters [1 through 139]) were selected from 161 submissions to be considered for presentation during November 4 – 8, 2011, at the Hilton Baltimore Hotel, to pathologists, cytopathologists, cytotechnologists, residents, fellows, students, and other members of cytopathology-related medical and scientific fields.

  15. Cytopathologic characteristics of the primary strumal carcinoid tumor of the ovary: a case report with emphasis on differential diagnostic considerations.

    PubMed

    Hayashi, Toshitetsu; Haba, Reiji; Kushida, Yoshio; Kadota, Kyuichi; Katsuki, Naomi; Miyai, Yumi; Shibuya, Shinsuke; Sasaki, Makiko; Bando, Kenji; Matsunaga, Toru; Hata, Toshiyuki

    2013-09-01

    Primary strumal carcinoid tumor of the ovary (SCTO) is an extremely rare entity, though the survival rate is excellent if the disease is confined to one ovary. A case is presented here in which intraoperative squash smears in a 45-year-old woman with a left adnexal mass revealed dispersed or small clusters of neoplastic cells forming loosely cohesive gland-like structures with abundant cytoplasm. The nuclear chromatin was finely granular with a "salt and pepper" appearance and occasional tiny nucleoli. The position of the nucleus presented a vaguely plasmacytoid appearance. Small fragments of thyroidal colloid-like structures were also identified. A cytopathologic diagnosis of a SCTO was suggested. Further evaluation and immunohistochemical studies were conducted on formalin-fixed, paraffin-embedded material. Cords or nests of uniform cells with abundant cytoplasm, and eccentric nuclei with coarse chromatin and occasional colloidal tissue were identified on H&E sections. The tumor cells showed diffuse and strong cytoplasmic staining for chromogranin A, synaptophysin, CD56, and vimentin but were negative for calretinin, α-inhibin or CDX2. The proliferative index with MIB-1 was around 3%. Thyroidal colloid-like structures were immunoreactive for thyroglobulin and TTF-1 stains. The diagnosis of primary SCTO was confirmed based on cytopathologic, histopathological, and immunohistochemical results, and the location of the tumor. Awareness of the cytopathological findings of SCTO can assist in diagnosing this rare entity correctly.

  16. Flow cytometry: retrospective, fundamentals and recent instrumentation.

    PubMed

    Picot, Julien; Guerin, Coralie L; Le Van Kim, Caroline; Boulanger, Chantal M

    2012-03-01

    Flow cytometry is a complete technology given to biologists to study cellular populations with high precision. This technology elegantly combines sample dimension, data acquisition speed, precision and measurement multiplicity. Beyond the statistical aspect, flow cytometry offers the possibility to physically separate sub-populations. These performances come from the common endeavor of physicists, biophysicists, biologists and computer engineers, who succeeded, by providing new concepts, to bring flow cytometry to current maturity. The aim of this paper is to present a complete retrospective of the technique and remind flow cytometry fundamentals before focusing on recent commercial instrumentation.

  17. Guidelines for resident training in veterinary clinical pathology. III: cytopathology and surgical pathology.

    PubMed

    Kidney, Beverly A; Dial, Sharon M; Christopher, Mary M

    2009-09-01

    The Education Committee of the American Society for Veterinary Clinical Pathology has identified a need for improved structure and guidance of training residents in clinical pathology. This article is the third in a series of articles that address this need. The goals of this article are to describe learning objectives and competencies in knowledge, abilities, and skills in cytopathology and surgical pathology (CSP); provide options and ideas for training activities; and identify resources in veterinary CSP for faculty, training program coordinators, and residents. Guidelines were developed in consultation with Education Committee members and peer experts and with evaluation of the literature. The primary objectives of training in CSP are: (1) to develop a thorough, extensive, and relevant knowledge base of biomedical and clinical sciences applicable to the practice of CSP in domestic animals, laboratory animals, and other nondomestic animal species; (2) to be able to reason, think critically, investigate, use scientific evidence, and communicate effectively when making diagnoses and consulting and to improve and advance the practice of pathology; and (3) to acquire selected technical skills used in CSP and pathology laboratory management. These guidelines define expected competencies that will help ensure proficiency, leadership, and the advancement of knowledge in veterinary CSP and will provide a useful framework for didactic and clinical activities in resident-training programs.

  18. Cytometry in Cell Necrobiology Revisited. Recent Advances and New Vistas

    PubMed Central

    Wlodkowic, Donald; Skommer, Joanna; Darzynkiewicz, Zbigniew

    2010-01-01

    Over a decade has passed since publication of the last review on “Cytometry in cell necrobiology.” During these years we have witnessed many substantial developments in the field of cell necrobiology such as remarkable advancements in cytometric technologies and improvements in analytical biochemistry. The latest innovative platforms such as laser scanning cytometry, multispectral imaging cytometry, spectroscopic cytometry, and microfluidic Lab-on-a-Chip solutions rapidly emerge as highly advantageous tools in cell necrobiology studies. Furthermore, we have recently gained substantial knowledge on alternative cell demise modes such as caspase-independent apoptosis-like programmed cell death (PCD), autophagy, necrosis-like PCD, or mitotic catastrophe, all with profound connotations to pathogenesis and treatment. Although detection of classical, caspase-dependent apoptosis is still the major ground for the advancement of cytometric techniques, there is an increasing demand for novel analytical tools to rapidly quantify noncanonical modes of cell death. This review highlights the key developments warranting a renaissance and evolution of cytometric techniques in the field of cell necrobiology. PMID:20235235

  19. Flow cytometry: basic principles and applications.

    PubMed

    Adan, Aysun; Alizada, Günel; Kiraz, Yağmur; Baran, Yusuf; Nalbant, Ayten

    2017-03-01

    Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.

  20. Flow Cytometry: Impact On Early Drug Discovery

    PubMed Central

    Edwards, Bruce S.; Sklar, Larry A.

    2015-01-01

    Summary Modern flow cytometers can make optical measurements of 10 or more parameters per cell at tens-of-thousands of cells per second and over five orders of magnitude dynamic range. Although flow cytometry is used in most drug discovery stages, “sip-and-spit” sampling technology has restricted it to low sample throughput applications. The advent of HyperCyt sampling technology has recently made possible primary screening applications in which tens-of-thousands of compounds are analyzed per day. Target-multiplexing methodologies in combination with extended multi-parameter analyses enable profiling of lead candidates early in the discovery process, when the greatest numbers of candidates are available for evaluation. The ability to sample small volumes with negligible waste reduces reagent costs, compound usage and consumption of cells. Improved compound library formatting strategies can further extend primary screening opportunities when samples are scarce. Dozens of targets have been screened in 384- and 1536-well assay formats, predominantly in academic screening lab settings. In concert with commercial platform evolution and trending drug discovery strategies, HyperCyt-based systems are now finding their way into mainstream screening labs. Recent advances in flow-based imaging, mass spectrometry and parallel sample processing promise dramatically expanded single cell profiling capabilities to bolster systems level approaches to drug discovery. PMID:25805180

  1. Digital imaging in pathology.

    PubMed

    Park, Seung; Pantanowitz, Liron; Parwani, Anil Vasdev

    2012-12-01

    Advances in computing speed and power have made a pure digital work flow for pathology. New technologies such as whole slide imaging (WSI), multispectral image analysis, and algorithmic image searching seem poised to fundamentally change the way in which pathology is practiced. This article provides the practicing pathologist with a primer on digital imaging. Building on this primer, the current state of the art concerning digital imaging in pathology is described. Emphasis is placed on WSI and its ramifications, showing how it is useful in both anatomic (histology, cytopathology) and clinical (hematopathology) pathology. Future trends are also extrapolated.

  2. In vitro micronucleus assay for the analysis of total particulate matter in cigarette smoke: comparison of flow cytometry and laser scanning cytometry with microscopy.

    PubMed

    Yao, Jianhua; Gao, Qian; Mi, Qili; Li, Xuemei; Miao, Mingming; Cheng, Peng; Luo, Ying

    2013-08-15

    The possible genotoxicity of the total particulate matter (TPM) in cigarette smoke has typically been evaluated using the in vitro micronucleus assay. In recent years, automated scoring techniques have been developed to replace the manual counting process in this assay. However, these automated scoring techniques have not been applied in routine genotoxicity assays for the analysis of TPM to improve the assay efficiency. Chinese hamster ovary (CHO) cells were treated with TPM produced from 14 types of cigarettes at five concentrations (25-200μg/ml) without exogenous metabolic activation. The three following methods were used to score the micronucleus (MN) frequency: (a) flow cytometry with SYTOX and EMA dyes, which differentially stain micronuclei and apoptotic/necrotic chromatin to enhance assay reliability; (b) laser scanning cytometry with FITC and PI dyes, which is a system that combines the analytical capabilities of flow and image cytometry; and (c) visual microcopy with Giemsa dye. The test results obtained using the three methods were compared using correlation analysis. The key findings for this set of compounds include the following: (a) both flow cytometry- and laser scanning cytometry-based methods were effective for MN identification, (b) the three scoring methods could detect dose-dependent micronucleus formation for the 14 types of TPM, and (c) the MN frequencies that were measured in the same samples by flow cytometry, laser scanning cytometry, and visual microscopy were highly correlated, and there were no significant differences (p>0.05). In conclusion, both flow cytometry and laser scanning cytometry can be used to evaluate the MN frequency induced by TPM without exogenous metabolic activation. The simpler and faster processing and the high correlation of the results make these two automatic methods appropriate tools for use in in vitro micronucleus assays for the analysis of TPM using CHO cells.

  3. Centromeric index measurement by slit-scan flow cytometry

    SciTech Connect

    Lucas, J.N.; Gray, J.W.; Peters, D.C.; Van Dilla, M.A.

    1983-01-01

    A report is given of the application of slit-scan flow cytometry (SSFCM) in the classification of muntjac, Chinese hamster, and human chromosomes according to centromeric index (CI) and total fluorescence. Chromosomes were isolated from mitotic cells, stained with propidium iodide and processed through the SSFCM where fluorescence profiles were measured. The centromere for each profile was taken as the point of maximum difference between the measured profile and a standard profile having no centromeric dip. The areas under the profile on either side of the centromere were then calculated and the CI was calculated as the ratio of the larger area to the total area under the profile. Relative DNA contents for each chromosome were taken to be proportional to the total fluorescence. Mean CI's for muntjac chromosomes 1, 2, and X + 3 were 0.52, 0.88, and 0.73, respectively; CI's for Chinese hamster M3-1 chromosomes 1, 2, 5, 8, and M2 were 0.53, 0.55, 0.57, 0.77, and 0.86, respectively; and average CI's for chromosome groups 4 + t (X;5), 6 + 7 + Y, 9 + M1, and 10 + 11 were 0.56, 0.82, 0.58, and 0.60, respectively. These results were, on average, within 4.4% of CI measurements made by image cytometry. CI's measured for human chromosomes 9 through 12, were, on average, within 2.0% of those made by image cytometry.

  4. Convention on nomenclature for DNA cytometry

    SciTech Connect

    Hiddemann, W.; Schumann, J.; Andreeff, M.; Barlogie, B.; Herman, C.J.; Leif, R.C.; Mayall, B.H.; Murphy, R.F.; Sandberg, A.A.

    1984-01-01

    The Committee on Nomenclature of the Society for Analytical Cytology presents guidelines for the analysis of DNA content by cytometry. These guidelines cover: staining of DNA; cytogenetic and cytometric terminology; DNA index; resolution of measurements; and cytometric standards.

  5. DNA polymorphism identity determination using flow cytometry

    DOEpatents

    Nolan, John P.; White, P. Scott; Cai, Hong

    2001-01-01

    DNA polymorphism identity determination using flow cytometry. Primers designed to be immobilized on microspheres are allowed to anneal to the DNA strand under investigation, and are extended by either DNA polymerase using fluorescent dideoxynucleotides or ligated by DNA ligase to fluorescent reporter oligonucleotides. The fluorescence of either the dideoxynucleotide or the reporter oligonucleotide attached to the immobilized primer is measured by flow cytometry, thereby identifying the nucleotide polymorphism on the DNA strand.

  6. Radiation effects on late cytopathological parameters in the murine lens relative to particle fluence

    NASA Astrophysics Data System (ADS)

    Tao, F.; Powers-Risius, P.; Alpen, E. L.; Medvedovsky, C.; David, J.; Worgul, B. V.

    1994-10-01

    Lenses of mice irradiated with 250 MeV protons, 670 MeV/amu20Ne, 600 MeV/amu 56Fe, 600 MeV/amu 93Nb and 593 MeV/amu 139La ions were evaluated by analyzing cytopathological indicators which have been implicated in the cataractogenic process. The LETs ranged from 0.40 keV/μm to 953 keV/μm and fluences from 1.31 × 103/mm2 to 4.99 × 107/mm2. 60Co γ-rays were used as the reference radiation. The doses ranged from 10 to 40 cGy. The lenses were assessed 64 weeks post irradiation in order to observe the late effects of LET and dose on the target cell population of the lens epithelium. Our study shows that growth dependent pathological changes occur at the cellular level as a function of dose and LET. The shapes of the RBE-LET and RBE-dose curves are consistent with previous work on eye and other biological systems done in both our laboratory and others. The RBEmax's were estimated, for the most radiation cataract related cytological changes, MN frequency and MR disorganization, by calculating the ratio of the initial slopes of dose effect curve for various heavy ions to that of 60Co γ-ray. For each ion studied, the RBEmax derived from micronucleus (MN) frequency is similar to that derived from meridional row (MR) disorganization, suggesting that heavy ions are equally efficient at producing each type of damage. Furthermore, on a per particle basis (particle/cell nucleus), both MN frequency and MR disorganization are LET dependent indicating that these classic precataractogenic indicators are multi-gene effects. Poisson probability analysis of the particle number traversing cell nuclei (average area = 24 μm2)suggested that single nuclear traversals determine these changes. By virtue of their precataractogenic nature the data on these endpoints intimate that radiation cataract may also be the consequence of single hits. In any case, these observations are consistent with the current theory of the mechanism of radiation cataractogenesis, which proposes that genomic

  7. The Intersection of Flow Cytometry with Microfluidics and Microfabrication

    PubMed Central

    Piyasena, Menake E.; Graves, Steven W.

    2014-01-01

    A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry. PMID:24488050

  8. Flow cytometry applications in the food industry.

    PubMed

    Comas-Riu, Jaume; Rius, Núria

    2009-08-01

    Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.

  9. Spaceflight Flow Cytometry: Design Challenges and Applications

    NASA Technical Reports Server (NTRS)

    Pappas, Dimitri; Kao, Shih-Hsin; Jeevarajan, Antony S.

    2004-01-01

    Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health and a wide array of biochemistry and immunology assays. While flow cytometry has been widely used for cellular analysis in both clinical and research settings, the requirements for proper operation in spaceflight impose constraints on any instrument designs. The challenges of designing a spaceflight-ready flow cytometer are discussed, as well as some preliminary results using a prototype system.

  10. Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

    PubMed

    Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

    2016-06-02

    Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

  11. Dissociation of the vacuolar and macroautophagic cytopathology from the cytotoxicity induced by the lipophilic local anesthetic bupivacaine.

    PubMed

    Morissette, Guillaume; Bawolak, Marie-Thérèse; Marceau, François

    2011-07-01

    Local anesthetics, like many other cationic drugs, induce a vacuolar and macroautophagic cytopathology that has been observed in vivo and in various cell types; some also induce cytotoxicity of mitochondrial origin (apoptosis and necrosis) and it is not known whether the 2 types of toxicity overlap or interact. We compared bupivacaine with a more hydrophilic agent, lidocaine, for morphological, functional, and toxicological responses in a previously exploited nonneuronal system, primary smooth muscle cells. Bupivacaine induced little vacuolization (≥2.5 mmol/L, 4 h), but elicited autophagic accumulation (≥0.5 mmol/L, 4 h) and was massively cytotoxic at 2.5-5 mmol/L (4-24 h), the latter effect being unabated by the V-ATPase inhibitor bafilomycin A1. Lidocaine exerted little cytotoxicity at and below 5 mmol/L for 24 h, but intensely induced the V-ATPase-dependent vacuolar and autophagic cytopathology. Bupivacaine was more potent than lidocaine in disrupting mitochondrial potential, as judged by Mitotracker staining (significant proportions of cells affected in the 1-5 and 5-10 mmol/L concentration ranges, respectively). The addition of mitochondrial-inactivating toxins antimycin A and oligomycin to lidocaine (2.5 mmol/L) reproduced the profile of bupivacaine action (low intensity of vacuolization and retained autophagic accumulation). The high potency of bupivacaine as a mitochondrial toxicant eclipses the benign vacuolar and autophagic response seen with more hydrophilic local anesthetics.

  12. Diffraction Phase Cytometry: blood on a CD-ROM.

    PubMed

    Mir, Mustafa; Wang, Zhuo; Tangella, Krishnarao; Popescu, Gabriel

    2009-02-16

    We demonstrate Diffraction Phase Cytometry (DPC) as a single shot, full-field, high throughput quantitative phase imaging modality, dedicated to analyzing whole blood smears. Utilizing a commercial CD as a sample substrate, along with dynamic spatial filtering via a liquid crystal spatial light modulator, we have developed a compact instrument capable of making quantitative, physiologically relevant measurements. To illustrate the ability of the system to function as a highly sensitive cytometer we imaged a large number (N=1,537) of live human erythrocytes in whole blood without preparation. We retrieved a comprehensive set of geometrical parameters including cell volume and surface area, which are not directly available using existing cytometers. Furthermore, we retrieved the minimum cylindrical diameter, through which red blood cells can pass, and deliver oxygen. These initial results prove the concept for an inexpensive lab-on-a-chip blood screening device.

  13. Flow Cytometry Analyses of Adipose Tissue Macrophages

    PubMed Central

    Cho, Kae Won; Morris, David L.; Lumeng, Carey N.

    2014-01-01

    Within adipose tissue, multiple leukocyte interactions contribute to metabolic homeostasis in health as well as to the pathogenesis of insulin resistance with obesity. Adipose tissue macrophages (ATMs) are the predominant leukocyte population in fat and contribute to obesity-induced inflammation. Characterization of ATMs and other leukocytes in the stromal vascular fraction from fat has benefited from the use of flow cytometry and flow-assisted cell sorting techniques. These methods permit the immunophenotyping, quantification, and purification of these unique cell populations from multiple adipose tissue depots in rodents and humans. Proper isolation, quantification, and characterization of ATM phenotypes are critical for understanding their role in adipose tissue function and obesity-induced metabolic diseases. Here, we present the flow cytometry protocols for phenotyping ATMs in lean and obese mice employed by our laboratory. PMID:24480353

  14. Fluorescence lifetime excitation cytometry by kinetic dithering.

    PubMed

    Li, Wenyan; Vacca, Giacomo; Castillo, Maryann; Houston, Kevin D; Houston, Jessica P

    2014-07-01

    Flow cytometers are powerful high-throughput devices that capture spectroscopic information from individual particles or cells. These instruments provide a means of multi-parametric analyses for various cellular biomarkers or labeled organelles and cellular proteins. However, the spectral overlap of fluorophores limits the number of fluorophores that can be used simultaneously during experimentation. Time-resolved parameters enable the quantification of fluorescence decay kinetics, thus circumventing common issues associated with intensity-based measurements. This contribution introduces fluorescence lifetime excitation cytometry by kinetic dithering (FLECKD) as a method to capture multiple fluorescence lifetimes using a hybrid time-domain approach. The FLECKD approach excites fluorophores by delivering short pulses of light to cells or particles by rapid dithering and facilitates measurement of complex fluorescence decay kinetics by flow cytometry. Our simulations demonstrated a resolvable fluorescence lifetime value as low as 1.8 ns (±0.3 ns) with less than 20% absolute error. Using the FLECKD instrument, we measured the shortest average fluorescence lifetime value of 2.4 ns and found the system measurement error to be ±0.3 ns (SEM), from hundreds of monodisperse and chemically stable fluorescent microspheres. Additionally, we demonstrate the ability to detect two distinct excited state lifetimes from fluorophores in single cells using FLECKD. This approach presents a new ability to resolve multiple fluorescence lifetimes while retaining the fluidic throughput of a cytometry system. The ability to discriminate more than one average fluorescence lifetime expands the current capabilities of high-throughput and intensity-based cytometry assays as the need to tag one single cell with multiple fluorophores is now widespread.

  15. Supercontinuum white light lasers for flow cytometry

    PubMed Central

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

    2009-01-01

    Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric analysis. The white light lasers used in this study were integrated into a commercial flow cytometry platform, and a series of high-transmission bandpass filters used to select wavelength ranges from the blue (~480 nm) to the long red (>700 nm). Cells labeled with a variety of fluorescent probes or expressing fluorescent proteins were then analyzed, in comparison with traditional lasers emitting at wavelengths similar to the filtered SC source. Based on a standard sensitivity metric, the white light laser bandwidths produced similar excitation levels to traditional lasers for a wide variety of fluorescent probes and expressible proteins. Sensitivity assessment using fluorescent bead arrays confirmed that the SC laser and traditional sources resulted in similar levels of detection sensitivity. Supercontinuum white light laser sources therefore have the potential to remove a significant barrier in flow cytometric analysis, namely the limitation of excitation wavelengths. Almost any visible wavelength range can be made available for excitation, allowing access to virtually any fluorescent probe, and permitting “fine-tuning” of excitation wavelength to particular probes. PMID:19072836

  16. Highly multiparametric analysis by mass cytometry.

    PubMed

    Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A; Tanner, Scott

    2010-09-30

    This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays.

  17. Supercontinuum white light lasers for flow cytometry.

    PubMed

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

    2009-05-01

    Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric analysis. The white light lasers used in this study were integrated into a commercial flow cytometry platform, and a series of high-transmission bandpass filters used to select wavelength ranges from the blue (approximately 480 nm) to the long red (>700 nm). Cells labeled with a variety of fluorescent probes or expressing fluorescent proteins were then analyzed, in comparison with traditional lasers emitting at wavelengths similar to the filtered SC source. Based on a standard sensitivity metric, the white light laser bandwidths produced similar excitation levels to traditional lasers for a wide variety of fluorescent probes and expressible proteins. Sensitivity assessment using fluorescent bead arrays confirmed that the SC laser and traditional sources resulted in similar levels of detection sensitivity. Supercontinuum white light laser sources therefore have the potential to remove a significant barrier in flow cytometric analysis, namely the limitation of excitation wavelengths. Almost any visible wavelength range can be made available for excitation, allowing access to virtually any fluorescent probe, and permitting "fine-tuning" of excitation wavelength to particular probes.

  18. Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

    EPA Science Inventory

    Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple perform...

  19. Optimized flow cytometry isolation of murine spermatocytes

    PubMed Central

    Gaysinskaya, Valeriya; Soh, Ina Y.; van der Heijden, Godfried W.; Bortvin, Alex

    2014-01-01

    Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages. Enrichment for Pachytene (P) and Diplotene (D) substages of late MPI was previously accomplished using flow cytometry. However, separation of early MPI spermatocytes, specifically, of Leptotene (L) and Zygotene (Z) substages, has been a challenge due to these cells’ similar characteristics. In this report, we describe an optimized Hoechst-33342 (Hoechst)-based flow cytometry approach for isolating individual MPI populations from adult murine testis. We get significant enrichment for individual L and Z spermatocytes, previously inseparable from each other, and optimize the isolation of other MPI substages. Our flow cytometry approach is a combination of three optimized strategies. The first is optimization of testis dissociation protocol that yields more consistent and reproducible testicular single cell suspension. The second involves optimization of flow cytometric gating protocol where a critical addition to the standard protocol for cell discrimination based on Hoechst fluorescence, involves a back-gating technique based on light scattering parameters. This step specifies selection of individual MPI substages. The third, is an addition of DNA content restriction to the gating protocol to minimize contamination from non-meiotic cells. Finally, we confirm significant enrichment of high-purity Preleptotene (PreL), L, Z, P and D MPI spermatocytes using stage-specific marker distribution. The technique will facilitate understanding of the molecular events underlying meiotic prophase I. PMID:24664803

  20. Quantitative Three-Dimensional Tissue Cytometry to Study Kidney Tissue and Resident Immune Cells.

    PubMed

    Winfree, Seth; Khan, Shehnaz; Micanovic, Radmila; Eadon, Michael T; Kelly, Katherine J; Sutton, Timothy A; Phillips, Carrie L; Dunn, Kenneth W; El-Achkar, Tarek M

    2017-02-02

    Analysis of the immune system in the kidney relies predominantly on flow cytometry. Although powerful, the process of tissue homogenization necessary for flow cytometry analysis introduces bias and results in the loss of morphologic landmarks needed to determine the spatial distribution of immune cells. An ideal approach would support three-dimensional (3D) tissue cytometry: an automated quantitation of immune cells and associated spatial parameters in 3D image volumes collected from intact kidney tissue. However, widespread application of this approach is limited by the lack of accessible software tools for digital analysis of large 3D microscopy data. Here, we describe Volumetric Tissue Exploration and Analysis (VTEA) image analysis software designed for efficient exploration and quantitative analysis of large, complex 3D microscopy datasets. In analyses of images collected from fixed kidney tissue, VTEA replicated the results of flow cytometry while providing detailed analysis of the spatial distribution of immune cells in different regions of the kidney and in relation to specific renal structures. Unbiased exploration with VTEA enabled us to discover a population of tubular epithelial cells that expresses CD11C, a marker typically expressed on dendritic cells. Finally, we show the use of VTEA for large-scale quantitation of immune cells in entire human kidney biopsies. In summary, we show that VTEA is a simple and effective tool that supports unique digital interrogation and analysis of kidney tissue from animal models or biobanked human kidney biopsies. We have made VTEA freely available to interested investigators via electronic download.

  1. In vitro hematocrit measurement using spectrally encoded flow cytometry

    PubMed Central

    Zeidan, Adel; Golan, Lior; Yelin, Dvir

    2016-01-01

    Measuring key physiological parameters of small blood samples extracted from patients could be useful for real-time clinical diagnosis at the point of care. An important parameter required from all blood tests is the blood hematocrit, a measure of the fractional volume occupied by the red cells within the blood. In this work, we present a method for in vitro evaluation of hematocrit based on the data acquired using spectrally encoded flow cytometry. Analysis of the reflectance confocal images of blood within a flow chamber resulted in an error as low as 1.7% in the measured hematocrit. The technique could be used as part of an in vitro diagnostic system that measures important blood parameters at the point of care. PMID:27867734

  2. Multiparameter Flow Cytometry For Clinical Applications

    NASA Astrophysics Data System (ADS)

    Stewart, Carleton C.

    1989-06-01

    Flow Cytometry facilities are well established and provide immunophenotyping and DNA content measurement services. The application of immunophenotyping has been primarily in monitoring therapy and in providing further information to aid in the definitive diagnosis of immunological and neoplastic disease such as: immunodeficiency disease, auto immune disease, organ transplantation, and leukemia and lymphoma. DNA content measurements have been particularly important in determining the fraction of cycling cells and presence of aneuploid cells in neoplasia. This information has been useful in the management of patients with solid tumors.

  3. Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

    NASA Astrophysics Data System (ADS)

    Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

    2016-06-01

    Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of

  4. Mixture modeling approach to flow cytometry data.

    PubMed

    Boedigheimer, Michael J; Ferbas, John

    2008-05-01

    Flow Cytometry has become a mainstay technique for measuring fluorescent and physical attributes of single cells in a suspended mixture. These data are reduced during analysis using a manual or semiautomated process of gating. Despite the need to gate data for traditional analyses, it is well recognized that analyst-to-analyst variability can impact the dataset. Moreover, cells of interest can be inadvertently excluded from the gate, and relationships between collected variables may go unappreciated because they were not included in the original analysis plan. A multivariate non-gating technique was developed and implemented that accomplished the same goal as traditional gating while eliminating many weaknesses. The procedure was validated against traditional gating for analysis of circulating B cells in normal donors (n = 20) and persons with Systemic Lupus Erythematosus (n = 42). The method recapitulated relationships in the dataset while providing for an automated and objective assessment of the data. Flow cytometry analyses are amenable to automated analytical techniques that are not predicated on discrete operator-generated gates. Such alternative approaches can remove subjectivity in data analysis, improve efficiency and may ultimately enable construction of large bioinformatics data systems for more sophisticated approaches to hypothesis testing.

  5. Future clinical role for flow cytometry.

    PubMed

    Ashcroft, R G

    1988-01-01

    It appears that there is an essential, not just supportive, role for flow cytometry in the clinical context, particularly in providing early information in clinical oncology. High flow rate enumeration and sorting of rare cells, combined with microscopy, offer immediate benefits in the clinical processing of at least some cancers. These benefits would be in diagnosis (perhaps very early detection of metastatic cells in the present prediagnostic phase of solid tumor growth), monitoring, prognosis, and therapy. Importantly, flow cytometric measures can be implemented immediately, and measurement times are short. The value of high flow rate operation of existing facilities in clinical, "supportive" flow cytometry should be better appreciated, if only because shorter measurement times and on-line analysis would make the existing facilities more cost effective: higher throughput for the same overheads. Finally, the wisdom of employing nonsorting cytometers for clinical use should be strongly questioned. Thus, what future impact will the application of flow sorting have in clinical fields old and new, e.g., in bacterial infection measurements in peripheral blood? In particular, nonsorting machines will be unable to adopt the "essential" clinical role I have proposed here.

  6. Application of flow cytometry to wine microorganisms.

    PubMed

    Longin, Cédric; Petitgonnet, Clément; Guilloux-Benatier, Michèle; Rousseaux, Sandrine; Alexandre, Hervé

    2017-04-01

    Flow cytometry (FCM) is a powerful technique allowing detection and enumeration of microbial populations in food and during food process. Thanks to the fluorescent dyes used and specific probes, FCM provides information about cell physiological state and allows enumeration of a microorganism in a mixed culture. Thus, this technique is increasingly used to quantify pathogen, spoilage microorganisms and microorganisms of interest. Since one decade, FCM applications to the wine field increase greatly to determine population and physiological state of microorganisms performing alcoholic and malolactic fermentations. Wine spoilage microorganisms were also studied. In this review we briefly describe FCM principles. Next, a deep revision concerning enumeration of wine microorganisms by FCM is presented including the fluorescent dyes used and techniques allowing a yeast and bacteria species specific enumeration. Then, the last chapter is dedicated to fluorescent dyes which are used to date in fluorescent microscopy but applicable in FCM. This chapter also describes other interesting "future" techniques which could be applied to study the wine microorganisms. Thus, this review seeks to highlight the main advantages of the flow cytometry applied to wine microbiology.

  7. Honey Bee Hemocyte Profiling by Flow Cytometry

    PubMed Central

    Marringa, William J.; Krueger, Michael J.; Burritt, Nancy L.; Burritt, James B.

    2014-01-01

    Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure. PMID:25285798

  8. International Society for Advancement of Cytometry (ISAC) flow cytometry shared resource laboratory (SRL) best practices.

    PubMed

    Barsky, Lora W; Black, Michele; Cochran, Matthew; Daniel, Benjamin J; Davies, Derek; DeLay, Monica; Gardner, Rui; Gregory, Michael; Kunkel, Desiree; Lannigan, Joanne; Marvin, James; Salomon, Robert; Torres, Carina; Walker, Rachael

    2016-11-01

    The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.

  9. Opposite effects of two different strains of equine herpesvirus 1 infection on cytoskeleton composition in equine dermal ED and African green monkey kidney Vero cell lines: application of scanning cytometry and confocal-microscopy-based image analysis in a quantitative study.

    PubMed

    Turowska, A; Pajak, B; Godlewski, M M; Dzieciatkowski, T; Chmielewska, A; Tucholska, A; Banbura, M

    2010-05-01

    Viruses can reorganize the cytoskeleton and restructure the host cell transport machinery. During infection viruses use different cellular cues and signals to enlist the cytoskeleton for their mission. However, each virus specifically affects the cytoskeleton structure. Thus, the aim of our study was to investigate the cytoskeletal changes in homologous equine dermal (ED) and heterologous Vero cell lines infected with either equine herpesvirus 1 (EHV-1) strain Rac-H or Jan-E. We found that Rac-H strain disrupted actin fibers and reduced F-actin level in ED cells, whereas the virus did not influence Vero cell cytoskeleton. Conversely, the Jan-E strain induced polymerization of both F-actin and MT in Vero cells, but not in ED cells. Confocal-microscopy analysis revealed that alpha-tubulin colocalized with viral antigen in ED cells infected with either Rac-H or Jan-E viruses. Alterations in F-actin and alpha-tubulin were evaluated by confocal microscopy, Microimage analysis and scanning cytometry. This unique combination allowed precise interpretation of confocal-based images showing the cellular events induced by EHV-1. We conclude that examination of viral-induced pathogenic effects in species specific cell lines is more symptomatic than in heterologous cell lines.

  10. Diagnosis of Fanconi's anemia by flow cytometry.

    PubMed

    Miglierina, R; Le Coniat, M; Gendron, M; Berger, R

    1990-01-01

    FA is a progressive bone marrow aplasia genetically transmitted by a recessive autosomal gene or genes. In our laboratory, cytogenetic diagnosis is based on evaluation of the chromosomal breakage of mitotic cell derived from patient blood-cell cultures and sensitized by nitrogen mustard (NM). We have observed, in parallel with this test, fluctuations of the cell cycle of PHA- stimulated peripheral blood lymphocytes from FA patients as compared with controls. FA cells treated with NM show a dramatic and significant increase in G2/M phase after 72 hr in vitro culture, compared with untreated or control cells (normal controls and non-FA patients). This test is rapid and simple, as it consists in staining cells with a DNA dye (propidium iodide), followed by a flow cytometry analysis of the cell cycle phases. Our results in twelve patients are correlated with the cytogenetic results.

  11. Blood screening using diffraction phase cytometry.

    PubMed

    Mir, Mustafa; Ding, Huafeng; Wang, Zhuo; Reedy, Jason; Tangella, Krishnarao; Popescu, Gabriel

    2010-01-01

    Blood smear analysis has remained a crucial diagnostic tool for pathologists despite the advent of automatic analyzers such as flow cytometers and impedance counters. Though these current methods have proven to be indispensible tools for physicians and researchers alike, they provide limited information on the detailed morphology of individual cells, and merely alert the operator to manually examine a blood smear by raising flags when abnormalities are detected. We demonstrate an automatic interferometry-based smear analysis technique known as diffraction phase cytometry (DPC), which is capable of providing the same information on red blood cells as is provided by current clinical analyzers, while rendering additional, currently unavailable parameters on the 2-D and 3-D morphology of individual red blood cells. To validate the utility of our technique in a clinical setting, we present a comparison between tests generated from 32 patients by a state of the art clinical impedance counter and DPC.

  12. Blood screening using diffraction phase cytometry

    NASA Astrophysics Data System (ADS)

    Mir, Mustafa; Ding, Huafeng; Wang, Zhuo; Reedy, Jason; Tangella, Krishnarao; Popescu, Gabriel

    2010-03-01

    Blood smear analysis has remained a crucial diagnostic tool for pathologists despite the advent of automatic analyzers such as flow cytometers and impedance counters. Though these current methods have proven to be indispensible tools for physicians and researchers alike, they provide limited information on the detailed morphology of individual cells, and merely alert the operator to manually examine a blood smear by raising flags when abnormalities are detected. We demonstrate an automatic interferometry-based smear analysis technique known as diffraction phase cytometry (DPC), which is capable of providing the same information on red blood cells as is provided by current clinical analyzers, while rendering additional, currently unavailable parameters on the 2-D and 3-D morphology of individual red blood cells. To validate the utility of our technique in a clinical setting, we present a comparison between tests generated from 32 patients by a state of the art clinical impedance counter and DPC.

  13. Flow cytometry for health monitoring in space

    SciTech Connect

    Jett, J.H.; Martin, J.C.; Saunders, G.C.; Stewart, C.C.

    1984-01-01

    Monitoring the health of space station or lunar base residents will be necessary to provide knowledge of the physiological status of astronauts. Flow cytometric techniques are uniquely capable of providing cellular, chromosome, hormone level and enzyme level information. The use of dyes provides the basis for fluorescently labeling specific cellular components. Laser induced fluorescence from stained cells is quantitated in a flow cytometer to measure cellular components such as DNA, RNA and protein. One major application of a flow cytometer will be to perform a complete blood count including hematocrit, hemoglobin content, and numbers of platelets, erythrocytes, granulocytes, lymphocytes and monocytes. A newly developed flow cytometry based fluoroimmunoassay will be able to measure levels of serum enzymes and hormones. It will also be possible to quantitate radiation exposure and some forms of chromosome damage with flow cytometric measurements. With relatively simple modifications to existing technology, it will be possible to construct a flight rated cytometer. 11 references, 6 figures, 2 tables.

  14. Evaluation of platelet turnover by flow cytometry.

    PubMed

    Salvagno, G L; Montagnana, M; Degan, M; Marradi, P L; Ricetti, M M; Riolfi, P; Poli, G; Minuz, P; Santonastaso, C L; Guidi, G C

    2006-05-01

    The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K(2)EDTA, was incubated with 0.6 microg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n = 23) was 6.13 +/- 3.09%. RPs were 10.41 +/- 9.02% in patients (n = 10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45 +/- 6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81 +/- 18.79 (P < 0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n = 21; 21.04 +/- 16.21%, P < 0.001 vs. controls) or systemic lupus erythematosus (n = 6, 29.08 +/- 15.57%; P < 0.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.

  15. In Vivo Photoswitchable Flow Cytometry for Direct Tracking of Single Circulating Tumor Cells

    PubMed Central

    Nedosekin, Dmitry A.; Verkhusha, Vladislav V.; Melerzanov, Alexander V.; Zharov, Vladimir P.; Galanzha, Ekaterina I.

    2014-01-01

    SUMMARY Photoswitchable fluorescent proteins (PSFPs) that change their color in response to light have led to breakthroughs in studying static cells. However, using PSFPs to study cells in dynamic conditions is challenging. Here we introduce a method for in vivo ultrafast photoswitching of PSFPs that provides labeling and tracking of single circulating cells. Using in vivo multicolor flow cytometry, this method demonstrated the capability for studying recirculation, migration, and distribution of circulating tumor cells (CTCs) during metastasis progression. In tumor-bearing mice, it enabled monitoring of real-time dynamics of CTCs released from primary tumor, identifying dormant cells, and imaging of CTCs colonizing a primary tumor (self-seeding) or existing metastasis (reseeding). Integration of genetically encoded PSFPs, fast photoswitching, flow cytometry, and imaging makes in vivo single cell analysis in the circulation feasible to provide insights into the behavior of CTCs and potentially immune-related and bacterial cells in circulation. PMID:24816228

  16. An active, collaborative approach to learning skills in flow cytometry.

    PubMed

    Fuller, Kathryn; Linden, Matthew D; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N; Röhrig, Kimberley J

    2016-06-01

    Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula.

  17. Dynamic proliferation assessment in flow cytometry.

    PubMed

    Diermeier-Daucher, Simone; Brockhoff, Gero

    2010-09-01

    Dynamic proliferation assessment via flow cytometry is legitimately supposed to be the most powerful tool for recording cell cycle kinetics in-vitro. The preeminent feature is a single cell-based multi-informative analysis by temporal high-resolution. Flow cytometric approaches are based on labeling of proliferating cells via thymidine substitution by a base analog (e.g., 5-bromo-2'-deoxyuridine, BrdU) that is added to cell cultures either for a short period of time (pulse labeling) or continuously until cell harvesting. This unit describes the alternative use of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) in place of BrdU for three different applications: (1) dynamic proliferation assessment by EdU pulse cell labeling; (2) the same approach as (1) but in combination with live/dead cell discrimination; and (3) dynamic cell cycle analysis based on continuous cell labeling with EdU and Hoechst fluorochrome quenching. In contrast to the detection of BrdU incorporation, EdU-positive cells can be identified by taking advantage of click chemistry, which facilitates a simplified and fast cell preparation. Further analysis options but also limitations of the utilization of EdU are discussed.

  18. Total Internal Reflection Fluorescence Flow Cytometry

    PubMed Central

    Wang, Jun; Bao, Ning; Paris, Leela L.; Geahlen, Robert L.; Lu, Chang

    2009-01-01

    Total internal reflection fluorescence microscopy (TIRFM) has been widely used to explore biological events that are close to the cell membrane by illuminating fluorescent molecules using the evanescent wave. However, TIRFM is typically limited to the examination of a low number of cells and the results do not reveal potential heterogeneity in the cell population. In this report, we develop an analytical tool referred to as total internal reflection fluorescence flow cytometry (TIRF-FC) to examine the region of the cell membrane with a throughput of ~100–150 cells/s and single cell resolution. We use an elastomeric valve that is partially closed to force flowing cells in contact with the glass surface where the evanescent field resides. We demonstrate that TIRF-FC is able to detect the differences in the subcellular location of an intracellular fluorescent protein. Proper data processing and analysis allows TIRF-FC to be quantitative. With the high throughput, TIRF-FC will be a very useful tool for generating information on cell populations with events and dynamics close to the cell surface. PMID:19007249

  19. Applications of Flow Cytometry to Clinical Microbiology†

    PubMed Central

    Álvarez-Barrientos, Alberto; Arroyo, Javier; Cantón, Rafael; Nombela, César; Sánchez-Pérez, Miguel

    2000-01-01

    Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory. PMID:10755996

  20. Immunological techniques: ELISA, flow cytometry, and immunohistochemistry.

    PubMed

    Ford, Pauline J

    2010-01-01

    Techniques to analyze the host immune response elicited by the presence of oral microorganisms and their products are central to our understanding of the local and systemic effects of oral diseases. This immune response has been extensively investigated for periodontal disease. The local response may result in lesions involving the gingival tissues and depending upon host susceptibility and microbial virulence may lead to local tissue destruction. More recently, however, the importance of the systemic inflammatory and immune response to oral organisms has been recognized. These systemic responses have been associated with an increased risk for cardiovascular disease, diabetes, and preterm low birth weight. A number of techniques are used extensively by researchers investigating humoral and cellular immune responses to oral organisms both in local oral tissues and fluids and systemically in peripheral blood. These are enzyme-linked immunosorbent assay (ELISA) to quantify specific antibody and cytokines in serum, gingival crevicular fluid (GCF), and saliva; characterization of T cells from peripheral blood and gingival tissues using flow cytometry; and immunohistological analysis of the inflammatory cell infiltrate in gingival tissues.

  1. Flow cytometry: A powerful technology for measuring biomarkers

    SciTech Connect

    Jett, J.H.

    1994-09-01

    A broad definition of a biomarker is that it is a measurable characteristic of a biological system that changes upon exposure to a physical or chemical insult. While the definition can be further refined, it is sufficient for the purposes of demonstrating the advantages of flow cytometry for making quantitative measurements of biomarkers. Flow cytometry and cell sorting technologies have emerged during the past 25 years to take their place alongside other essential tools used in biology such as optical and electron microscopy. This paper describes the basics of flow cytometry technology, provides illustrative examples of applications of the technology in the field of biomarkers, describes recent developments in flow cytometry that have not yet been applied to biomarker measurements, and projects future developments of the technology. The examples of uses of flow cytometry for biomarker quantification cited in this paper are meant to be illustrative and not exhaustive in the sense of providing a review of the field.

  2. An introduction to mass cytometry: fundamentals and applications.

    PubMed

    Tanner, Scott D; Baranov, Vladimir I; Ornatsky, Olga I; Bandura, Dmitry R; George, Thaddeus C

    2013-05-01

    Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

  3. Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

    PubMed Central

    Galanzha, Ekaterina I; Tuchin, Valery V; Zharov, Vladimir P

    2007-01-01

    Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell’s functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed. PMID:17226898

  4. Accelerated heavy ions and the lens. IV. Biomicroscopic and cytopathological analyses of the lenses of mice irradiated with 600 MeV/amu sup 56 Fe ions

    SciTech Connect

    Worgul, B.V.; Medvedovsky, C.; Powers-Risius, P.; Alpen, E. )

    1989-11-01

    The lenses of mice exposed to 600 MeV/amu iron ions were evaluated by slit-lamp biomicroscopy and cytopathological analyses. The doses ranged from 0.05 to 1.6 Gy, and the lenses were assessed at several intervals postirradiation. Cataract, the development of which is dependent on both time and dose, is significantly more advanced in all of the exposed mice when compared to the unirradiated controls. The great difference between the severity of the cataracts caused by 0.05 Gy (the lowest dose used) and those that developed spontaneously in the control animals is an indication that 0.05 Gy may far exceed the threshold dose for the production of cataracts by accelerated iron ions. Cytopathologically, a similar dose dependence was observed for a number of end points including micronucleation, interphase death, and meridional row disorganization. In addition the exposure to the 56Fe ions produced a long-term effect on the mitotic population and a pronounced focal loss of epithelial cytoarchitecture. The microscopic changes support the view that the mechanism of heavy-ion-induced cataractogenesis is the same as that for cataracts caused by low-LET radiation.

  5. Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2015-01-01

    Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform

  6. Detection of circulating breast cancer cells using photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Kiran

    According to the American Cancer Society, more than 200,000 new cases of breast cancer are expected to be diagnosed this year. Moreover, about 40,000 women died from breast cancer last year alone. As breast cancer progresses in an individual, it can transform from a localized state to a metastatic one with multiple tumors distributed through the body, not necessarily contained within the breast. Metastasis is the spread of cancer through the body by circulating tumor cells (CTCs) which can be found in the blood and lymph of the diagnosed patient. Diagnosis of a metastatic state by the discovery of a secondary tumor can often come too late and hence, significantly reduce the patient's chance of survival. There is a current need for a CTC detection method which would diagnose metastasis before the secondary tumor occurs or reaches a size resolvable by current imaging systems. Since earlier detection would improve prognosis, this study proposes a method of labeling of breast cancer cells for detection with a photoacoustic flow cytometry system as a model for CTC detection in human blood. Gold nanoparticles and fluorescent polystyrene nanoparticles are proposed as contrast agents for T47D, the breast cancer cell line of choice. The labeling, photoacoustic detection limit, and sensitivity are first characterized and then applied to a study to show detection from human blood.

  7. Laser-scan cytometry: a new tool for clinical diagnostics

    NASA Astrophysics Data System (ADS)

    Maerz, Holger K.; Baumgartner, Adolf; Hambsch, Joerg; Hennig, Bert; Nuesse, Michael; Schmid, Thomas; Schneider, Peter; Zotz, Rainer; Tarnok, Attila

    1999-04-01

    The common usage of flow cytometry (FCM) in research and clinical diagnostic is limited by the lack visualizing the fluorescence labelled cells. The Laser Scanning Cytometer (LSC) enables multicolor cytometric measurements on a slide featuring relocation of single cells for further investigation via brightfield and fluorescence microscopy. Additionally, it is possible to capture these images for documentation. In a FISH application, the LSC was successfully used for automated scoring techniqeus for evaluating the frequency of aneuploid sperm in humans and mice. In just 30 minutes, we were able to acquire more than 15,000 sperms, a task which normally takes more than a day. After relocation, genetic defects were identified and confirmed via fluorescence microscopy. In an on going study, we investigate via the LSC the remain of a new radiopaque material for high resolution echocardiography in the blood circulation. At first the result exhibited that the radiopaque material is endocysed by leukocytes just after application but is still detectable via echocardiography for up to 40 minutes. In conclusion, with the additional data acquisition by the LSC, it is possible to perform further detailed information from very small samples. Therefore, we are working up to now on developing new methods to introduce the LSC in our clinical diagnostic of neonates undergoing cardiac surgery.

  8. Authors attain comparable or slightly higher rates of citation publishing in an open access journal (CytoJournal) compared to traditional cytopathology journals - A five year (2007-2011) experience

    PubMed Central

    Frisch, Nora K.; Nathan, Romil; Ahmed, Yasin K.; Shidham, Vinod B.

    2014-01-01

    Background: The era of Open Access (OA) publication, a platform which serves to better disseminate scientific knowledge, is upon us, as more OA journals are in existence than ever before. The idea that peer-reviewed OA publication leads to higher rates of citation has been put forth and shown to be true in several publications. This is a significant benefit to authors and is in addition to another relatively less obvious but highly critical component of the OA charter, i.e. retention of the copyright by the authors in the public domain. In this study, we analyzed the citation rates of OA and traditional non-OA publications specifically for authors in the field of cytopathology. Design: We compared the citation patterns for authors who had published in both OA and traditional non-OA peer-reviewed, scientific, cytopathology journals. Citations in an OA publication (CytoJournal) were analyzed comparatively with traditional non-OA cytopathology journals (Acta Cytologica, Cancer Cytopathology, Cytopathology, and Diagnostic Cytopathology) using the data from web of science citation analysis site (based on which the impact factors (IF) are calculated). After comparing citations per publication, as well as a time adjusted citation quotient (which takes into account the time since publication), we also analyzed the statistics after excluding the data for meeting abstracts. Results: Total 28 authors published 314 publications as articles and meeting abstracts (25 authors after excluding the abstracts). The rate of citation and time adjusted citation quotient were higher for OA in the group where abstracts were included (P < 0.05 for both). The rates were also slightly higher for OA than non-OA when the meeting abstracts were excluded, but the difference was statistically insignificant (P = 0.57 and P = 0.45). Conclusion We observed that for the same author, the publications in the OA journal attained a higher rate of citation than the publications in the traditional non

  9. Immunophenotyping by slide-based cytometry and by flow cytometry are comparable

    NASA Astrophysics Data System (ADS)

    Gerstner, Andreas O.; Laffers, Wiebke; Mittag, Anja; Daehnert, Ingo; Lenz, Domnik; Bootz, Friedrich; Bocsi, Jozsef; Tarnok, Attila

    2005-03-01

    Immunophenotyping of peripheral blood leukocytes (PBLs) is performed by flow cytometry (FCM) as the golden standard. Slide based cytometry systems for example laser scanning cytometer (LSC) can give additional information (repeated staining and scanning, morphology). In order to adequately judge on the clinical usefulness of immunophenotyping by LSC it is obligatory to compare it with the long established FCM assays. We performed this study to systematically compare the two methods, FCM and LSC for immunophenotyping and to test the correlation of the results. Leucocytes were stained with directly labeled monoclonal antibodies with whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in a FACScan (BD-Biosciences) using standard protocols and parallel with LSC (CompuCyte) after placing to glass slide, drying and fixation by aceton and 7-AAD staining. Calculating the percentage distribution of PBLs obtained by LSC and by FCM shows very good correlation with regression coefficients close to 1.0 for the major populations (neutrophils, lymphocytes, and monocytes), as well as for the lymphocyte sub-populations (T-helper-, T-cytotoxic-, B-, NK-cells). LSC can be recommended for immunophenotyping of PBLs especially in cases where only very limited sample volumes are available or where additional analysis of the cells" morphology is important. There are limitations in the detection of rare leucocytes or weak antigens where appropriate amplification steps for immunofluorescence should be engaged.

  10. The early fluidic and optical physics of cytometry.

    PubMed

    Watson, J V

    1999-02-15

    All forms of cytometry, depend on the basic laws of physics, including those of fluidics, optics, and electronics, most of which were established centuries ago. Flow cytometry depends critically on the fluidics presenting each individual cell with precision to the sensing volume. This is intersected by a high-intensity light source, and light scattering and fluorescence from suitably stained constituents in each cell are captured by the light-collecting optics and measured. The works and observations of Bernoulli and Euler in the 18th century, Reynolds in the 19th century, and Crosland-Taylor in the 20th century in the field of fluid dynamics laid the foundations for hydrodynamic focussing, which is the primary prerequisite for presenting individual cells to the sensing volume. In addition, electrostatic cell sorters must have the ability to generate stable droplet formation in the jet-stream issuing from the flow chamber nozzle. The origins here can be traced to work carried out in the early to mid-19th century by Savart, Magnus, and Thomson. Flow, image, and confocal cytometry are all dependent on the laws of optics, including those of reflection and refraction as well as numerous other optical principles. The observations and works of Socrates, Ptolemy, Snel, and Descartes between about BC 370 and 1637 were of seminal importance in developing the laws of reflection and refraction. In the mid-17th century Hooke illustrated the power of magnifying glasses and microscopy in his Micrographia and Newton was responsible for explaining colours in the spectrum. Huygens, toward the end of the 17th century, put forward the concept of point source light propagation contributing to a wave front. Finally, Thomas Young, early in the 19th century, established the wave form of light from interference patterns. Most people will be familiar with some of these discoveries and the investigators who carried out the work; some people will be familiar with all of these. However, very

  11. Tracking tissue section surfaces for automated 3D confocal cytometry

    NASA Astrophysics Data System (ADS)

    Agustin, Ramses; Price, Jeffrey H.

    2002-05-01

    Three-dimensional cytometry, whereby large volumes of tissue would be measured automatically, requires a computerized method for detecting the upper and lower tissue boundaries. In conventional confocal microscopy, the user interactively sets limits for axial scanning for each field-of-view. Biological specimens vary in section thickness, thereby driving the requirement for setting vertical scan limits. Limits could be set arbitrarily large to ensure the entire tissue is scanned, but automatic surface identification would eliminate storing undue numbers of empty optical sections and forms the basis for incorporating lateral microscope stage motion to collect unlimited numbers of stacks. This walk-away automation of 3D confocal scanning for biological imaging is the first sep towards practical, computerized statistical sampling from arbitrarily large tissue volumes. Preliminary results for automatic tissue surface tracking were obtained for phase-contrast microscopy by measuring focus sharpness (previously used for high-speed autofocus by our group). Measurements were taken from 5X5 fields-of-view from hamster liver sections, varying from five to twenty microns in thickness, then smoothed to lessen variations of in-focus information at each axial position. Because image sharpness (as the power of high spatial frequency components) drops across the axial boundaries of a tissue section, mathematical quantities including the full-width at half-maximum, extrema in the first derivative, and second derivative were used to locate the proximal and distal surfaces of a tissue. Results from these tests were evaluated against manual (i.e., visual) determination of section boundaries.

  12. Laser scanning cytometry as a tool for biomarker validation

    NASA Astrophysics Data System (ADS)

    Mittag, Anja; Füldner, Christiane; Lehmann, Jörg; Tarnok, Attila

    2013-03-01

    Biomarkers are essential for diagnosis, prognosis, and therapy. As diverse is the range of diseases the broad is the range of biomarkers and the material used for analysis. Whereas body fluids can be relatively easily obtained and analyzed, the investigation of tissue is in most cases more complicated. The same applies for the screening and the evaluation of new biomarkers and the estimation of the binding of biomarkers found in animal models which need to be transferred into applications in humans. The latter in particular is difficult if it recognizes proteins or cells in tissue. A better way to find suitable cellular biomarkers for immunoscintigraphy or PET analyses may be therefore the in situ analysis of the cells in the respective tissue. In this study we present a method for biomarker validation using Laser Scanning Cytometry which allows the emulation of future in vivo analysis. The biomarker validation is exemplarily shown for rheumatoid arthritis (RA) on synovial membrane. Cryosections were scanned and analyzed by phantom contouring. Adequate statistical methods allowed the identification of suitable markers and combinations. The fluorescence analysis of the phantoms allowed the discrimination between synovial membrane of RA patients and non-RA control sections by using median fluorescence intensity and the "affected area". As intensity and area are relevant parameters of in vivo imaging (e.g. PET scan) too, the presented method allows emulation of a probable outcome of in vivo imaging, i.e. the binding of the target protein and hence, the validation of the potential of the respective biomarker.

  13. Mass cytometry: blessed with the curse of dimensionality.

    PubMed

    Newell, Evan W; Cheng, Yang

    2016-07-19

    Immunologists are being compelled to develop new high-dimensional perspectives of cellular heterogeneity and to determine which applications best exploit the power of mass cytometry and associated multiplex approaches.

  14. Visible and near infrared fluorescence spectral flow cytometry.

    PubMed

    Nolan, John P; Condello, Danilo; Duggan, Erika; Naivar, Mark; Novo, David

    2013-03-01

    There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection. Customized software allows the single cell spectral data to be displayed and analyzed to produce new spectra-derived parameters. We show that familiar reference and calibration beads can be employed to quantitatively assess instrument performance. We use microspheres stained with six different quantum dots to compare a virtual bandpass filter approach with classic least squares (CLS) spectral unmixing, and then use antibody capture beads and CLS unmixing to demonstrate immunophenotyping of peripheral blood mononuclear cells using spectral flow cytometry. Finally, we characterize and evaluate several near infrared (NIR) emitting fluorophores for use in spectral flow cytometry. Spectral flow cytometry offers a number of attractive features for single cell analysis, including a simplified optical path, high spectral resolution, and streamlined approaches to quantitative multiparameter measurements. The availability of robust instrumentation, software, and analysis approaches will facilitate the development of spectral flow cytometry applications.

  15. Visible and Near Infrared Fluorescence Spectral Flow Cytometry

    PubMed Central

    Nolan, John P.; Condello, Danilo; Duggan, Erika; Naivar, Mark; Novo, David

    2013-01-01

    There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection. Customized software allows the single cell spectral data to be displayed and analyzed to produce new spectra-derived parameters. We show that familiar reference and calibration beads can be employed to quantitatively assess instrument performance. We use microspheres stained with six different quantum dots to compare a virtual bandpass filter approach with classic least squares (CLS) spectral unmixing, and then use antibody capture beads and CLS unmixing to demonstrate immunophenotyping of peripheral blood mononuclear cells using spectral flow cytometry. Finally, we characterize and evaluate several near infrared (NIR) emitting fluorophores for use in spectral flow cytometry. Spectral flow cytometry offers a number of attractive features for single cell analysis, including a simplified optical path, high spectral resolution, and streamlined approaches to quantitative multiparameter measurements. The availability of robust instrumentation, software, and analysis approaches will facilitate the development of spectral flow cytometry applications. PMID:23225549

  16. How fruit developmental biology makes use of flow cytometry approaches.

    PubMed

    Pirrello, Julien; Bourdon, Matthieu; Cheniclet, Catherine; Bourge, Mickaël; Brown, Spencer C; Renaudin, Jean-Pierre; Frangne, Nathalie; Chevalier, Christian

    2014-02-01

    Fleshy fruit species such as tomato are important because of their nutritional and economic value. Several stages of fruit development such as ovary formation, fruit set, and fruit maturation have already been the subject of many developmental studies. However, fruit growth per se has been much less addressed. Fruit growth like all plant organs depends upon the developmental processes of cell division and cell expansion. The activity of cell divisions sets the number of cells that will compose the fruit; the cell expansion activity then determines its final size. Among the various mechanisms that may influence the determination of cell size, endopolyploidy by the means of endoreduplication, i.e. genome amplification in the absence of mitosis, appears to be of great importance in fleshy fruits. In tomato fruit, endoreduplication is associated with DNA-dependent cell expansion: cell size can reach spectacular levels such as hundreds of times its initial size (e.g. >0.5 mm in diameter), with as much as a 256-fold increase in nuclear DNA content. Using tomato fruit development as a model, recent investigations combining the use of flow cytometry, cellular imaging and molecular analyses have provided new data in favor of the long-standing karyoplasmic ratio theory, stating that cells tend to adjust their cytoplasmic volume to the nuclear DNA content. By establishing a highly structured cellular system where multiple physiological functions are integrated, endoreduplication acts as a morphogenetic factor supporting cell growth during tomato fruit development. In the context of plant breeding, deciphering the mechanisms controlling fruit growth, in particular those connecting the process of nuclear endoreduplication with modulation of gene expression, the regulation of cell size and final fruit size and composition, is necessary to understand better the establishment of fleshy fruit quality traits.

  17. Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

    PubMed Central

    Gohar, Ali Vaziri; Cao, Ruofan; Jenkins, Patrick; Li, Wenyan; Houston, Jessica P.; Houston, Kevin D.

    2013-01-01

    Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime. PMID:24010001

  18. Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies.

    PubMed Central

    McClelland, R G; Pinder, A C

    1994-01-01

    Flow cytometry, combined with fluorescently labelled monoclonal antibodies, offers advantages of speed and sensitivity for the detection of specific pathogenic bacteria in foods. We investigated the detection of Salmonella typhimurium in eggs and milk. Using a sample clearing procedure, we determined that the detection limit was on the order of 10(3) cells per ml after a total analysis time of 40 min. After 6 h of nonselective enrichment, the detection limits were 10 cells per ml for milk and 1 cell per ml for eggs, even in the presence of a 10,000-fold excess of Escherichia coli cells. Images PMID:7811064

  19. Cell-based screening using high-throughput flow cytometry.

    PubMed

    Black, Christopher B; Duensing, Thomas D; Trinkle, Linda S; Dunlay, R Terry

    2011-02-01

    This review describes the use of high-throughput flow cytometry for performing multiplexed cell-based and bead-based screens. With the many advances in cell-based analysis and screening, flow cytometry has historically been underutilized as a screening tool largely due to the limitations in handling large numbers of samples. However, there has been a resurgence in the use of flow cytometry due to a combination of innovations around instrumentation and a growing need for cell-based and bead-based applications. The HTFC™ Screening System (IntelliCyt Corporation, Albuquerque, NM) is a novel flow cytometry-based screening platform that incorporates a fast sample-loading technology, HyperCyt®, with a two-laser, six-parameter flow cytometer and powerful data analysis capabilities. The system is capable of running multiplexed screening assays at speeds of up to 40 wells per minute, enabling the processing of a 96- and 384-well plates in as little as 3 and 12 min, respectively. Embedded in the system is HyperView®, a data analysis software package that allows rapid identification of hits from multiplexed high-throughput flow cytometry screening campaigns. In addition, the software is incorporated into a server-based data management platform that enables seamless data accessibility and collaboration across multiple sites. High-throughput flow cytometry using the HyperCyt technology has been applied to numerous assay areas and screening campaigns, including efflux transporters, whole cell and receptor binding assays, functional G-protein-coupled receptor screening, in vitro toxicology, and antibody screening.

  20. Lymphoid Neoplasia: Correlations Between Morphology and Flow Cytometry.

    PubMed

    Rout, Emily D; Avery, Paul R

    2017-01-01

    Cytology is commonly used to diagnose lymphoma and leukemia. Frequently, a diagnosis of lymphoproliferative disease can be obtained via cytology, and some of the common subtypes of canine lymphoma and leukemia can have characteristic cytologic features. Flow cytometry is a critical tool in the objective diagnosis and further characterization of lymphoma and leukemia. Features of the immunophenotype, such as expression of certain cell surface proteins or cell size, can provide important prognostic information. This review describes the cytologic features, flow cytometry immunophenotype, and immunophenotypic prognostic information for 6 major types of canine lymphoma and leukemia.

  1. Multispectral flow cytometry: The consequences of increased light collection.

    PubMed

    Feher, Kristen; von Volkmann, Konrad; Kirsch, Jenny; Radbruch, Andreas; Popien, Jan; Kaiser, Toralf

    2016-07-01

    In recent years, multispectral flow cytometry systems have come to attention. They differ from conventional flow cytometers in two key ways: a multispectral flow cytometer collects the full spectral information at the single cell level and the detector configuration is fixed and not explicitly tuned to a particular staining panel. This brings about clear hardware advantages, as a closed system should be highly stable, and ease-of-use should be improved if used in conjunction with custom unmixing software. An open question remains: what are the benefits of multispectral over conventional flow cytometry in terms of sensitivity and resolution? To probe this, we use Q (detection efficiency) and B (background) values and develop a novel "multivariate population overlap factor" to characterize the cytometer performance. To verify the usefulness of our factor, we perform representative experiments and compare our overlap factor to Q and B. Finally, we conclude that the increased light collection of multispectral flow cytometry does indeed lead to increased sensitivity, an improved detection limit, and a higher resolution. © 2016 International Society for Advancement of Cytometry.

  2. Microfluidic impedance cytometry of tumour cells in blood

    PubMed Central

    Spencer, Daniel; Morgan, Hywel

    2014-01-01

    The dielectric properties of tumour cells are known to differ from normal blood cells, and this difference can be exploited for label-free separation of cells. Conventional measurement techniques are slow and cannot identify rare circulating tumour cells (CTCs) in a realistic timeframe. We use high throughput single cell microfluidic impedance cytometry to measure the dielectric properties of the MCF7 tumour cell line (representative of CTCs), both as pure populations and mixed with whole blood. The data show that the MCF7 cells have a large membrane capacitance and size, enabling clear discrimination from all other leukocytes. Impedance analysis is used to follow changes in cell viability when cells are kept in suspension, a process which can be understood from modelling time-dependent changes in the dielectric properties (predominantly membrane conductivity) of the cells. Impedance cytometry is used to enumerate low numbers of MCF7 cells spiked into whole blood. Chemical lysis is commonly used to remove the abundant erythrocytes, and it is shown that this process does not alter the MCF7 cell count or change their dielectric properties. Combining impedance cytometry with magnetic bead based antibody enrichment enables MCF7 cells to be detected down to 100 MCF7 cells in 1 ml whole blood, a log 3.5 enrichment and a mean recovery of 92%. Microfluidic impedance cytometry could be easily integrated within complex cell separation systems for identification and enumeration of specific cell types, providing a fast in-line single cell characterisation method. PMID:25553198

  3. Solid state yellow and orange lasers for flow cytometry.

    PubMed

    Kapoor, Veena; Karpov, Vladimir; Linton, Claudette; Subach, Fedor V; Verkhusha, Vladislav V; Telford, William G

    2008-06-01

    Diode and DPSS lasers emitting a variety of wavelengths are now commonly incorporated into flow cytometers, greatly increasing our capacity to excite a wide variety of fluorochromes. Until recently, however, virtually no practical technology existed for generating yellow or orange laser light for flow cytometry that was compatible with smaller instrumentation. In this study, we evaluate several new solid state laser systems that emit from the 570 to 600 nm as excitation sources for flow cytometry. DPSS 580, 589, and 592 nm sources were integrated into a cuvette-based flow cytometer (BD LSR II) and a stream-in-air cell sorter (FACSVantage DiVa), and used to excite a variety of yellow, orange, and red excited fluorochromes, including Texas Red, APC, and its tandem conjugates, and the genetically encoded red fluorescent protein HcRed and the more recently developed Katushka. All laser sources were successfully incorporated into the indicated flow cytometry platforms. The yellow and orange sources (particularly 592 nm) were ideal for exciting Texas Red, and provided excitation of APC and its tandems that was comparable to a traditional red laser source, albeit at higher power levels than red sources. Yellow and orange laser light was optimal for exciting HcRed and Katushka. Practical yellow and orange laser sources are now available for flow cytometry. This technology fills an important gap in the laser wavelengths available for flow, now almost any fluorochrome requiring visible light excitation can be accommodated.

  4. An Active, Collaborative Approach to Learning Skills in Flow Cytometry

    ERIC Educational Resources Information Center

    Fuller, Kathryn; Linden, Matthew D.; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N.; Röhrig, Kimberley J.

    2016-01-01

    Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow…

  5. In vivo flow cytometry and time-resolved near-IR angiography and lymphography

    NASA Astrophysics Data System (ADS)

    Galanzha, Ekaterina I.; Tuchin, Valery V.; Brock, Robert W.; Zharov, Vladimir P.

    2007-05-01

    Integration of photoacoustic and photothermal techniques with high-speed, high-resolution transmission and fluorescence microscopy shows great potential for in vivo flow cytometry and indocyanine green (ICG) near-infrared (IR) angiography of blood and lymph microvessels. In particular, the capabilities of in vivo flow cytometry using rat mesentery and nude mouse ear models are demonstrated for real-time quantitative detection of circulating and migrating individual blood and cancer cells in skin, mesentery, lymph nodes, liver, kidney; studying vascular dynamics with a focus on lymphatics; monitoring cell traffic between blood and lymph systems; high-speed imaging of cell deformability in flow; and label-free real-time monitoring of single cell extravasation from blood vessel lumen into tissue. As presented, the advantages of ICG IR-angiography include estimation of time resolved dye dynamics (appearance and clearance) in blood and lymph microvessels using fluorescent and photoacoustic modules of the integrated technique. These new approaches are important for monitoring and quantifying metastatic and apoptotic cells; comparative measurements of plasma and cell velocities; analysis of immune responses; monitoring of circulating macromolecules, chylomicrons, bacteria, viruses and nanoparticles; molecular imaging. In the future, we believe that the integrated technique presented will have great potential for translation to early disease diagnoses (e.g. cancer) or assessment of innovative therapeutic interventions in humans.

  6. Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

    PubMed Central

    Gavin, Carson T; Ali, Sohrab N; Tailly, Thomas; Olvera-Posada, Daniel; Alenezi, Husain; Power, Nicholas E; Hou, Jinqiang; St. Amant, Andre H; Luyt, Leonard G; Wood, Stephen; Wu, Charles; Razvi, Hassan; Leong, Hon S

    2016-01-01

    Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden. PMID:26771074

  7. Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

    PubMed Central

    Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

    2015-01-01

    This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

  8. Cytopathology of the nasal mucosa in chronic exposure to diesel engine emission: a five-year survey of Swiss customs officers.

    PubMed Central

    Glück, Ulrich; Schütz, Rudolf; Gebbers, Jan-Olaf

    2003-01-01

    The simple and cheap technique of nasal cytology was used to assess possible adverse effects of chronic exposure to diesel engine emission (DEE) on respiratory mucous membranes. Brush cytology probes were taken from the noses of 194 male, nonsmoking customs officers twice a year (January and July) over a period of 5 years. The study group of 136 officers was solely occupied with clearing of diesel trucks (8.4 hr/day, 42 hr/week). Measured DEE concentrations varied between 31 and 60 microg/m3) and of benzo[a]pyrene concentrations were between 10 and 15 ng/m3). The control group of 58 officers worked only in the office. Over the 5-year period, similar results were obtained in summer and winter. In contrast to those not exposed to DEE, those who were had clear goblet cell hyperplasia with increased metaplastic and dysplastic epithelia and an increase in leukocytes. We found no evidence of progression of the cytopathologic changes. The findings may be described as a chronic inflammation of the nasal mucous membrane in the presence of chronic DEE exposure (chemical-induced rhinitis). Additionally, the findings of metaplastic and dysplastic nasal epithelia in the exposed subjects may indicate a genotoxic effect of chronic DEE exposure in humans. PMID:12782493

  9. Fine Needle Aspiration of Thyroid Nodules Using the Bethesda System for Reporting Thyroid Cytopathology: An Institutional Experience in a Rural Setting

    PubMed Central

    Kaminoh, Yuuki; Forward, Terra; Schwartz, Frank L.; Jenkinson, Scott

    2017-01-01

    Background. Fine needle aspiration (FNA) remains the first-line diagnostic in management of thyroid nodules and reduces unnecessary surgeries. However, it is still challenging since cytological results are not always straightforward. This study aimed to examine the results of thyroid FNA using the Bethesda system for reporting thyroid cytopathology (TBSRTC) to establish the level of accuracy of FNA procedures in a rural practice setting. Method. A retrospective chart review was conducted on existing thyroid FNA performed in a referral endocrine center between December 2011 and November 2015. Results. A total of 159 patients (18–88 years old) and 236 nodule aspirations were performed and submitted for evaluation. 79% were benign, 3% atypia/follicular lesion of unknown significance (AUS/FLUS), 5% follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), 4% suspicious for malignancy (one case was indeed an atypical parathyroid neoplasm by surgical pathology), 2% malignant, and 7% nondiagnostic. Two cases also had advanced molecular analysis on FNA specimens before thyroidectomy. Conclusion. The diagnostic yield of FNA cytology from our practice in a rural setting suggests that accuracy and specificity are comparable to results from larger centers. PMID:28280507

  10. Step-specific Sorting of Mouse Spermatids by Flow Cytometry.

    PubMed

    Simard, Olivier; Leduc, Frédéric; Acteau, Geneviève; Arguin, Mélina; Grégoire, Marie-Chantal; Brazeau, Marc-André; Marois, Isabelle; Richter, Martin V; Boissonneault, Guylain

    2015-12-31

    The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

  11. CRITICAL ASSESSMENT OF AUTOMATED FLOW CYTOMETRY DATA ANALYSIS TECHNIQUES

    PubMed Central

    Aghaeepour, Nima; Finak, Greg; Hoos, Holger; Mosmann, Tim R.; Gottardo, Raphael; Brinkman, Ryan; Scheuermann, Richard H.

    2013-01-01

    Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks – mammalian cell population identification to determine if automated algorithms can reproduce expert manual gating, and sample classification to determine if analysis pipelines can identify characteristics that correlate with external variables (e.g., clinical outcome). This analysis presents the results of the first of these challenges. Several methods performed well compared to manual gating or external variables using statistical performance measures, suggesting that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis. PMID:23396282

  12. Analyzing Schizosaccharomyces pombe DNA Content by Flow Cytometry.

    PubMed

    Boye, Erik; Anda, Silje; Rothe, Christiane; Stokke, Trond; Grallert, Beáta

    2016-06-01

    Flow cytometry can be used to measure the DNA content of individual cells. The data are usually presented as DNA histograms that can be used to examine the cells' progression through the cell cycle. Under standard growth conditions, fission yeast cells do not complete cytokinesis until after G1 phase; therefore, DNA histograms show one major peak representing cells in G1 (2×1C DNA) and G2 phase (1×2C DNA). By analysis of the duration of the fluorescence signal as well as the intensity of the DNA-related signal, it is possible to discriminate between cells in M/G1, S, and G2 This protocol describes how to prepare cells for flow cytometry and analyze them. We also describe the application of barcoding for more accurate comparison of samples.

  13. Recent advances of flow cytometry in fundamental and applied microbiology.

    PubMed

    Fouchet, P; Jayat, C; Héchard, Y; Ratinaud, M H; Frelat, G

    1993-01-01

    This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated: light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology: fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

  14. Flow cytometry as a tool to quantify oyster defence mechanisms.

    PubMed

    Goedken, Michael; De Guise, Sylvain

    2004-04-01

    The fast growing oyster aquaculture industry is greatly hindered by Perkinsus marinus and Haplosporidium nelsoni which can kill up to 80% of the production. The relationship between parasites and oyster defence mechanisms is unclear. Two defence mechanisms of the Eastern Oyster (Crassostrea virginica) were quantified at the single cell level utilising flow cytometry. Phagocytosis was measured using fluorescent beads. Respiratory burst activity was quantified as the H2O2-specific increase in dichlorofluorescein-associated fluorescence upon stimulation. These two assays distinguished three populations of haemocytes (granulocytes, hyalinocytes and intermediate cells) with unique functional characteristics. Granulocytes were most active at phagocytosis and H2O2 production while hyalinocytes were relatively inactive. The intermediate cells had moderate phagocytic and respiratory burst activity. Flow cytometry can rapidly, accurately and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the bivalve immune system.

  15. Critical assessment of automated flow cytometry data analysis techniques.

    PubMed

    Aghaeepour, Nima; Finak, Greg; Hoos, Holger; Mosmann, Tim R; Brinkman, Ryan; Gottardo, Raphael; Scheuermann, Richard H

    2013-03-01

    Traditional methods for flow cytometry (FCM) data processing rely on subjective manual gating. Recently, several groups have developed computational methods for identifying cell populations in multidimensional FCM data. The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of these methods on two tasks: (i) mammalian cell population identification, to determine whether automated algorithms can reproduce expert manual gating and (ii) sample classification, to determine whether analysis pipelines can identify characteristics that correlate with external variables (such as clinical outcome). This analysis presents the results of the first FlowCAP challenges. Several methods performed well as compared to manual gating or external variables using statistical performance measures, which suggests that automated methods have reached a sufficient level of maturity and accuracy for reliable use in FCM data analysis.

  16. Microsphere cytometry to interrogate microenvironment-dependent cell signaling.

    PubMed

    Ertsås, Henriette Christie; Nolan, Garry P; LaBarge, Mark A; Lorens, James B

    2017-02-20

    Microenvironmental cues comprising surface-mediated and soluble factors control cellular signaling mechanisms underlying normal cellular responses that define homeostatic and diseased cell states. In order to measure cell signaling in single adherent cells, we developed a novel microsphere-based flow cytometry approach. Single normal or neoplastic cells were adhered to uniform microspheres that display mimetic-microenvironments comprising surface combinations of extracellular matrix (ECM) in the presence of soluble agonists/antagonists. Temporal signaling responses were measured with fluorophore-conjugated antibodies that recognize response-dependent epitopes by multiparametric flow cytometry. Using this approach we demonstrate that microenvironment-mimetic combinations of growth factors and extracellular matrix proteins generate distinct cellular signal networks that reveal unique cell signatures in normal and patient biopsy-derived neoplastic cells.

  17. Using Flow Cytometry to Measure Phagocytic Uptake in Earthworms†

    PubMed Central

    Fuller-Espie, Sheryl L.

    2010-01-01

    This laboratory module familiarizes students with flow cytometry while acquiring quantitative reasoning skills during data analysis. Leukocytes, also known as coelomocytes (including hyaline and granular amoebocytes, and chloragocytes), from Eisenia hortensis (earthworms) are isolated from the coelomic cavity and used for phagocytosis of fluorescent Escherichia coli. Students learn how to set up in vitro cellular assays and become familiar with theoretical principles of flow cytometry. Histograms based on fluorescence and scatter properties combined with gating options permit students to restrict their analyses to particular subsets of coelomocytes when measuring phagocytosis, a fundamentally important innate immune mechanism used in earthworms. Statistical analysis of data is included in laboratory reports which serve as the primary assessment instrument. PMID:23653715

  18. Managing Multi-center Flow Cytometry Data for Immune Monitoring.

    PubMed

    White, Scott; Laske, Karoline; Welters, Marij Jp; Bidmon, Nicole; van der Burg, Sjoerd H; Britten, Cedrik M; Enzor, Jennifer; Staats, Janet; Weinhold, Kent J; Gouttefangeas, Cécile; Chan, Cliburn

    2014-01-01

    With the recent results of promising cancer vaccines and immunotherapy1-5, immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization21-23, as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables automated

  19. Managing Multi-center Flow Cytometry Data for Immune Monitoring

    PubMed Central

    White, Scott; Laske, Karoline; Welters, Marij JP; Bidmon, Nicole; van der Burg, Sjoerd H; Britten, Cedrik M; Enzor, Jennifer; Staats, Janet; Weinhold, Kent J; Gouttefangeas, Cécile; Chan, Cliburn

    2014-01-01

    With the recent results of promising cancer vaccines and immunotherapy1–5, immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization21–23, as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables

  20. Analysis of repetitive DNA in chromosomes by flow cytometry.

    PubMed

    Brind'Amour, Julie; Lansdorp, Peter M

    2011-06-01

    We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.

  1. Ploidy Determination in Agrostis Using Flow Cytometry and Morphological Traits.

    PubMed

    Bonos, Stacy A.; Plumley, Karen A.; Meyer, William A.

    2002-01-01

    The taxonomic classification of the genus Agrostis is one of the most complicated of the grass genera. Classification based upon morphological and anatomical characters is difficult and complicated by the presence of intermediate forms and the misapplication of names. Determining ploidy levels of new germplasm can assist in species determination and is necessary before initiating breeding or genetics studies. The objectives of this study were to (i) evaluate the use of laser flow cytometry as a quick, reliable tool to determine ploidy level and aid in Agrostis species determination, and (ii) identify morphological characters associated with DNA content or ploidy level. The six Agrostis species evaluated were A. canina L. subsp. canina, A. canina L. subsp. montana (Hartm.) Hartm., A. palustris Huds. [= A. stolonifera var. palustris (Huds.) Farw.], A. tenuis Sibth. (= A. capillaris L.), A. castellana Boiss. & Reut., and A. alba L. Ploidy level was determined by flow cytometry and root tip chromosome counts. Plant height, panicle height, flag leaf length, flag leaf width, and highest internode length of mature field-grown spaced plants were measured. Significant differences in 2C DNA content were found between species (P < 0.01) differing in ploidy level. Flow cytometry was effective in differentiating between diploid, tetraploid, and hexaploid species. Chromosome numbers previously reported and those observed in this study were positively correlated with 2C nuclear DNA content (r = 0.98, P < 0.01). Flag leaf length was the only morphological measurement taken that was significantly positively correlated to DNA content (r = 0.98, P < 0.001). The results of this study indicate that laser flow cytometry is a quick, reliable tool to determine ploidy levels and infer certain species of AGROSTIS: This technique will aid breeders to quickly and accurately determine ploidy levels of new germplasm collections.

  2. Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry.

    PubMed

    Fine, Noah; Barzilay, Oriyah; Glogauer, Michael

    2017-01-01

    Neutrophils are primary phagocytes that recognize their targets through surface chemistry, either through Pattern Recognition Receptor (PPR) interaction with Pathogen-Associated Molecular Patterns (PAMPs) or through immunoglobulin (Ig) or complement mediated recognition. Opsonization can be important for target recognition, and phagocytosis by neutrophils in whole blood can be greatly enhanced due to the presence of blood serum components and platelets. Powerful and sensitive flow cytometry based methods are presented to measure phagocytosis by human blood neutrophils and mouse peritoneal neutrophils.

  3. A CLIPS expert system for clinical flow cytometry data analysis

    NASA Technical Reports Server (NTRS)

    Salzman, G. C.; Duque, R. E.; Braylan, R. C.; Stewart, C. C.

    1990-01-01

    An expert system is being developed using CLIPS to assist clinicians in the analysis of multivariate flow cytometry data from cancer patients. Cluster analysis is used to find subpopulations representing various cell types in multiple datasets each consisting of four to five measurements on each of 5000 cells. CLIPS facts are derived from results of the clustering. CLIPS rules are based on the expertise of Drs. Stewart, Duque, and Braylan. The rules incorporate certainty factors based on case histories.

  4. Performance of computer vision in vivo flow cytometry with low fluorescence contrast

    NASA Astrophysics Data System (ADS)

    Markovic, Stacey; Li, Siyuan; Niedre, Mark

    2015-03-01

    Detection and enumeration of circulating cells in the bloodstream of small animals are important in many areas of preclinical biomedical research, including cancer metastasis, immunology, and reproductive medicine. Optical in vivo flow cytometry (IVFC) represents a class of technologies that allow noninvasive and continuous enumeration of circulating cells without drawing blood samples. We recently developed a technique termed computer vision in vivo flow cytometry (CV-IVFC) that uses a high-sensitivity fluorescence camera and an automated computer vision algorithm to interrogate relatively large circulating blood volumes in the ear of a mouse. We detected circulating cells at concentrations as low as 20 cells/mL. In the present work, we characterized the performance of CV-IVFC with low-contrast imaging conditions with (1) weak cell fluorescent labeling using cell-simulating fluorescent microspheres with varying brightness and (2) high background tissue autofluorescence by varying autofluorescence properties of optical phantoms. Our analysis indicates that CV-IVFC can robustly track and enumerate circulating cells with at least 50% sensitivity even in conditions with two orders of magnitude degraded contrast than our previous in vivo work. These results support the significant potential utility of CV-IVFC in a wide range of in vivo biological models.

  5. Separating the signal from the noise: Expanding flow cytometry into the sub-micron range.

    EPA Science Inventory

    Cytometry Part A Special Section: Separating the signal from the noise: Expanding flow cytometry into the sub-micron range. The current Cytometry Part A Special Section presents three studies that utilize cytometers to study sub-micron particles. The three studies involve the 1...

  6. Fluorescein Isothiocyanate-Labeled Lectin Analysis of the Surface of the Nitrogen-Fixing Bacterium Azospirillum brasilense by Flow Cytometry

    PubMed Central

    Yagoda-Shagam, Janet; Barton, Larry L.; Reed, William P.; Chiovetti, Robert

    1988-01-01

    The cell surface of Azospirillum brasilense was probed by using fluorescein isothiocyanate (FITC)-labeled lectins, with binding determined by fluorescence-activated flow cytometry. Cells from nitrogen-fixing or ammonium-assimilating cultures reacted similarly to FITC-labeled lectins, with lectin binding in the following order: Griffonia simplicifolia II agglutinin > Griffonia simplicifolia I agglutinin > Triticum vulgaris agglutinin > Glycine max agglutinin > Canavalia ensiformis agglutinin > Limax flavus agglutinin > Lotus tetragonolobus agglutinin. The fluorescence intensity of cells labeled with FITC-labeled G. simplicifolia I, C. ensiformis, T. vulgaris, and G. max agglutinins was influenced by lectin concentration. Flow cytometry measurements of lectin binding to cells was consistent with measurements of agglutination resulting from lectin-cell interaction. Capsules surrounding nitrogen-fixing and ammonium-assimilating cells were readily demonstrated by light and transmission electron microscopies. Images PMID:16347693

  7. A single institution experience with the new bethesda system for reporting thyroid cytopathology: correlation with existing cytologic, clinical, and histological data.

    PubMed

    McElroy, Michele K; Mahooti, Sepi; Hasteh, Farnaz

    2014-07-01

    Our goal was to evaluate the Bethesda system (TBS) in comparison to the previously used system at our institution. One hundred consecutive thyroid fine needle aspirations (FNAs) and 45 consecutive indeterminate FNAs were reviewed by two cytopathology-boarded pathologists, diagnosed based on TBS and correlated with management and follow-up. Re-evaluation led to a diagnosis change in 48% of cases. Thirty-nine percent of benign cases were unsatisfactory under TBS. For malignant diagnoses the positive predictive value (PPV) was unchanged, while the negative predictive value (NPV) was slightly improved using TBS. Both the PPV and NPV were improved for actionable diagnoses. Inter-observer variability across all categories was in moderate agreement. Clinical management of both follicular lesion (FL) and indeterminate cases ranged from none to immediate surgery. Repeat FNA resolved the diagnosis in 50% of indeterminate cases. Indeterminate cases had an overall malignancy rate of 27%; higher in pre- (46%) than post-TBS cases (8%). Inter-observer variability between the reviewing pathologists and the original pathologists for indeterminate cases was fair, and between the two reviewing pathologists was moderate. Using TBS criteria increased the unsatisfactory rate and led to improved prediction of malignancy and actionable diagnoses. The pre-Bethesda diagnosis of FL at our institution led to inconsistent clinical management. Clinical management of patients with indeterminate diagnoses was essentially unchanged following adoption of TBS. The moderate inter-observer agreement between the reviewing pathologists may be related to level of cytology experience, strict adherence to TBS, and the exclusive use of cytomorphology for diagnosis.

  8. Identification of contact and respiratory sensitizers using flow cytometry

    SciTech Connect

    Goutet, Michele . E-mail: michele.goutet@inrs.fr; Pepin, Elsa; Langonne, Isabelle; Huguet, Nelly; Ban, Masarin

    2005-06-15

    Identification of the chemicals responsible for respiratory and contact allergies in the industrial area is an important occupational safety issue. This study was conducted in mice to determine whether flow cytometry is an appropriate method to analyze and differentiate the specific immune responses to the respiratory sensitizer trimellitic anhydride (TMA) and to the contact sensitizer dinitrochlorobenzene (DNCB) used at concentrations with comparable immunogenic potential. Mice were exposed twice on the flanks (days 0, 5) to 10% TMA or 1% DNCB and challenged three times on the ears (days 10, 11, 12) with 2.5% TMA or 0.25% DNCB. Flow cytometry analyses were conducted on draining lymph node cells harvested on days 13 and 18. Comparing TMA and DNCB immune responses on day 13, we found obvious differences that persisted for most of them on day 18. An increased proportion of IgE+ cells correlated to total serum IgE level and an enhancement of MHC II molecule expression were observed in the lymph node B lymphocytes from TMA-treated mice. The percentage of IL-4-producing CD4+ lymphocytes and the IL-4 receptor expression were clearly higher following TMA exposure. In contrast, higher proportions of IL-2-producing cells were detected in CD4+ and CD8+ cells from DNCB-treated mice. Both chemicals induced a significant increase in the percentage of IFN-{gamma}-producing cells among CD8+ lymphocytes but to a greater proportion following TMA treatment. In conclusion, this study encourages the use of flow cytometry to discriminate between contact and respiratory sensitizers by identifying divergent expression of immune response parameters.

  9. Ultraviolet 320 nm laser excitation for flow cytometry.

    PubMed

    Telford, William; Stickland, Lynn; Koschorreck, Marco

    2017-02-27

    Although multiple lasers and high-dimensional analysis capability are now standard on advanced flow cytometers, ultraviolet (UV) lasers (usually 325-365 nm) remain an uncommon excitation source for cytometry. This is primarily due to their cost, and the small number of applications that require this wavelength. The development of the Brilliant Ultraviolet (BUV fluorochromes, however, has increased the importance of this formerly niche excitation wavelength. Historically, UV excitation was usually provided by water-cooled argon- and krypton-ion lasers. Modern flow cytometers primary rely on diode pumped solid state lasers emitting at 355 nm. While useful for all UV-excited applications, DPSS UV lasers are still large by modern solid state laser standards, and remain very expensive. Smaller and cheaper near UV laser diodes (NUVLDs) emitting at 375 nm make adequate substitutes for 355 nm sources in many situations, but do not work as well with very short wavelength probes like the fluorescent calcium chelator indo-1. In this study, we evaluate a newly available UV 320 nm laser for flow cytometry. While shorter in wavelength that conventional UV lasers, 320 is close to the 325 nm helium-cadmium wavelength used in the past on early benchtop cytometers. A UV 320 nm laser was found to excite almost all Brilliant Ultraviolet dyes to nearly the same level as 355 nm sources. Both 320 nm and 355 nm sources worked equally well for Hoechst and DyeCycle Violet side population analysis of stem cells in mouse hematopoetic tissue. The shorter wavelength UV source also showed excellent excitation of indo-1, a probe that is not compatible with NUVLD 375 nm sources. In summary, a 320 nm laser module made a suitable substitute for conventional 355 nm sources. This laser technology is available in a smaller form factor than current 355 nm units, making it useful for small cytometers with space constraints. © 2017 International Society for Advancement of Cytometry.

  10. Stochastic Measurement Models for Quantifying Lymphocyte Responses Using Flow Cytometry

    PubMed Central

    Kan, Andrey; Pavlyshyn, Damian; Markham, John F.; Dowling, Mark R.; Heinzel, Susanne; Zhou, Jie H. S.; Marchingo, Julia M.; Hodgkin, Philip D.

    2016-01-01

    Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus

  11. Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry.

    PubMed

    Furman, Jennifer L; Holmes, Brandon B; Diamond, Marc I

    2015-12-08

    Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as α-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

  12. Waveguide detection of right-angle-scattered light in flow cytometry

    DOEpatents

    Mariella, Jr., Raymond P.

    2000-01-01

    A transparent flow cell is used as an index-guided optical waveguide. A detector for the flow cell but not the liquid stream detects the Right-Angle-Scattered (RAS) Light exiting from one end of the flow cell. The detector(s) could view the trapped RAS light from the flow cell either directly or through intermediate optical light guides. If the light exits one end of the flow cell, then the other end of the flow cell can be given a high-reflectivity coating to approximately double the amount of light collected. This system is more robust in its alignment than the traditional flow cytometry systems which use imaging optics, such as microscope objectives.

  13. Laser rastering flow cytometry: fast cell counting and identification

    NASA Astrophysics Data System (ADS)

    Vacca, G.; Junnarkar, M. R.; Goldblatt, N. R.; Yee, M. W.; Van Slyke, B. M.; Briese, T. C.

    2009-02-01

    We describe the concept of laser rastering flow cytometry, where a rapidly scanning laser beam allows counting and classification of cells at much higher rates than currently possible. Modifications to existing flow cytometers to implement the concept include an acousto-optic deflector, fast analog-to-digital conversion, and a two-step digital-signal-processing scheme that handles the high data rates and provides key assay information. Results are shown that prove the concept, demonstrating the ability to resolve closely spaced cells and to measure cells at rates more than an order of magnitude faster than on conventional flow-cytometer-based hematology analyzers.

  14. Pitfalls in the use of multicolour flow cytometry in haematology.

    PubMed

    Johansson, Ulrika; Macey, Marion

    2011-07-01

    Multicolour flow cytometry in haematology has developed considerably in recent years. The ability to analyse eight or more colours of fluorescence on millions of cells in a matter of minutes has enabled the provision of rapid and reliable measures of minimal residual disease for clinicians. The use of multicolour analysis has also enabled more specific characterisation of presenting leukaemias and lymphomas. However, there has not been a concomitant increase in the knowledge and experience of the flow cytometrists to deal with certain problems associated with this more complex analysis.

  15. CymeR: cytometry analysis using KNIME, Docker and R.

    PubMed

    Muchmore, B; Alarcon-Riquelme, M E

    2016-12-20

    Here we present open-source software for the analysis of high-dimensional cytometry data using state of the art algorithms. Importantly, use of the software requires no programming ability, and output files can either be interrogated directly in CymeR or they can be used downstream with any other cytometric data analysis platform. Also, because we use Docker to integrate the multitude of components that form the basis of CymeR, we have additionally developed a proof-of-concept of how future open-source bioinformatic programs with graphical user interfaces could be developed.

  16. Cell-cooling in flow cytometry by Peltier elements.

    PubMed

    Göttlinger, C; Meyer, K L; Weichel, W; Müller, W; Raftery, B; Radbruch, A

    1986-05-01

    We have built a cooling device for cell suspensions in flow cytometry that makes use of the Peltier effect (Barnard RD, Thermo electricity in Metals and Alloys, Taylor and Francis, London; Siemens-Z 34:383-88, 1963). The prototype described here is used for cooling collection tubes during long-duration cell sorting and is capable of maintaining a temperature of 2-5 degrees C in a cell suspension of up to 3 ml. In general, Peltier element-based cooling is useful for equilibrating the temperature of small volumes of fluids. Furthermore, Peltier element-based cooling devices are easy to build and handle.

  17. Zebrafish thrombocyte aggregation by whole blood aggregometry and flow cytometry.

    PubMed

    Sundaramoorthi, Hemalatha; Panapakam, Rekha; Jagadeeswaran, Pudur

    2015-01-01

    Zebrafish has become an excellent model system to study mammalian hemostasis. Despite our extensive efforts to develop technologies to measure zebrafish hemostasis and even with previously established thrombocyte qualitative and quantitative functional assays, quantifying thrombocyte function for high throughput applications has been a challenge. In this paper, we have developed two quantitative methods to estimate thrombocyte aggregation: one by whole blood aggregometry and the other by flow cytometry. We found that it is possible to conduct whole blood aggregometry using only 2 µl of blood and the currently available aggregometer. Each of three agonists, arachidonic acid, ADP, and collagen yielded impedance curves similar to those obtained with human blood. We were also able to use flow cytometry to indirectly quantify the extent of thrombocyte aggregation by labeling whole blood with mepacrine, aggregating in the presence of each of the above agonists, separating the aggregates from the white blood cells by centrifugation, and then sorting the resulting white cell fraction for thrombocyte numbers. These methods have high throughput capabilities and have the potential to be used in large scale screens to detect and characterize mutants with thrombocyte functional defects or to identify genes involved in thrombocyte function by large scale knockdowns.

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

    PubMed

    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.

  19. Beam shaping in flow cytometry with diffractive optical elements

    NASA Astrophysics Data System (ADS)

    Qu, Weidong; Li, Derong; Jian, Peng

    2016-10-01

    Focusing elements are usually employed in the flow cytometry to focus the input laser beam into elliptically shaped Gaussian beam in order to increase power for excitation of fluorescence for high signal-to-noise ratio (SNR). While in order to ensure repeatable and reliable signal generation for accurate population discrimination - despite slight deviations of the cell from the flow centre, the shaped beam should be a cubic diffraction region with uniform power intensity across the cell flow stream. However, it is hard for beam shaping with refractive optical elements. In this paper, we present a beam shaping system in flow cytometry with diffractive optical elements (DOEs) to shape the input laser beam to a cubic diffraction region with uniform power intensity. The phase distribution of the DOE is obtained by using the inverse Fresnel diffraction based layered holographic stereogram, and the cubic diffraction region with uniform power intensity within the cell flow channel is well reconstructed. Simulation results demonstrate the good performance of the new beam shaping system.

  20. High-throughput vibrational cytometry based on nonlinear Raman microspectroscopy

    NASA Astrophysics Data System (ADS)

    Arora, R.; Petrov, G. I.; Yakovlev, V. V.

    2010-02-01

    Flow cytometry is a technology that allows a single cell or particle to be measured for a variety of characteristics, determined by looking at their properties while they flow in a liquid stream. High speed of flow and huge number of objects to be analyzed imposed some strict criteria on which methods can be used for analysis. All the known commercial instruments are currently using light scattering for particle sizing and fluorescence detection for chemical recognition. However, vibrational spectroscopy is the only non-invasive optical spectroscopy tool, which has proven to provide chemically-specific information about the interrogated sample. It is proposed that vibrational spectroscopy, based on nonlinear Raman scattering can be used to serve as an analytical tool for cytometry by providing rapid and accurate chemical recognition of flowing materials. To achieve a desired speed (>10,000 cell/particles per second), we have substantially upgraded our previous system for nonlinear Raman microspectroscopy. By increasing the size of the excitation volume to the size of a cell and by keeping the incident intensity at the same level, a dramatic increase of the nonlinear Raman signal is achieved. This allows high-quality vibrational spectra to be acquired within 10-100 microsecond from a single yeast cell without any observable damage to the irradiated cell. This is four orders of magnitude better than any previous attempts involving Raman microspectroscopy.

  1. Sample handling for kinetics and molecular assembly in flow cytometry

    SciTech Connect

    Sklar, L.A. |; Seamer, L.C.; Kuckuck, F.; Prossnitz, E.; Edwards, B.; Posner, G.

    1998-07-01

    Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrounding medium. It permits assemblies of macromolecular complexes on beads or cells to be detected in real-time with precision and specificity. The authors have investigated two types of robust sample handling systems which provide sub-second resolution and high throughput: (1) mixers which use stepper-motor driven syringes to initiate chemical reactions in msec time frames; and (2) flow injection controllers with valves and automated syringes used in chemical process control. In the former system, the authors used fast valves to overcome the disparity between mixing 100 {micro}ls of sample in 100 msecs and delivering sample to a flow cytometer at 1 {micro}l/sec. Particles were detected within 100 msec after mixing, but turbulence was created which lasted for 1 sec after injection of the sample into the flow cytometer. They used optical criteria to discriminate particles which were out of alignment due to the turbulent flow. Complex sample handling protocols involving multiple mixing steps and sample dilution have also been achieved. With the latter system they were able to automate sample handling and delivery with intervals of a few seconds. The authors used a fluidic approach to defeat turbulence caused by sample introduction. By controlling both sheath and sample with individual syringes, the period of turbulence was reduced to {approximately} 200 msecs. Automated sample handling and sub-second resolution should permit broad analytical and diagnostic applications of flow cytometry.

  2. Use of laser scanning cytometry to study tumor microenvironment.

    PubMed

    Mocellin, S; Wang, E; Panelli, M; Rossi, C R; Marincola, F M

    2003-04-01

    The study of phenomena occurring in the tumor microenvironment is a challenging task because of technical difficulties, particularly when dealing with hypocellular specimens. Laser scanning cytometry (LSC) is a new laboratory technology that has been recently introduced to overcome the limitations of other traditional technologies. By combining the properties and the advantages of flow cytometry (FC) and immunohistochemistry (IHC), LSC allows the investigator to obtain objective information on DNA content, protein expression and cellular localization is combination with morphological features. It has been already shown that LSC results are reliable compared to more traditional technologies, and its implementation in the clinical routine is under way. Its use in oncology, which is rapidly expanding, spans from apoptosis analysis to DNA content quantitation and tumor cell phenotyping. Here we describe the technology underlying this novel fluorescence-based device, review its use in oncology by dissecting the phenomena occurring in the tumor microenvironment and propose its application for the immunological follow-up of malignant lesions undergoing immunotherapeutic manipulation.

  3. Measurement and Characterization of Apoptosis by Flow Cytometry.

    PubMed

    Telford, William; Tamul, Karen; Bradford, Jolene

    2016-07-01

    Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti-cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal-transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation. These assays are particularly powerful when combined into multicolor assays determining several apoptotic characteristics simultaneously. The multiparametric nature of flow cytometry makes this technology particularly suited to measuring apoptosis. In this unit, we will describe the four main techniques for analyzing caspase activity in apoptotic cells, combined with annexin V and cell permeability analysis. These relatively simple multiparametric assays are powerful techniques for assessing cell death. © 2016 by John Wiley & Sons, Inc.

  4. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  5. Metal-Containing Polystyrene Beads as Standards for Mass Cytometry.

    PubMed

    Abdelrahman, Ahmed I; Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Kinach, Robert; Dai, Sheng; Thickett, Stuart C; Tanner, Scott; Winnik, Mitchell A

    2010-01-01

    We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells.

  6. Defining human dendritic cell progenitors by multiparametric flow cytometry

    PubMed Central

    Breton, Gaëlle; Lee, Jaeyop; Liu, Kang; Nussenzweig, Michel C

    2015-01-01

    Human dendritic cells (DCs) develop from progressively restricted bone marrow (BM) progenitors: these progenitor cells include granulocyte, monocyte and DC progenitor (GMDP) cells; monocyte and DC progenitor (MDP) cells; and common DC progenitor (CDP) and DC precursor (pre-DC) cells. These four DC progenitors can be defined on the basis of the expression of surface markers such as CD34 and hematopoietin receptors. In this protocol, we describe five multiparametric flow cytometry panels that can be used as a tool (i) to simultaneously detect or phenotype the four DC progenitors, (ii) to isolate DC progenitors to enable in vitro differentiation or (iii) to assess the in vitro differentiation and proliferation of DC progenitors. The entire procedure from isolation of cells to flow cytometry can be completed in 3–7 h. This protocol provides optimized antibody panels, as well as gating strategies, for immunostaining of BM and cord blood specimens to study human DC hematopoiesis in health, disease and vaccine settings. PMID:26292072

  7. The College of American Pathologists guidelines for whole slide imaging validation are feasible for pediatric pathology: a pediatric pathology practice experience.

    PubMed

    Arnold, Michael A; Chenever, Emily; Baker, Peter B; Boué, Daniel R; Fung, Bonita; Hammond, Sue; Hendrickson, Brett W; Kahwash, Samir B; Pierson, Christopher R; Prasad, Vinay; Nicol, Kathleen K; Barr, Thomas

    2015-01-01

    Whole slide imaging (WSI) is rapidly transforming educational and diagnostic pathology services. Recently, the College of American Pathologists Pathology and Laboratory Quality Center (CAP-PLQC) published recommended guidelines for validating diagnostic WSI. We prospectively evaluated the guidelines to determine their utility in validating pediatric surgical pathology and cytopathology specimens. Our validation included varied pediatric specimen types, including complex or less common diagnoses, in accordance with the guidelines. We completed WSI review of 60 surgical pathology cases and attempted WSI review of 21 cytopathology cases. For surgical pathology cases, WSI diagnoses were highly concordant with glass slide diagnoses; a discordant diagnosis was observed in 1 of 60 cases (98.3% concordance). We found that nucleated red blood cells and eosinophilic granular bodies represented specific challenges to WSI review of pediatric specimens. Cytology specimens were more frequently discordant or failed for technical reasons, with overall concordance of 66.7%. Review of pediatric cytopathology specimens will likely require image capture in multiple focal planes. This study is the first to specifically evaluate WSI review for pediatric specimens and demonstrates that specimens representing the spectrum of pediatric surgical pathology practice can be reviewed using WSI. Our application of the proposed CAP-PLQC guidelines to pediatric surgical pathology specimens is, to our knowledge, the first prospective implementation of the CAP-PLQC guidelines.

  8. Cytopathologic features of an unusual case of multiple eccrine spiradenomas misdiagnosed as a malignant round cell tumor

    PubMed Central

    Rekhi, Bharat; Agarwal, Archi

    2017-01-01

    A 28-year-old lady presented with multiple swellings in her left shoulder, associated with intermittent pain since last one and a half years. Radiologic imaging revealed multiple, well-defined, subcutaneous lesions in her left supraclavicular region. Fine needle aspiration cytology (FNAC) smears were initially reported as Ewing sarcoma, elsewhere. On review, the smears showed cohesive clusters of round-to-oval cells with scant cytoplasm, which were focally arranged in an acinar-rosetting pattern around hyaline “droplets/bodies,” along with few scattered lymphocytes against a background of red blood cells. The diagnosis considered was adnexal tumor. Subsequent biopsy from the multiple lesions confirmed the diagnosis of eccrine spiradenoma. By immunohistochemistry, tumor cells were positive for CK7, epithelial membrane antigen (focally), S100 protein, and tyrosine-protein kinase Kit(C-KIT) /Cluster of differentiation (CD117). This case underscores the value of FNAC in skin adnexal tumors and constitutes as the first case report of multiple eccrine spiradenomas, initially misdiagnosed as Ewing sarcoma. Literature review of similar reported cases with treatment implications are presented. PMID:28182081

  9. Assessment of erythrocyte shape by flow cytometry techniques

    PubMed Central

    Piagnerelli, M; Boudjeltia, K Zouaoui; Brohee, D; Vereerstraeten, A; Piro, P; Vincent, J‐L; Vanhaeverbeek, M

    2007-01-01

    Background Red blood cell (RBC) rheology is altered in different diseases, including acute conditions such as patients in intensive care units (ICU) with sepsis or with an inflammatory reaction due to postoperative states or intracerebral haemorrhage, or chronic conditions such as diabetes mellitus or terminal renal failure. Several techniques are available to assess alterations in RBC rheology, especially deformability, but they are too cumbersome to be used on a large number of cells. Objective To develop a new, rapid flow cytometry technique for easy assessment of RBC shape in patients. Methods In flow cytometry, healthy human RBC shape shows a bimodal distribution related to the biconcave form. On this histogram, the second Pearson coefficient of dissymmetry (PCD) representing the asymmetry of this histogram and the spherical index (M2:M1) were calculated, both representing the spherical shape. This technique was used in healthy volunteers (n = 17) and in diseases characterised by abnormalities in RBC rheology, including terminal renal failure requiring haemodialysis (n = 28), diabetes mellitus (n = 18), sepsis (n = 19) and acute inflammatory states (postoperative, intracerebral haemorrhage, chronic obstructive pulmonary disease, epilepsy or severe drug intoxication; n = 21). Multivariate analysis was performed to determine the factors influencing RBC shape. Results Measurement of RBC shape was highly reproducible. A good correlation was observed between the PCD and the spherical index, except in the critically ill patients without sepsis. RBCs were more spherical in patients with terminal renal failure (PCD −0.56 (0.14), p<0.05), diabetes mellitus (PCD −0.59 (0.23), p<0.05), sepsis (PCD −0.58 (0.22), p<0.05) or an acute inflammatory state (PCD −0.65 (0.29), p<0.05) than in healthy volunteers (PCD −0.89 (0.12)). The spherical index was also increased in all populations compared with healthy volunteers (terminal renal failure 2

  10. A simple diagnostic test for Fanconi anemia by flow cytometry.

    PubMed

    Miglierina, R; Le Coniat, M; Berger, R

    1991-03-01

    A simple diagnostic test for Fanconi anemia (FA) by flow cytometry is proposed. It is based on the cell cycle disturbances of FA cells and their sensitisation by alkylating agents. Following PHA-stimulation of whole blood cell cultures in the presence or absence of nitrogen mustard, the accumulation of cells in G2/M phase was measured. A sharp increase of cells in G2/M was observed in cultures from FA patients when nitrogen mustard was added. This increase allows one to distinguish FA patients from patients with anemias of other origin, healthy controls, and FA heterozygotes, as effectively as chromosome breakage studies. The rapidity of the test and its reliability as demonstrated on the ten FA patients studied, will make the diagnosis of FA easier in centers without cytogenetic laboratory facilities.

  11. An improved flow cytometry assay to monitor phagosome acidification.

    PubMed

    Colas, Chloé; Menezes, Shinelle; Gutiérrez-Martínez, Enric; Péan, Claire B; Dionne, Marc S; Guermonprez, Pierre

    2014-10-01

    Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidification is required for many enzymatic processes taking place in phago-lysosomes, like proteolysis, and supports the microbicidal activity of macrophages. Here we present a new quantitative methodology to assess phagosome acidification by flow cytometry based on the use of bi-fluorescent particles. This method relies on the use of UV polystyrene beads labelled with the acid sensor pHrodo-succinimidyl ester (pHrodo(TM) SE red) and enables us to dissociate particle association with phagocytes from their engulfment in acidified compartments. This methodology is well suited to monitor the acidification of phagosomes formed in vivo after fluorescent bead administration.

  12. A Deep Profiler’s Guide to Cytometry

    PubMed Central

    Bendall, Sean C.; Nolan, Garry P.; Roederer, Mario; Chattopadhyay, Pratip K.

    2012-01-01

    In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to simultaneously measure multiple genes on the same cell is necessary to resolve the incredible diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10,000 cells per second. “Mass cytometry” is a new technology that promises to significantly extend these capabilities. Immunophenotyping by mass spectrometry provides the ability to measure more than three dozen proteins at a rate of 1,000 cells per second. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays. PMID:22476049

  13. Applications of Flow Cytometry to Characterize Bacterial Physiological Responses

    PubMed Central

    Contreras-Garduño, Jorge A.; Pedraza-Reyes, Mario

    2014-01-01

    Although reports of flow cytometry (FCM) applied to bacterial analysis are increasing, studies of FCM related to human cells still vastly outnumber other reports. However, current advances in FCM combined with a new generation of cellular reporter probes have made this technique suitable for analyzing physiological responses in bacteria. We review how FCM has been applied to characterize distinct physiological conditions in bacteria including responses to antibiotics and other cytotoxic chemicals and physical factors, pathogen-host interactions, cell differentiation during biofilm formation, and the mechanisms governing development pathways such as sporulation. Since FCM is suitable for performing studies at the single-cell level, we describe how this powerful technique has yielded invaluable information about the heterogeneous distribution of differently and even specialized responding cells and how it may help to provide insights about how cell interaction takes place in complex structures, such as those that prevail in bacterial biofilms. PMID:25276788

  14. Detection of Apoptotic Versus Autophagic Cell Death by Flow Cytometry.

    PubMed

    Sica, Valentina; Maiuri, M Chiara; Kroemer, Guido; Galluzzi, Lorenzo

    2016-01-01

    Different modes of regulated cell death (RCD) can be initiated by distinct molecular machineries and their morphological manifestations can be difficult to discriminate. Moreover, cells responding to stress often activate an adaptive response centered around autophagy, and whether such a response is cytoprotective or cytotoxic cannot be predicted based on morphological parameters only. Molecular definitions are therefore important to understand various RCD subroutines from a mechanistic perspective. In vitro, various forms of RCD including apoptosis and autophagic cell death can be easily discriminated from each other with assays that involve chemical or pharmacological interventions targeting key components of either pathway. Here, we detail a straightforward method to discriminate apoptosis from autophagic cell death by flow cytometry, based on the broad-spectrum caspase inhibitor Z-VAD-fmk and the genetic inhibition of ATG5.

  15. Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry

    PubMed Central

    Houston, Jessica P.; Naivar, Mark A.; Freyer, James P.

    2010-01-01

    Frequency-domain flow cytometry techniques are combined with modifications to the digital signal processing capabilities of the Open Reconfigurable Cytometric Acquisition System (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency modulated detector signals, implementing Fourier analysis programming with ORCAS’ digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5–25 ns simulated lifetime), pulse widths ranging from 2 to 15 µs, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142° to 1.6°. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 µs and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a radiofrequency modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to ~98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively

  16. Label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoling; Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

    2016-03-01

    Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

  17. Optical analysis of nanomaterial-cell interactions: flow cytometry and digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Mues, Sarah; Antunovic, Jan; Ossig, Rainer; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    The in vitro cytotoxicity assessment of engineered nanoparticles commonly involves the measurement of different endpoints like the formation of reactive oxygen species, cell viability or cell death. Usually these parameters are determined by optical readouts of enzymatically converted substrates that often interfere with the tested nanomaterials. Using cell viability (WST-8) and cell death (LDH) as parameter we have initially investigated the toxic effects of spherical (NM 300) and rod shaped (NM 302) silver nanomaterials with a matrix of four cell lines representing different functions: lung and kidney epithelial cells, macrophages and fibroblasts. In addition, we have used a label-free flow cytometer configuration to investigate interactions of particles and macrophages by side scatter signal analysis. Finally, we explored digital holographic microscopy (DHM) for multimodal label-free analysis of nanomaterial toxicity. Quantitative DHM phase images were analyzed for cell thickness, volume, density, dry mass and refractive index. We could demonstrate that silver spheres lead to more cytotoxic effects than rods in all four examined cell lines and both assay. Exemplarily a dose dependent interaction increase of cells with NM 300 and NM 302 analyzed by flow cytometry is shown. Furthermore, we found that the refractive index of cells is influenced by incubation with NM 300 in a decreasing manner. A 24 hours time-lapse measurement revealed a dose dependent decrease of dry mass and surface area development indicating reduced cell viability and cell death. Our results demonstrate digital holographic microscopy and flow cytometry as valuable label-free tools for nanomaterial toxicity and cell interaction studies.

  18. Spectral Cytometry Has Unique Properties Allowing Multicolor Analysis of Cell Suspensions Isolated from Solid Tissues

    PubMed Central

    Schmutz, Sandrine; Valente, Mariana

    2016-01-01

    Flow cytometry, initially developed to analyze surface protein expression in hematopoietic cells, has increased in analytical complexity and is now widely used to identify cells from different tissues and organisms. As a consequence, data analysis became increasingly difficult due the need of large multi-parametric compensation matrices and to the eventual auto-fluorescence frequently found in cell suspensions obtained from solid organs. In contrast with conventional flow cytometry that detects the emission peak of fluorochromes, spectral flow cytometry distinguishes the shapes of emission spectra along a large range of continuous wave lengths. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable to discriminate fluorochromes with similar emission peaks and provide multi-parametric analysis without compensation requirements. Here we show that spectral flow cytometry achieves a 21-parametric (19 fluorescent probes) characterization and deals with auto-fluorescent cells, providing high resolution of specifically fluorescence-labeled populations. Our results showed that spectral flow cytometry has advantages in the analysis of cell populations of tissues difficult to characterize in conventional flow cytometry, such as heart and intestine. Spectral flow cytometry thus combines the multi-parametric analytical capacity of the highest performing conventional flow cytometry without the requirement for compensation and enabling auto-fluorescence management. PMID:27500930

  19. Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

    PubMed Central

    Correia, Rodolfo Patussi; Bento, Laiz Cameirão; Bortolucci, Ana Carolina Apelle; Alexandre, Anderson Marega; Vaz, Andressa da Costa; Schimidell, Daniela; Pedro, Eduardo de Carvalho; Perin, Fabricio Simões; Nozawa, Sonia Tsukasa; Mendes, Cláudio Ernesto Albers; Barroso, Rodrigo de Souza; Bacal, Nydia Strachman

    2016-01-01

    ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. PMID:27759825

  20. Photoacoustic and photothermal cytometry for monitoring multiple blood rheology parameters in vivo

    PubMed Central

    Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2012-01-01

    Alterations of blood rheology (hemorheology) are important for the early diagnosis, prognosis, and prevention of many diseases, including myocardial infarction, stroke, sickle cell anemia, thromboembolism, trauma, inflammation, and malignancy. However, real-time in vivo monitoring of hemorheological status using multiple parameters over long periods of time has not been reported. Here we describe the capability of label-free photoacoustic (PA) and photothermal (PT) flow cytometry in detection and imaging modes for dynamic monitoring of rheological parameters in circulating blood. We show that this integrated platform can simultaneously measure the main rheological parameters and may improve their diagnostic value. Using phenomenological approaches, we analyze correlations of PT and PA signal characteristics in the dynamic modes with red blood cell (RBC) aggregation, deformability, shape (e.g., as in sickle cells), intracellular hemoglobin distribution, individual cell velocity, flux of RBCs, and likely shear rate. Proof of concept is demonstrated in ex vivo and in vivo tests, including high-speed PT imaging of RBC shape in pathological conditions and identification of sickle cells in a mouse model of human sickle cell disease. These studies revealed the potential of this new platform integrating PT, PA, and conventional optical techniques for translation to use in humans using safe, portable, laser-based medical devices for point-of-care screening of disease progression and therapy efficiency. PMID:21948731

  1. Photoacoustic Flow Cytometry for Single Sickle Cell Detection In Vitro and In Vivo

    PubMed Central

    Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Proskurnin, Mikhail A.

    2016-01-01

    Control of sickle cell disease (SCD) stage and treatment efficiency are still time-consuming which makes well-timed prevention of SCD crisis difficult. We show here that in vivo photoacoustic (PA) flow cytometry (PAFC) has a potential for real-time monitoring of circulating sickled cells in mouse model. In vivo data were verified by in vitro PAFC and photothermal (PT) and PA spectral imaging of sickle red blood cells (sRBCs) expressing SCD-associated hemoglobin (HbS) compared to normal red blood cells (nRBCs). We discovered that PT and PA signal amplitudes from sRBCs in linear mode were 2–4-fold lower than those from nRBCs. PT and PA imaging revealed more profound spatial hemoglobin heterogeneity in sRBCs than in nRBCs, which can be associated with the presence of HbS clusters with high local absorption. This hypothesis was confirmed in nonlinear mode through nanobubble formation around overheated HbS clusters accompanied by spatially selective signal amplification. More profound differences in absorption of sRBCs than in nRBCs led to notable increase in PA signal fluctuation (fluctuation PAFC mode) as an indicator of SCD. The obtained data suggest that noninvasive label-free fluctuation PAFC has a potential for real-time enumeration of sRBCs both in vitro and in vivo. PMID:27699143

  2. Digital analysis and sorting of fluorescence lifetime by flow cytometry.

    PubMed

    Houston, Jessica P; Naivar, Mark A; Freyer, James P

    2010-09-01

    Frequency-domain flow cytometry techniques are combined with modifications to the digital signal-processing capabilities of the open reconfigurable cytometric acquisition system (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency (RF)-modulated detector signals, implementing Fourier analysis programming with ORCAS' digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5-25 ns simulated lifetime), pulse widths ranging from 2 to 15 micros, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142 degrees to 1.6 degrees. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 micros and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells, and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a RF-modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to approximately 98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to

  3. Intraocular tumors. A cytopathologic study.

    PubMed

    Scroggs, M W; Johnston, W W; Klintworth, G K

    1990-01-01

    The cytologic characteristics and histopathologic correlates of common ocular tumors were examined using (1) cytologic and histologic specimens prepared from enucleated eyes with retinoblastoma and melanoma, (2) cytologic specimens prepared from clinically obtained intraocular fluids from eyes with lymphoma, metastatic adenocarcinoma and retinoblastoma and (3) cytologic specimens prepared from orbital aspirates and cerebrospinal fluids from a patient in whom retinoblastoma had spread to the orbit and central nervous system. Retinoblastoma cells occurred singly and in clusters and were associated with abundant necrotic debris and portions of capillaries with perivascular tumor infiltrates. Melanoma cells frequently had prominent nucleoli and variable amounts of fine cytoplasmic pigmentation and were found individually and in groups. Lymphoma cells were noncohesive, with scant cytoplasm. Metastatic intraocular adenocarcinoma cells had well-defined borders, multiple nucleoli and vacuolated cytoplasm. In general, the cellular morphology in the cytologic and tissue preparations of the intraocular tumors correlated well with each other. The findings suggest that common primary and metastatic intraocular tumors can be differentiated in cytologic preparations.

  4. Regression analysis of cytopathological data

    SciTech Connect

    Whittemore, A.S.; McLarty, J.W.; Fortson, N.; Anderson, K.

    1982-12-01

    Epithelial cells from the human body are frequently labelled according to one of several ordered levels of abnormality, ranging from normal to malignant. The label of the most abnormal cell in a specimen determines the score for the specimen. This paper presents a model for the regression of specimen scores against continuous and discrete variables, as in host exposure to carcinogens. Application to data and tests for adequacy of model fit are illustrated using sputum specimens obtained from a cohort of former asbestos workers.

  5. Hardware-software complex for diagnostics of breast cancer on the basis of flow cytometry

    NASA Astrophysics Data System (ADS)

    Selchuk, V. Y.; Shamilov, F. A.; Beznos, O. A.; Vorotnikov, I. K.; Polyakov, E. V.; Tupitsyn, N. N.

    2017-01-01

    The method of multicolor flow cytometry makes it possible to quantify the disseminated tumor cells (DTC) in patients with solid tumors. Application of the method multi-color flow cytometry showed its efficiency and accuracy in the detection of DTC in patients with breast cancer.

  6. Modeling flow cytometry data for cancer vaccine immune monitoring.

    PubMed

    Frelinger, Jacob; Ottinger, Janet; Gouttefangeas, Cécile; Chan, Cliburn

    2010-09-01

    Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories.

  7. Reevaluating multicolor flow cytometry to assess microbial viability.

    PubMed

    Buysschaert, Benjamin; Byloos, Bo; Leys, Natalie; Van Houdt, Rob; Boon, Nico

    2016-11-01

    Flow cytometry is a rapid and quantitative method to determine bacterial viability. Although different stains can be used to establish viability, staining protocols are inconsistent and lack a general optimization approach. Very few "true" multicolor protocols, where dyes are combined in one sample, have been developed for microbiological applications. In this mini-review, the discrepancy between protocols for cell-permeant nucleic acid and functional stains are discussed as well as their use as viability dyes. Furthermore, optimization of staining protocols for a specific setup are described. Original data using the red-excitable SYTO dyes SYTO 59 to 64 and SYTO 17, combined with functional stains, for double and triple staining applications is also included. As each dye and dye combination behaves differently within a certain combination of medium matrix, microorganism, and instrument, protocols need to be tuned to obtain reproducible results. Therefore, single, double, and triple stains are reviewed, including the different parameters that influence staining such as stain kinetics, optimal stain concentration, and the effect of the chelator EDTA as membrane permeabilizer. In the last section, we highlight the need to investigate the stability of multicolor assays to ensure correct results as multiwell autoloaders are now commonly used.

  8. Electric impedance microflow cytometry for characterization of cell disease states.

    PubMed

    Du, E; Ha, Sungjae; Diez-Silva, Monica; Dao, Ming; Suresh, Subra; Chandrakasan, Anantha P

    2013-10-07

    The electrical properties of biological cells have connections to their pathological states. Here we present an electric impedance microflow cytometry (EIMC) platform for the characterization of disease states of single cells. This platform entails a microfluidic device for a label-free and non-invasive cell-counting assay through electric impedance sensing. We identified a dimensionless offset parameter δ obtained as a linear combination of a normalized phase shift and a normalized magnitude shift in electric impedance to differentiate cells on the basis of their pathological states. This paper discusses a representative case study on red blood cells (RBCs) invaded by the malaria parasite Plasmodium falciparum. Invasion by P. falciparum induces physical and biochemical changes on the host cells throughout a 48-h multi-stage life cycle within the RBC. As a consequence, it also induces progressive changes in electrical properties of the host cells. We demonstrate that the EIMC system in combination with data analysis involving the new offset parameter allows differentiation of P. falciparum infected RBCs from uninfected RBCs as well as among different P. falciparum intraerythrocytic asexual stages including the ring stage. The representative results provided here also point to the potential of the proposed experimental and analysis platform as a valuable tool for non-invasive diagnostics of a wide variety of disease states and for cell separation.

  9. Quantitative assessment of Mycoplasma hemadsorption activity by flow cytometry.

    PubMed

    García-Morales, Luis; González-González, Luis; Costa, Manuela; Querol, Enrique; Piñol, Jaume

    2014-01-01

    A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.

  10. Applications of flow cytometry in environmental microbiology and biotechnology.

    PubMed

    Bergquist, Peter L; Hardiman, Elizabeth M; Ferrari, Belinda C; Winsley, Tristrom

    2009-05-01

    Flow cytometry (FCM) is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It uses the principles of light scattering, light excitation and the emission from fluorescent molecules to generate specific multiparameter data from particles and cells. The cells are hydrodynamically focussed in a sheath solution before being intercepted by a focused light source provided by a laser. FCM has been used primarily in medical applications but is being used increasingly for the examination of individual cells from environmental samples. It has found uses in the isolation of both culturable and hitherto non-culturable bacteria present infrequently in environmental samples using appropriate growth conditions. FCM lends itself to high-throughput applications in directed evolution for the analysis of single cells or cell populations carrying mutant genes. It is also suitable for encapsulation studies where individual bacteria are compartmentalised with substrate in water-in-oil-in-water emulsions or with individual genes in transcriptional/translational mixtures for the production of mutant enzymes. The sensitivity of the technique has allowed the examination of gene optimisation by a procedure known as random or neutral drift where screening and selection is based on the retention of some predetermined level of activity through multiple rounds of mutagenesis.

  11. Monitoring circulating apoptotic cells by in-vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Wei, Xunbin; Tan, Yuan; Chen, Yun; Zhang, Li; Li, Yan; Liu, Guangda; Wu, Bin; Wang, Chen

    2008-02-01

    Chemotherapies currently constitute one main venue of cancer treatment. For a large number of adult and elderly patients, however, treatment options are poor. These patients may suffer from disease that is resistant to conventional chemotherapy or may not be candidates for curative therapies because of advanced age or poor medical conditions. To control disease in these patients, new therapies must be developed that are selectively targeted to unique characteristics of tumor cell growth and metastasis. A reliable early evaluation and prediction of response to the chemotherapy is critical to its success. Chemotherapies induce apoptosis in tumor cells and a portion of such apoptotic cancer cells may be present in the circulation. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method that enables real-time detection and quantification of circulating cells without blood extraction. Apoptotic cells are rapidly cleared from the circulation with a half-life of ~10 minutes. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention.

  12. Impedance Flow Cytometry: A Novel Technique in Pollen Analysis

    PubMed Central

    Lambalk, Joep; Ottiger, Marcel

    2016-01-01

    Introduction An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen. Method Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays. Results Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population. Conclusion The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner. PMID:27832091

  13. Electric Impedance Microflow Cytometry for Characterization of Cell Disease States†

    PubMed Central

    Diez-Silva, Monica; Dao, Ming; Suresh, Subra; Chandrakasan, Anantha P.

    2013-01-01

    The electrical properties of biological cells have connections to their pathological states. Here we present an electric impedance microflow cytometry (EIMC) platform for the characterization of disease states of single cells. This platform entails a microfluidic device for a label-free and non-invasive cell-counting assay through electric impedance sensing. We identified a dimensionless offset parameter δ obtained as a linear combination of a normalized phase shift and a normalized magnitude shift in electric impedance to differentiate cells on the basis of their pathological states. This paper discusses a representative case study on red blood cells (RBCs) invaded by Plasmodium falciparum malaria parasites. Invasion of P. falciparum induces physical and biochemical changes on the host cells throughout a 48-h multi-stage life cycle within the RBC. As a consequence, it also induces progressive changes in electrical properties of the host cells .We demonstrate that the EIMC system in combination with data analysis involving the new offset parameter allows differentiation of Pf–invaded RBCs from uninfected RBCs as well as among different P. falciparum intraerythrocytic asexual stages including the ring stage. The representative results provided here also point to the potential of the proposed experimental and analysis platform as a valuable tool for non-invasive diagnostics of a wide variety of disease states and for cell separation. PMID:23925122

  14. Resonant-cavity apparatus for cytometry or particle analysis

    DOEpatents

    Gourley, Paul L.

    1998-01-01

    A resonant-cavity apparatus for cytometry or particle analysis. The apparatus comprises a resonant optical cavity having an analysis region within the cavity for containing one or more biological cells or dielectric particles to be analyzed. In the presence of a cell or particle, a light beam in the form of spontaneous emission or lasing is generated within the resonant optical cavity and is encoded with information about the cell or particle. An analysis means including a spectrometer and/or a pulse-height analyzer is provided within the apparatus for recovery of the information from the light beam to determine a size, shape, identification or other characteristics about the cells or particles being analyzed. The recovered information can be grouped in a multi-dimensional coordinate space for identification of particular types of cells or particles. In some embodiments of the apparatus, the resonant optical cavity can be formed, at least in part, from a vertical-cavity surface-emitting laser. The apparatus and method are particularly suited to the analysis of biological cells, including blood cells, and can further include processing means for manipulating, sorting, or eradicating cells after analysis thereof.

  15. Quantification of telomere length by FISH and laser scanning cytometry

    NASA Astrophysics Data System (ADS)

    Mahoney, John E.; Sahin, Ergun; Jaskelioff, Mariela; Chin, Lynda; DePinho, Ronald A.; Protopopov, Alexei I.

    2008-02-01

    Telomeres play a critical role in the maintenance of chromosomal stability. Telomere erosion, coupled with loss of DNA damage checkpoint function, results in genomic instability that promotes the development of cancer. The critical role of telomere dynamics in cancer has motivated the development of technologies designed to monitor telomere reserves in a highly quantitative and high-throughput manner in humans and model organisms. To this end, we have adapted and modified two established technologies, telomere-FISH and laser scanning cytometry. Specifically, we have produced a number of enhancements to the iCys LSC (CompuCyte) package including software updates, use of 60X dry objectives, and increased spatial resolution by 0.2 um size of stage steps. In addition, the 633 nm HeNe laser was replaced with a 532 nm green diode laser to better match the viewing options. Utilization of telomere-deficient mouse cells with short dysfunctional telomeres and matched telomerase reconstituted cultures demonstrated significantly higher mean integral specific fluorescence values for mTR transfectants relative to empty vector controls: 4.485M vs. 1.362M (p<0.0001). Histograms of average telomere intensities for individual cells were obtained and demonstrated intercellular heterogeneity in telomere lengths. The validation of the approach derives from a strong correlation between iCys LSC values and Southern blotting. This validated method greatly increases our experimental throughput and objectivity.

  16. Microchip-based flow cytometry for effective detection and count

    NASA Astrophysics Data System (ADS)

    Mu, Canjun; Zhang, Zhiyi; Lin, Min; Cao, Xudong; Zhang, Feiling

    2009-06-01

    High-throughput detection and identification of foodborne pathogens are in increasing demand for rapid bacteria detections in food safety and quality monitoring. As an effective method, microchip-based flow cytometry (microcytometery) has a potential to be less expensive and high throughout, and requires less bulky instrumentation than conventional methods. In this work, a low-cost and robust microcytometer with a simple optical setup was developed for demonstrating the high-throughput identification of foodborne bacterial pathogens that integrate sample flow focusing and detection into one testing procedure. High performance identification capability was achieved through simultaneously detecting the fluorescence and scatter light emitted from micro-fabricated channel, after designing and optimizing the laser shaping optical system and the micro-channel structure to improve the excitation light intensity as well as the detection sensitivity. In our configuration, the simple testing configuration with the collection angle of 42° in the orthogonal plane to micro chip presents the best SNR for both signals through simulation and systematic measurements. As a result, the maximum throughput of 83particles/s for the fluorescence-labelled bead with diameter of 1.013μm was obtained as well as the high detection efficiency (above 99%) and the correlation percentage (above 99.5%). Apart from the high detection sensitivity and identification power, this microcytometer also has the advantages of simple optical structure, compactness and ease in building.

  17. Investigation of platelet function and platelet disorders using flow cytometry.

    PubMed

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

    2016-01-01

    Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard-Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard-Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.

  18. Resonant-cavity apparatus for cytometry or particle analysis

    DOEpatents

    Gourley, P.L.

    1998-08-11

    A resonant-cavity apparatus for cytometry or particle analysis is described. The apparatus comprises a resonant optical cavity having an analysis region within the cavity for containing one or more biological cells or dielectric particles to be analyzed. In the presence of a cell or particle, a light beam in the form of spontaneous emission or lasing is generated within the resonant optical cavity and is encoded with information about the cell or particle. An analysis means including a spectrometer and/or a pulse-height analyzer is provided within the apparatus for recovery of the information from the light beam to determine a size, shape, identification or other characteristics about the cells or particles being analyzed. The recovered information can be grouped in a multi-dimensional coordinate space for identification of particular types of cells or particles. In some embodiments of the apparatus, the resonant optical cavity can be formed, at least in part, from a vertical-cavity surface-emitting laser. The apparatus and method are particularly suited to the analysis of biological cells, including blood cells, and can further include processing means for manipulating, sorting, or eradicating cells after analysis. 35 figs.

  19. Diagnostic flow cytometry for low-grade myelodysplastic syndromes.

    PubMed

    Ogata, Kiyoyuki

    2008-12-01

    It has long been considered that flow cytometry (FCM) has little role in clinical practice in the diagnosis of myelodysplastic syndromes (MDS). However, recent advances in the analytical method and knowledge of MDS FCM are changing this stereotype. This paper reviews the concept and current status of FCM in the diagnosis of low-grade MDS. The diagnosis of low-grade MDS in the absence of ringed sideroblasts and chromosomal aberration is not always straightforward, and a report from a recent international working conference has proposed FCM as an adjunctive diagnostic test for such cases. Currently, only a limited number of laboratories are applying FCM to the diagnosis of MDS. Furthermore, standard analytical methods in FCM for MDS have not been established, and no single FCM parameter is sufficiently sensitive and specific to make the diagnosis of MDS. To establish MDS FCM as a widely accepted, dependable diagnostic tool, prospective studies should increase flow parameters that can be analysed reproducibly and determine their sensitivity and specificity, either alone or in combination. CD34+ cell-related parameters that are applicable for diagnosing low-grade MDS in many laboratories are introduced here.

  20. Detecting endotoxin with a flow cytometry-based magnetic aptasensor.

    PubMed

    Zuo, Ming-Yan; Chen, Li-Juan; Jiang, Hao; Tan, Lin; Luo, Zhao-Feng; Wang, Yan-Mei

    2014-12-01

    Endotoxin, which is also known as lipopolysaccharide (LPS), is a marker for intruding gram-negative pathogens. It is essential to detect endotoxin quickly and sensitively in a complex milieu. A new flow cytometry (FCM)-based magnetic aptasensor assay that employs two endotoxin-binding aptamers and magnetic beads has been developed to detect endotoxin. The endotoxin-conjugated sandwich complex on magnetic beads was observed by scanning confocal laser microscopy. The resulting magnetic aptasensor rapidly detected (<1 min) endotoxin within a broad dynamic detection range of 10(-8) to 10(0)mg/ml in the presence of bovine serum albumin (BSA), RNA, sucrose, and glucose, which are most likely to coexist with endotoxin in the majority of biological liquids. Only 2 μl of magnetic aptasensor was required to quantify the endotoxin solution. Furthermore, the magnetic aptasensor could be regenerated seven times and still presented an outstanding response to the endotoxin solution. Therefore, the magnetic aptasensor exhibited high sensitivity, selectivity, and reproducibility, thereby serving as a powerful tool for the quality control and high-throughput detection of endotoxin in the food and pharmaceutical industries.

  1. In Vivo Flow Cytometry of Circulating Tumor-Associated Exosomes

    PubMed Central

    Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Jamshidi-Parsian, Azemat; Kore, Rajshekhar A.

    2016-01-01

    Circulating tumor cells (CTCs) demonstrated the potential as prognostic markers of metastatic development. However, the incurable metastasis can already be developed at the time of initial diagnosis with the existing CTC assays. Alternatively, tumor-associated particles (CTPs) including exosomes can be a more valuable prognostic marker because they can be released from the primary tumor long before CTCs and in larger amount. However, little progress has been made in high sensitivity detection of CTPs, especially in vivo. We show here that in vivo integrated photoacoustic (PA) and fluorescence flow cytometry (PAFFC) platform can provide the detection of melanoma and breast-cancer-associated single CTPs with endogenously expressed melanin and genetically engineered proteins or exogenous dyes as PA and fluorescent contrast agents. The two-beam, time-of-light PAFFC can measure the sizes of CTCs and CTPs and identify bulk and rolling CTCs and CTC clusters, with no influence on blood flow instability. This technique revealed a higher concentration of CTPs than CTCs at an early cancer stage. Because a single tumor cell can release many CTPs and in vivo PAFFC can examine the whole blood volume, PAFFC diagnostic platform has the potential to dramatically improve (up to 105-fold) the sensitivity of cancer diagnosis. PMID:27965916

  2. Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes

    PubMed Central

    Della Porta, Matteo G.; Picone, Cristina

    2017-01-01

    The pathological hallmark of myelodysplastic syndromes (MDS) is marrow dysplasia, which represents the basis of the WHO classification of these disorders. This classification provides clinicians with a useful tool for defining the different subtypes of MDS and individual prognosis. The WHO proposal has raised some concern regarding minimal diagnostic criteria particularly in patients with normal karyotype without robust morphological markers of dysplasia (such as ring sideroblasts or excess of blasts). Therefore, there is clearly need to refine the accuracy to detect marrow dysplasia. Flow cytometry (FCM) immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia. The rationale for the application of FCM in the diagnostic work up of MDS is that immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells and that MDS have been found to have abnormal expression of several cellular antigens. To become applicable in clinical practice, FCM analysis should be based on parameters with sufficient specificity and sensitivity, data should be reproducible between different operators, and the results should be easily understood by clinicians. In this review, we discuss the most relevant progresses in detection of marrow dysplasia by FCM in MDS PMID:28293405

  3. Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry.

    PubMed

    Maskow, Thomas; Müller, Susann; Lösche, Andreas; Harms, Hauke; Kemp, Richard

    2006-02-20

    The substrate-carbon flow can be controlled in continuous bioreactor cultures by the medium composition, for example, by the C/N ratio. The carbon distribution is optimal when a maximum fraction flows into the desired product and the residual is just sufficient to compensate for the dilution of the microbial catalyst. Undershooting of the latter condition is reflected immediately by changes in the Gibbs energy dissipation and cellular states. Two calorimetric measurement principles were applied to optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065 during growth with constantly increasing supply rates of fructose or toxic phenol. Firstly, the changed slope of the heat production rate in a complete heat balanced bioreactor (CHB) indicated optimum carbon channeling into PHB. The extent of the alteration depended directly on the toxic properties of the substrate. Secondly, a flow through calorimeter was connected with the bioreactor as a "measurement loop." The optimum substrate carbon distribution was indicated by a sudden change in the heat production rate independent of substrate toxicity. The sudden change was explained mathematically and exploited for the long-term control of phenol conversion into PHB. LASER flow cytometry measurements distinguished between subpopulations with completely different PHB-content. Populations grown on fructose preserved a constant ratio of two subpopulations with double and quadruple sets of DNA. Cells grown on phenol comprised a third subpopulation with a single DNA set. Rising phenol concentrations caused this subpopulation to increase. It may thus be considered as an indicator of chemostress.

  4. Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

    PubMed Central

    Inglis, Heather; Norris, Philip; Danesh, Ali

    2015-01-01

    Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal–to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes. PMID:25867010

  5. Understanding microbial/DOM interactions using fluorescence and flow cytometry

    NASA Astrophysics Data System (ADS)

    Fox, Bethany; Rushworth, Cathy; Attridge, John; Anesio, Alexandre; Cox, Tim; Reynolds, Darren

    2015-04-01

    The transformation and movement of dissolved organic carbon (DOC) within freshwater aquatic systems is an important factor in the global cycling of carbon. DOC within aquatic systems is known to underpin the microbial food web and therefore plays an essential role in supporting and maintaining the aquatic ecosystem. Despite this the interactions between bacteria and dissolved organic matter (DOM) are not well understood, although the literature indicates that the microbial processing of bioavailable DOM is essential during the production of autochthonous, labile, DOM. DOM can be broadly characterised by its fluorescing properties and Coble et al. (2014) define terrestrially derived DOM as exhibiting "peak C" fluorescence, whilst labile microbially derived DOM is defined as showing "peak T" fluorescence. Our work explores the microbial/DOM interactions by analysing aquatic samples using fluorescence excitation and emission matrices (EEMs) in conjunction with microbial consumption of dissolved oxygen. Environmental and synthetic water samples were subjected to fluorescence characterisation using both fluorescence spectroscopy and in situ fluorescence sensors (Chelsea Technologies Group Ltd.). PARAFAC analysis and peak picking were performed on EEMs and compared with flow cytometry data, used to quantify bacterial numbers present within samples. Synthetic samples were created using glucose, glutamic acid, nutrient-rich water and a standard bacterial seed. Synthetic samples were provided with terrestrially derived DOM via the addition of an aliquot of environmental water. Using a closed system approach, samples were incubated over time (up to a maximum of 20 days) and analysed at pre-defined intervals. The main focus of our work is to improve our understanding of microbial/DOM interactions and how these interactions affect both the DOM characteristics and microbial food web in freshwater aquatic systems. The information gained, in relation to the origin, microbial

  6. Alkaline unwinding flow cytometry assay to measure nucleotide excision repair.

    PubMed

    Thyagarajan, Bharat; Anderson, Kristin E; Lessard, Christopher J; Veltri, Gregory; Jacobs, David R; Folsom, Aaron R; Gross, Myron D

    2007-03-01

    Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50,000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G(1), S and G(2)/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being approximately 43% for cells in the G(1) phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.

  7. Early reprogramming regulators identified by prospective isolation and mass cytometry

    PubMed Central

    Lujan, Ernesto; Zunder, Eli R.; Ng, Yi Han; Goronzy, Isabel N.; Nolan, Garry P.; Wernig, Marius

    2015-01-01

    In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1–11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. PMID:25830878

  8. Evaluation of a green laser pointer for flow cytometry.

    PubMed

    Habbersett, Robert C; Naivar, Mark A; Woods, Travis A; Goddard, Gregory R; Graves, Steven W

    2007-10-01

    Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise < or = 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and < or =1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times.

  9. Impedance microflow cytometry for viability studies of microorganisms

    NASA Astrophysics Data System (ADS)

    Di Berardino, Marco; Hebeisen, Monika; Hessler, Thomas; Ziswiler, Adrian; Largiadèr, Stephanie; Schade, Grit

    2011-02-01

    Impedance-based Coulter counters and its derivatives are widely used cell analysis tools in many laboratories and use normally DC or low frequency AC to perform these electrical analyses. The emergence of micro-fabrication technologies in the last decade, however, provides a new means of measuring electrical properties of cells. Microfluidic approaches combined with impedance spectroscopy measurements in the radio frequency (RF) range increase sensitivity and information content and thus push single cell analyses beyond simple cell counting and sizing applications towards multiparametric cell characterization. Promising results have been shown already in the fields of cell differentiation and blood analysis. Here we emphasize the potential of this technology by presenting new data obtained from viability studies on microorganisms. Impedance measurements of several yeast and bacteria strains performed at frequencies around 10 MHz enable an easy discrimination between dead and viable cells. Moreover, cytotoxic effects of antibiotics and other reagents, as well as cell starvation can also be monitored easily. Control analyses performed with conventional flow cytometers using various fluorescent dyes (propidium iodide, oxonol) indicate a good correlation and further highlight the capability of this device. The label-free approach makes on the one hand the use of usually expensive fluorochromes obsolete, on the other hand practically eliminates laborious sample preparation procedures. Until now, online cell monitoring was limited to the determination of viable biomass, which provides rather poor information of a cell culture. Impedance microflow cytometry, besides other aspects, proposes a simple solution to these limitations and might become an important tool for bioprocess monitoring applications in the biotech industry.

  10. Aequorea green fluorescent protein analysis by flow cytometry.

    PubMed

    Ropp, J D; Donahue, C J; Wolfgang-Kimball, D; Hooley, J J; Chin, J Y; Hoffman, R A; Cuthbertson, R A; Bauer, K D

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263: 802-805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663-664, 1995) have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T-GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm.

  11. Aequorea green fluorescent protein analysis by flow cytometry

    SciTech Connect

    Ropp, J.D.; Cuthbertson, R.A.; Donahue, C.J.; Wolfgang-Kimball, D.

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types. The longer wavelength peak (470 nm) of GFP`s bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T- GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm. 29 refs., 5 figs.

  12. Staging and monitoring of childhood rhabdomyosarcoma with flow cytometry

    PubMed Central

    SHEN, HONGQIANG; TANG, YONGMIN; DONG, AO; LI, HUAMEI; SHEN, DIYING; YANG, SHILONG; TANG, HONGFENG; GU, WEIZHONG; SHU, QIANG

    2014-01-01

    Patients with metastatic rhabdomyosarcoma (RMS) have a poor prognosis. The detection of contaminating RMS cells in the bone marrow (BM) is important in clinical staging and risk assessment. The cytological examination of the BM remains the gold standard for the diagnosis of RMS, but has a limited sensitivity. In the present study, 32 BM and two cerebrospinal fluid (CSF) samples from 11 patients with suspected metastasis were analyzed by flow cytometry (FCM) with ganglioside D2 (GD2) conjugated with fluorescein isothiocyanate, cluster of differentiation (CD)90-phycoerythrin, CD45-peridinin chlorophyll protein and CD56-allophycocyanin monoclonal antibody cocktail in parallel to morphological examination at diagnosis or during treatment. Five samples (14.7%) were positive for RMS onup morphological examination. By FCM, 16 samples (47.1%) were positive for RMS. A significant difference was identified between the two methods. The four-color FCM assay successfully detected RMS cells in BM samples to a level of 0.01% (1 per 104 cells). RMS cells demonstrated a phenotype with CD56+/CD90+/CD45−/GD2− expression, which is different from the CD56+/CD90+/CD45−/GD2+ expression phenotype in neuroblastoma cells. The follow-up of four patients by FCM demonstrated that two patients became minimal residual disease-negative following two and four cycles of chemotherapy, respectively, and survived. The other two cases remained FCM-positive despite receiving four courses of chemotherapy and consequently succumbed to progressive disease. In addition, FCM analysis of the CSF samples from one patient confirmed a diagnosis of CSF metastasis with RMS. In conclusion, FCM may have a role not only in staging and monitoring the effects of therapy, but also in providing diagnostic confirmation of CSF metastasis with RMS. PMID:24944652

  13. Generalized unmixing model for multispectral flow cytometry utilizing nonsquare compensation matrices.

    PubMed

    Novo, David; Grégori, Gérald; Rajwa, Bartek

    2013-05-01

    Multispectral and hyperspectral flow cytometry (FC) instruments allow measurement of fluorescence or Raman spectra from single cells in flow. As with conventional FC, spectral overlap results in the measured signal in any given detector being a mixture of signals from multiple labels present in the analyzed cells. In contrast to traditional polychromatic FC, these devices utilize a number of detectors (or channels in multispectral detector arrays) that is larger than the number of labels, and no particular detector is a priori dedicated to the measurement of any particular label. This data-acquisition modality requires a rigorous study and understanding of signal formation as well as unmixing procedures that are employed to estimate labels abundance. The simplest extension of the traditional compensation procedure to multispectral data sets is equivalent to an ordinary least-square (LS) solution for estimating abundance of labels in individual cells. This process is identical to the technique employed for unmixing spectral data in various imaging fields. The present study shows that multispectral FC data violate key assumptions of the LS process, and use of the LS method may lead to unmixing artifacts, such as population distortion (spreading) and the presence of negative values in biomarker abundances. Various alternative unmixing techniques were investigated, including relative-error minimization and variance-stabilization transformations. The most promising results were obtained by performing unmixing using Poisson regression with an identity-link function within a generalized linear model framework. This formulation accounts for the presence of Poisson noise in the model of signal formation and subsequently leads to superior unmixing results, particularly for dim fluorescent populations. The proposed Poisson unmixing technique is demonstrated using simulated 8-channel, 2-fluorochrome data and real 32-channel, 6-fluorochrome data. The quality of unmixing is

  14. Good Cell, Bad Cell: Flow Cytometry Reveals T-cell Subsets Important in HIV Disease

    PubMed Central

    Chattopadhyay, Pratip K.; Roederer, Mario

    2010-01-01

    Flow cytometry is a key technology in the study of HIV disease. In this article, we review various cellular markers that can be measured in the setting of pathogenesis or vaccination studies, including markers of activation, differentiation, senescence, immune suppression, and function. In addition, we discuss important considerations for making these measurements. Finally, we examine how flow cytometry studies have taught researchers about the disease process, and the potential for flow cytometry technology to guide treatment decisions and evaluate vaccine candidates in the future. PMID:20583275

  15. [Assessment of bactericidal and growth-inhibiting activity of blood serum using flow cytometry and photometry].

    PubMed

    Budikhina, A S; Mikhaĭlova, N A; Bitkova, E E; Khvatov, V B; Pinegin, B V

    2007-01-01

    Method of measurement of biological fluids bactericidal activity against Staphylococcus aureus using laser flow cytometry has been developed and proposed for clinical use. Overall bactericidal activity of sera of healthy donors has been assessed by this method. Strong positive correlation between bactericidal activity measured by flow cytometry and ability of the sera of healthy donors to inhibit bacterial growth assessed by photometric method was determined. High degree of positive correlation between results of cytometry and classical microbiological method of measurement of mentioned parameters has been shown.

  16. High-throughput quantitation of inorganic nanoparticle biodistribution at the single-cell level using mass cytometry

    PubMed Central

    Yang, Yu-Sang Sabrina; Atukorale, Prabhani U.; Moynihan, Kelly D.; Bekdemir, Ahmet; Rakhra, Kavya; Tang, Li; Stellacci, Francesco; Irvine, Darrell J.

    2017-01-01

    Inorganic nanoparticles (NPs) are studied as drug carriers, radiosensitizers and imaging agents, and characterizing nanoparticle biodistribution is essential for evaluating their efficacy and safety. Tracking NPs at the single-cell level with current technologies is complicated by the lack of reliable methods to stably label particles over extended durations in vivo. Here we demonstrate that mass cytometry by time-of-flight provides a label-free approach for inorganic nanoparticle quantitation in cells. Furthermore, mass cytometry can enumerate AuNPs with a lower detection limit of ∼10 AuNPs (3 nm core size) in a single cell with tandem multiparameter cellular phenotyping. Using the cellular distribution insights, we selected an amphiphilic surface ligand-coated AuNP that targeted myeloid dendritic cells in lymph nodes as a peptide antigen carrier, substantially increasing the efficacy of a model vaccine in a B16-OVA melanoma mouse model. This technology provides a powerful new level of insight into nanoparticle fate in vivo. PMID:28094297

  17. Preparing a Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) compliant manuscript using the International Society for Advancement of Cytometry (ISAC) FCS file repository (FlowRepository.org).

    PubMed

    Spidlen, Josef; Breuer, Karin; Brinkman, Ryan

    2012-07-01

    FlowRepository.org is a Web-based flow cytometry data repository provided by the International Society for Advancement of Cytometry (ISAC). It supports storage, annotation, analysis, and sharing of flow cytometry datasets. A fundamental tenet of scientific research is that published results should be open to independent validation and refutation. With FlowRepository, researchers can annotate their datasets in compliance with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, thus greatly facilitating third-party interpretation of their data. In this unit, we will mainly focus on the deposition, sharing, and annotation of flow cytometry data.

  18. Flow cytometry in the exploration of the physiopathology of occupational lung disease

    PubMed Central

    Curran, A. D.

    1999-01-01

    Flow cytometry is a simple analytical technique used for the investigation of cells taken from various sources. Cells are identified by both their physical characteristics and the presence or absence of specific molecules on the cell surface. These molecules may be either phenotypic, or induced by a specific stimulus. Flow cytometry has been used to identify the nature and extent of the immune response in several occupational respiratory conditions including occupational asthma, irritant induced respiratory problems, and asbestos related lung disease. Also, it may be of value in monitoring workplace exposure to some hazardous materials. Although of limited diagnostic value at present, the technique has provided an insight into the modulation of immune cells, and their function, in people exposed to hazardous materials in the workplace. In this review, the principals of flow cytometry will be explored and the use of flow cytometry to investigate occupational respiratory disease will be discussed.   PMID:10658559

  19. The end of gating? An introduction to automated analysis of high dimensional cytometry data.

    PubMed

    Mair, Florian; Hartmann, Felix J; Mrdjen, Dunja; Tosevski, Vinko; Krieg, Carsten; Becher, Burkhard

    2016-01-01

    Ever since its invention half a century ago, flow cytometry has been a major tool for single-cell analysis, fueling advances in our understanding of a variety of complex cellular systems, in particular the immune system. The last decade has witnessed significant technical improvements in available cytometry platforms, such that more than 20 parameters can be analyzed on a single-cell level by fluorescence-based flow cytometry. The advent of mass cytometry has pushed this limit up to, currently, 50 parameters. However, traditional analysis approaches for the resulting high-dimensional datasets, such as gating on bivariate dot plots, have proven to be inefficient. Although a variety of novel computational analysis approaches to interpret these datasets are already available, they have not yet made it into the mainstream and remain largely unknown to many immunologists. Therefore, this review aims at providing a practical overview of novel analysis techniques for high-dimensional cytometry data including SPADE, t-SNE, Wanderlust, Citrus, and PhenoGraph, and how these applications can be used advantageously not only for the most complex datasets, but also for standard 14-parameter cytometry datasets.

  20. Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

    PubMed Central

    Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

    2015-01-01

    Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving

  1. Multiplexed microbead immunoassays by flow cytometry for molecular profiling: Basic concepts and proteomics applications.

    PubMed

    Krishhan, V V; Khan, Imran H; Luciw, Paul A

    2009-01-01

    Flow cytometry was originally established as an automated method for measuring optical or fluorescence characteristics of cells or particles in suspension. With the enormous increase in development of reliable electronics, lasers, micro-fluidics, as well as many advances in immunology and other fields, flow cytometers have become user-friendlier, less-expensive instruments with an increasing importance for both basic research and clinical applications. Conventional uses of flow cytometry include immunophenotyping of blood cells and the analysis of the cell cycle. Importantly, methods for labeling microbeads with unique combinations of fluorescent spectral signatures have made multiplex analysis of soluble analytes (i.e. the ability to detect multiple targets in a single test sample) feasible by flow cytometry. The result is a rapid, high-throughput, sensitive, and reproducible detection technology for a wide range of biomedical applications requiring detection of proteins (in cells and biofluids) and nucleic acids. Thus, novel methods of flow cytometry are becoming important for diagnostic purposes (e.g. identifying multiple clinical biomarkers for a wide range of diseases) as well as for developing novel therapies (e.g. elucidating drug mechanisms and potential toxicities). In addition, flow cytometry for multiplex analysis, coupled with automated sample handling devices, has the potential to significantly enhance proteomics research, particularly analysis of post-translational modifications of proteins, on a large scale. Inherently, flow cytometry methods are strongly rooted in the laws of the physics of optics, fluidics, and electromagnetism. This review article describes principles and early sources of flow cytometry, provides an introduction to the multiplex microbead technology, and discusses its applications and advantages in comparison to other methods. Anticipated future directions, particularly for translational research in medicine, are also discussed.

  2. High throughput image cytometry for detection of suspicious lesions in the oral cavity

    NASA Astrophysics Data System (ADS)

    MacAulay, Calum; Poh, Catherine F.; Guillaud, Martial; Michele Williams, Pamela; Laronde, Denise M.; Zhang, Lewei; Rosin, Miriam P.

    2012-08-01

    The successful management of oral cancer depends upon early detection, which relies heavily on the clinician's ability to discriminate sometimes subtle alterations of the infrequent premalignant lesions from the more common reactive and inflammatory conditions in the oral mucosa. Even among experienced oral specialists this can be challenging, particularly when using new wide field-of-view direct fluorescence visualization devices clinically introduced for the recognition of at-risk tissue. The objective of this study is to examine if quantitative cytometric analysis of oral brushing samples could facilitate the assessment of the risk of visually ambiguous lesions. About 369 cytological samples were collected and analyzed: (1) 148 samples from pathology-proven sites of SCC, carcinoma in situ or severe dysplasia; (2) 77 samples from sites with inflammation, infection, or trauma, and (3) 144 samples from normal sites. These were randomly separated into training and test sets. The best algorithm correctly recognized 92.5% of the normal samples, 89.4% of the abnormal samples, 86.2% of the confounders in the training set as well as 100% of the normal samples, and 94.4% of the abnormal samples in the test set. These data suggest that quantitative cytology could reduce by more than 85% the number of visually suspect lesions requiring further assessment by biopsy.

  3. Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry.

    PubMed

    Fürnrohr, Barbara G; Stein, Merle; Rhodes, Benjamin; Chana, Prabhjoat S; Schett, Georg; Vyse, Timothy J; Herrmann, Martin; Mielenz, Dirk

    2015-07-01

    Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FL-integral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry.

  4. Detection of Intracellular ADAMTS13, a Secreted Zinc-metalloprotease, via Flow Cytometry

    PubMed Central

    S., Geetha; Allen, Courtni E.; Hunt, Ryan; Plum, Elizabeth; Garfield, Susan; Friedman, Scott L.; Soejima, Kenji; Sauna, Zuben E.; Kimchi-Sarfaty, Chava

    2009-01-01

    Background ADAMTS13 is a secreted metalloprotease that cleaves von Willebrand Factor multimers and maintains proper homeostasis. A severe deficiency in ADAMTS13 triggers a disorder known as thrombotic thrombocytopenic purpura (TTP). At present, ADAMTS13 expression levels are determined by immunoblotting. Methods We established a flow cytometry methodology to detect intracellular ADAMTS13 in liver and kidney cells using a polyclonal antibody, BL154G, and several monoclonal antibodies previously used to detect ADAMTS13 by immunoblotting. Results were validated using confocal microscopy, immunoblotting and an activity assay (FRETS-VWF73). Results We show that labeling ADAMTS13 with specific antibodies and detection by flow cytometry yields results that are comparable to previously established methods for ADAMTS13 detection. Specifically, we compared the endogenous expression levels of ADAMTS13 in various liver cell lines using flow cytometry and obtained results that parallel immunoblot analysis. Knock-down of ADAMTS13 expression via targeted siRNA resulted in significantly reduced median signal, displaying the sensitivity of this detection method. A further analysis of reliability and specificity was achieved through plasmid DNA and transfection reagent dose response studies. Conclusions The flow cytometry method described here is useful in determining the expression of both endogenous and recombinant forms of intracellular ADAMTS13. Flow cytometry is a convenient, efficient and cost effective way to measure the expression levels of ADAMTS13. PMID:19526483

  5. Use of flow cytometry for rapid and accurate enumeration of live pathogenic Leptospira strains.

    PubMed

    Fontana, Célia; Crussard, Steve; Simon-Dufay, Nathalie; Pialot, Daniel; Bomchil, Natalia; Reyes, Jean

    2017-01-01

    Enumeration of Leptospira, the causative agent of leptospirosis, is arduous mainly because of its slow growth rate. Rapid and reliable tools for numbering leptospires are still lacking. The current standard for Leptospira cultures is the count on Petroff-Hausser chamber under dark-field microscopy, but this method remains time-consuming, requires well-trained operators and lacks reproducibility. Here we present the development of a flow-cytometry technique for counting leptospires. We showed that upon addition of fluorescent dyes, necessary to discriminate the bacterial population from debris, several live Leptospira strains could be enumerated at different physiologic states. Flow cytometry titers were highly correlated to counts with Petroff-Hausser chambers (R(2)>0.99). Advantages of flow cytometry lie in its rapidity, its reproducibility significantly higher than Petroff-Hausser method and its wide linearity range, from 10(4) to 10(8)leptospires/ml. Therefore, flow cytometry is a fast, reproducible and sensitive tool representing a promising technology to replace current enumeration techniques of Leptospira in culture. We were also able to enumerate Leptospira in artificially infected urine and blood with a sensitivity limit of 10(5)leptospires/ml and 10(6)leptospires/ml, respectively, demonstrating the feasibility to use flow cytometry as first-line tool for diagnosis or bacterial dissemination studies.

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

    PubMed Central

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

    2017-01-01

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

  7. A Different Perspective on Evaluating the Malignancy Rate of the Non-Diagnostic Category of the Bethesda System for Reporting Thyroid Cytopathology: A Single Institute Experience and Review of the Literature

    PubMed Central

    Onenerk, Mine; Erkan, Murat; Gursan, Nilufer; Kilinc, Emine; Kilicoglu, Gamze Zeynep

    2016-01-01

    Objective To determine the malignancy rate in the non-diagnostic (ND) category of the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) based on a different approach in relation to histopathology diagnoses. Study Design All ND fine needle aspirations (FNAs) that were performed under ultrasound guidance by an interventional radiologist with rapid on-site evaluation were included in the study. Slides were reevaluated to identify the cause of inadequacy as “qualitative” or “quantitative.” The malignancy rate of the ND category was assessed. Nodule/patient characteristics were compared between benign and malignant cases within the study cohort. Results The study cohort consisted of 192 ND aspirations. Overall there were 156 (81.3%) women and 36 (18.7%) men with a mean age of 50.6 years (range 24–82 years). The malignancy rate was 4.7%. None of the nodules (size, consistency, and number) or patient characteristics (gender and age) were found to be predictive of malignancy. Conclusion The malignancy rate of the ND category was high when compared to BSRTC predictions, but at the low end of the reported malignancy rates in the literature. Our results revealed that cyto-histopathologic correlation and method of malignancy rate estimation could have an effect on a wide range of reported malignancy rates. Furthermore, patient/nodule dependent factors were not statistically found to be predictive of malignancy. PMID:27627674

  8. Indigenous development of an imaging flow cytometer for clinical and biological applications

    NASA Astrophysics Data System (ADS)

    J, Veerendra Kalyan; Srinivasan, Rajesh; Gorthi, Sai Siva

    2014-10-01

    Flow cytometry is a benchmark technique used for basic research and clinical diagnosis of various diseases. Despite being a high-throughput technique, it fails in capturing the morphology of cells being analyzed. Imaging flow cytometry is a combination of flow-cytometry and digital microscopy, which offers advantages of both the techniques. In this paper, we report on the development of an indigenous Imaging Flow Cytometer, realized with the combination of Optics, Microfluidics, and High-speed imaging. A custom-made bright-field transmission microscope is used to capture images of cells flowing across the microfluidic device. High-throughput morphological analysis on suspension of yeast cells is presented.

  9. First proposed panels on acute leukemia for four-color immunophenotyping by flow cytometry from the Brazilian group of flow cytometry-GBCFLUX.

    PubMed

    Ikoma, Maura R V; Sandes, Alex F; Thiago, Leandro S; Cavalcanti Júnior, Geraldo B; Lorand-Metze, Irene G H; Costa, Elaine S; Pimenta, Glicinia; Santos-Silva, Maria C; Bacal, Nydia S; Yamamoto, Mihoko; Souto, Elizabeth X

    2015-01-01

    Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2).

  10. Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development

    PubMed Central

    Schwickart, Martin; Schneider, Amy K.; Vainshtein, Inna; Del Nagro, Christopher; Standifer, Nathan; Roskos, Lorin K.

    2015-01-01

    Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on the cell surface and are frequently used to generate pharmacodynamic (PD) biomarker data in nonclinical and clinical studies of biopharmaceuticals. When combined with the pharmacokinetic (PK) profile, RO data can establish PKPD relationships, which are crucial for informing dose decisions. RO is commonly measured by flow cytometry on fresh blood specimens and is subject to numerous technical and logistical challenges. To ensure that reliable and high quality results are generated from RO assays, careful assay design, key reagent characterization, data normalization/reporting, and thorough planning for implementation are of critical importance during development. In this article, the authors share their experiences and perspectives in these areas and discuss challenges and potential solutions when developing and implementing a flow cytometry‐based RO method in support of biopharmaceutical drug development. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc. PMID:26054054

  11. 2D light scattering label-free cytometry using light-sheet illumination

    NASA Astrophysics Data System (ADS)

    Lin, Meiai; Su, Xuantao

    2016-10-01

    Two-dimensional (2D) light scattering cytometry has been demonstrated as an effective label-free technology for cell analysis. Here we develop the light-sheet illumination in 2D light scattering static cytometry. In our cytometer, a cylindrical lens is used to form the light-sheet for better excitation of the static cells under an inverted microscope. The thickness of the light-sheet measured in fluorescent solution is about 13 μm. Two-dimensional light scattering patterns of standard microspheres and yeast cells are obtained by using a complementary metal oxide semiconductor (CMOS) detector via a low numerical aperture (NA 0.4) optical objective. The experimental patterns characterized with fringe structures agree well with Mie theory simulated ones. Our results suggest that the light-sheet illumination is an effective excitation method for 2D light scattering label-free cytometry.

  12. Methodology and Application of Flow Cytometry for Investigation of Human Malaria Parasites

    PubMed Central

    Grimberg, Brian T.

    2011-01-01

    Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries. PMID:21296083

  13. An integrated, multiparametric flow cytometry chip using "microfluidic drifting" based three-dimensional hydrodynamic focusing.

    PubMed

    Mao, Xiaole; Nawaz, Ahmad Ahsan; Lin, Sz-Chin Steven; Lapsley, Michael Ian; Zhao, Yanhui; McCoy, J Philip; El-Deiry, Wafik S; Huang, Tony Jun

    2012-06-01

    In this work, we demonstrate an integrated, single-layer, miniature flow cytometry device that is capable of multi-parametric particle analysis. The device integrates both particle focusing and detection components on-chip, including a "microfluidic drifting" based three-dimensional (3D) hydrodynamic focusing component and a series of optical fibers integrated into the microfluidic architecture to facilitate on-chip detection. With this design, multiple optical signals (i.e., forward scatter, side scatter, and fluorescence) from individual particles can be simultaneously detected. Experimental results indicate that the performance of our flow cytometry chip is comparable to its bulky, expensive desktop counterpart. The integration of on-chip 3D particle focusing with on-chip multi-parametric optical detection in a single-layer, mass-producible microfluidic device presents a major step towards low-cost flow cytometry chips for point-of-care clinical diagnostics.

  14. Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies

    PubMed Central

    Aubert, Geraldine

    2016-01-01

    Genetic defects of the immune system are referred to as primary immunodeficiencies (PIDs). These immunodeficiencies are clinically and immunologically heterogeneous and, therefore, pose a challenge not only for the clinician but also for the diagnostic immunologist. There are several methodological tools available for evaluation and monitoring of patients with PIDs, and of these tools, flow cytometry has gained prominence, both for phenotyping and functional assays. Flow cytometry allows real-time analysis of cellular composition, cell signaling, and other relevant immunological pathways, providing an accessible tool for rapid diagnostic and prognostic assessment. This minireview provides an overview of the use of flow cytometry in disease-specific diagnosis of PIDs, in addition to other broader applications, which include immune phenotyping and cellular functional measurements. PMID:26912782

  15. Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

    PubMed

    Käser, T; Pasternak, J A; Hamonic, G; Rieder, M; Lai, K; Delgado-Ortega, M; Gerdts, V; Meurens, F

    2016-05-01

    Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry.

  16. High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

    2016-02-01

    Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

  17. Circulation times of cancer cells by in vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Zhang, Li; Li, Yan; Gu, Zhengqin; Chen, Tong; Wang, Cheng; Wei, Xunbin

    2012-03-01

    Liver cancer is one of the most common malignancies in the world, with approximately 1,000,000 cases reported every year. Hepatocellular carcinoma may metastasize to lung, bones, kidney, and many other organs. Surgical resection, liver transplantation, chemotherapy and radiation therapy are the foundation of current HCC therapies. However the outcomes are poor: the survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. A recently developed "in vivo flow cytometer" combined with real-time confocal fluorescence imaging are used to assess spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labeled cells in vivo in real time without extracting blood sample. We have measured the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which were from the same origin and obtained by repetitive screenings in mice. >60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

  18. Flow cytometry data analysis: comparing large multivariate data sets using classification trees

    SciTech Connect

    Norman, J.

    1994-12-31

    This paper describes a method to compare flow cytometry data sets, which typically contain 50,000 six-parameter measurements each. By this method, the data points in two such data sets are divided into subpopulations using a binary classification tree generated from the data. The {chi}{sup 2} test is then used to establish the homogeneity of the two data sets based on how their data are distributed across these subpopulations. Preliminary results indicate that this comparison method is sufficiently sensitive to detect differences between flow cytometry data sets that are too subtle for human investigators to notice.

  19. Flow cytometry in mastocytosis: utility as a diagnostic and prognostic tool.

    PubMed

    Sánchez-Muñoz, Laura; Teodosio, Cristina; Morgado, Jose Mario T; Perbellini, Omar; Mayado, Andrea; Alvarez-Twose, Ivan; Matito, Almudena; Jara-Acevedo, María; García-Montero, Andrés C; Orfao, Alberto; Escribano, Luis

    2014-05-01

    This article presents information for the identification and characterization of mast cells from bone marrow and other tissues using multiparametric flow cytometry. In addition, it provides guidelines for the application of this technique in the subclassification of systemic mastocytosis and assessment of the long-term prognosis of patients individually.

  20. Application of flow cytometry to monitor assimilable organic carbon (AOC) and microbial community changes in water.

    PubMed

    Elhadidy, Ahmed M; Van Dyke, Michele I; Peldszus, Sigrid; Huck, Peter M

    2016-11-01

    Flow cytometry is an efficient monitoring tool for rapid cell counting, and can be applied to research on water quality and treatment. In this study, a method that employs flow cytometry and a natural microbial inoculum to determine assimilable organic carbon (AOC) was adapted for use with challenging surface waters that have a high organic and particle content, and subsequently applied in a long term river water study. AOC method optimization showed that river water bacteria could pass through a 0.2μm membrane filter, and therefore membrane filtration combined with heat treatment was required for sample sterilization. Preparation of the natural river inoculum with an acceptable yield value could only be achieved when grown using the natural water source, since growth was limited on different types of inorganic minimal media and in natural spring water. The resulting flow cytometry AOC method was reliable and reproducible, and results were comparable to the standard plate count AOC method. Size exclusion chromatography showed that both high and low molecular weight organic matter fractions were utilized by the natural AOC inoculum. Flow cytometry was used to measure both AOC levels and total cell counts in a long term study to monitor the water quality of a river which was used as a drinking water source. The method could distinguish between high nucleic acid (HNA) and low nucleic acid (LNA) groups of bacteria, and HNA bacteria were found to respond faster than LNA bacteria to seasonal changes in nutrients and water temperature.

  1. Computational prediction of manually gated rare cells in flow cytometry data1

    PubMed Central

    Qiu, Peng

    2015-01-01

    Rare cell identification is an interesting and challenging question in flow cytometry data analysis. In the literature, manual gating is a popular approach to distill flow cytometry data and drill down to the rare cells of interest, based on prior knowledge of measured protein markers and visual inspection of the data. Several computational algorithms have been proposed for rare cell identification. To compare existing algorithms and promote new developments, FlowCAP-III put forward one computational challenge that focused on this question. The challenge provided flow cytometry data for 202 training samples and two manually gated rare cell types for each training sample, roughly 0.02% and 0.04% of the cells, respectively. In addition, flow cytometry data for 203 testing samples were provided, and participants were invited to computationally identify the rare cells in the testing samples. Accuracy of the identification results was evaluated by comparing to manual gating of the testing samples. We participated in the challenge, and developed a method that combined the Hellinger divergence, a downsampling trick and the ensemble SVM. Our method achieved the highest accuracy in the challenge. PMID:25755118

  2. In vivo plant flow cytometry: A first proof-of-concept

    PubMed Central

    Nedosekin, Dmitry A.; Khodakovskaya, Mariya V.; Biris, Alexandru S.; Wang, Daoyuan; Xu, Yang; Villagarcia, Hector; Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2011-01-01

    In vivo flow cytometry has facilitated advances in the ultrasensitive detection of tumor cells, bacteria, nanoparticles, dyes, and other normal and abnormal objects directly in blood and lymph circulatory systems. Here, we propose in vivo plant flow cytometry for the real-time noninvasive study of nanomaterial transport in xylem and phloem plant vascular systems. As a proof of this concept, we demonstrate in vivo real-time photoacoustic monitoring of quantum dot-carbon nanotube conjugate uptake and uptake by roots and spreading through stem to leaves in a tomato plant. In addition, in vivo scanning cytometry using multimodal photoacoustic, photothermal, and fluorescent detection schematics provided multiplex detection and identification of nanoparticles accumulated in plant leaves in the presence of intensive absorption, scattering, and autofluorescent backgrounds. The use of a portable fiber-based photoacoustic flow cytometer for studies of plant vasculature was demonstrated. These integrated cytometry modalities using both endogenous and exogenous contrast agents have a potential to open new avenues of in vivo study of the nutrients, products of photosynthesis and metabolism, nanoparticles, infectious agents, and other objects transported through plant vasculature. PMID:21905208

  3. Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways**

    EPA Science Inventory

    Rationale: The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. ...

  4. 78 FR 5186 - Clinical Flow Cytometry in Hematologic Malignancies; Public Workshop; Request for Comments

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-24

    ... in the diagnosis of leukemia and lymphoma and more recently in the detection of minimal residual... leukemia (CLL); (3) Third-party flow cytometry data analysis software; and (4) Overview of FDA regulation... (77 FR 76051, December 26, 2012). An FDA workshop for acute lymphocytic leukemia (ALL) MRD was...

  5. Mass cytometry panel optimization through the designed distribution of signal interference.

    PubMed

    Takahashi, Chikara; Au-Yeung, Amelia; Fuh, Franklin; Ramirez-Montagut, Teresa; Bolen, Chris; Mathews, William; O'Gorman, William E

    2017-01-01

    Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making it a promising technology for innovating biomarker discovery. However, in order for this potential to be fully realized, best practices in panel design need to be further defined in order to achieve consistency and reproducibility in data analysis. Of particular importance are controls that reveal, and panel design principles that mitigate the effects of signal interference or overlap. We observed a disparity between the staining profiles of two noncompeting anti- integrin β7 mAbs and hypothesized that signal interference was responsible. A mass-minus-one (MMO) control was applied and demonstrated that signal overlap caused the perceived interclonal discrepancy in β7 expression. Panel redesign in consideration of mass-cytometry specific interference dynamics dramatically improved concordance between both mAbs by redistributing background signals caused by overlap. These studies visualize how signal overlap can complicate mass cytometry data interpretation and demonstrate how the rational distribution of interference can greatly improve panel design and data quality. © 2016 International Society for Advancement of Cytometry.

  6. Minimal residual disease analysis by eight-color flow cytometry in relapsed childhood acute lymphoblastic leukemia.

    PubMed

    Karawajew, Leonid; Dworzak, Michael; Ratei, Richard; Rhein, Peter; Gaipa, Giuseppe; Buldini, Barbara; Basso, Giuseppe; Hrusak, Ondrej; Ludwig, Wolf-Dieter; Henze, Günter; Seeger, Karl; von Stackelberg, Arend; Mejstrikova, Ester; Eckert, Cornelia

    2015-07-01

    Multiparametric flow cytometry is an alternative approach to the polymerase chain reaction method for evaluating minimal residual disease in treatment protocols for primary acute lymphoblastic leukemia. Given considerable differences between primary and relapsed acute lymphoblastic leukemia treatment regimens, flow cytometric assessment of minimal residual disease in relapsed leukemia requires an independent comprehensive investigation. In the present study we addressed evaluation of minimal residual disease by flow cytometry in the clinical trial for childhood relapsed acute lymphoblastic leukemia using eight-color flow cytometry. The major challenge of the study was to reliably identify low amounts of residual leukemic cells against the complex background of regeneration, characteristic of follow-up samples during relapse treatment. In a prospective study of 263 follow-up bone marrow samples from 122 patients with B-cell precursor acute lymphoblastic leukemia, we tested various B-cell markers, adapted the antibody panel to the treatment protocol, and evaluated its performance by a blinded parallel comparison with the polymerase chain reaction data. The resulting eight-color single-tube panel showed a consistently high overall concordance (P<0.001) and, under optimal conditions, sensitivity similar to that of the reference polymerase chain reaction method. Overall, evaluation of minimal residual disease by flow cytometry can be successfully integrated into the clinical management of relapsed childhood acute lymphoblastic leukemia either as complementary to the polymerase chain reaction or as an independent risk stratification tool. ALL-REZ BFM 2002 clinical trial information: NCT00114348.

  7. Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants

    PubMed Central

    Khalil, Jacques Y. B.; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

    2017-01-01

    Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus, we sorted and re-cultured a new, slow-growing virus, which we named “Cedratvirus.” The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry. PMID:28111619

  8. Teaching the Microbial Growth Curve Concept Using Microalgal Cultures and Flow Cytometry

    ERIC Educational Resources Information Center

    Forget, Nathalie; Belzile, Claude; Rioux, Pierre; Nozais, Christian

    2010-01-01

    The microbial growth curve is widely studied within microbiology classes and bacteria are usually the microbial model used. Here, we describe a novel laboratory protocol involving flow cytometry to assess the growth dynamics of the unicellular microalgae "Isochrysis galbana." The algal model represents an appropriate alternative to…

  9. In vivo plant flow cytometry: a first proof-of-concept.

    PubMed

    Nedosekin, Dmitry A; Khodakovskaya, Mariya V; Biris, Alexandru S; Wang, Daoyuan; Xu, Yang; Villagarcia, Hector; Galanzha, Ekaterina I; Zharov, Vladimir P

    2011-10-01

    In vivo flow cytometry has facilitated advances in the ultrasensitive detection of tumor cells, bacteria, nanoparticles, dyes, and other normal and abnormal objects directly in blood and lymph circulatory systems. Here, we propose in vivo plant flow cytometry for the real-time noninvasive study of nanomaterial transport in xylem and phloem plant vascular systems. As a proof of this concept, we demonstrate in vivo real-time photoacoustic monitoring of quantum dot-carbon nanotube conjugates uptake by roots and spreading through stem to leaves in a tomato plant. In addition, in vivo scanning cytometry using multimodal photoacoustic, photothermal, and fluorescent detection schematics provided multiplex detection and identification of nanoparticles accumulated in plant leaves in the presence of intensive absorption, scattering, and autofluorescent backgrounds. The use of a portable fiber-based photoacoustic flow cytometer for studies of plant vasculature was demonstrated. These integrated cytometry modalities using both endogenous and exogenous contrast agents have a potential to open new avenues of in vivo study of the nutrients, products of photosynthesis and metabolism, nanoparticles, infectious agents, and other objects transported through plant vasculature.

  10. The analysis of optical signals in response to various cells using a microflow cell cytometry

    NASA Astrophysics Data System (ADS)

    Byun, Insoo; Kim, Sunghyun; Kim, Yiseok; Yang, Jooran; Park, Sekwang

    2005-01-01

    The fabricated system is advantageous due to its low cost and simple structure. This is possible because it replaces many optical lenses and expensive equipment with optical fibers. We tested various optical fibers to select a suitable fiber for effective micro flow cell cytometry. In order to align between micro nozzle and optical fibers, a guide channel was fabricated by Si wafer etching with MEMS (Micro Electro-Mechanical System) technology. The micro flow cell cytometry using optical science and MEMS technology. The optical science using multi mode optical fiber and He-Ne laser (20mW, 658nm). The guide channel was fabricated by MEMS technology. The output voltage was as high as about 300 mV, so we are going to use a light source which has relatively small output power. By injecting various cells, we were able to detect cells. In addition, we can use the micro flow cell cytometry for analyzing cells (1, 2). We have no doubt that this micro flow cell cytometry can contribute to the development of biological engineering and clinical testing and it can be practically used in diagnoses of particular diseases and biological symptoms and invitro diagnostics (7, 8).

  11. Evaluating the spore genome sizes of ferns and lycophytes: a flow cytometry approach.

    PubMed

    Kuo, Li-Yaung; Huang, Yi-Jia; Chang, JenYu; Chiou, Wen-Liang; Huang, Yao-Moan

    2017-03-01

    Ferns and lycophytes produce spores to initiate the gametophyte stage for sexual reproduction. Approximately 10% of these seedless vascular plants are apomictic, and produce genomic unreduced spores. Genome size comparisons between spores and leaves are a reliable, and potentially easier way to determine their reproductive mode compared to traditional approaches. However, estimation of the spore genome sizes of these plants has not been attempted. We attempted to evaluate the spore genome sizes of ferns and lycophytes using flow cytometry, collected spores from selected species representing different spore physical properties and taxonomic groups, and sought to optimize bead-vortexing conditions. By evaluating the spore and sporophyte genome sizes, we examined whether reproductive modes could be ascertained from these flow cytometry results. We proposed two separate sets of optimized bead-vortexing conditions for the nuclear extraction of green and nongreen spores. We further successfully extracted spore nuclei of 19 families covering most orders, and the qualities and quantities of these extractions satisfied the C-value criteria. These evaluated genome sizes further supported the reproductive modes reported previously. In the current study, flow cytometry was used for the first time to evaluate the spore genome sizes of ferns and lycophytes. This use of spore flow cytometry provides a new, efficient approach to ascertaining the reproductive modes of these plants.

  12. Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants.

    PubMed

    Khalil, Jacques Y B; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

    2016-01-01

    Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus, we sorted and re-cultured a new, slow-growing virus, which we named "Cedratvirus." The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry.

  13. Flow Cytometry To Assess Cerebrospinal Fluid Fungal Burden in Cryptococcal Meningitis

    PubMed Central

    Graham, Lisa M.; Schutz, Charlotte; Scriba, Thomas J.; Wilkinson, Robert J.; Boulware, David R.; Meintjes, Graeme; Lalloo, David G.; Urban, Britta C.

    2015-01-01

    Fungal burden in the cerebrospinal fluid is an important determinant of mortality in cryptococcal meningitis, but its use in aiding clinical decision making is hampered by the time involved to perform quantitative cultures. Here, we demonstrate the potential of flow cytometry as a novel and rapid technique to address this issue. PMID:26719441

  14. Data File Standard for Flow Cytometry, Version FCS 3.1

    SciTech Connect

    Spidlen, Josef; Moore, Wayne; Parks, David; Goldberg, Michael; Bray, Chris; Gorombey, Peter; Hyun, Bill; Hubbard, Mark; Lange, Simon; Lefebvre, Ray; Leif, Robert; Novo, David; Ostruszka, Leo; Treister, Adam; Wood, James; Murphy, Robert F.; Roederer, Mario; Sudar, Damir; Zigon, Robert; Brinkman, Ryan R.; Brierre, Pierre

    2009-11-10

    The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type. The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.

  15. Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry.

    PubMed

    Peng, Yan; Lee-Pullen, Tracey F; Heel, Kathy; Millar, A Harvey; Baer, Boris

    2014-05-01

    Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n = 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods.

  16. Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data.

    PubMed

    Weber, Lukas M; Robinson, Mark D

    2016-12-01

    Recent technological developments in high-dimensional flow cytometry and mass cytometry (CyTOF) have made it possible to detect expression levels of dozens of protein markers in thousands of cells per second, allowing cell populations to be characterized in unprecedented detail. Traditional data analysis by "manual gating" can be inefficient and unreliable in these high-dimensional settings, which has led to the development of a large number of automated analysis methods. Methods designed for unsupervised analysis use specialized clustering algorithms to detect and define cell populations for further downstream analysis. Here, we have performed an up-to-date, extensible performance comparison of clustering methods for high-dimensional flow and mass cytometry data. We evaluated methods using several publicly available data sets from experiments in immunology, containing both major and rare cell populations, with cell population identities from expert manual gating as the reference standard. Several methods performed well, including FlowSOM, X-shift, PhenoGraph, Rclusterpp, and flowMeans. Among these, FlowSOM had extremely fast runtimes, making this method well-suited for interactive, exploratory analysis of large, high-dimensional data sets on a standard laptop or desktop computer. These results extend previously published comparisons by focusing on high-dimensional data and including new methods developed for CyTOF data. R scripts to reproduce all analyses are available from GitHub (https://github.com/lmweber/cytometry-clustering-comparison), and pre-processed data files are available from FlowRepository (FR-FCM-ZZPH), allowing our comparisons to be extended to include new clustering methods and reference data sets. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

  17. Avian reovirus-induced syncytium formation is independent of infectious progeny virus production and enhances the rate, but is not essential, for virus-induced cytopathology and virus egress.

    PubMed

    Duncan, R; Chen, Z; Walsh, S; Wu, S

    1996-10-15

    The nonenveloped avian reoviruses represent a distinct antigenic subgroup of orthoreoviruses. Unlike their mammalian counterparts, the avian reoviruses exhibit the unusual property of inducing rapid and extensive syncytium formation in cell cultures, a cytopathic effect more commonly associated with enveloped virus replication. While the syncytium-inducing capability of avian reovirus has been known for quite some time, the relationship between cell fusion and the virus replication cycle has not been determined. The conservation of the syncytial phenotype among all field isolates of avian reovirus suggests that avian reovirus-induced syncytium formation either reflects an essential step in the virus replication cycle involving intracellular membrane interactions or that cell fusion contributes to enhanced virus replication in infected animals. In order to distinguish between these possibilities, we have examined several aspects of virus replication in the presence of inhibitors of syncytium formation. Inhibitors of intracellular vesicle transport and O-linked glycosylation eliminated or markedly reduced syncytium formation with little effect on the rate or extent of virus macromolecular synthesis and infectious progeny virus production. Moreover, syncytium formation was not required for virus-induced cytopathology or virus egress but did significantly enhance the rate of both of these processes. The data indicate that, unlike the syncytium-inducing enveloped viruses, the membrane interactions and protein trafficking required for avian reovirus-induced syncytium formation do not reflect the sequelae of an essential step in the virus replication cycle. These results suggest that the conservation of the avian reovirus syncytial phenotype may reflect a fortuitous aspect of virus replication which confers advantages associated with the rapid spread of the virus within an infected host.

  18. National flow cytometry and sorting research resource. Annual progress report, July, 1, 1994--June 30, 1995, Year 12

    SciTech Connect

    Jett, J.H.

    1995-04-27

    Research progress utilizing flow cytometry is described. Topics include: rapid kinetics flow cytometry; characterization of size determinations for small DNA fragments; statistical analysis; energy transfer measurements of molecular confirmation in micelles; and enrichment of Mus spretus chromosomes by dual parameter flow sorting and identification of sorted fractions by fluorescence in-situ hybridization onto G-banded mouse metaphase spreads.

  19. Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment.

    PubMed

    Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

    2015-11-17

    Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible.

  20. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    PubMed Central

    Pandey, Gouri Shankar; Tseng, Sandra C.; Howard, Tom E.; Sauna, Zuben E.

    2013-01-01

    Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels. PMID:23555096

  1. Establishment and application of a flow cytometry-based method for detecting histone acetylation levels.

    PubMed

    Jianhui, Li; Chunfu, Wang; Fan, Bai; Yan, Zhuang; Zhuojun, Mao; Yongtao, Sun

    2016-06-20

    Histone deacetylase inhibitors, which have also received attention in AIDS and other diseases, are a new class of anticancer drugs developed in recent years. However, there is still a lack of a unified and reliable method for detecting histone acetylation levels in basic and clinical research. In this study, we developed a flow cytometry-based method to detect histone acetylation levels by comparing different sample processing temperature (on ice vs. room temperature), permeabilization method (intracellular vs. nuclear), antibody dose (antibody titration) and antibody incubation time (time gradient) using whole blood and peripheral blood mononuclear cells. In addition, we applied this optimized method in in vitro experiment and clinical trial of Chidamide (the only China FDA approved HDACi), the result of which confirmed that the flow cytometry-based method for detecting histone acetylation levels is a reliable, fast and convenient method which can be used in basic and clinical research.

  2. Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet.

    PubMed

    Collier, Bradley B; Awasthi, Samir; Lieu, Deborah K; Chan, James W

    2015-05-29

    Modern flow cytometry instruments have become vital tools for high-throughput analysis of single cells. However, as issues with the cellular labeling techniques often used in flow cytometry have become more of a concern, the development of label-free modalities for cellular analysis is increasingly desired. Non-linear optical phenomena (NLO) are of growing interest for label-free analysis because of the ability to measure the intrinsic optical response of biomolecules found in cells. We demonstrate that a light-sheet consisting of a scanned Bessel beam is an optimal excitation geometry for efficiently generating NLO signals in a microfluidic environment. The balance of photon density and cross-sectional area provided by the light-sheet allowed significantly larger two-photon fluorescence intensities to be measured in a model polystyrene microparticle system compared to measurements made using other excitation focal geometries, including a relaxed Gaussian excitation beam often used in conventional flow cytometers.

  3. Acoustic tweezing cytometry for live-cell subcellular modulation of intracellular cytoskeleton contractility

    PubMed Central

    Fan, Zhenzhen; Sun, Yubing; Di Chen; Tay, Donald; Chen, Weiqiang; Deng, Cheri X.; Fu, Jianping

    2013-01-01

    Mechanical forces are critical to modulate cell spreading, contractility, gene expression, and even stem cell differentiation. Yet, existing tools that can apply controllable subcellular forces to a large number of single cells simultaneously are still limited. Here we report a novel ultrasound tweezing cytometry utilizing ultrasound pulses to actuate functionalized lipid microbubbles covalently attached to single live cells to exert mechanical forces in the pN - nN range. Ultrasonic excitation of microbubbles could elicit a rapid and sustained reactive intracellular cytoskeleton contractile force increase in different adherent mechanosensitive cells. Further, ultrasound-mediated intracellular cytoskeleton contractility enhancement was dose-dependent and required an intact actin cytoskeleton as well as RhoA/ROCK signaling. Our results demonstrated the great potential of ultrasound tweezing cytometry technique using functionalized microbubbles as an actuatable, biocompatible, and multifunctional agent for biomechanical stimulations of cells. PMID:23846290

  4. MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions

    NASA Astrophysics Data System (ADS)

    Shipunova, V. O.; Nikitin, M. P.; Nikitin, P. I.; Deyev, S. M.

    2016-06-01

    Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method

  5. Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment

    PubMed Central

    Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

    2015-01-01

    Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible. PMID:26588562

  6. Monitoring of microbial communities by flow cytometry and rRNA-targeted hybridization probes

    SciTech Connect

    Wallner, G.; Amann, R. |

    1995-12-31

    Flow cytometry in combination with ribosomal RNA (rRNA) based fluorescence in situ hybridization is a new technique for the analysis of microbial communities. Oligonucleotide probes directed against ribosomal RNA allow the identification of species or groups of microorganisms. Combined with flow cytometry, up to several thousand cells per second can be classified. In addition to the identification and specific enumeration of microorganisms, further information on the distribution of cell size, DNA and ribosome content -- and therefore an assessment of activity -- within the entire community of subpopulations can be obtained. This technique is much more accurate, informative, and rapid than classical culture-dependent methods. Data of activated sludge samples hybridized with fluorescein labelled oligonucleotides and counterstained with the DNA-specific dye Hoechst 33342 are presented as examples for its applicability to complex microbial communities.

  7. Fixation and long-term storage of human lymphocytes for surface marker analysis by flow cytometry.

    PubMed

    Lal, R B; Edison, L J; Chused, T M

    1988-05-01

    A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4 degrees C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analyzing lymphocytes that express T-cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.

  8. Considerations for the control of background fluorescence in clinical flow cytometry.

    PubMed

    Hulspas, Ruud; O'Gorman, Maurice R G; Wood, Brent L; Gratama, Jan W; Sutherland, D Robert

    2009-11-01

    Accurate measurement of antigen-positive cells by flow cytometry can be hampered by background fluorescence of antigen-negative cells and other particles (e.g., debris). This article focuses on three major causes of background (autofluorescence, spectral overlap, and undesirable antibody binding) by reviewing individual aspects of flow cytometric measurements that contribute to these causes. The appropriate use of controls facilitates a thorough understanding of these contributing factors as well as the development of robust cell labeling protocols intended for routine flow cytometric analysis. We present a set of recommendations that enables the user to develop an optimized cell labeling protocol that minimizes background and maximizes the ability to reliably distinguish between a positive and a negative population of cells. These recommendations are also intended to augment existing guidelines designed to aid in the formulation of a consensus regarding the utility of flow cytometry for the analysis of clinical samples.

  9. Sources of error in screening by cytometry for the effects of environmental mutagens

    SciTech Connect

    Tiersch, T.R. ); Wachtel, S.S. )

    1993-01-01

    Flow cytometry is often used to detect DNA aneuploidy and mosaicism associated with malignancy or genetic damage. Yet DNA aneuploidy and mosaicism detected by flow cytometry may be more apparent than real. In contrast to the DNA mass observed for blood, the authors consistently found markedly different values and higher variability for DNA mass among other tissues collected from the same animal. Prepared mixtures of blood and other cells generated multiple fluorescence peaks identical to those that might be expected for aneuploid mosaicism. Moreover, analysis of tissues such as feather pulp, which contains a combination of cell types, yielded multiple fluorescence peaks that were not observed when blood alone was analyzed. Thus care should be exercised in classifying DNA values from different tissues as normal or abnormal, because the appearance of supernumerary fluorescence peaks might not always indicate the presence of abnormal cell populations.

  10. Analysis of polyethylene glycol (PEG) fusion in cultured neuroblastoma cells via flow cytometry: Techniques & optimization.

    PubMed

    Hoffman, Ashley N; Bamba, Ravinder; Pollins, Alonda C; Thayer, Wesley P

    2017-02-01

    Polyethylene glycol (PEG) has long been used as a membrane fusogen, but recently it has been adopted as a technique for peripheral nerve repair. Vertebrate models using PEG fusion have shown improved outcomes when PEG is applied during repair of severed peripheral nerves. The cellular mechanism of PEG fusion in the peripheral nerve repair model has not previously been assessed via flow cytometry. PEG fusion was assessed in this experiment by dying B35 rat neuroblastoma cells with different color fluorescent labels. The different color cells were combined and PEG was applied in concentrations of 50%, 75% and 100%. The amount of cell fusion was assessed via flow cytometry as the percentage of double positive cells. Results showed increasing fusion and decreasing viability with increasing concentrations of PEG.

  11. Monitoring of microbial communities by flow cytometry and rRNA-targeted hybridization probes

    NASA Astrophysics Data System (ADS)

    Wallner, Guenter; Amann, Rudolf

    1995-10-01

    Flow cytometry in combination with ribosomal RNA (rRNA) based fluorescence in situ hybridization is a new technique for the analysis of microbial communities. Oligonucleotide probes directed against ribosomal RNA allow the identification of species or groups of micro- organisms. Combined with flow cytometry, up to several thousand cells per second can be classified. In addition to the identification and specific enumeration of micro-organisms, further information on the distribution of cell size, DNA and ribosome content -- and therefore an assessment of activity -- within the entire community or subpopulations can be obtained. This technique is much more accurate, informative, and rapid than classical culture-dependent methods. Data of activated sludge samples hybridized with fluorescein labeled oligonucleotides and counterstained with the DNA-specific dye Hoechst 33342 are presented as examples for its applicability to complex microbial communities.

  12. Exploring Glucocorticoid Receptor Agonists Mechanism of Action Through Mass Cytometry and Radial Visualizations.

    PubMed

    Abraham, Yann; Gerrits, Bertran; Ludwig, Marie-Gabrielle; Rebhan, Michael; Gubser Keller, Caroline

    2017-01-01

    Recent advances in combining flow cytometry and mass spectrometry have led to the development of mass cytometry, allowing for the interrogation of complex cell populations on an unprecedented scale. The volumes and high dimensionality of mass cytometry data pose significant challenges in terms of analysis and visualization. We implement a method called Radviz, where multidimensional single cell data can be visualized as a projection that maintains the original dimensions and data complexity whilst facilitating analysis and visualization. This enables identification of changes in populations, focusing the analysis on the most relevant aspect of large multidimensional datasets. To highlight the potential of Radviz, we profiled peripheral mononuclear blood cells (PBMCs) from three healthy donors and showed donor-specific differences in the number and composition of cell populations. In a second study, we explored the anti-inflammatory effects of two glucocorticoid receptor (GR) ligands (cpd6 and cpd11) compared to dexamethasone (Dex) on human primary macrophages. Standard analysis at the population level showed that cpd6 and cpd11 have an overall anti-inflammatory profile similar to that of Dex. CyTOF profiling and Radviz-driven analysis at the single cell level confirmed this observation, and identified a concentration-dependent effect of cpd6 that was not detected at the population level. Altogether, Radviz combines the strengths of a projection method, reducing the dimensionality of datasets, with that of a scatter plot, where the identity of each point can be inferred from the distance to the axis. This enables the visual exploration, analysis, and interpretation of complex, high dimensional data. © 2016 International Clinical Cytometry Society.

  13. Rapid parallel flow cytometry assays of active GTPases using effector beads.

    PubMed

    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela

    2013-11-15

    We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.

  14. [Flux cytometry of the cellular cycle of leukemic cells of the blood].

    PubMed

    Pierrez, J; Guerci, A; Guerci, O

    1988-01-01

    The coordination of flux cytometry and of a techniques of leukoconcentration allowed to determine the cellular cycle of nucleated cells of circulating blood, without logs nor enrichment of cellular type on a definitive moment. The study of acute leukemias allow to conclude that: 1) it exists in peripheral blood a synthetic activity of ADN bound to the presence of leukemic or blastic cells; 2) this activity allows to appreciate the spontaneous variations of synthesis and the incidence of chemotherapy.

  15. Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization.

    PubMed

    Nassar, Ala F; Wisnewski, Adam V; Raddassi, Khadir

    2017-03-01

    Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

  16. Detection of Antineutrophil Autoantibodies by Flow Cytometry: Use of Unfixed Neutrophils as Antigenic Targets

    DTIC Science & Technology

    1993-01-01

    Coombs’ Sera from neutropenic patients with a variety of associated positive hemolytic anemia , chronic hepatitis, and lympho- clinical conditions...Liss, Inc.* for enhanced binding of IgG to normal target Key words: neutrophils, immunology, flow cytometry, autoimmunity , irnunoglobulins, neutro- A...however, can be time-consuming, te- autoimmune neutropenia and from normal volunteers by ven- dious, difficult to quantitate, and of limited

  17. Strategies for the synthesis and screening of glycoconjugates. 2. Covalent immobilization for flow cytometry.

    PubMed

    Vetter, D; Tate, E M; Gallop, M A

    1995-01-01

    Glycosylamines are readily available carbohydrate derivatives that undergo acylation reactions with homobifunctional N-hydroxysuccinimidyl esters. The product glycosylamides carry a spacer group equipped with one active ester functionality. This route provides well-defined glycoconjugates, which may be cross-linked to various amino-functionalized resins. Carbohydrate recognition of the resulting sugar-bead conjugates is probed by lectin immunostaining or flow cytometry using a fluorescently labeled lectin.

  18. In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues

    PubMed Central

    Biris, Alexandru S.; Galanzha, Ekaterina I.; Li, Zhongrui; Mahmood, Meena; Xu, Yang; Zharov, Vladimir P.

    2016-01-01

    Nanoparticles are intensively being explored as contrast agents for medical diagnostics and therapies using various optical methods. We present the first demonstration of the use of time-resolved Raman spectroscopy for in vivo real-time detection of circulating carbon nanotubes (CNTs) or cancer cells labeled with CNTs in the lymph, blood, and tissues of live animals with fast spectral acquisition times of down to few milliseconds. After intravenously administering CNTs in the tail vein of the rat, this technique provides the ability to detect the circulation of CNTs in the blood microvessels of the intact rat ear. The capability of Raman spectroscopy is also demonstrated to monitor, identify, and image the CNTs during their transportation by lymphatics in the rat ear and mesentery. The strong and specific Raman scattering properties of CNTs make it possible to detect in vitro and in vivo single cancer cells (HeLa) tagged with CNTs. In vivo Raman flow cytometry opens a new avenue for multiparameter analysis of circulating nanoparticles with strong Raman scattering properties and their pharmokinetics in blood and lymph systems. Moreover, this technology has the potential for molecular detection and identification of circulating tumor cells, and infections labeled with CNTs. PMID:19405719

  19. Discovering cell types in flow cytometry data with random matrix theory

    NASA Astrophysics Data System (ADS)

    Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

    Flow cytometry is a widely used experimental technique in immunology research. During the experiments, peripheral blood mononuclear cells (PBMC) from a single patient, labeled with multiple fluorescent stains that bind to different proteins, are illuminated by a laser. The intensity of each stain on a single cell is recorded and reflects the amount of protein expressed by that cell. The data analysis focuses on identifying specific cell types related to a disease. Different cell types can be identified by the type and amount of protein they express. To date, this has most often been done manually by labelling a protein as expressed or not while ignoring the amount of expression. Using a cross correlation matrix of stain intensities, which contains both information on the proteins expressed and their amount, has been largely ignored by researchers as it suffers from measurement noise. Here we present an algorithm to identify cell types in flow cytometry data which uses random matrix theory (RMT) to reduce noise in a cross correlation matrix. We demonstrate our method using a published flow cytometry data set. Compared with previous analysis techniques, we were able to rediscover relevant cell types in an automatic way. Department of Physics, University of Maryland, College Park, MD 20742.

  20. Identification and visualization of multidimensional antigen-specific T-cell populations in polychromatic cytometry data.

    PubMed

    Lin, Lin; Frelinger, Jacob; Jiang, Wenxin; Finak, Greg; Seshadri, Chetan; Bart, Pierre-Alexandre; Pantaleo, Giuseppe; McElrath, Julie; DeRosa, Steve; Gottardo, Raphael

    2015-07-01

    An important aspect of immune monitoring for vaccine development, clinical trials, and research is the detection, measurement, and comparison of antigen-specific T-cells from subject samples under different conditions. Antigen-specific T-cells compose a very small fraction of total T-cells. Developments in cytometry technology over the past five years have enabled the measurement of single-cells in a multivariate and high-throughput manner. This growth in both dimensionality and quantity of data continues to pose a challenge for effective identification and visualization of rare cell subsets, such as antigen-specific T-cells. Dimension reduction and feature extraction play pivotal role in both identifying and visualizing cell populations of interest in large, multi-dimensional cytometry datasets. However, the automated identification and visualization of rare, high-dimensional cell subsets remains challenging. Here we demonstrate how a systematic and integrated approach combining targeted feature extraction with dimension reduction can be used to identify and visualize biological differences in rare, antigen-specific cell populations. By using OpenCyto to perform semi-automated gating and features extraction of flow cytometry data, followed by dimensionality reduction with t-SNE we are able to identify polyfunctional subpopulations of antigen-specific T-cells and visualize treatment-specific differences between them.

  1. Unfold High-Dimensional Clouds for Exhaustive Gating of Flow Cytometry Data.

    PubMed

    Qiu, Peng

    2014-01-01

    Flow cytometry is able to measure the expressions of multiple proteins simultaneously at the single-cell level. A flow cytometry experiment on one biological sample provides measurements of several protein markers on or inside a large number of individual cells in that sample. Analysis of such data often aims to identify subpopulations of cells with distinct phenotypes. Currently, the most widely used analytical approach in the flow cytometry community is manual gating on a sequence of nested biaxial plots, which is highly subjective, labor intensive, and not exhaustive. To address those issues, a number of methods have been developed to automate the gating analysis by clustering algorithms. However, completely removing the subjectivity can be quite challenging. This paper describes an alternative approach. Instead of automating the analysis, we develop novel visualizations to facilitate manual gating. The proposed method views single-cell data of one biological sample as a high-dimensional point cloud of cells, derives the skeleton of the cloud, and unfolds the skeleton to generate 2D visualizations. We demonstrate the utility of the proposed visualization using real data, and provide quantitative comparison to visualizations generated from principal component analysis and multidimensional scaling.

  2. International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards

    PubMed Central

    Holmes, Kevin L.; Fontes, Benjamin; Hogarth, Philip; Konz, Richard; Monard, Simon; Pletcher, Charles H.; Wadley, Robert B.; Schmid, Ingrid; Perfetto, Stephen P.

    2014-01-01

    Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. PMID:24634405

  3. A novel procedure of quantitation of virus based on microflow cytometry analysis.

    PubMed

    Vazquez, Diego; López-Vázquez, Carmen; Cutrín, Juan Manuel; Dopazo, Carlos P

    2016-03-01

    The accurate and fast titration of viruses is a critical step in research laboratories and biotechnology industries. Different approaches are commonly applied which either are time consuming (like the plaque and endpoint dilution assays) or do not ensure quantification of only infective particles (like quantitative real-time PCR). In the last decade, a methodology based on the analysis of infected cells by flow cytometry and fluorescence-activated cell sorting (FACS) has been reported as a fast and reliable test for the titration of some viruses. However, this technology needs expensive equipment and expert technicians to operate it. Recently, the "lab on a chip" integrated devices have brought about the miniaturization of this equipment, turning this technology into an affordable and easy-to-use alternative to traditional flow cytometry. In the present study, we have designed a microflow cytometry (μFC) procedure for the quantitation of viruses, using the infectious pancreatic necrosis virus (IPNV) as a model. The optimization of conditions and validation of the method are reported here.

  4. Characterisation of the green turtle's leukocyte subpopulations by flow cytometry and evaluation of their phagocytic activity.

    PubMed

    Muñoz, F A; Franco-Noguez, S Y; Gonzalez-Ballesteros, E; Negrete-Philippe, A C; Flores-Romo, L

    2014-06-01

    Phagocytosis is a fundamental aspect of innate immunity that is conserved across many species making it a potentially useful health-assessment tool for wildlife. In non-mammalian vertebrates, heterophils, monocytes, macrophages, melanomacrophages, and thrombocytes all have phagocytic properties. Recently, B lymphocytes from fish, amphibians, and aquatic turtles have also showed phagocytic capacity. Phagocytes can be studied by flow cytometry; however, the use of this tool is complicated in reptiles partly because nucleated erythrocytes complicate the procedure. We separated green turtle leukocytes by density gradient centrifugation and identified subpopulations by flow cytometry and confocal microscopy. Additionally, we assessed their ability to phagocytize Fluorspheres and Ovoalbumin-Alexa. We found that heterophils and lymphocytes but not monocytes could be easily identified by flow cytometry. While heterophils from adults and juvenile turtles were equally able to phagocytize fluorspheres, adults had significantly more phagocytic ability for OVA-Alexa. Lymphocytes had a mild phagocytic activity with fluorospheres (27-38 %; 39-45 %) and OVA-Alexa (35-46 %; 14-22 %) in juvenile and adult green turtles, respectively. Confocal microscopy confirmed phagocytosis of fluorospheres in both heterophils and lymphocytes. This provides the first evidence that green turtle lymphocytes have phagocytic activity and that this assay could potentially be useful to measure one aspect of innate immunity in this species.

  5. In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

    PubMed Central

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

    2015-01-01

    During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection. PMID:25867202

  6. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    NASA Astrophysics Data System (ADS)

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-09-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

  7. RBC aggregation in dextran solutions can be measured by flow cytometry.

    PubMed

    Zhao, Lian; Kaewprayoon, Waraporn; Zhou, Hong; Georgieva, Radostina; Bäumler, Hans

    2017-01-01

    The impact of macromolecules on RBC aggregation continues to be of interest, nevertheless present measurements still have limitations and need improvement. We applied flow cytometry to measure RBC aggregation in dextran T500 (Dx500) solution. The samples were fixed in the aggregated state by glutaraldehyde. Fixed RBC exhibit auto fluorescence, which can be detected by flow cytometry. Single cells, doublets, triplets and larger aggregates can be distinguished quantitatively and quickly due to the correlation between auto fluorescence intensity and number of RBC per measured event. With the increase in concentration of Dx500, percentages of all aggregates and bigger aggregates increased significantly at concentration of 2%, 4% and 6%, while decreased when the concentration reached 8% and 10%. The percentage of bigger aggregates in concentration of 4% was higher than that in 2% and 6%. The data of flow cytometry was confirmed by microscopic observation and are in good agreement with the literature. The method provide additional advantages to the conventional measurement of RBC aggregation. It gets the distribution of single cells and aggregates as derived from the microscopic observation with hematocrit of physiological level. It uses sample volume as 1/5∼1/10 as needed in sendimentation and photometricmethods.

  8. Flow cytometry community fingerprinting and amplicon sequencing for the assessment of landfill leachate cellulolytic bioaugmentation.

    PubMed

    Kinet, R; Dzaomuho, P; Baert, J; Taminiau, B; Daube, G; Nezer, C; Brostaux, Y; Nguyen, F; Dumont, G; Thonart, P; Delvigne, F

    2016-08-01

    Flow cytometry (FCM) is a high throughput single cell technology that is actually becoming widely used for studying phenotypic and genotypic diversity among microbial communities. This technology is considered in this work for the assessment of a bioaugmentation treatment in order to enhance cellulolytic potential of landfill leachate. The experimental results reveal the relevant increase of leachate cellulolytic potential due to bioaugmentation. Cytometric monitoring of microbial dynamics along these assays is then realized. The flow FP package is used to establish microbial samples fingerprint from initial 2D cytometry histograms. This procedure allows highlighting microbial communities' variation along the assays. Cytometric and 16S rRNA gene sequencing fingerprinting methods are then compared. The two approaches give same evidence about microbial dynamics throughout digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at genus or species level. Same phenotypical profiles of microbiota during assays matched to several 16S rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses.

  9. In vivo, label-free, and noninvasive detection of melanoma metastasis by photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Liu, Rongrong; Wang, Cheng; Hu, Cheng; Wang, Xueding; Wei, Xunbin

    2014-02-01

    Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC's capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).

  10. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    PubMed Central

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-01-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis. PMID:27596736

  11. Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity

    PubMed Central

    Ayers, Lisa; Harrison, Paul; Kohler, Malcolm; Ferry, Berne

    2014-01-01

    Background Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results. Methods One hundred samples from healthy individuals and patients with obstructive sleep apnoea were analysed by conventional flow cytometry (FACSCalibur) and by three functional MV assays: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA® Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation. Results Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001); correlated with ETP (r=0.7444, p<0.0001); negatively correlated with STA® Phospholipid Procoag Assay clotting time (−0.7872, p<0.0001), reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results. Conclusions Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive

  12. Flow Cytometry with Gold Nanoparticles and their Clusters as scattering Contrast Agents: FDTD Simulation of Light-Cell Interaction

    PubMed Central

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

    2010-01-01

    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 a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. PMID:19670359

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

    PubMed

    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.

  14. Belgian consensus recommendations for flow cytometric immunophenotyping. The Belgian Association for Cytometry/Belgische Vereniging voor Cytometrie/Association Belge de Cytométrie.

    PubMed

    Van Bockstaele, D R; Deneys, V; Philippé, J; Bernier, M; Kestens, L; Chatelain, B; De Waele, M; Demanet, C

    1999-04-01

    This paper summarises the guidelines and recommendations that were generated during a number of discussion forums attended by the majority of Belgian cytometry laboratory professionals. These forums focused on the rational and optimal use of flow cytometric evaluations in the clinical laboratory setting. The aim was to improve the coherence of the testing panels and the quality of the results and--as such--the clinical diagnostic information. It was also the aim to provide the Belgian prescribing physician and interested laymen with an updated overview of the flow cytometric possibilities. Emphasis is placed on immunophenotyping of haematological malignancies, hematopoietic progenitor cell counting and follow-up of the viral infection caused by the human immunodeficiency virus.

  15. Does FDG uptake measure proliferative activity of human cancer cells? In vitro comparison with DNA flow cytometry and tritiated thymidine uptake.

    PubMed

    Higashi, K; Clavo, A C; Wahl, R L

    1993-03-01

    The relationship between 3H-2-fluoro-2-deoxy-D-glucose (FDG) uptake and the proliferative rate of a human ovarian adenocarcinoma cell line (HTB77IP3) was examined in vitro. HTB77IP3 cells were plated and allowed to grow through lag, exponential and plateau phases. Proliferative rate assessed by DNA flow cytometry and 3H-thymidine incorporation was highest in the lag phase and fell significantly as the cells progressed from the exponential through plateau phases. By DNA flow cytometry, the proliferation index (% of S+G2/M phase cells) fell from 65% to 23%. Thymidine uptake per cell also declined, by 82%, from lag to plateau phase. By contrast, 3H-FDG uptake per cell was largely unchanged as the cells progressed through the cell growth cycle. Total 3H-FDG uptake was strongly correlated with the number of viable cancer cells present (r = 0.957). Total thymidine uptake, however, substantially underestimated the number of viable cancer cells present. These in vitro differences in tracer uptake suggest that in this adenocarcinoma cell line, FDG measures a substantially different parameter (viable cell number) than thymidine (proliferative rate) and that these differences may result in disparate findings on PET imaging of cancers using these two tracers. Our data for this in vitro system indicate that FDG uptake does not relate to the proliferative activity of cancer cells. However, FDG uptake is strongly related to the number of viable tumor cells.

  16. Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

    PubMed Central

    Heinonen, J T; Sidhu, J S; Reilly, M T; Farin, F M; Omiecinski, C J; Eaton, D L; Kavanagh, T J

    1996-01-01

    Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur. Images Figure 1. Figure 2. Figure 3. Figure 3. Figure 4. Figure 4. Figure 5. Figure 6. PMID:8743442

  17. A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action

    PubMed Central

    Hendon-Dunn, Charlotte Louise; Doris, Kathryn Sarah; Thomas, Stephen Richard; Allnutt, Jonathan Charles; Marriott, Alice Ann Neville; Hatch, Kim Alexandra; Watson, Robert James; Bottley, Graham; Marsh, Philip David; Taylor, Stephen Charles

    2016-01-01

    Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic. PMID:26902767

  18. High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA.

    PubMed

    Torres, Alex José Leite; Brígido, Luis Fernando de Macedo; Abrahão, Marcos Herculano Nunes; Angelo, Ana Luiza Dias; de Jesus Ferreira, Gilcivaldo; Coelho, Luana Portes; Ferreira, João Leandro; Jorge, Célia Regina Mayoral Pedroso; Netto, Eduardo Martins; Brites, Carlos

    2015-01-01

    Use of CCR5 antagonists requires previous viral tropism determination. The available methods have high cost, are time-consuming, or require highly trained personnel, and sophisticated equipment. We compared a flow cytometry-based tropism assay with geno2pheno method to determine HIV-1 tropism in AIDS patients, in Bahia, Brazil. We tested peripheral blood mononuclear cells of 102 AIDS patients under antiretroviral therapy by using a cytometry-based tropism assay and geno2pheno assay. Cellular membrane receptors were identified by using CXCR4, CCR5 and CD4 monoclonal antibodies, while detection of cytoplasmic mRNAs for gag and pol HIV regions was achieved by using a labeled probe. Genotypic identification of X4 and R5 tropic viruses was attempted by geno2pheno algorithm. There was a high degree of concordance between cytometry-based tropism assay and geno2pheno algorithm in determination of HIV-1 tropism. Cytometry-based tropism assay demonstrated higher sensitivity and specificity in comparison to geno2pheno, which was used as a gold-standard. One sample could not be amplified by geno2pheno method, but was classified as duotropic by cytometry-based tropism assay. We did not find any association between CD4+ count or plasma HIV-1 RNA viral load and tropism results. The overall performances of cytometry-based tropism assay and geno2pheno assay were almost identical in determination of HIV-1 tropism.

  19. A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action.

    PubMed

    Hendon-Dunn, Charlotte Louise; Doris, Kathryn Sarah; Thomas, Stephen Richard; Allnutt, Jonathan Charles; Marriott, Alice Ann Neville; Hatch, Kim Alexandra; Watson, Robert James; Bottley, Graham; Marsh, Philip David; Taylor, Stephen Charles; Bacon, Joanna

    2016-07-01

    Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

  20. Multi-parametric imaging of cell heterogeneity in apoptosis analysis.

    PubMed

    Vorobjev, Ivan A; Barteneva, Natasha S

    2017-01-01

    Apoptosis is a multistep process of programmed cell death where different morphological and molecular events occur simultaneously and/or consequently. Recent progress in programmed cell death analysis uncovered large heterogeneity in response of individual cells to the apoptotic stimuli. Analysis of the complex and dynamic process of apoptosis requires a capacity to quantitate multiparametric data obtained from multicolor labeling and/or fluorescent reporters of live cells in conjunction with morphological analysis. Modern methods of multiparametric apoptosis study include but are not limited to fluorescent microscopy, flow cytometry and imaging flow cytometry. In the current review we discuss the image-based evaluation of apoptosis on the single-cell and population level by imaging flow cytometry in parallel with other techniques. The advantage of imaging flow cytometry is its ability to interrogate multiparametric morphometric and fluorescence quantitative data in statistically robust manner. Here we describe the current status and future perspectives of this emerging field, as well as some challenges and limitations. We also highlight a number of assays and multicolor labeling probes, utilizing both microscopy and different variants of imaging cytometry, including commonly based assays and novel developments in the field.

  1. Development of wide-angle 2D light scattering static cytometry

    NASA Astrophysics Data System (ADS)

    Xie, Linyan; Liu, Qiao; Shao, Changshun; Su, Xuantao

    2016-10-01

    We have recently developed a 2D light scattering static cytometer for cellular analysis in a label-free manner, which measures side scatter (SSC) light in the polar angular range from 79 to 101 degrees. Compared with conventional flow cytometry, our cytometric technique requires no fluorescent labeling of the cells, and static cytometry measurements can be performed without flow control. In this paper we present an improved label-free static cytometer that can obtain 2D light scattering patterns in a wider angular range. By illuminating the static microspheres on chip with a scanning optical fiber, wide-angle 2D light scattering patterns of single standard microspheres with a mean diameter of 3.87 μm are obtained. The 2D patterns of 3.87 μm microspheres contain both large-angle forward scatter (FSC) and SSC light in the polar angular range from 40 to 100 degrees, approximately. Experimental 2D patterns of 3.87 μm microspheres are in good agreement with Mie theory simulated ones. The wide-angle light scattering measurements may provide a better resolution for particle analysis as compared with the SSC measurements. Two dimensional light scattering patterns of HL-60 human acute leukemia cells are obtained by using our static cytometer. Compared with SSC 2D light scattering patterns, wide-angle 2D patterns contain richer information of the HL-60 cells. The obtaining of 2D light scattering patterns in a wide angular range could help to enhance the capabilities of our label-free static cytometry for cell analysis.

  2. Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species

    PubMed Central

    Loureiro, João; Rodriguez, Eleazar; Doležel, Jaroslav; Santos, Conceição

    2007-01-01

    Background and Aims After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry. Methods GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species. Key Results In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed. Conclusions WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high

  3. Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry.

    PubMed

    Schulz, Danae; Mugnier, Monica R; Boothroyd, Catherine E; Papavasiliou, F Nina

    2016-10-19

    Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

  4. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    PubMed Central

    Pospichalova, Vendula; Svoboda, Jan; Dave, Zankruti; Kotrbova, Anna; Kaiser, Karol; Klemova, Dobromila; Ilkovics, Ladislav; Hampl, Ales; Crha, Igor; Jandakova, Eva; Minar, Lubos; Weinberger, Vit; Bryja, Vitezslav

    2015-01-01

    Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for

  5. Cytology, immunopathology and flow cytometry in the diagnosis of pleural and peritoneal effusions.

    PubMed

    Croonen, A M; van der Valk, P; Herman, C J; Lindeman, J

    1988-06-01

    There were 106 pleural and peritoneal effusions studied in order to investigate the contribution of immunocytochemistry and flow cytometry to routine cytologic diagnosis. A panel of antibodies (to cytokeratin, vimentin, human milk fat globule, epithelial membrane antigen and carcinoembryonic antigen) was applied to aceton-fixed slides, using immunoperoxydase and immunofluorescence methods. Flow cytometry was performed using a double labeling method, i.e., propidium iodide for DNA staining and keratin for labeling of epithelial cells. In this way DNA aneuploidy was more evident in the histograms when the fluid contained many reactive nonepithelial cells (lymphocytes). A designation of marker profiles was made for the three most frequently occurring diagnoses, i.e., reactive mesothelial proliferation (I), adenocarcinoma (II), and malignant mesothelioma (III). For the differentiation between adenocarcinoma and malignant mesothelioma, carcinoembryonic antigen was the most useful marker as 75% of the adenocarcinomas was carcinoembryonic antigen-positive and the malignant mesotheliomas were consistently negative. Furthermore, evident DNA-aneuploidy strongly supported the diagnosis of adenocarcinoma, as most malignant mesotheliomas were DNA-euploid, even though occasional DNA-aneuploidy was found in malignant mesotheliomas when different effusions from the same patient were examined. For the differentiation between reactive mesothelial cells and malignant mesothelioma human milk fat globule and/or epithelial membrane antigen, in this study proved to be most reliable, their presence strongly indicating malignancy. It is stressed that the method used (fixation, antibodies, washing procedures) can influence these findings. In 16 patients (17%) performing immunopathology and/or flow cytometry meant an important contribution to diagnosis.

  6. An Improved Flow Cytometry Method For Precise Quantitation Of Natural-Killer Cell Activity

    NASA Technical Reports Server (NTRS)

    Crucian, Brian; Nehlsen-Cannarella, Sandra; Sams, Clarence

    2006-01-01

    The ability to assess NK cell cytotoxicity using flow cytometry has been previously described and can serve as a powerful tool to evaluate effector immune function in the clinical setting. Previous methods used membrane permeable dyes to identify target cells. The use of these dyes requires great care to achieve optimal staining and results in a broad spectral emission that can make multicolor cytometry difficult. Previous methods have also used negative staining (the elimination of target cells) to identify effector cells. This makes a precise quantitation of effector NK cells impossible due to the interfering presence of T and B lymphocytes, and the data highly subjective to the variable levels of NK cells normally found in human peripheral blood. In this study an improved version of the standard flow cytometry assay for NK activity is described that has several advantages of previous methods. Fluorescent antibody staining (CD45FITC) is used to positively identify target cells in place of membranepermeable dyes. Fluorescent antibody staining of target cells is less labor intensive and more easily reproducible than membrane dyes. NK cells (true effector lymphocytes) are also positively identified by fluorescent antibody staining (CD56PE) allowing a simultaneous absolute count assessment of both NK cells and target cells. Dead cells are identified by membrane disruption using the DNA intercalating dye PI. Using this method, an exact NK:target ratio may be determined for each assessment, including quantitation of NK target complexes. Backimmunoscatter gating may be used to track live vs. dead Target cells via scatter properties. If desired, NK activity may then be normalized to standardized ratios for clinical comparisons between patients, making the determination of PBMC counts or NK cell percentages prior to testing unnecessary. This method provides an exact cytometric determination of NK activity that highly reproducible and may be suitable for routine use in the

  7. A Case of Blastic Plasmacytoid Dendritic Cell Neoplasm Extensively Studied by Flow Cytometry and Immunohistochemistry

    PubMed Central

    Cesana, Clara; Cittone, Micol Giulia; Bandiera, Laura; Scarpati, Barbara; Mancini, Valentina; Soriani, Silvia; Veronese, Silvio; Truini, Mauro; Rossini, Silvano; Cairoli, Roberto

    2017-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with aggressive clinical course and poor prognosis. Diagnosis is based on detection of CD4+ CD56+, CD123high, TCL-1+, and blood dendritic cell antigen-2/CD303+ blasts, together with the absence of lineage specific antigens on tumour cells. In this report we present a case of BPDCN presenting with extramedullary and bone marrow involvement, extensively studied by flow cytometry and immunohistochemistry, who achieved complete remission after acute lymphoblastic leukemia like chemotherapy and allogeneic hematopoietic stem cell transplantation.

  8. Micro Flow Cytometry Miniaturisation - Towards in-situ Optical Phytoplankton Analysis

    NASA Astrophysics Data System (ADS)

    Zmijan, R.; Abi Kaed Bey, S.; Mowlem, M. C.; Morgan, H.

    2012-04-01

    The use of flow cytometry for studies of temporal and spatial variability of phytoplankton populations is a valuable tool contributing to research relating carbon biogeochemistry and climate change. Early designs and marine deployments of such devices started over two decades ago [1-3]. Miniaturisation and cost reduction without sacrificing performance remains a major challenge but would enable mass production and deployment. Large numbers of measurement nodes (e.g. as part of a global ocean observation system) would be possible which would increase data available over both spatial and temporal scales. This research presents two different design approaches for miniaturisation and integration of optics into a microfluidic cytometer chip. The proposed solutions are suitable for micro cytometers with external components coupled with optical fibres and were simulated and optimised using ray tracing software (Zemax). The two designs address light delivery for excitation of particles within the measurement region of the cytometer. One uses an integrated micro lens (fabricated in the chip) and the other a ball shaped micro lens manufactured separately and then inserted into the chip. Both approaches collimate the excitation light beam (from an off chip diode laser coupled with an optical fibre) into the fluidic channel. The predicted (by ray tracing) excitation beam widths are 70 and 80 µm for the integrated and the ball lens respectively, and are in agreement with experimental data presented. The proposed cytometer chip design is compatible with low cost materials (acrylic glass, cyclo-olefines) and manufacturing methods (micro milling, hot embossing, injection moulding). 1. Dubelaar, G.B.J. and P.L. Gerritzen, CytoBuoy: a step forward towards using flow cytometry in operational oceanography. Scientia Marina, 2000. 64(2): p. 255-265. 2. Peeters, J.C.H., et al., Optical Plankton Analyzer - a Flow Cytometer for Plankton Analysis .1. Design Considerations. Cytometry, 1989

  9. [Research progress on multiple myeloma immunophenotyping and minimal residual disease detected by flow cytometry].

    PubMed

    Li, Han-Qing; Zhai, Yong-Ping

    2015-02-01

    Multiple myeloma (MM) is a haematological malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow and remained incurable. Flow cytometry has been widely used in the detection of immunophenotype and minimal residual disease, diagnosis, monitoring and prognosis of MM. Normal plasma cells and malignant plasma cells can be distinguished according to different cell surface antigen expression. The clinical significane of many immune markes has been elucidated. However, the clinical significance of some phenotype remains controversial, the detection scheme and gating strategy are not unified. This review discusses the recent research progress on detection of MM immunophenotype and minimal residual disease by flow cytovetry.

  10. A structured population modeling framework for quantifying and predicting gene expression noise in flow cytometry data.

    PubMed

    Flores, Kevin B

    2013-07-01

    We formulated a structured population model with distributed parameters to identify mechanisms that contribute to gene expression noise in time-dependent flow cytometry data. The model was validated using cell population-level gene expression data from two experiments with synthetically engineered eukaryotic cells. Our model captures the qualitative noise features of both experiments and accurately fit the data from the first experiment. Our results suggest that cellular switching between high and low expression states and transcriptional re-initiation are important factors needed to accurately describe gene expression noise with a structured population model.

  11. Comparison of nucleolar organiser regions and DNA flow cytometry in the evaluation of pleural effusion.

    PubMed Central

    Huang, M. S.; Tsai, M. S.; Hwang, J. J.; Wang, T. H.

    1994-01-01

    BACKGROUND--In conventional cytological diagnosis of pleural effusions the assessment of morphological features plays an important part. However, false negative and false positive results may occur. In this study conventional cytology was compared with flow cytometric DNA analysis and the argyrophil staining technique for nucleolar organiser regions (AgNOR) to characterise benign and malignant effusions. METHODS--Pleural effusions from 71 patients (38 with benign lung disease, 33 with proven adenocarcinoma of lung) were studied by conventional cytology, flow cytometric DNA analysis, and the AgNOR technique. Tumour cell ploidy was determined by flow cytometry. In an attempt to detect the cell proliferative state, flow cytometric S phase fraction and the AgNOR technique were used. The correlations among conventional cytology, flow cytometric DNA ploidy, S phase fraction analysis, and nucleolar organiser regions were investigated. RESULTS--All the 38 benign pleural effusions were diploid. There were 17 (52%) aneuploid and 16 (48%) diploid malignant pleural effusions. Based on these results this type of DNA analysis had a sensitivity of 52% and a specificity of 100%. The mean (SD) numbers of flow cytometric S phase fractions of benign and malignant cases were 5.32 (1.67)% and 12.45 (3.93)% respectively. The mean numbers of S phase fractions of diploid malignant cases were higher than diploid benign cases. In each case the number of AgNORs was counted in 100 cells. The mean number of AgNOR dots per nucleus was 12.57 (3.64) for malignant pleural effusion cells and 3.96 (1.39) for benign pleural effusion cells. The mean number of AgNOR dots was 14.45 (3.36) for aneuploid malignant pleural effusion cells and 10.57 (2.82) for diploid malignant pleural effusion cells. The AgNOR numbers were higher in diploid malignant cells than in diploid benign cells. There was a significant correlation between the S phase fraction determined by flow cytometry and the mean number of Ag

  12. A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum.

    PubMed

    Bei, Amy K; Desimone, Tiffany M; Badiane, Aida S; Ahouidi, Ambroise D; Dieye, Tandakha; Ndiaye, Daouda; Sarr, Ousmane; Ndir, Omar; Mboup, Souleymane; Duraisingh, Manoj T

    2010-04-01

    Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry-based method for simultaneously calculating both the parasitemia and the number of multiply-infected erythrocytes. Staining with the DNA-specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion.

  13. Flow cytometry analysis of porcine platelets: optimized methods for best results.

    PubMed

    Krajewski, Stefanie; Kurz, Julia; Wendel, Hans Peter; Straub, Andreas

    2012-01-01

    Animal models are essential tools for the in vivo evaluation of pharmacological modulation of platelet function and the mechanisms underlying thrombosis. In particular, pigs are being increasingly used in cardiovascular and platelet research. One standard method for the investigation of platelet function under experimental conditions is flow cytometry. However, this approach is limited by a shortage of feasible antibodies and a lack of incubation protocols with regard to porcine platelets. This study aimed to establish a method for the investigation of porcine platelets in flow cytometry. Platelets from pigs and human donors were stained with various commercially available specific antibodies against platelet receptors CD41a, CD42bα, CD62P, activated CD41/CD61, and platelet-bound fibrinogen. Staining procedures were performed in undiluted or diluted whole blood (WB) or platelet-rich plasma (PRP). Samples were treated with PBS buffer as control or with adenosine diphosphate (ADP) to induce platelet activation. Flow cytometry was performed using standard methodology. Furthermore, platelet counts were determined and ADP-induced platelet aggregations of both species were examined to confirm that the agonist ADP reliably activates human as well as porcine platelets. Five of the investigated antibodies bound to human, but not to porcine platelets only. However, two chicken-derived antibodies directed against CD62P (09-143) and fibrinogen (09-038) as well as a monoclonal mouse anti-CD62P (KO2.5) and a polyclonal rabbit anti-fibrinogen antibody (F0111) allowed reliable detection of porcine platelet activation. Moreover, binding intensity of the 09-143 antibody was increased when incubated in porcine PRP compared to WB, whereas antibody binding of both anti-fibrinogen antibodies to porcine platelets was only observed when incubated in a WB-buffer solution. KO2.5 antibody binding was detectable employing PRP as well as the WB-buffer solution. The feasibility of our new

  14. A double-coated magnetite-based magnetic fluid evaluation by cytometry and genetic tests

    NASA Astrophysics Data System (ADS)

    Freitas, M. L. L.; Silva, L. P.; Azevedo, R. B.; Garcia, V. A. P.; Lacava, L. M.; Grisólia, C. K.; Lucci, C. M.; Morais, P. C.; Da Silva, M. F.; Buske, N.; Curi, R.; Lacava, Z. G. M.

    2002-11-01

    Magnetite nanoparticles pre-coated with dodecanoic acid and ethoxylated alcohol (DE) were used to obtain a physiologically stable magnetic fluid (DE-MF) sample. Three different doses of DE-MF were intraperitoneally applied to mice. Blood and peritoneum cytometry and micronucleus test were performed for 1-21 days after injection to investigate the DE-MF toxicity. Changes in cell population, peritoneum inflammation, and potential DE-MF genotoxic action were all time and dose dependent. At the lowest dose (5×10 15 particles/kg), DE-MF seems to be useful as a drug precursor with both diagnostic and therapeutic values.

  15. Organizing the Cellular and Molecular Heterogeneity in High-Grade Serous Ovarian Cancer by Mass Cytometry

    DTIC Science & Technology

    2013-10-01

    Appendix A — Single-cell mass cytometry for analysis of immune system functional states Zach B Bjornson, Garry P Nolan and Wendy J Fantl 14...lymphocytes and primary human bone marrow, Behbehani et al. ident- ified all cell cycle phases with a core panel: p-Rb (pS807/ S811), IdU, cyclin B and p...applications of how they have been used to decipher complex cellular systems. 29. Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK: A deep profiler’s

  16. Using Lanthanide Nanoparticles as Isotopic Tags for Biomarker Detection by Mass Cytometry

    NASA Astrophysics Data System (ADS)

    Cao, Pengpeng

    The development of robust, versatile, and high-throughput biosensing techniques has widespread implications for early disease detection and accurate diagnosis. An innovative technology, mass cytometry, has been developed to use isotopically-labelled antibodies to simultaneously study multiple parameters of single cells. The current detection sensitivity of mass cytometry is limited by the number of copies of a given isotope that can be attached to a given antibody. This thesis describes research on the synthesis, characterization, and bioconjugation of a new class of nanoparticle-based labelling agents to be employed for the detection of low-abundance biomarkers by mass cytometry. Hydrophobic lanthanide nanoparticles (Ln NPs) have been prepared by the Winnik group. To render the NPs water-soluble for biological applications, we coated the NP surface with a first generation of multidentate poly(ethylene glycol) (PEG)-based ligands via ligand exchange. We measured the size, morphology, and polydispersity of these hydrophilic NPs by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The colloidal stability of the NPs was determined at various pH and in phosphate buffered saline (PBS) solutions. Tetradentate-PEG-coated NPs (Tetra-NPs) exhibited the best stability at pH 3 to 9, and in PBS. However, when cells were treated with Tetra-NPs in preliminary in vitro studies, significant undesirable non-specific binding (NSB) was observed. In order to tackle the NSB issue presented in the Tetra-NPs, we prepared a second generation of polymer-based ligands using ring-opening metathesis polymerization (ROMP). A small library of ROMP polymers was synthesized, characterized, and used to stabilize NPs in aqueous solutions. The ROMP-NPs were found to have significantly reduced NSB to cells by inductively coupled plasma-mass spectrometry (ICP-MS). To further modify the NPs, amine groups were introduced as functional handles to both the tetradentate-PEG and

  17. Ray tracing analysis of the image quality of a high collection efficiency mirror system

    SciTech Connect

    Seitzinger, N.K.; Martin, J.C.; Keller, R.A. )

    1990-10-01

    Recently, a high collection efficiency mirror system was developed by Watson (Cytometry, {bold 10}, 681--688 (1989)) to increase the sensitivity of low level fluorescence detection. The mirror system consists of an ellipsoidal imaging mirror and spherical backreflecting mirror. The fluorescing sample is located at one focus of the ellipsoid, and its image is formed at the other focus. In this paper we evaluate the image quality of this geometry using a PC-based ray tracing program. The analysis demonstrates high collection efficiency but poor image quality. The effect of poor image quality on single molecule detection is discussed. Keywords: Fluorescence, ellipsoidal mirror, spherical mirror, single molecule detection, flow cytometry.

  18. Lensless Imaging and Sensing.

    PubMed

    Ozcan, Aydogan; McLeod, Euan

    2016-07-11

    High-resolution optical microscopy has traditionally relied on high-magnification and high-numerical aperture objective lenses. In contrast, lensless microscopy can provide high-resolution images without the use of any focusing lenses, offering the advantages of a large field of view, high resolution, cost-effectiveness, portability, and depth-resolved three-dimensional (3D) imaging. Here we review various approaches to lensless imaging, as well as its applications in biosensing, diagnostics, and cytometry. These approaches include shadow imaging, fluorescence, holography, superresolution 3D imaging, iterative phase recovery, and color imaging. These approaches share a reliance on computational techniques, which are typically necessary to reconstruct meaningful images from the raw data captured by digital image sensors. When these approaches are combined with physical innovations in sample preparation and fabrication, lensless imaging can be used to image and sense cells, viruses, nanoparticles, and biomolecules. We conclude by discussing several ways in which lensless imaging and sensing might develop in the near future.

  19. Recognition of DAF and αvβ3 by inactivated Hantaviruses, towards the development of HTS flow cytometry assays

    PubMed Central

    Buranda, Tione; Wu, Yang; Perez, Dominique; Jett, Stephen D.; BonduHawkins, Virginie; Ye, Chunyan; Edwards, Bruce; Hall, Pamela; Larson, Richard S.; Lopez, Gabriel P.; Sklar, Larry A.; Hjelle, Brian

    2010-01-01

    Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardio-pulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS, and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study we have developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then use flow cytometry and live cell confocal fluorescence microscopy imaging to show that UV-killed SNV bind to the cognate receptors of live virions, namely, decay accelerating factor (CD55/DAF) expressed on Tanoue B cells and αvβ3 integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (Kd ≈ 26pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of ∼1:14000. We have configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high throughput flow cytometer. In this way, we establish a proof of principle high throughput screening assay for binding inhibition. This is a first step towards the development of HTS format assays for small molecule inhibitors of viral-cell interactions, as well as dissecting the mechanism of infection in a BSL-2 environment. PMID:20363206

  20. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    PubMed

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J; Vaulot, Daniel

    2011-04-28

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  1. Quantitative studies of chicken somatotrophs during growth and development by morphometry, immunocytochemistry, and flow cytometry.

    PubMed

    Malamed, S; Deaver, D; Perez, F; Radecki, S; Gibney, J; Scanes, C G

    1997-10-01

    Changes in the male chicken somatotroph during growth and maturation have been examined by morphometric and immunocytochemical (ICC) analysis of serial sections of the anterior pituitary gland and by flow cytometry of dispersed anterior pituitary cells. ICC showed that somatotrophs are confined to the middle and caudal thirds of the anterior pituitary gland at all ages from 5 to 26 weeks. At a given age somatotrophs are of equal size at all positions along the cephalocaudal axis of the anterior pituitary gland. However, there are age-related changes: from 5 to 11 weeks rises occur in both the mean total somatotroph volume per gland (64%) and the mean number of somatotrophs (78%), while the mean volume of the single somatotroph is unchanged. From 11 to 18 weeks the mean volume of the single somatotroph decreases 41%. From 18 to 26 weeks the mean volume of the somatotroph, the mean total somatotroph volume, and the mean number per gland do not change. Flow cytometry studies suggested that somatotrophs from adults have less growth hormone (GH) than somatotrophs from young birds. The increases in total somatotroph volume and number from 5 to 11 weeks are consistent with the rise in anterior pituitary GH reported previously. Basic quantitative morphological information about age-related changes in somatotrophs is reported here. When combined with additional facts from future work, they may explain the well-documented sharp decline in circulating GH from 5 to 11 weeks.

  2. Using dual laser flow cytometry for monitoring phytoplankton composition and integrity

    SciTech Connect

    Schaefer, H.; Beisker, W.; Steinberg, C.

    1995-12-31

    Dual laser flow cytometry can be used for determining phytoplankton populations in lakes and lowland rivers and streams. Apart from answering basic limnological questions such as the time course of algal blooms or the annual succession of phytoplankton composition further investigations can be made for estimating the integrity of phytoplankton community using biomass distribution spectra. Thus anthropogenic influence such as eutrophication, acidification or effects of xenobiotica can be monitored. Dual laser flow cytometry with excitation wavelengths of 458 and 528 nm was used to measure photosynthesis pigment fluorescence (chlorophyll a (CHLa), Em>665 nm) and phycoerythrin (PE, Em 575 nm) and cell density of phytoplankton organisms in water samples. CHLa is excited directly by 458 nm and by energy transfer from carotenoids (Ex 528 nm). The ratio of the two fluorescence parameters (CFR) allows to identify pigment groups in the phytoplankton population (chlorophytes and euglenophytes from chrysophytes, diatoms and dinophytes). PE-containing cyanophytes and cryptophytes can be detected by their PE fluorescence (Ex 528 nm). As a result of preliminary studies for preparing biomass spectra of phytoplankton communities measurements of protein content by staining with fluorescein isothiocyanate (FITC, ex 488 nm, Em 530 nm) are also shown.

  3. Bead-Based Assays for Biodetection: From Flow-Cytometry to Microfluidics

    SciTech Connect

    Ozanich, Richard M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby J.

    2009-05-04

    ABSTRACT The potential for the use of biological agents by terrorists is a real threat. Two approaches for detection of biological species will be described: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. The methods and automated fluidic systems used for trapping functionalized microbeads will be described. This approach allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive assays. The automated fluidic approach resulted in up to five-fold improvements in assay sensitivity/speed as compared to identical assays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based assays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (> 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100’s of picomolar range (10’s of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach. Video taping magnetic nanoparticle capture and release was used to improve understanding of the process and revealed interesting behavior.

  4. Integrated optical waveguides and inertial focussing microfluidics in silica for microflow cytometry applications

    NASA Astrophysics Data System (ADS)

    Butement, Jonathan T.; Hunt, Hamish C.; Rowe, David J.; Sessions, Neil P.; Clark, Owain; Hua, Ping; Senthil Murugan, G.; Chad, John E.; Wilkinson, James S.

    2016-10-01

    A key challenge in the development of a microflow cytometry platform is the integration of the optical components with the fluidics as this requires compatible micro-optical and microfluidic technologies. In this work a microflow cytometry platform is presented comprising monolithically integrated waveguides and deep microfluidics in a rugged silica chip. Integrated waveguides are used to deliver excitation light to an etched microfluidic channel and also collect transmitted light. The fluidics are designed to employ inertial focussing, a particle positioning technique, to reduce signal variation by bringing the flowing particles onto the same plane as the excitation light beam. A fabrication process is described which exploits microelectronics mass production techniques including: sputtering, ICP etching and PECVD. Example devices were fabricated and the effectiveness of inertial focussing of 5.6 µm fluorescent beads was studied showing lateral and vertical confinement of flowing beads within the microfluidic channel. The fluorescence signals from flowing calibration beads were quantified demonstrating a CV of 26%. Finally the potential of this type of device for measuring the variation in optical transmission from input to output waveguide as beads flowed through the beam was evaluated.

  5. Detection and capture of breast cancer cells with photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Kiran; Goldschmidt, Benjamin S.; Viator, John A.

    2016-08-01

    According to the Centers for Disease Control and Prevention, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis-the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems-significantly worsens the prognosis of any breast cancer patient. A technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser is used to interrogate thousands of blood cells with one pulse as they flow through the beam path. Cells that are optically absorbing, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to enhance optical absorption. After which, the PA cytometry device is calibrated to demonstrate the ability to detect single cells. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25 to 45 breast cancer cells per 1 mL of blood. An in vitro PA flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy but also it can be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

  6. Using dual-laser flow cytometry for monitoring phytoplankton composition and integrity

    NASA Astrophysics Data System (ADS)

    Schaefer, Harald; Beisker, Wolfgang; Steinberg, Christian

    1995-10-01

    Dual laser flow cytometry can be used for determining phytoplankton populations in lakes and lowland rivers and streams. Apart from answering basic limnological questions such as the time course of algal blooms or the annual succession of phytoplankton composition further investigations can be made for estimating the integrity of a phytoplankton community using biomass distribution spectra. Thus anthropogenic influence such as eutrophication, acidification or effects of xenobiotica can be monitored. Dual laser flow cytometry with excitation wavelengths of 458 and 528 nm was used to measure photosynthesis pigment fluorescence (chlorophyll a (CHLa), Em greater than 665 nm) and phycoerythrin (PE, Em 575 nm) and cell density of phytoplankton organisms in water samples. CHLa is excited directly by 458 nm and by energy transfer from carotenoids (Ex 528 nm). The ratio of the two fluorescence parameters (CFR) allows users to identify pigment groups in the phytoplankton population (chlorophytes and euglenophytes from chrysophytes, diatoms and dinophytes). PE- containing cyanophytes and cryptophytes can be detected by their PE fluorescence (Ex 528 nm). As a result of preliminary studies for preparing biomass spectra of phytoplankton communities measurements of protein content by staining with fluorescein isothiocyanate (FITC, Ex 488 nm, Em 530 nm) are also shown.

  7. Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

    PubMed Central

    Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

    2013-01-01

    The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

  8. Determination of aneuploids in hop (Humulus lupulus L.) using flow cytometry.

    PubMed

    Šesek, Predrag; Šuštar-Vozlič, Jelka; Bohanec, Borut

    2000-01-01

    In order to study the possibility that high-resolution flow cytometry can be used for determination of aneuploids, different genotypes of Humulus lupulus were analyzed. Triploid cultivars are bred by hybridization between diploid and tetraploid lines, and as the result of this process, some aneuploids are occasionally also formed. We analyzed eight triploid cultivars and seven putative aneuploids. Triploid cultivars Cerera, Cicero, Celeia, Cekin, Blisk, Mt. Hood, Huller Bit. and Willamette (3x = 30) were measured for nuclear DNA content using Trifolium repens as reference. No significant differences among peak positions of triploid cultivars (having an average CV value per peak of 1.94%) were found. Measurement of nuclear DNA content was also performed for seven lines: 175/75, 89/113, 89/154, 91/215, 175/17, 89/87 and 91/74 previously determined by chromosome counting to be aneuploids (CV per peak was 1.41%). A statistically lower DNA content was found for line 175/75 and higher values were measured for lines 89/154, 89/113 and 91/215. Repeated chromosome counting revealed that the number of chromosomes in line 175/75 was 29, while lines 89/154, 89/113 and 91/215 possessed 31 chromosomes. The other lines were identified as triploids. We conclude that flow cytometry can be efficiently used for determination of aneuploidy in Humulus lupulus.

  9. Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

    2015-03-01

    Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

  10. A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination

    PubMed Central

    van der Gaast—de Jongh, Christa E.; Diavatopoulos, Dimitri A.; de Jonge, Marien I.

    2017-01-01

    The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner. PMID:28288168

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Recent Advances on Multi-Parameter Flow Cytometry to Characterize Antimicrobial Treatments

    PubMed Central

    Léonard, Lucie; Bouarab Chibane, Lynda; Ouled Bouhedda, Balkis; Degraeve, Pascal; Oulahal, Nadia

    2016-01-01

    The investigation on antimicrobial mechanisms is a challenging and crucial issue in the fields of food or clinical microbiology, as it constitutes a prerequisite to the development of new antimicrobial processes or compounds, as well as to anticipate phenomenon of microbial resistance. Nowadays it is accepted that a cells population exposed to a stress can cause the appearance of different cell populations and in particular sub-lethally compromised cells which could be defined as viable but non-culturable (VBNC). Recent advances on flow cytometry (FCM) and especially on multi-parameter flow cytometry (MP-FCM) provide the opportunity to obtain high-speed information at real time on damage at single-cell level. This review gathers MP-FCM methodologies based on individual and simultaneous staining of microbial cells employed to investigate their physiological state following different physical and chemical antimicrobial treatments. Special attention will be paid to recent studies exploiting the possibility to corroborate MP-FCM results with additional techniques (plate counting, microscopy, spectroscopy, molecular biology techniques, membrane modeling) in order to elucidate the antimicrobial mechanism of action of a given antimicrobial treatment or compound. The combination of MP-FCM methodologies with these additional methods is namely a promising and increasingly used approach to give further insight in differences in microbial sub-population evolutions in response to antimicrobial treatments. PMID:27551279

  13. The role of multiparametric flow cytometry in the detection of minimal residual disease in acute leukaemia.

    PubMed

    Lee, Denise; Grigoriadis, George; Westerman, David

    2015-12-01

    Flow cytometry is the most accessible method for minimal residual disease (MRD) detection due to its availability in most haematological centres. Using a precise combination of different antibodies, immunophenotypic detection of MRD in acute leukaemia can be performed by identifying abnormal combinations or expressions of antigens on malignant cells at diagnosis, during and post treatment. These abnormal phenotypes, referred to as leukaemia-associated immunophenotypes (LAIPs) are either absent or expressed at low frequency in normal bone marrow (BM) cells and are used to monitor the behaviour and quantitate the amount of residual disease following treatment. In paediatric acute lymphoblastic leukaemia (ALL), the level of MRD by multiparametric flow cytometry (MPFC) during therapy is recognised as an important predictor of outcome. Although less extensively studied, adult ALL and adult and paediatric acute myeloid leukaemia (AML) have also demonstrated similar findings. The challenge now is incorporating this information for risk-stratification so that therapy can be tailored individually and ultimately improve outcome while also limiting treatment-related toxicity. In this review we will elaborate on the current and future role of MPFC in MRD in acute leukaemia while also addressing its limitations.

  14. Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.

    PubMed

    Menon, Vishal; Thomas, Ria; Ghale, Arun R; Reinhard, Christina; Pruszak, Jan

    2014-12-18

    Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

  15. Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation1

    PubMed Central

    Escoffier, Jessica; Navarrete, Felipe; Haddad, Doug; Santi, Celia M.; Darszon, Alberto; Visconti, Pablo E.

    2015-01-01

    To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K+ channels. PMID:25855261

  16. Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

    PubMed Central

    Wang, F I; Williams, T J; el-Awar, F Y; Pang, V F; Hahn, E C

    1987-01-01

    As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry. PMID:3453262

  17. Optimization of buffer solutions to analyze inflammatory cytokines in gingival crevicular fluid by multiplex flow cytometry

    PubMed Central

    Ríos-Lugo, María-Judith; Martin, Conchita; Alarcón, José-Antonio; Esquifino, Ana; Solano, Patricia; Sanz, Mariano

    2015-01-01

    Objective: the aim of this study was to test two buffer solutions in order to attain a reliable and reproducible analysis of inflammatory cytokines (IL-1β, IL-6, TNF-α, OPG, OPN and OC), in gingival crevicular fluid (GCF) by flow cytometry. Material and Methods: GCF samples from healthy volunteers were collected with perio-paper strips and diluted either in phosphate buffered saline (PBS) or Tris-HCl buffer, with and without protease inhibitors (PI). Cytokine immunoassays were carried out by flow cytometry (Luminex Xmap 200) generating standard curves. Results: standards curves generated with the use of phosphate-buffered saline (PBS) demonstrated best adjustment for cytokines IL-1ß, IL-6 and TNF- α levels, when using Tris-HCl (p<0.05). Conclusions: The use of PBS buffer with the addition of PI provided reliable measurements of inflammatory biomarkers in GCF samples of healthy volunteers. Key words:Curve fitting, flow cytometer, immunoassay buffer, crevicular fluid, cytokines. PMID:24880451

  18. In vitro flow cytometry-based screening platform for cellulase engineering

    PubMed Central

    Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

  19. Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

    PubMed Central

    Pickles, Sarah; Arbour, Nathalie; Vande Velde, Christine

    2014-01-01

    Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver. PMID:25285411

  20. Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L. using flow cytometry.

    PubMed

    Loureiro, J; Pinto, G; Lopes, T; Dolezel, J; Santos, C

    2005-08-01

    Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somatic embryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P< or =0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of "true-to-type" propagation of cork oak using somatic embryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value.

  1. Bead-based assays for biodetection: from flow-cytometry to microfluidics

    NASA Astrophysics Data System (ADS)

    Ozanich, Richard M., Jr.; Antolick, Kathryn; Bruckner-Lea, Cynthia J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby; Warner, Cynthia L.; Warner, Marvin G.

    2009-05-01

    The potential for the use of biological agents by terrorists is a real threat. Two approaches for antibody-based detection of biological species are described in this paper: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. These approaches both involve the use of automated fluidic systems for trapping antibody-functionalized microbeads, which allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive immunoassays. The automated fluidic approach resulted in up to five-fold improvements in immunoassay sensitivity/speed as compared to identical immunoassays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based immunoassays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (>= 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100's of picomolar range (10's of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach.

  2. Enumerating Microorganism Surrogates for Groundwater Transport Studies Using Solid-Phase Cytometry.

    PubMed

    Stevenson, Margaret E; Blaschke, A Paul; Schauer, Sonja; Zessner, Matthias; Sommer, Regina; Farnleitner, Andreas H; Kirschner, Alexander K T

    2014-01-01

    Investigations on the pollution of groundwater with pathogenic microorganisms, e.g. tracer studies for groundwater transport, are constrained by their potential health risk. Thus, microspheres are often used in groundwater transport studies as non-hazardous surrogates for pathogenic microorganisms. Even though pathogenic microorganisms occur at low concentrations in groundwater, current detection methods of microspheres (spectrofluorimetry, flow cytometry and epifluorescence microscopy) have rather high detection limits and are unable to detect rare events. Solid-phase cytometry (SPC) offers the unique capability of reliably quantifying extremely low concentrations of fluorescently labelled microorganisms or microspheres in natural waters, including groundwater. Until now, microspheres have been used in combination with SPC only for instrument calibration purposes and not for environmental applications. In this study, we explored the limits of the SPC methodology for its applicability to groundwater transport studies. The SPC approach proved to be a highly sensitive and reliable enumeration system for microorganism surrogates down to a minimum size of 0.5 μm, in up to 500 ml of groundwater, and 0.75 μm, in up to 1 ml of turbid surface water. Hence, SPC is proposed to be a useful method for enumerating microspheres for groundwater transport studies in the laboratory, as well as in the field when non-toxic, natural products are used.

  3. Deformation of double emulsions under conditions of flow cytometry hydrodynamic focusing.

    PubMed

    Ma, Shaohua; Huck, Wilhelm T S; Balabani, Stavroula

    2015-11-21

    Water-in-oil-in-water (w/o/w) microfluidics double emulsions offer a new route to compartmentalise reagents into isolated aqueous microenvironments while maintaining an aqueous carrier fluid phase; this enables compatibility with commercial flow cytometry systems such as fluorescence-activated cell sorting (FACS). Double emulsion (inner core) deformation under hydrodynamic focusing conditions that mimic the environment double emulsions experience in flow cytometry applications is of particular importance for droplet stability and cell viability. This paper reports on an experimental study of the dynamic deformation of aqueous cores of w/o/w double emulsions under hydrodynamic focusing, with the sheath flow directed at 45° to the sample flow. A number of factors affecting the inner core deformation and recovery were examined. Deformation was found to depend significantly on the core or shell viscosity, the droplet-to-sheath flow velocity ratio, and core and shell sizes. Core deformation was found to depend more on the type of surfactant rather concentration with high molecular weight surfactant exhibiting a negligible effect on deformation whereas low molecular weight surfactant enhancing deformation at low concentrations due to their lateral mobility at the interface.

  4. Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction

    PubMed Central

    Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela

    2016-01-01

    Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascular manifestations. A number of studies highlighted the role of these cells in age-related diseases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentiation markers in healthy subjects and in patients affected by peripheral vascular manifestations associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accurately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vascular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context. PMID:27560136

  5. Analysis of molecular assemblies by flow cytometry: determinants of Gi1 and by binding

    NASA Astrophysics Data System (ADS)

    Sarvazyan, Noune A.; Neubig, Richard R.

    1998-05-01

    We report here a novel application of flow cytometry for the quantitative analysis of the high affinity interaction between membrane proteins both in detergent solutions and when reconstituted into lipid vesicles. The approach is further advanced to permit the analysis of binding to expressed protein complexes in native cell membranes. The G protein heterotrimer signal transduction function links the extracellularly activated transmembrane receptors and intracellular effectors. Upon activation, (alpha) and (beta) (gamma) subunits of G protein undergo a dissociation/association cycle on the cell membrane interface. The binding parameters of solubilized G protein (alpha) and (beta) (gamma) subunits have been defined but little is known quantitatively about their interactions in the membrane. Using a novel flow cytometry approach, the binding of low nanomolar concentrations of fluorescein-labeled G(alpha) i1 (F- (alpha) ) to (beta) (gamma) both in detergent solution and in a lipid environment was quantitatively compared. Unlabeled (beta) $gama reconstituted in biotinylated phospholipid vesicles bound F-(alpha) tightly (Kd 6 - 12 nM) while the affinity for biotinylated-(beta) (gamma) in Lubrol was even higher (Kd of 2.9 nM). The application of this approach to proteins expressed in native cell membranes will advance our understanding of G protein function in context of receptor and effector interaction. More generally, this approach can be applied to study the interaction of any fluorescently labeled protein with a membrane protein which can be expressed in Sf9 plasma membranes.

  6. Alternative flow cytometry strategies to analyze stem cells and cell death in planarians

    PubMed Central

    Peiris, Tanuja Harshani; García‐Ojeda, Marcos E.

    2016-01-01

    Abstract Planarians possess remarkable stem cell populations that continuously support cellular turnover and are instrumental in the regeneration of tissues upon injury. Cellular turnover and tissue regeneration in planarians rely on the proper integration of local and systemic signals that regulate cell proliferation and cell death. Thus, understanding the signals controlling cellular proliferation and cell death in planarians could provide valuable insights for maintenance of adult body homeostasis and the biology of regeneration. Flow cytometry techniques have been utilized widely to identify, isolate, and characterize planarian stem cell populations. We developed alternative flow cytometry strategies that reduce the number of reagents and the time of sample preparation to analyze stem cells and cell death in planarians. The sensitivity of these methods is validated with functional studies using RNA interference and treatment with  γ irradiation or stressful conditions that are known to trigger cell death. Altogether, we provide a community resource intended to minimize adverse effects during ex vivo studies of stem cells and cell death in planarians. PMID:27307993

  7. Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

    PubMed

    Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

    2014-01-01

    The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis.

  8. Evaluating the morphology of erythrocyte population: An approach based on atomic force microscopy and flow cytometry.

    PubMed

    Ghosh, Sayari; Chakraborty, Ishita; Chakraborty, Monojit; Mukhopadhyay, Ashis; Mishra, Raghwendra; Sarkar, Debasish

    2016-04-01

    Erythrocyte morphology is gaining importance as a powerful pathological index in identifying the severity of any blood related disease. However, the existing technique of quantitative microscopy is highly time consuming and prone to personalized bias. On the other hand, relatively unexplored, complementary technique based on flow cytometry has not been standardized till date, particularly due to the lack of a proper morphological scoring scale. In this article, we have presented a new approach to formulate a non-empirical scoring scale based on membrane roughness (R(rms)) data obtained from atomic force microscopy. Subsequently, the respective morphological quantifier of the whole erythrocyte population, commonly known as morphological index, was expressed as a function of highest correlated statistical parameters of scattered signal profiles generated by flow cytometry. Feed forward artificial neural network model with multilayer perceptron architecture was used to develop the intended functional form. High correlation coefficient (R(2) = 0.95), even for model-formulation exclusive samples, clearly indicates the universal validity of the proposed model. Moreover, a direct pathological application of the proposed model has been illustrated in relation to patients, diagnosed to be suffering from a wide variety of cancer.

  9. Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays

    PubMed Central

    de Almeida Santiago, Marta; de Paula Fonseca e Fonseca, Bruna; da Silva Marques, Christiane de Fátima; Domingos da Silva, Edimilson

    2016-01-01

    The use of antibodies in immunodiagnostic kits generally implies the conjugation of these proteins with other molecules such as chromophores or fluorochromes. The development of more sensitive quality control procedures than spectrophotometry is essential to assure the use of better fluorescent conjugates since the fluorescent conjugates are critical reagents for a variety of immunodiagnostic kits. In this article, we demonstrate a new flow cytometric protocol to evaluate conjugates by molecules of equivalent soluble fluorochromes (MESF) and by traditional flow cytometric analysis. We have coupled microspheres with anti-IgG-PE and anti-HBSAg-PE conjugates from distinct manufactures and/or different lots and evaluated by flow cytometry. Their fluorescence intensities were followed for a period of 18 months. Our results showed that there was a great difference in the fluorescence intensities between the conjugates studied. The differences were observed between manufactures and lots from both anti-IgG-PE and anti-HBSAg-PE conjugates. Coefficients of variation (CVs) showed that this parameter can be used to determine better coupling conditions, such as homogenous coupling. The MESF analysis, as well as geometric mean evaluation by traditional flow cytometry, showed a decrease in the values for all conjugates during the study and were indispensable tools to validate the results of stability tests. Our data demonstrated the feasibility of the flow cytometric method as a standard quality control of immunoassay kits. PMID:27936034

  10. NetFCM: a semi-automated web-based method for flow cytometry data analysis.

    PubMed

    Frederiksen, Juliet; Buggert, Marcus; Karlsson, Annika C; Lund, Ole

    2014-11-01

    Multi-parametric flow cytometry (FCM) represents an invaluable instrument to conduct single cell analysis and has significantly increased our understanding of the immune system. However, due to new techniques allowing us to measure an increased number of phenotypes within the immune system, FCM data analysis has become more complex and labor-intensive than previously. We have therefore developed a semi-automatic gating strategy (NetFCM) that uses clustering and principal component analysis (PCA) together with other statistical methods to mimic manual gating approaches. NetFCM is an online tool both for subset identification as well as for quantification of differences between samples. Additionally, NetFCM can classify and cluster samples based on multidimensional data. We tested the method using a data set of peripheral blood mononuclear cells collected from 23 HIV-infected individuals, which were stimulated with overlapping HIV Gag-p55 and CMV-pp65 peptides or medium alone (negative control). NetFCM clustered the virus-specific CD8+ T cells based on IFNγ and TNF responses into distinct compartments. Additionally, NetFCM was capable of identifying HIV- and CMV-specific responses corresponding to those obtained by manual gating strategies. These data demonstrate that NetFCM has the potential to identify relevant T cell populations by mimicking classical FCM data analysis and reduce the subjectivity and amount of time associated with such analysis. © 2014 International Society for Advancement of Cytometry.

  11. A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

    PubMed Central

    García-García, Erick; Uribe-Querol, Eileen; Rosales, Carlos

    2013-01-01

    Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types. PMID:23603868

  12. Minimal residual disease monitoring by 8-color flow cytometry in mantle cell lymphoma: an EU-MCL and LYSA study

    PubMed Central

    Cheminant, Morgane; Derrieux, Coralie; Touzart, Aurore; Schmit, Stéphanie; Grenier, Adrien; Trinquand, Amélie; Delfau-Larue, Marie-Hélène; Lhermitte, Ludovic; Thieblemont, Catherine; Ribrag, Vincent; Cheze, Stéphane; Sanhes, Laurence; Jardin, Fabrice; Lefrère, François; Delarue, Richard; Hoster, Eva; Dreyling, Martin; Asnafi, Vahid; Hermine, Olivier; Macintyre, Elizabeth

    2016-01-01

    Quantification of minimal residual disease may guide therapeutic strategies in mantle cell lymphoma. While multiparameter flow cytometry is used for diagnosis, the gold standard method for minimal residual disease analysis is real-time quantitative polymerase chain reaction (RQ-PCR). In this European Mantle Cell Lymphoma network (EU-MCL) pilot study, we compared flow cytometry with RQ-PCR for minimal residual disease detection. Of 113 patients with at least one minimal residual disease sample, RQ-PCR was applicable in 97 (86%). A total of 284 minimal residual disease samples from 61 patients were analyzed in parallel by flow cytometry and RQ-PCR. A single, 8-color, 10-antibody flow cytometry tube allowed specific minimal residual disease assessment in all patients, with a robust sensitivity of 0.01%. Using this cut-off level, the true-positive-rate of flow cytometry with respect to RQ-PCR was 80%, whereas the true-negative-rate was 92%. As expected, RQ-PCR frequently detected positivity below this 0.01% threshold, which is insufficiently sensitive for prognostic evaluation and would ideally be replaced with robust quantification down to a 0.001% (10-5) threshold. In 10 relapsing patients, the transition from negative to positive by RQ-PCR (median 22.5 months before relapse) nearly always preceded transition by flow cytometry (4.5 months), but transition to RQ-PCR positivity above 0.01% (5 months) was simultaneous. Pre-emptive rituximab treatment of 2 patients at minimal residual disease relapse allowed re-establishment of molecular and phenotypic complete remission. Flow cytometry minimal residual disease is a complementary approach to RQ-PCR and a promising tool in individual mantle cell lymphoma therapeutic management. PMID:26703963

  13. Minimal residual disease monitoring by 8-color flow cytometry in mantle cell lymphoma: an EU-MCL and LYSA study.

    PubMed

    Cheminant, Morgane; Derrieux, Coralie; Touzart, Aurore; Schmit, Stéphanie; Grenier, Adrien; Trinquand, Amélie; Delfau-Larue, Marie-Hélène; Lhermitte, Ludovic; Thieblemont, Catherine; Ribrag, Vincent; Cheze, Stéphane; Sanhes, Laurence; Jardin, Fabrice; Lefrère, François; Delarue, Richard; Hoster, Eva; Dreyling, Martin; Asnafi, Vahid; Hermine, Olivier; Macintyre, Elizabeth

    2016-03-01

    Quantification of minimal residual disease may guide therapeutic strategies in mantle cell lymphoma. While multiparameter flow cytometry is used for diagnosis, the gold standard method for minimal residual disease analysis is real-time quantitative polymerase chain reaction (RQ-PCR). In this European Mantle Cell Lymphoma network (EU-MCL) pilot study, we compared flow cytometry with RQ-PCR for minimal residual disease detection. Of 113 patients with at least one minimal residual disease sample, RQ-PCR was applicable in 97 (86%). A total of 284 minimal residual disease samples from 61 patients were analyzed in parallel by flow cytometry and RQ-PCR. A single, 8-color, 10-antibody flow cytometry tube allowed specific minimal residual disease assessment in all patients, with a robust sensitivity of 0.01%. Using this cut-off level, the true-positive-rate of flow cytometry with respect to RQ-PCR was 80%, whereas the true-negative-rate was 92%. As expected, RQ-PCR frequently detected positivity below this 0.01% threshold, which is insufficiently sensitive for prognostic evaluation and would ideally be replaced with robust quantification down to a 0.001% (10-5) threshold. In 10 relapsing patients, the transition from negative to positive by RQ-PCR (median 22.5 months before relapse) nearly always preceded transition by flow cytometry (4.5 months), but transition to RQ-PCR positivity above 0.01% (5 months) was simultaneous. Pre-emptive rituximab treatment of 2 patients at minimal residual disease relapse allowed re-establishment of molecular and phenotypic complete remission. Flow cytometry minimal residual disease is a complementary approach to RQ-PCR and a promising tool in individual mantle cell lymphoma therapeutic management. (clinicaltrials identifiers: 00209209 and 00209222).

  14. Molecular biology and cytopathology. Principles and applications.

    PubMed

    Schmitt, Fernando C; Vielh, Philippe

    2012-12-01

    Some of the main applications of molecular techniques using cellular materials obtained from tumors by means of non-gynecological exfoliative cytology or fine-needle aspiration are briefly described in this review.

  15. Pulmonary Hamartoma Mimicking Malignancy: A Cytopathological Diagnosis

    PubMed Central

    Kishore, Manjari; Preeti; Deepak, Desh

    2016-01-01

    Pulmonary Hamartomas (PH) are benign tumour-like lesions of lung with an uncommon occurrence and pose a diagnostic challenge on chest radiograph. Fine Needle Aspiration Cytology (FNAC) can lead to a definitive diagnosis as well as distinguishes these from malignant lung mass. Most of the patients are asymptomatic and incidentally detected on routine chest radiographs. We report a case of pulmonary hamartoma where the patient was symptomatic and a possibility of malignant neoplasm was considered until the FNAC concluded the diagnosis. PMID:28050379

  16. 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

    PubMed Central

    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 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. PMID:26267664

  17. Femtosecond laser pulse optimization for multiphoton cytometry and control of fluorescence

    NASA Astrophysics Data System (ADS)

    Tkaczyk, Eric Robert

    This body of work encompasses optimization of near infrared femtosecond laser pulses both for enhancement of flow cytometry as well as adaptive pulse shaping to control fluorescence. A two-photon system for in vivo flow cytometry is demonstrated, which allows noninvasive quantification of circulating cell populations in a single live mouse. We monitor fluorescently-labeled red blood cells for more than two weeks, and are also able to noninvasively measure circulation times of two distinct populations of breast cancer cells simultaneously in a single mouse. We build a custom laser excitation source in the form of an extended cavity mode-locked oscillator, which enables superior detection in whole blood or saline of cell lines expressing fluorescent proteins including the green fluorescent protein (GFP), tdTomato and mPlum. A mathematical model explains unique features of the signals. The ability to distinguish different fluorescent species is central to simultaneous measurement of multiple molecular targets in high throughput applications including the multiphoton flow cytometer. We demonstrate that two dyes which are not distinguishable to one-photon measurements can be differentiated and in fact quantified in mixture via phase-shaped two-photon excitation pulses found by a genetic algorithm. We also selectively enhance or suppress two-photon fluorescence of numerous common dyes with tailored pulse shapes. Using a multiplicative (rather than ratiometric) fitness parameter, we are able to control the fluorescence while maintaining a strong signal. With this method, we control the two-photon fluorescence of the blue fluorescent protein (BFP), which is of particular interest in investigations of protein-protein interactions, and has frustrated previous attempts of control. Implementing an acousto-optic interferometer, we use the same experimental setup to measure two-photon excitation cross-sections of dyes and prove that photon-photon interferences are the

  18. Flow cytometry methods for the study of cell-cycle parameters of planarian stem cells.

    PubMed

    Kang, Hara; Sánchez Alvarado, Alejandro

    2009-05-01

    Due to their characteristic inaccessibility and low numbers, little is known about the cell-cycle dynamics of most stem cells in vivo. A powerful, established methodology to study cell-cycle dynamics is flow cytometry, which is used routinely to study the cell-cycle dynamics of proliferating cells in vitro. Its use in heterogeneous mixtures of cells obtained from whole animals, however, is complicated by the relatively low abundance of cycling to non-cycling cells. We report on flow cytometric methods that take advantage of the abundance of proliferating stem cells in the planarian Schmidtea mediterranea. The optimized protocols allow us to measure cell-cycle dynamics and follow BrdU-labeled cells specifically in complex mixtures of cells. These methods expand on the growing toolkit being developed to study stem cell biology in planarians, and open the door to detailed cytometric studies of a collectively totipotent population of adult stem cells in vivo.

  19. [Multi-parametric Flow Cytometry for Neuroblastoma, a new and possible diagnostic tool: case report].

    PubMed

    Manrique, Belén; López Marti, Jessica; Cacciavillano, Walter; Rossi, Jorge

    2016-04-01

    Neuroblastoma is the most frequent extracranial solid tumor in childhood, representing 5.6% according to the "Registro Oncopediatrico Hospitalario Argentino". For its diagnosis, several complementary methods (radiological, biological and biochemical) are required, and Multi-parametric Flow Cytometry (MFC) arises as a potential diagnostic method, despite not having been so far extensively explored. MFC is a method that allows to obtain several information about size, internal complexity and antigenic expression by the use of a laser and fluorescent monoclonal antibodies. There are an increasing number of reports in the literature, which reveal the importance of using MFC for diagnosis and monitoring of solid tumors. The aim in this presentation is to highlight the fundamental role that MFC had in the case of a patient affected by neuroblastoma, in which an early diagnosis using this methodology allowed prompt administration of adequate treatment.

  20. A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity

    PubMed Central

    O’Brien-Simpson, Neil M.; Pantarat, Namfon; Attard, Troy J.; Walsh, Katrina A.; Reynolds, Eric C.

    2016-01-01

    We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy. PMID:26986223

  1. Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes.

    PubMed

    Tracy, Bryan P; Gaida, Stefan M; Papoutsakis, Eleftherios T

    2010-02-01

    Flow cytometry (FC) and FC-based cell sorting have been established as critical tools in modern cell and developmental biology. Yet, their applications in bacteria, especially in the multiparametric mode, remain limited. We argue that FC technologies have the potential to greatly accelerate the analysis and development of microbial complex phenotypes through applications of metabolic engineering, synthetic biology, and evolutionary engineering. We demonstrate the importance of FC for elucidating population heterogeneity because of developmental processes or epigenetic regulation. FC can be engaged for both synthetic and analytical applications of complex phenotypes within a single species, multispecies, and microbial-library populations. Examples include methods to identify developmental microbial stages associated with productive metabolic phenotypes, select desirable promoters from a single species or metagenomic libraries, and to screen designer riboswitches for synthetic-biology applications.

  2. CASP3 protein expression by flow cytometry in Down's syndrome subjects.

    PubMed

    Salemi, Michele; Condorelli, Rosita A; Romano, Corrado; Concetta, Barone; Romano, Carmelo; Salluzzo, Maria Grazia; Bosco, Paolo; Calogero, Aldo E

    2014-01-01

    Down's syndrome (DS), the most common chromosomal disorder, is caused by 21 trisomy and is featured by intellectual disability. Subjects with DS can develop some traits of Alzheimer disease (AD) at an earlier age than subjects without trisomy 21. Apoptosis is a programmed cell death process under both normal physiological and pathological conditions. Caspase-3 (CASP3) plays an important role in neuronal death during nervous system development and under certain pathological conditions. Furthermore, in vitro and in vivo studies report elevated expression and activation of CASP3 in models of AD. On this account, the expression of CASP3 gene was evaluated in cultures of fibroblasts of DS and normal subjects by flow cytometry. CASP3 protein was up-regulated in fibroblasts of DS. The data obtained from this study strengthen the hypothesis that the over-expression of CASP3 gene could have a role in the activation of the apoptotic pathways acting in the neurodegenerative processes in DS.

  3. Flow cytometry evidence of human granulocytes interaction with polyhedral oligomeric silsesquioxanes: effect of nanoparticle charge

    NASA Astrophysics Data System (ADS)

    Renò, Filippo; Carniato, Fabio; Rizzi, Manuela; Olivero, Francesco; Pittarella, Pamela; Marchese, Leonardo

    2013-05-01

    Nanoparticles (NPs) entering the human body are immediately confronted with the innate part of human immune system. In particular, monocyte and neutrophil granulocytes readily clear particles by phagocytosis, even if in the case of NPs the uptake mechanism may be classified as macropinocytosis. Among engineered nanoparticles, in the last years, siliceous materials have emerged as promising materials for several applications ranging from catalysis to biomedical. The polyhedral oligomeric silsesquioxanes (POSS) are nanodimensional, easily synthesizable molecular compounds and POSS-based systems are promising carriers for biological molecules. In this work, the ability of human granulocytes to uptake positively and negatively charged POSS was measured using a simple flow cytometry analysis based on cell size modifications. The data obtained showed that after a 30 min exposure only positive NPs were uptaken by human granulocyte using both macropinocytosis and clathrin-mediated mechanisms as demonstrated by uptake inhibition mediated by amiloride and chlorpromazine.

  4. Impact of recent innovations in the use of mass cytometry in support of drug development

    PubMed Central

    Ogura, Hideki; Wisnewski, Adam V.

    2015-01-01

    Cytometry by time-of-flight (CyTOF) is a novel technology for the real-time analysis of single cells. CyTOF is a significant advance in fields including immunology, hematology, and oncology. It resolves multiple metal-conjugated probes per cell with minimal signal overlap, which maximizes the information obtained from each individual sample. CyTOF provides the ability to phenotypically and functionally profile cells from normal and diseased states. Single cell technologies enable researchers to measure the effects of a drug at the single cell level and better understand its mechanism of action. Here, we discuss novel instruments for the analysis of individual biological cells, the impact of recent innovations in support of drug development, and the important roles of CyTOF in drug profiling. PMID:26092491

  5. BeadCons: detection of nucleic acid sequences by flow cytometry.

    PubMed

    Horejsh, Douglas; Martini, Federico; Capobianchi, Maria Rosaria

    2005-11-01

    Molecular beacons are single-stranded nucleic acid structures with a terminal fluorophore and a distal, terminal quencher. These molecules are typically used in real-time PCR assays, but have also been conjugated with solid matrices. This unit describes protocols related to molecular beacon-conjugated beads (BeadCons), whose specific hybridization with complementary target sequences can be resolved by cytometry. Assay sensitivity is achieved through the concentration of fluorescence signal on discrete particles. By using molecular beacons with different fluorophores and microspheres of different sizes, it is possible to construct a fluid array system with each bead corresponding to a specific target nucleic acid. Methods are presented for the design, construction, and use of BeadCons for the specific, multiplexed detection of unlabeled nucleic acids in solution. The use of bead-based detection methods will likely lead to the design of new multiplex molecular diagnostic tools.

  6. Flow cytometry analysis of drug transport mechanisms in Haemonchus contortus susceptible or resistant to anthelmintics.

    PubMed

    Kerboeuf, D; Chambrier, P; Le Vern, Y; Aycardi, J

    1999-02-01

    The role of membrane drug-transport mechanisms in resistance to anthelmintics was examined using a flow cytometry method. This method was adapted from assays developed for the study of similar mechanisms in tumor cells. Rhodamine 123, a P-glycoprotein transport probe, associated with the reversal agent verapamil gave a significantly higher level of green fluorescence in Haemonchus contortus-resistant eggs as compared with that of susceptible eggs. In the same way, verapamilbodipy, a new fluorescent probe for the detection of multidrug resistance in cells, showed a significantly higher degree of binding to resistant eggs. The results confirm those obtained with biological drug assays using both anthelmintics and verapamil and provide a quantitative and effective methodology for the functional study of multidrug resistance in nematodes.

  7. Assessment of immune parameters of manila clam Ruditapes philippinarum in different physiological conditions using flow cytometry

    NASA Astrophysics Data System (ADS)

    Park, Kyung-Il; Donaghy, Ludovic; Kang, Hyun-Sil; Hong, Hyun-Ki; Kim, Young-Ok; Choi, Kwang-Sik

    2012-03-01

    Cellular and humoral immune parameters are often used as biomarkers to trace environmental and physiological stresses in marine bivalves. In this study, we compared various immune parameters of Manila clams ( Ruditapes philippinarum) under normal conditions and under a high level of desiccation, using flow cytometry. The immune parameters analyzed included, total hemocyte count, hemocyte mortality, hemocyte DNA damage, reactive oxygen species (ROS) production, and phagocytosis activity. Total hemocyte count, hemocyte DNA damage, and hemocyte mortality were significantly elevated among clams under high desiccation stress, while phagocytosis activity and spontaneous ROS production were significantly lower compared to those parameters of the control clams ( p<0.05). These data suggest that the immune parameters analyzed in this study well reflect the physiological status of clams.

  8. Monitoring of Dynamic Microbiological Processes Using Real-Time Flow Cytometry

    PubMed Central

    Arnoldini, Markus; Heck, Tobias; Blanco-Fernández, Alfonso; Hammes, Frederik

    2013-01-01

    We describe a straightforward approach to continuously monitor a variety of highly dynamic microbiological processes in millisecond resolution with flow cytometry, using standard bench-top instrumentation. Four main experimental examples are provided, namely: (1) green fluorescent protein expression by antibiotic-stressed Escherichia coli, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary E. coli cells inoculated into fresh growth media, and (4) oxidative disinfection of a mixed culture of auto-fluorescent microorganisms. These examples demonstrate the broad applicability of the method to diverse biological experiments, showing that it allows the collection of detailed, time-resolved information on complex processes. PMID:24244624

  9. Rapid flow cytometry analysis of antimicrobial properties of nettle powder and cranberry powder

    NASA Astrophysics Data System (ADS)

    Hattuniemi, Maarit; Korhonen, Johanna; Jaakkola, Mari; Räty, Jarkko; Virtanen, Vesa

    2010-11-01

    Both nettle (Urtica dioica) and cranberry (Vaccinium oxycoccus) are widely known to have good influence on health. The aim of this study was to investigate antimicrobial properties of nettle powder and cranberry powder against Escherichia coli (E. coli) and monitor the growth of the bacteria by a rapid flow cytometry (FCM) method. For FCM measurements samples were stained with fluorescent dyes. The inhibitory effects of plant material on growth of E. coli were estimated by comparing the results of control sample (E. coli) to E. coli samples with plant material. FCM offers both a brilliant tool to investigate the kinetics of the growth of bacterium, since subsamples can be taken from the same liquid medium during the growing period and with fluorescent dyes a rapid method to investigate viability of the bacterium.

  10. Artificial neural network study of whole-cell bacterial bioreporter response determined using fluorescence flow cytometry.

    PubMed

    Busam, Sirisha; McNabb, Maia; Wackwitz, Anke; Senevirathna, Wasana; Beggah, Siham; Meer, Jan Roelof van der; Wells, Mona; Breuer, Uta; Harms, Hauke

    2007-12-01

    Genetically engineered bioreporters are an excellent complement to traditional methods of chemical analysis. The application of fluorescence flow cytometry to detection of bioreporter response enables rapid and efficient characterization of bacterial bioreporter population response on a single-cell basis. In the present study, intrapopulation response variability was used to obtain higher analytical sensitivity and precision. We have analyzed flow cytometric data for an arsenic-sensitive bacterial bioreporter using an artificial neural network-based adaptive clustering approach (a single-layer perceptron model). Results for this approach are far superior to other methods that we have applied to this fluorescent bioreporter (e.g., the arsenic detection limit is 0.01 microM, substantially lower than for other detection methods/algorithms). The approach is highly efficient computationally and can be implemented on a real-time basis, thus having potential for future development of high-throughput screening applications.

  11. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons.

    PubMed

    Custer, T W; Bickham, J W; Lyne, T B; Lewis, T; Ruedas, L A; Custer, C M; Melancon, M J

    1994-08-01

    The flow cytometry methods (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a side in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. The CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

  12. Quality control in the application of flow cytometry to studies of environmentally-induced genetic damage

    SciTech Connect

    McCreedy, C.D.; Robinson, J.P.; Dallas, C.E.; Jagoe, C.H.

    1999-07-01

    Flow cytometry (FCM) has been used to demonstrate altered DNA content in fish, reptiles, birds and mammals exposed to radionuclides, PAHs and other contaminants. However, artifacts resulting from sample preparation, handling, variations in instrument parameters or other factors may confound such measurements. Some artifacts resemble genotoxic responses and so could lead to erroneous positive conclusions. As part of ongoing studies of effects of various pollutants on DNA content in fishes, the authors tested sample handling and preparation methods for the induction of artifacts. The authors describe QA/QC methods, including control of staining, conditions, doublet discrimination by comparison of peak versus integral fluorescence, internal DNA standards, and the use of time versus fluorescence plots. Consistent application of these practices is essential to obtain valid measurements of DNA content in environmental samples, and neglect of these can result in poor quality data and the acceptance of incorrect hypotheses.

  13. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons

    USGS Publications Warehouse

    Custer, T.W.; Bickham, J.W.; Lyne, T.B.; Lewis, T.; Ruedas, L.A.; Custer, Christine M.; Melancon, M.J.

    1994-01-01

    The flow cytometry method (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a site in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. The CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

  14. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons

    USGS Publications Warehouse

    Custer, T.W.; Bickham, J.W.; Lyne, T.B.; Lewis, T.; Ruedas, L.A.; Custer, Christine M.; Melancon, M.J.

    1994-01-01

    The flow cytometry method (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a site in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. the CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

  15. FRET and Flow Cytometry Assays to Measure Proteopathic Seeding Activity in Biological Samples.

    PubMed

    Furman, Jennifer L; Diamond, Marc I

    2017-01-01

    Transcellular propagation of protein aggregates-or seeds-is increasingly implicated as a mechanism for disease progression in many neurodegenerative disorders, including Alzheimer's disease and the related tauopathies. While neuropathology generally originates in one discrete brain region, pathology progresses as disease severity advances, often along discrete neural networks. The stereotypical spread of tau pathology suggests that cell-to-cell transfer of toxic protein aggregates could underlie disease progression, and recent studies implicate seeding as a proximal marker of disease, as compared to standard histological and biochemical analyses. Commonly used metrics for protein aggregation detection, however, lack sensitivity, are not quantitative, and/or undergo subjective classification. Here, we describe a FRET and flow cytometry cell-based assay that allows for rapid and quantitative detection of protein aggregates from human and rodent biological specimens.

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

    PubMed

    Chesney, Alden; Good, David; Reis, Marciano

    2011-01-01

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

  17. Hierarchical modeling for rare event detection and cell subset alignment across flow cytometry samples.

    PubMed

    Cron, Andrew; Gouttefangeas, Cécile; Frelinger, Jacob; Lin, Lin; Singh, Satwinder K; Britten, Cedrik M; Welters, Marij J P; van der Burg, Sjoerd H; West, Mike; Chan, Cliburn

    2013-01-01

    Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less). Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to id