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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. Diagnosis of leptomeningeal disease in diffuse large B-cell lymphomas of the central nervous system by flow cytometry and cytopathology.

    PubMed

    Schroers, Roland; Baraniskin, Alexander; Heute, Christoph; Vorgerd, Matthias; Brunn, Anna; Kuhnhenn, Jan; Kowoll, Annika; Alekseyev, Andriy; Schmiegel, Wolff; Schlegel, Uwe; Deckert, Martina; Pels, Hendrik

    2010-12-01

    Reliable detection of leptomeningeal disease has the potential of facilitating the diagnosis of central nervous system (CNS) lymphoma and is important for therapeutic considerations. Currently, the standard diagnostic procedure for the detection of lymphoma in the cerebrospinal fluid is cytopathology. To improve the limited specificity and sensitivity of cytopathology, flow cytometry has been suggested as an alternative. Here, we evaluated multi-parameter flow cytometry in combination with conventional cytopathology in cerebrospinal fluid (CSF) samples from 30 patients with primary CNS lymphoma and seven patients with secondary CNS lymphoma. Overall, in 11 of 37 (29.7%) patients with CNS lymphoma, lymphoma cells were detected in CSF by flow cytometry, while cytopathology was less sensitive displaying unequivocally malignant CSF cells in only seven of all 37 (18.9%) patients. Six (16.2%) patients showed cytopathological results suspicious of lymphoma; however, in only one of these patients, the diagnosis of CSF lymphoma cells could be confirmed by flow cytometry. In primary CNS lymphomas (PCNSL), seven of 30 (23.3%) patients were positive for CSF lymphoma cells in flow cytometry, in contrast to four (13.3%) patients with PCNSL with definitely positive cytopathology. In summary, our results suggest that multi-parameter flow cytometry increases the sensitivity and specificity of leptomeningeal disease detection in CNS lymphomas. Both methods should be applied concurrently for complementary diagnostic assessment in patients with CNS lymphoma. © 2010 John Wiley & Sons A/S.

  5. 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.

  6. 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.

  7. Cytopathology whole slide images and adaptive tutorials for senior medical students: a randomized crossover trial.

    PubMed

    Van Es, Simone L; Kumar, Rakesh K; Pryor, Wendy M; Salisbury, Elizabeth L; Velan, Gary M

    2016-01-08

    Diagnostic cytopathology is an essential part of clinical decision-making. However, due to a combination of factors including curriculum reform and shortage of pathologists to teach introductory cytopathology, this area of pathology receives little or no formal attention in most medical school curricula. We have previously described the successful use of efficient and effective digital learning resources, including whole slide images (WSI) and virtual microscopy adaptive tutorials (VMATs), to teach cytopathology to pathology specialist trainees - a group that had prior exposure to cytopathology in their day to day practice. Consequently, in the current study we attempted to demonstrate the efficiency and efficacy of this eLearning resource in a cohort of senior medical students that was completely naïve to the subject matter (cytopathology). We evaluated both the quantitative and qualitative impact of these digital educational materials for learning cytopathology compared with existing resources (e-textbooks and online atlases). The senior medical students were recruited from The University of New South Wales Australia for a randomized cross-over trial. Online assessments, administered after each arm of the trial, contained questions which related directly to a whole slide image. Two categories of questions in the assessments (focusing on either diagnosis or identification of cellular features) were utilized to determine efficacy. User experience and perceptions of efficiency were evaluated using online questionnaires containing Likert scale items and open-ended questions. For this cohort of senior medical students, virtual microscopy adaptive tutorials (VMATs) proved to be at least as effective as existing digital resources for learning cytopathology. Importantly, virtual microscopy adaptive tutorials had superior efficacy in facilitating accurate diagnosis on whole slide images. Student perceptions of VMATs were positive, particularly regarding the immediate

  8. Cytopathology whole slide images and adaptive tutorials for postgraduate pathology trainees: a randomized crossover trial.

    PubMed

    Van Es, Simone L; Kumar, Rakesh K; Pryor, Wendy M; Salisbury, Elizabeth L; Velan, Gary M

    2015-09-01

    To determine whether cytopathology whole slide images and virtual microscopy adaptive tutorials aid learning by postgraduate trainees, we designed a randomized crossover trial to evaluate the quantitative and qualitative impact of whole slide images and virtual microscopy adaptive tutorials compared with traditional glass slide and textbook methods of learning cytopathology. Forty-three anatomical pathology registrars were recruited from Australia, New Zealand, and Malaysia. Online assessments were used to determine efficacy, whereas user experience and perceptions of efficiency were evaluated using online Likert scales and open-ended questions. Outcomes of online assessments indicated that, with respect to performance, learning with whole slide images and virtual microscopy adaptive tutorials was equivalent to using traditional methods. High-impact learning, efficiency, and equity of learning from virtual microscopy adaptive tutorials were strong themes identified in open-ended responses. Participants raised concern about the lack of z-axis capability in the cytopathology whole slide images, suggesting that delivery of z-stacked whole slide images online may be important for future educational development. In this trial, learning cytopathology with whole slide images and virtual microscopy adaptive tutorials was found to be as effective as and perceived as more efficient than learning from glass slides and textbooks. The use of whole slide images and virtual microscopy adaptive tutorials has the potential to provide equitable access to effective learning from teaching material of consistently high quality. It also has broader implications for continuing professional development and maintenance of competence and quality assurance in specialist practice. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. 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.

  10. Quantitative Functional Morphology by Imaging Flow Cytometry.

    PubMed

    Vorobjev, Ivan A; Barteneva, Natasha S

    2016-01-01

    This chapter describes advantages and limitations of imaging flow cytometry (IFC) based on Imagestream instrumentation using a hybrid approach of morphometric measurement and quantitation of multiparametric fluorescent intensities' distribution in cells and particles. Brief comparison is given of IFC with conventional flow cytometry and fluorescent microscopy. Some future directions of the IFC technology are described and discussed.

  11. 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

  12. Diffusion tensor imaging with direct cytopathological validation: characterisation of decorin treatment in experimental juvenile communicating hydrocephalus.

    PubMed

    Aojula, Anuriti; Botfield, Hannah; McAllister, James Patterson; Gonzalez, Ana Maria; Abdullah, Osama; Logan, Ann; Sinclair, Alexandra

    2016-05-31

    In an effort to develop novel treatments for communicating hydrocephalus, we have shown previously that the transforming growth factor-β antagonist, decorin, inhibits subarachnoid fibrosis mediated ventriculomegaly; however decorin's ability to prevent cerebral cytopathology in communicating hydrocephalus has not been fully examined. Furthermore, the capacity for diffusion tensor imaging to act as a proxy measure of cerebral pathology in multiple sclerosis and spinal cord injury has recently been demonstrated. However, the use of diffusion tensor imaging to investigate cytopathological changes in communicating hydrocephalus is yet to occur. Hence, this study aimed to determine whether decorin treatment influences alterations in diffusion tensor imaging parameters and cytopathology in experimental communicating hydrocephalus. Moreover, the study also explored whether diffusion tensor imaging parameters correlate with cellular pathology in communicating hydrocephalus. Accordingly, communicating hydrocephalus was induced by injecting kaolin into the basal cisterns in 3-week old rats followed immediately by 14 days of continuous intraventricular delivery of either human recombinant decorin (n = 5) or vehicle (n = 6). Four rats remained as intact controls and a further four rats served as kaolin only controls. At 14-days post-kaolin, just prior to sacrifice, routine magnetic resonance imaging and magnetic resonance diffusion tensor imaging was conducted and the mean diffusivity, fractional anisotropy, radial and axial diffusivity of seven cerebral regions were assessed by voxel-based analysis in the corpus callosum, periventricular white matter, caudal internal capsule, CA1 hippocampus, and outer and inner parietal cortex. Myelin integrity, gliosis and aquaporin-4 levels were evaluated by post-mortem immunohistochemistry in the CA3 hippocampus and in the caudal brain of the same cerebral structures analysed by diffusion tensor imaging. Decorin significantly

  13. 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

  14. 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.

  15. Untutored discrimination training on paired cell images influences visual learning in cytopathology.

    PubMed

    Evered, Andrew; Walker, Darren; Watt, Andrew A; Perham, Nick

    2014-03-01

    Cytologists must learn how to discriminate cells that might be visually very similar but have different neoplastic potential. The mechanism by which trainees learn this task is poorly researched and is the focus of the current investigation. Cognitive science offers a theoretical platform from which to design meaningful experiments that could lead to novel training strategies. The interpretation of a cell image is a category-discrimination task, and the process by which discrimination improves with practice is called perceptual learning. The study authors operationalized this concept by training 150 naive observers on paired cell images without providing explicit tuition, employing cervical cytology as a model system. Six strategies were tested, which differed according to the diagnostic category and level of interpretive difficulty of each image. Participants were tested before and after training to determine the extent to which visual learning had occurred. Diagnostic accuracy improved for participants who were trained on normal/abnormal image pairs in which at least one member of the pair was "easy" to interpret (P < .05). Training was not effective when image pairs were drawn from the same diagnostic category or when both members of the pair were "difficult" to interpret (P > .05). Training on paired cell images without explicit tuition can be an efficient and effective means of visual learning in cytopathology, but only if care is taken to avoid image pairs from category boundaries. Training on same-category image pairs is ineffective. This study is a step toward the development of perceptual learning modules for cytopathology. © 2013 American Cancer Society.

  16. 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.

  17. 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.

  18. 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

  19. [Image cytometry appliance in chronic tonsillitis].

    PubMed

    Zabel-Olejnik, Joanna; Grzegorowski, Michał; Warchoł, Jerzy

    2003-01-01

    Enlargement of palatinar tonsils in the course of chronic inflammatory process has been thought till now to reflect pronounced lymphocyte proliferation even if such mechanism fails to explain the persistent increase in size of the organ. The studies were performed on 92 tonsils obtained from 4 to 16 years old children in whom the tonsils were removed due to chronic inflammatory processes in upper respiratory tract or in ears. Cell proliferation and apoptosis were evaluated by flow cytometry and by image analysis. The data show that apoptosis in the tonsils is accompanying lymphocyte proliferation in the course of chronic inflammatory process. Evaluation of homeostasis in palatine tonsils could be helpful in referring children for tonsillectomy. Imagine analysis may objectivize and specify diagnosis of chronic tonsillitis.

  20. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Moving pictures: imaging flow cytometry for drug development.

    PubMed

    Elliott, Gary S

    2009-11-01

    As technologies for high throughput and high content screening continue to evolve, new platforms for quantitative cellular imaging will play an increasingly important role in identifying and profiling lead compounds. To gain insight into the effects of a compound on cell morphology or intracellular events, it is necessary to have quality images and the ability to enumerate thousands of data points for statistical relevance. Imaging flow cytometry combines many of the features of flow cytometry, microscopy and imaging as well as a number of unique characteristics. The result is an instrument capable of highly quantitative analysis of cellular behaviors such as receptor internalization, phagocytosis, cell-cell communication, apoptosis and nuclear translocation. This promising new technology and unique type of flow cytometry provides enhanced capabilities for highly multiplexed assays. Here, we review the capabilities of the ImageStream imaging cytometer and discuss several applications relevant to compound screening and profiling.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. Lensfree holographic imaging for on-chip cytometry and diagnostics†

    PubMed Central

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

    2014-01-01

    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 ~2 µm 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. PMID:19255659

  9. 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.

  10. Imaging cytometry in a plastic ultra-mobile system

    NASA Astrophysics Data System (ADS)

    Martínez Vázquez, R.; Trotta, G.; Paturzo, M.; Volpe, A.; Bernava, G.; Basile, V.; Ancona, A.; Ferraro, P.; Fassi, I.; Osellame, R.

    2017-03-01

    We present a cost-effective and highly-portable plastic prototype that can be interfaced with a cell phone to implement an optofluidic imaging cytometry platform. It is based on a PMMA microfluidic chip that fits inside an opto-mechanical platform fabricated by a 3D printer. The fluorescence excitation and imaging is performed using the LED and the CMOS from the cell phone increasing the compactness of the system. A custom developed application is used to analyze the images and provide a value of particle concentration.

  11. 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

  12. Optofluidic Fluorescent Imaging Cytometry on a Cell Phone

    PubMed Central

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

    2012-01-01

    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

  13. Framework for morphometric classification of cells in imaging flow cytometry.

    PubMed

    Gopakumar, G; Jagannadh, Veerendra Kalyan; Gorthi, Sai Siva; Subrahmanyam, Gorthi R K Sai

    2016-03-01

    Imaging flow cytometry is an emerging technology that combines the statistical power of flow cytometry with spatial and quantitative morphology of digital microscopy. It allows high-throughput imaging of cells with good spatial resolution, while they are in flow. This paper proposes a general framework for the processing/classification of cells imaged using imaging flow cytometer. Each cell is localized by finding an accurate cell contour. Then, features reflecting cell size, circularity and complexity are extracted for the classification using SVM. Unlike the conventional iterative, semi-automatic segmentation algorithms such as active contour, we propose a noniterative, fully automatic graph-based cell localization. In order to evaluate the performance of the proposed framework, we have successfully classified unstained label-free leukaemia cell-lines MOLT, K562 and HL60 from video streams captured using custom fabricated cost-effective microfluidics-based imaging flow cytometer. The proposed system is a significant development in the direction of building a cost-effective cell analysis platform that would facilitate affordable mass screening camps looking cellular morphology for disease diagnosis.

  14. 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

  15. 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.

  16. Detection and Quantification of Mitochondrial Fusion Using Imaging Flow Cytometry.

    PubMed

    Nascimento, Aldo; Lannigan, Joanne; Kashatus, David

    2017-07-05

    Mitochondria are dynamic organelles that perform several vital cellular functions. Requisite for these functions are mitochondrial fusion and fission. Despite the increasing importance of mitochondrial dynamics in a range of cellular processes, there exist limited methods for robust quantification of mitochondrial fission and fusion. Currently, the most widely used method to measure mitochondrial fusion is the polyethylene glycol (PEG) fusion assay. While this assay can provide useful information regarding fusion activity, the reliance on manual selection of rare fusion events is time consuming and may introduce selection bias. By utilizing the image-capture features and colocalization analysis of imaging flow cytometry in combination with the PEG fusion assay, we are able to develop a high-throughput method to detect and quantify mitochondrial fusion activity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  17. 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.

  18. 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.

  19. 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.

  20. Endometrial cytopathology. An image analysis approach using the Ki-67 biomarker.

    PubMed

    Apostolou, G; Apostolou, N; Moulos, P; Chatzipantelis, P

    2017-10-01

    To investigate the different identity and biological behaviour of endometrial benign epithelial and endometrial adenocarcinoma cell categories. For this study, the imprint smears from three groups, 10 cases of disordered proliferative/benign hyperplastic endometrium, 21 cases of low-grade and eight cases of high-grade endometrial adenocarcinoma, were examined using image analysis and the Ki-67 biomarker. The plastic stem cell model was also applied. Among the examined groups, the nuclear area major axis ranged statistically different in the digitally measured Ki-67 positive endometrial epithelial and adenocarcinoma cells (P<.0001). Moreover, higher values of the cycling nuclear area major axis were observed in high-grade, as compared with the low-grade endometrial adenocarcinomas (P<.0001) and the cases of disordered/benign hyperplastic endometrium (P<.0001). Additionally, a Ki-67 increase pathway was observed in the benign endometrial lesions, and a relatively stable pathway was noticed in low- and high-grade endometrial adenocarcinomas. The different range of the nuclear area major axis among cycling endometrial epithelial and adenocarcinoma cells may correlate with their specific identity and biological behaviour. The different values of the cycling nuclear area major dimension may also be connected with the biological behaviour of the three examined groups. Moreover, the endometrial epithelial cells may follow a Ki-67 increase pathway, instead of the relatively stable pathway which the rapidly proliferating adenocarcinoma cells may use. Finally, the studied cell categories may exhibit different biology, because their stem cells may reside in different states of stemness. © 2017 John Wiley & Sons Ltd.

  1. Malignant biliary stenosis: conventional cytology versus DNA image cytometry.

    PubMed

    Binek, Janek; Lindenmann, Nadja; Meyenberger, Christa M; Hell, Margarete; Ulmer, Hanno; Spieler, Peter; Borovicka, Jan

    2011-06-01

    The aim of this study is to evaluate the utility of image cytometry (ICM)-DNA analysis on cytological brush specimens in improving the sensitivity and diagnostic accuracy for biliary neoplasias. A total of 71 patients with 89 samples of biliary tree brushing from a stenosis were included in this prospective study. Conventional cytology (CC) and DNA ploidy using ICM of the brushing were performed. Benign or malignant findings were confirmed by surgical exploration or a clinical follow-up of at least 12 months. Diagnosis was confirmed by clinical follow-up in 44 cases and surgical investigation or histology in 41 cases. A definitive diagnosis of the smears resulted in 40 malignant and 49 benign diagnoses. The sensitivity was 0.666 for CC and 0.658 for ICM, and the specificity was 0.920 and 0.937, respectively. The positive predictive value (PPV) was 0.866 for CC and 0.900 for ICM. McNemar's test did not reveal a significant difference between CC and ICM (P=0.803). Agreement of the two methods was found in 73 samples, raising specificity to 0.998 but not sensitivity (0.725). ICM-DNA seems not to improve significantly the PPV and NPV for detecting neoplasias of the biliary tract compared to CC. Nevertheless a clinical advantage can be seen in the agreement of the two methods in diagnosing dysplasia or cancer, since it did not show false positive results.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. histoCAT: analysis of cell phenotypes and interactions in multiplex image cytometry data.

    PubMed

    Schapiro, Denis; Jackson, Hartland W; Raghuraman, Swetha; Fischer, Jana R; Zanotelli, Vito R T; Schulz, Daniel; Giesen, Charlotte; Catena, Raúl; Varga, Zsuzsanna; Bodenmiller, Bernd

    2017-09-01

    Single-cell, spatially resolved omics analysis of tissues is poised to transform biomedical research and clinical practice. We have developed an open-source, computational histology topography cytometry analysis toolbox (histoCAT) to enable interactive, quantitative, and comprehensive exploration of individual cell phenotypes, cell-cell interactions, microenvironments, and morphological structures within intact tissues. We highlight the unique abilities of histoCAT through analysis of highly multiplexed mass cytometry images of human breast cancer tissues.

  7. Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

    PubMed

    Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

    2017-06-28

    Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization

  8. 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.

  9. Accurate Assessment of Cell Death by Imaging Flow Cytometry.

    PubMed

    Rieger, Aja M; Barreda, Daniel R

    2016-01-01

    The number of investigators using cell death analysis applications has greatly expanded since the introduction of flow cytometry. The Annexin V/propidium iodide (PI) method is among the most commonly used procedures and allows users to determine if cells are viable, apoptotic, or necrotic, based on changes in membrane lipid composition, integrity, and permeability. Unfortunately, PI can intercalate into RNA, in addition to DNA, which contributes to a large number of events showing PI staining within the cytoplasmic compartment. We show that this occurs across a broad range of animal primary cells and commonly used cell lines, and is most prevalent in large cells (nuclear:cytoplasmic ratio <0.5). Any cellular system where RNA levels change throughout an experiment will be particularly affected, such as those that utilize virally infected cells. As two examples, we highlight our recent work on cells infected with vesicular stomatitis virus (VSV), an RNA virus, and herpes simplex virus-1 (HSV-1), a DNA virus. Similarly, these issues are relevant to experimental systems where cells have increased RNA content such as during genotoxic stress, following exposure to cell cycle arrest drugs such as thymidine or hydroxyurea, or where developmental progression promotes discrete changes in cellular RNA synthesis. This chapter outlines a modified Annexin V/PI method that addresses cytoplasmic RNA staining issues to allow for accurate assessment of cell death. This protocol takes advantage of an additional cellular permeability caused by fixation to promote RNase A entry into the cell. Based on our observations, cell morphological parameters are well maintained and less than 5 % of total cellular events exhibit cytoplasmic PI staining under this protocol.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. Automated segmentation and isolation of touching cell nuclei in cytopathology smear images of pleural effusion using distance transform watershed method

    NASA Astrophysics Data System (ADS)

    Win, Khin Yadanar; Choomchuay, Somsak; Hamamoto, Kazuhiko

    2017-06-01

    The automated segmentation of cell nuclei is an essential stage in the quantitative image analysis of cell nuclei extracted from smear cytology images of pleural fluid. Cell nuclei can indicate cancer as the characteristics of cell nuclei are associated with cells proliferation and malignancy in term of size, shape and the stained color. Nevertheless, automatic nuclei segmentation has remained challenging due to the artifacts caused by slide preparation, nuclei heterogeneity such as the poor contrast, inconsistent stained color, the cells variation, and cells overlapping. In this paper, we proposed a watershed-based method that is capable to segment the nuclei of the variety of cells from cytology pleural fluid smear images. Firstly, the original image is preprocessed by converting into the grayscale image and enhancing by adjusting and equalizing the intensity using histogram equalization. Next, the cell nuclei are segmented using OTSU thresholding as the binary image. The undesirable artifacts are eliminated using morphological operations. Finally, the distance transform based watershed method is applied to isolate the touching and overlapping cell nuclei. The proposed method is tested with 25 Papanicolaou (Pap) stained pleural fluid images. The accuracy of our proposed method is 92%. The method is relatively simple, and the results are very promising.

  15. Quantitative Assessment of Pap Smear Cells by PC-Based Cytopathologic Image Analysis System and Support Vector Machine

    NASA Astrophysics Data System (ADS)

    Huang, Po-Chi; Chan, Yung-Kuan; Chan, Po-Chou; Chen, Yung-Fu; Chen, Rung-Ching; Huang, Yu-Ruei

    Cytologic screening has been widely used for controlling the prevalence of cervical cancer. Errors from sampling, screening and interpretation, still concealed some unpleasant results. This study aims at designing a cellular image analysis system based on feasible and available software and hardware for a routine cytologic laboratory. Totally 1814 cellular images from the liquid-based cervical smears with Papanicolaou stain in 100x, 200x, and 400x magnification were captured by a digital camera. Cell images were reviewed by pathologic experts with peer agreement and only 503 images were selected for further study. The images were divided into 4 diagnostic categories. A PC-based cellular image analysis system (PCCIA) was developed for computing morphometric parameters. Then support vector machine (SVM) was used to classify signature patterns. The results show that the selected 13 morphometric parameters can be used to correctly differentiate the dysplastic cells from the normal cells (p<0.001). Additionally, SVM classifier has been demonstrated to be able to achieve a high accuracy for cellular classification. In conclusion, the proposed system provides a feasible and effective tool for the evaluation of gynecologic cytologic specimens.

  16. 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.

  17. 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.

  18. 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.

  19. 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}.

  20. 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.

  1. 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).

  2. 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.

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

    PubMed Central

    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. PMID:26492577

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. MRT letter: light sheet based imaging flow cytometry on a microfluidic platform.

    PubMed

    Regmi, Raju; Mohan, Kavya; Mondal, Partha P

    2013-11-01

    We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state-of-art techniques the proposed technique shows marked improvement. Using fluorescently-coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on-the-go. Overall, the proposed system is cost-effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Copyright © 2013 Wiley Periodicals, Inc.

  10. 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.

  11. Survey of medical training in cytopathology carried out by the journal Cytopathology.

    PubMed

    Anshu; Herbert, A; Cochand-Priollet, B; Cross, P; Desai, M; Dina, R; Duskova, J; Evered, A; Farnsworth, A; Gray, W; Gupta, S S; Kapila, K; Kardum-Skelin, I; Kloboves-Prevodnik, V; Kobayashi, T K; Koutselini, H; Olszewski, W; Onal, B; Pitman, M B; Marinsek, Z; Sauer, T; Schenck, U; Schmitt, F; Shabalova, I; Smith, J H F; Tani, E; Vass, L; Vielh, P; Wiener, H

    2010-06-01

    This report of the Editorial Advisory Board of Cytopathology gives the results of a survey of medical practitioners in cytopathology, which aimed to find out their views on the current situation in undergraduate and postgraduate training in their institutions and countries. The results show that training in cytopathology and histopathology are largely carried out at postgraduate level and tend to be organized nationally rather than locally. Histopathology was regarded as essential for training in cytopathology by 89.5% of respondents and was mandatory according to 83.1%. Mandatory cytopathology sections of histopathology were reported by 67.3% and specific examinations in cytopathology by 55.4%. The main deficiencies in training were due to its variability; there were insufficient numbers of pathologists interested in cytology and a consequent lack of training to a high level of competence. Pathologists without specific training in cytopathology signed out cytology reports according to 54.7% of responses, more often in centres where training was 3-6 months or less duration. Although 92.2% of respondents thought that specialist cytology should not be reported by pathologists without experience in general cytopathology, that practice was reported by 30.9%, more often in centres with small workloads. The survey report recommends that 6-12 months should be dedicated to cytopathology during histopathology training, with optional additional training for those wanting to carry out independent practice in cytopathology. Formal accreditation should be mandatory for independent practice in cytopathology. When necessary, temporary placements to centres of good practice should be available for trainees intending to practise independently in cytopathology. There should be adequate numbers of pathologists trained in cytopathology to a high level of competence; some of their time could be released by training cytotechnologists and trainee pathologists to prescreen cytology slides

  12. Trypan blue as an affordable marker for automated live-dead cell analysis in image cytometry.

    PubMed

    Melzer, Susanne; Nunes, Celio Siman Mafra; Endringer, Denise Coutinho; de Andrade, Tadeu Uggere; Tarnok, Attila; Lenz, Dominik

    2016-11-01

    The aim of the present study was to combine image cytometry and trypan blue (TB) exclusion staining for a reproducible high-throughput detection of dead cells, enabling TB as an inexpensive marker, to be affordable for many studies and creating the possibility to combine fluorochromes without or with less spectral overlap. Capillary blood was drawn from a healthy volunteer, red blood cells were lysed and leukocyte cell death was induced. Samples were stained with CD45-FITC, CD14-PE, TB and DAPI, and then analyzed using image cytometry (iCys). TB quenching control tests were performed using DAPI and CD45-FITC. Images were generated in .TIF and .JPEG format using iCys image cytometer. The images were analyzed using CellProfiler (CP) modules to optimize the analysis based on the aims of each phase of this study. CellProfiler Analyst (CPA) was used to classify cells throughout machine learning and to calculate sensibility of the classification. A sensitivity of 0.94 for dead cells and 0.99 for live cells was calculated using CPA. We did not see any quenching effects of the FITC staining. DAPI signal was reduced in the presence of TB. The results of the present study revealed that TB serves as a dead cell marker in an image cytometric analysis, being able to be combined with other fluorescence markers without loss of fluorescence intensity signal or overlapping emission spectrum. SCANNING 38:857-863, 2016. © 2016 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

  13. 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

  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. 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.

  16. 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.

  17. Characterization of Protein Particles in Therapeutic Formulations Using Imaging Flow Cytometry.

    PubMed

    Probst, Christine; Zeng, Yuanchun; Zhu, Rong-Rong

    2017-08-01

    Quantitation of particles >10 μm in therapeutic protein formulations is required by pharmacopeia guidelines, and characterization of particles <10 μm is increasingly expected. Established methods offer limited ability to detect or characterize small particles; consequently, new methods are needed to measure the sub-10 μm size range. Here, we evaluate imaging flow cytometry (IFC) as a new method for detection of protein aggregates, taking advantage of key enabling attributes including rapid multi-modal high-resolution imaging of individual particles, low sample volume, high sampling efficiency, wide dynamic size and concentration range, and low clog risk. IFC sensitivity was compared with dynamic imaging, a "gold standard" technique for analysis of particles in protein formulations. Both techniques yielded similar results for polystyrene beads ≥2 μm. However, IFC demonstrated greater protein particle detection sensitivity, especially for the sub-10 μm size range. Interestingly, for an aggregated lysozyme sample, IFC detected protein particles using fluorescence images, whereas dynamic imaging failed to detect even large particles >25 μm due to high transparency. The results corroborate implementation of IFC as an advanced technique for protein particle analysis, offering in-depth characterization of particle physical and chemical properties, and enhanced sensitivity for sub-10 μm and transparent particles. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Using in situ flow cytometry images of ciliates and dinoflagellates for aquatic system monitoring.

    PubMed

    Pereira, G C; Figueiredo, A R; Ebecken, N F F

    2017-08-17

    Short-period variability in plankton communities is poorly documented, especially for variations occurring in specific groups in the assemblage because traditional analysis is laborious and time-consuming. Moreover, it does not allow the high sampling frequency required for decision making. To overcome this limitation, we tested the submersible CytoSub flow cytometer. This device was anchored at a distance of approximately 10 metres from the low tide line at a depth of 1.5 metres for 12 hours to monitor the plankton at a site in the biological reserve of Barra da Tijuca beach, Rio de Janeiro. Data analysis was performed with two-dimensional scatter plots, individual pulse shapes and micro images acquisition. High-frequency monitoring results of two interesting groups are shown. The abundance and carbon biomass of ciliates were relatively stable, whereas those from dinoflagellates were highly variable along the day. The linear regression of biovolume measures between classical microscopy and in situ flow cytometry demonstrate high degree of adjustment. Despite the success of the trial and the promising results obtained, the large volume of images generated by the method also creates a need to develop pattern recognition models for automatic classification of in situ cytometric images.

  3. 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.

  4. 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

  5. Application of image flow cytometry for the characterization of red blood cell morphology

    NASA Astrophysics Data System (ADS)

    Pinto, Ruben N.; Sebastian, Joseph A.; Parsons, Michael; Chang, Tim C.; Acker, Jason P.; Kolios, Michael C.

    2017-02-01

    Red blood cells (RBCs) stored in hypothermic environments for the purpose of transfusion have been documented to undergo structural and functional changes over time. One sign of the so-called RBC storage lesion is irreversible damage to the cell membrane. Consequently, RBCs undergo a morphological transformation from regular, deformable biconcave discocytes to rigid spheroechinocytes. The spherically shaped RBCs lack the deformability to efficiently enter microvasculature, thereby reducing the capacity of RBCs to oxygenate tissue. Blood banks currently rely on microscope techniques that include fixing, staining and cell counting in order to morphologically characterize RBC samples; these methods are labor intensive and highly subjective. This study presents a novel, high-throughput RBC morphology characterization technique using image flow cytometry (IFC). An image segmentation template was developed to process 100,000 images acquired from the IFC system and output the relative spheroechinocyte percentage. The technique was applied on samples extracted from two blood bags to monitor the morphological changes of the RBCs during in vitro hypothermic storage. The study found that, for a given sample of RBCs, the IFC method was twice as fast in data acquisition, and analyzed 250-350 times more RBCs than the conventional method. Over the lifespan of the blood bags, the mean spheroechinocyte population increased by 37%. Future work will focus on expanding the template to segregate RBC images into more subpopulations for the validation of the IFC method against conventional techniques; the expanded template will aid in establishing quantitative links between spheroechinocyte increase and other RBC storage lesion characteristics.

  6. Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

    PubMed

    Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

    2017-09-14

    Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

  7. 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.

  8. 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.

  9. Avoidance of unnecessary fine-needle aspiration with the use of the Thyroid Imaging Reporting Data System classification and strain elastography based on The Bethesda System for Reporting Thyroid Cytopathology

    PubMed Central

    Erkan, Murat; Canberk, Sule; Kilicoglu, Gamze Z.; Onenerk, Mine; Uludokumaci, Atay; Gunes, Pembegul; Atasoy, Tugba

    2016-01-01

    Thyroid fine-needle aspiration (FNA) biopsy has been widely accepted as an accurate and cost-effective tool in the management of thyroid nodules. To avoid unnecessary FNAs and provide appropriate management, patient evaluation should be based on a multidisciplinary approach. For this purpose, the Thyroid Imaging Reporting and Data System (TI-RADS) and strain elastography (SE) were proposed as tools for the risk assessment of malignancy in thyroid nodules. The aim of the present study was to analyze the utility of TI-RADS system and SE, along with FNA, and prospectively evaluate 369 consecutive patients referred for FNA of a thyroid nodule. TI-RADS was tested against The Bethesda System for Reporting Thyroid Cytopathology to determine whether there was an agreement between the two classification systems; statistically, some agreement was observed. Medians of the maximum SE values (E-max) were obtained for benign and malignant FNA results and found to be 1.97 [interquartile range (IQR): 1.87] and 2.8 (IQR: 3.42), respectively (P=0.004). The number of studies investigating the utility of TI-RADS and SE along with TBSRCT is currently limited. Our study demonstrated that a multidisciplinary approach with the use of TI-RADS and SE may mildly improve the management of thyroid nodules. PMID:27900100

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. New insights into cell cycle and DNA damage response machineries through high-resolution AMICO quantitative imaging cytometry.

    PubMed

    Tarnok, A; Darzynkiewicz, Z

    2013-10-01

    Progress in biology and medicine research is being driven by development of new instrumentation and associated methodologies which open analytical capabilities that expand understanding of complexity of biological systems. Application of cytometry, which is now widely used in so many disciplines of biology, is the best example of such a progress. Recent publications push the envelope in expanding capabilities of cytometry by introducing a high resolution imaging cytometry defined as Automated Microscopy for Image CytOmetry (AMICO). This instrumentation is utilized to further elucidate mechanisms of the cell cycle progression and also the DNA damage response. This approach is going beyond the presently possible analytical technologies regarding throughput and depth of information. The possibility of multiparametric analysis combined with the high resolution mapping of individual constituents of cell cycle and DNA damage response machineries provides new tools to probe molecular mechanism of these processes. The capability of analysis of proximity of these constituents to each other offered by AMICO is a novel and potentially important approach that can be used to elucidate mechanisms of other biological processes. © 2013 John Wiley & Sons Ltd.

  15. 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

  16. Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood.

    PubMed

    Hewitt, Rachel E; Vis, Bradley; Pele, Laetitia C; Faria, Nuno; Powell, Jonathan J

    2017-09-20

    Pigment grade titanium dioxide is composed of sub-micron sized particles, including a nanofraction, and is widely utilized in food, cosmetic, pharmaceutical, and biomedical industries. Oral exposure to pigment grade titanium dioxide results in at least some material entering the circulation in humans, although subsequent interactions with blood immune cells are unknown. Pigment grade titanium dioxide is employed for its strong light scattering properties, and this work exploited that attribute to determine whether single cell-particle associations could be determined in immune cells of human whole blood at "real life" concentrations. In vitro assays, initially using isolated peripheral blood mononuclear cells, identified titanium dioxide associated with the surface of, and within, immune cells by darkfield reflectance in imaging flow cytometry. This was confirmed at the population level by side scatter measurements using conventional flow cytometry. Next, it was demonstrated that imaging flow cytometry could quantify titanium dioxide particle-bearing cells, within the immune cell populations of fresh whole blood, down to titanium dioxide levels of 10 parts per billion, which is in the range anticipated for human blood following titanium dioxide ingestion. Moreover, surface association and internal localization of titanium dioxide particles could be discriminated in the assays. Overall, results showed that in addition to the anticipated activity of blood monocytes internalizing titanium dioxide particles, neutrophil internalization and cell membrane adhesion also occurred, the latter for both phagocytic and nonphagocytic cell types. What happens in vivo and whether this contributes to activation of one or more of these different cells types in blood merits further attention. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  17. 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

  18. 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.

  19. Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

    PubMed

    Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

    2016-08-01

    Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. 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.

  1. Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error.

    PubMed

    Chan, Leo Li-Ying; Laverty, Daniel J; Smith, Tim; Nejad, Parham; Hei, Hillary; Gandhi, Roopali; Kuksin, Dmitry; Qiu, Jean

    2013-02-28

    Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs

  2. Fully automated on-chip imaging flow cytometry system with disposable contamination-free plastic re-cultivation chip.

    PubMed

    Hayashi, Masahito; Hattori, Akihiro; Kim, Hyonchol; Terazono, Hideyuki; Kaneko, Tomoyuki; Yasuda, Kenji

    2011-01-01

    We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA)) based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 10(6) particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics.

  3. Fully Automated On-Chip Imaging Flow Cytometry System with Disposable Contamination-Free Plastic Re-Cultivation Chip

    PubMed Central

    Hayashi, Masahito; Hattori, Akihiro; Kim, Hyonchol; Terazono, Hideyuki; Kaneko, Tomoyuki; Yasuda, Kenji

    2011-01-01

    We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA)) based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 106 particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics. PMID:21747698

  4. Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry-the fast alternative to microscopic image analysis.

    PubMed

    Ehgartner, Daniela; Herwig, Christoph; Fricke, Jens

    2017-09-14

    An important parameter in filamentous bioreactor cultivations is the morphology of the fungi, due to its interlink to productivity and its dependency on process conditions. Filamentous fungi show a large variety of morphological forms in submerged cultures. These range from dispersed hyphae, to interwoven mycelial aggregates, to denser hyphal aggregates, the so-called pellets. Depending on the objective function of the bioprocess, different characteristics of the morphology are favorable and need to be quantified accurately. The most common method to quantitatively characterize morphology is image analysis based on microscopy. This method is work intensive and time consuming. Therefore, we developed a faster, at-line applicable, alternative method based on flow cytometry. Within this contribution, this novel method is compared to microscopy for a penicillin production process. Both methods yielded in comparable distinction of morphological sub-populations and described their morphology in more detail. In addition to the appropriate quantification of size parameters and the description of the hyphal region around pellets, the flow cytometry method even revealed a novel compactness parameter for fungal pellets which is not accessible via light microscopy. Hence, the here presented flow cytometry method for morphological analysis is a fast and reliable alternative to common tools with some new insights in the pellet morphology, enabling at-line use in production environments.

  5. Utility of cytopathological specimens and an automated image analysis for the evaluation of HER2 status and intratumor heterogeneity in breast carcinoma.

    PubMed

    Arihiro, Koji; Oda, Miyo; Ogawa, Katsunari; Kaneko, Yoshie; Shimizu, Tomomi; Tanaka, Yuna; Marubashi, Yukari; Ishida, Katsunari; Takai, Chikako; Taoka, Chie; Kimura, Shuji; Shiroma, Noriyuki

    2016-12-01

    Although updated HER2 testing guidelines have been improved by a collaboration between the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) in 2013, HER2 evaluation is still problematic because of issues involving CEP17 polysomy, heterogeneity, and HER2 score 2+ cases. The aim of this retrospective study was to evaluate the relationship between HER2 gene heterogeneity, or so called CEP17 polysomy, using breast carcinoma cells sampled by scraping and the IHC score graded by automated image analysis using whole slide image. We randomly selected 23 breast carcinoma cases with a HER2 score 0, 24 cases with a HER2 score 1+, 24 cases with HER2 score 2+, and 23 cases with HER2 score 3+ from the records of patients with breast cancer at Hiroshima University Hospital. We compared the results of fluorescent in situ hybridization (FISH) using formalin-fixed, paraffin-embedded (FFPE) tissues and cytological samples and compared the HER2 score calculated using an automated image analysis using wholly scanned slide images and visual counting. We successfully performed the FISH assay in 78 of 94 cases (83%) using FFPE tissues and in all 94 (100%) cases using cytological samples. Frequency of both HER2 amplification and CEP17 polysomy was higher when cytological samples were used than when FFPE tissue was used. Frequency of HER2 heterogeneity using cytological samples was higher that than using FFPE tissue, except for the IHC score 3+ cases. When assessment of HER2 status based on FISH using FFPE tissue cannot be accomplished, FISH using cytological samples should be considered. When intensity of HER2 is heterogeneous in the tumor tissue, particularly in cases regarded as score 2+, they should be evaluated by automated image analysis using the whole slide image. Copyright © 2016 Elsevier GmbH. All rights reserved.

  6. Application of an Image Cytometry Protocol for Cellular and Mitochondrial Phenotyping on Fibroblasts from Patients with Inherited Disorders.

    PubMed

    Fernandez-Guerra, Paula; Lund, M; Corydon, T J; Cornelius, N; Gregersen, N; Palmfeldt, J; Bross, Peter

    2016-01-01

    Cellular phenotyping of human dermal fibroblasts (HDFs) from patients with inherited diseases provides invaluable information for diagnosis, disease aetiology, prognosis and assessing of treatment options. Here we present a cell phenotyping protocol using image cytometry that combines measurements of crucial cellular and mitochondrial parameters: (1) cell number and viability, (2) thiol redox status (TRS), (3) mitochondrial membrane potential (MMP) and (4) mitochondrial superoxide levels (MSLs). With our protocol, cell viability, TRS and MMP can be measured in one small cell sample and MSL on a parallel one. We analysed HDFs from healthy individuals after treatment with various concentrations of hydrogen peroxide (H2O2) for different intervals, to mimic the physiological effects of oxidative stress. Our results show that cell number, viability, TRS and MMP decreased, while MSL increased both in a time- and concentration-dependent manner. To assess the use of our protocol for analysis of HDFs from patients with inherited diseases, we analysed HDFs from two patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD), one with a severe clinical phenotype and one with a mild one. HDFs from both patients displayed increased MSL without H2O2 treatment. Treatment with H2O2 revealed significant differences in MMP and MSL between HDFs from the mild and the severe patient. Our results establish the capacity of our protocol for fast analysis of cellular and mitochondrial parameters by image cytometry in HDFs from patients with inherited metabolic diseases.

  7. 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.

  8. Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8(+) T-cells.

    PubMed

    Wabnitz, Guido H; Kirchgessner, Henning; Samstag, Yvonne

    2017-09-01

    The clearance of tumors or virus infected cells is a crucial task of the immune system. Cytotoxic T-cells (CTLs) are able to detect and to kill such altered host cells. Given the recent success of checkpoint inhibitors for tumor therapy, it becomes more and more important to understand the biology of T-cell mediated target cell killing. Tests that allow analyzing the biology of CTLs are either based on flow cytometry or fluorescence microscopy. Thus, they either lack image-based information or have a poor statistical robustness. Therefore, we describe an approach to quantify CTL-mediated cytotoxicity using imaging flow cytometry. Using activated primary human cytotoxic T-cells as CTLs and P815 as target cells, we show that both the evaluation of target cell death and the biology of CTLs can be evaluated in parallel. This enables to gain information about CTL-mediated cytotoxicity in samples from patients important for translational medicine. J. Cell. Biochem. 118: 2528-2533, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. MXS-Chaining: A Highly Efficient Cloning Platform for Imaging and Flow Cytometry Approaches in Mammalian Systems.

    PubMed

    Sladitschek, Hanna L; Neveu, Pierre A

    2015-01-01

    The continuous improvement of imaging technologies has driven the development of sophisticated reporters to monitor biological processes. Such constructs should ideally be assembled in a flexible enough way to allow for their optimization. Here we describe a highly reliable cloning method to efficiently assemble constructs for imaging or flow cytometry applications in mammalian cell culture systems. We bioinformatically identified a list of restriction enzymes whose sites are rarely found in human and mouse cDNA libraries. From the best candidates, we chose an enzyme combination (MluI, XhoI and SalI: MXS) that enables iterative chaining of individual building blocks. The ligation scar resulting from the compatible XhoI- and SalI-sticky ends can be translated and hence enables easy in-frame cloning of coding sequences. The robustness of the MXS-chaining approach was validated by assembling constructs up to 20 kb long and comprising up to 34 individual building blocks. By assessing the success rate of 400 ligation reactions, we determined cloning efficiency to be 90% on average. Large polycistronic constructs for single-cell imaging or flow cytometry applications were generated to demonstrate the versatility of the MXS-chaining approach. We devised several constructs that fluorescently label subcellular structures, an adapted version of FUCCI (fluorescent, ubiquitination-based cell cycle indicator) optimized to visualize cell cycle progression in mouse embryonic stem cells and an array of artificial promoters enabling dosage of doxycyline-inducible transgene expression. We made publicly available through the Addgene repository a comprehensive set of MXS-building blocks comprising custom vectors, a set of fluorescent proteins, constitutive promoters, polyadenylation signals, selection cassettes and tools for inducible gene expression. Finally, detailed guidelines describe how to chain together prebuilt MXS-building blocks and how to generate new customized MXS

  14. 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

  15. 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.

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

    PubMed

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

    2017-09-01

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

  17. 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

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

    PubMed Central

    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.

    2014-01-01

    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. PMID:24585634

  19. 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.

  20. 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.

  1. Changing Trends and Practices in Cytopathology.

    PubMed

    Gonzalez, Maria F; Akhtar, Israh; Manucha, Varsha

    2017-01-01

    To explore the current and anticipated changes in the practice of cytopathology. The present review is based on a review of recent literature and an evaluation of the authors' personal experiences. In recent years the practice of cytopathology, nationwide and in our institute, has witnessed a major change affecting gynecologic and nongynecologic cytology. There has been a decline in the number of Papanicolaou tests which has affected the utilization of cytotechnologists and provoked a reorganization of their work flow. The "need to do more with less" in the era of targeted therapy/personalized medicine has resulted in an increasing preference for needle core biopsy when performing a rapid on-site evaluation. We feel that this change is unavoidable. It is pertinent that cytopathologists as a group recognize this change and prepare themselves and the trainees not only to become adapt but also to use this as an opportunity to discover the yet unexplored world of cytology. © 2017 S. Karger AG, Basel.

  2. 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.

  3. 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.

  4. 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.

  5. Using a web-based system for the continuous distance education in cytopathology.

    PubMed

    Stergiou, Nikolaos; Georgoulakis, Giannis; Margari, Niki; Aninos, Dionisios; Stamataki, Melina; Stergiou, Efi; Pouliakis, Abraam; Karakitsos, Petros

    2009-12-01

    The evolution of information technologies and telecommunications has made the World Wide Web a low cost and easily accessible tool for the dissemination of information and knowledge. Continuous Medical Education (CME) sites dedicated in cytopathology field are rather poor, they do not succeed in following the constant changes and lack the ability of providing cytopathologists with a dynamic learning environment, adaptable to the development of cytopathology. Learning methods including skills such as decision making, reasoning and problem solving are critical in the development of such a learning environment. The objectives of this study are (1) to demonstrate on the basis of a web-based training system the successful application of traditional learning theories and methods and (2) to effectively evaluate users' perception towards the educational program, using a combination of observers, theories and methods. Trainees are given the opportunity to browse through the educational material, collaborate in synchronous and asynchronous mode, practice their skills through problems and tasks and test their knowledge using the self-evaluation tool. On the other hand, the trainers are responsible for editing learning material, attending students' progress and organizing the problem-based and task-based scenarios. The implementation of the web-based training system is based on the three-tier architecture and uses an Apache Tomcat web server and a MySQL database server. By December 2008, CytoTrainer's learning environment contains two courses in cytopathology: Gynaecological Cytology and Thyroid Cytology offering about 2000 digital images and 20 case sessions. Our evaluation method is a combination of both qualitative and quantitative approaches to explore how the various parts of the system and students' attitudes work together. Trainees approved of the course's content, methodology and learning activities. The triangulation of evaluation methods revealed that the training

  6. 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.

  7. Automated classification of oral premalignant lesions using image cytometry and Random Forests-based algorithms.

    PubMed

    Baik, Jonathan; Ye, Qian; Zhang, Lewei; Poh, Catherine; Rosin, Miriam; MacAulay, Calum; Guillaud, Martial

    2014-06-01

    A major challenge for the early diagnosis of oral cancer is the ability to differentiate oral premalignant lesions (OPL) at high risk of progressing into invasive squamous cell carcinoma (SCC) from those at low risk. Our group has previously used high-resolution image analysis algorithms to quantify the nuclear phenotypic changes occurring in OPLs. This approach, however, requires a manual selection of nuclei images. Here, we investigated a new, semi-automated algorithm to identify OPLs at high risk of progressing into invasive SCC from those at low risk using Random Forests, a tree-based ensemble classifier. We trained a sequence of classifiers using morphometric data calculated on nuclei from 29 normal, 5 carcinoma in situ (CIS) and 28 SCC specimens. After automated discrimination of nuclei from other objects (i.e., debris, clusters, etc.), a nuclei classifier was trained to discriminate abnormal nuclei (8,841) from normal nuclei (5,762). We extracted voting scores from this trained classifier and created an automated nuclear phenotypic score (aNPS) to identify OPLs at high risk of progression. The new algorithm showed a correct classification rate of 80% (80.6% sensitivity, 79.3% specificity) at the cellular level for the test set, and a correct classification rate of 75% (77.8% sensitivity, 71.4% specificity) at the tissue level with a negative predictive value of 76% and a positive predictive value of 74% for predicting progression among 71 OPLs, performed on par with the manual method in our previous study. We conclude that the newly developed aNPS algorithm serves as a crucial asset in the implementation of high-resolution image analysis in routine clinical pathology practice to identify lesions that require molecular evaluation or more frequent follow-up.

  8. High-Frequency Observations of Phytoplankton Spring Bloom Dynamics in Baffin Bay Using Imaging Flow Cytometry

    NASA Astrophysics Data System (ADS)

    Grondin, P. L.; Ferland, J.; Karp-Boss, L.; Babin, M.

    2016-02-01

    The FlowCytobot (IFCB) is a high-frequency submersible imaging flow cytometer that allows a detailed characterization of phytoplankton community composition. The IFCB was used to study the under-ice spring bloom dynamics at a fixed station (67˚28.774N, 63˚47.398W) in Baffin Bay from April 22nd until July 10th 2015. Seawater and sea-ice samples were collected every second day, at six different depths in water and at the bottom of ice cores. Preliminary analyses show an increase in algae abundance in sea-ice from the end of April to mid-June, reaching ca. 3000 cells mL-1. As spring sets in, the abundance decreased rapidly to 250 cells mL-1. Visual inspection of images showed the dominance of pennate diatoms such as Nitzschia frigida, Entomoneis spp. and Navicula spp. in the sea-ice biota from April to mid-June. Concurrently, we observed an abrupt increase in ice related algae abundance in the water column (ca. 25 to ca. 225 cells mL-1). This suggests a "flushing" of sympagic algae from sea ice. Inspection of images from the seawater samples supports this idea by showing the same community, with a substantial proportion of pennate diatoms debris. Data also shows the onset of a phytoplankton bloom at the beginning of July, with a maximum abundance near surface deepening over time. The data suggest a shift towards a phytoplankton community, largely dominated by Thalassiosira spp. and Chaetoceros spp., with limited occurrences of <10µm flagellates and dinoflagellates. Results match commonly used algal biomass proxies like chl a concentration as shown by a strong correlation with cell abundance from the IFCB. Further comparisons with irradiance, water masses properties, sea-ice cover and algal pigments will improve our understanding of the under-ice spring bloom dynamics. Together with automated classification of images, this new method allows reduced sampling costs, time effective species identification and real-time visualisation of phytoplankton communities

  9. Using Image-Based Flow Cytometry to Assess Monocyte Oxidized LDL Phagocytosis Capacity.

    PubMed

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

    2016-01-01

    The examination of monocyte phagocytosis of modified lipoproteins is important to the understanding of plaque deposition and the development of atherosclerosis. Current methods lack the high-throughput image-based analysis capabilities, which may yield novel information concerning monocyte activity in disease processes. Specifically, this method identifies monocyte phagocytosis of oxidized LDL along with a change in adhesion molecules and scavenger receptors. Our laboratory is currently implementing this method as a means to study how acute dietary modifications alter risk of developing atherosclerosis.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. Equivocal cytology in lung cancer diagnosis: improvement of diagnostic accuracy using adjuvant multicolor FISH, DNA-image cytometry, and quantitative promoter hypermethylation analysis.

    PubMed

    Schramm, Martin; Wrobel, Christian; Born, Ingmar; Kazimirek, Marietta; Pomjanski, Natalia; William, Marina; Kappes, Rainer; Gerharz, Claus Dieter; Biesterfeld, Stefan; Böcking, Alfred

    2011-06-25

    Sometimes, cytological lung cancer diagnosis is challenging because equivocal diagnoses are common. To enhance diagnostic accuracy, fluorescent in situ hybridization (FISH), DNA-image cytometry, and quantitative promoter hypermethylation analysis have been proposed as adjuncts. Bronchial washings and/or brushings or transbronchial fine-needle aspiration biopsies were prospectively collected from patients who were clinically suspected of having lung carcinoma. After routine cytological diagnosis, 70 consecutive specimens, each cytologically diagnosed as negative, equivocal, or positive for cancer cells, were investigated with adjuvant methods. Suspicious areas on the smears were restained with the LAVysion multicolor FISH probe set (Abbott Molecular, Des Plaines, Illinois) or according to the Feulgen Staining Method for DNA-image cytometry analysis. DNA was extracted from residual liquid material, and frequencies of aberrant methylation of APC, p16(INK4A) , and RASSF1A gene promoters were determined with quantitative methylation-specific polymerase chain reaction (QMSP) after bisulfite conversion. Clinical and histological follow-up according to a reference standard, defined in advance, were available for 198 of 210 patients. In the whole cohort, cytology, FISH, DNA-image cytometry, and QMSP achieved sensitivities of 83.7%, 78%, 79%, and 49.6%, respectively (specificities of 69.8%, 98.2%, 98.2%, and 98.4%, respectively). Subsequent to cytologically equivocal diagnoses, FISH, DNA-image cytometry, and QMSP definitely identified malignancy in 79%, 83%, and 49%, respectively. With QMSP, 4 of 22 cancer patients with cytologically negative diagnoses were correctly identified. Thus, adjuvant FISH or DNA-image cytometry in cytologically equivocal diagnoses improves diagnostic accuracy at comparable rates. Adjuvant QMSP in cytologically negative cases with persistent suspicion of lung cancer would enhance sensitivity. Copyright © 2011 American Cancer Society.

  15. 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.

  16. Cytopathology of parasitic dermatitis in dogs.

    PubMed

    Sood, N K; Mekkib, Berhanu; Singla, L D; Gupta, K

    2012-04-01

    Out of 44 cases of dermatitis in dogs, 11 cases of parasitic origin were analyzed by cytopathology. Histopathologic examination of punch biopsies was also done for correlation with cytologic findings. Sarcoptic dermatitis was recorded in six cases, wherein, besides sarcoptic mites, neutrophils, macrophages, and plasma cells and keratinizing epithelial cells were also seen. Hematology revealed a relative neutrophilia and mild eosinophilia. Four cases of severe and generalized demodicosis complicated with bacteria and/or Malassezia sp. infection were also recorded. Histopathologically numerous Demodex sp. mites in varying stage of maturation were found damaging the hair follicles along with associated pathological changes and foreign body granulomas in one case. In addition, flea allergy dermatitis was also observed in one dog. In nutshell, cytology was found to be unequivocally effective in diagnosing parasitic dermatitis.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting.

    PubMed

    Slaninová, Iva; López-Sánchez, Noelia; Šebrlová, Kristýna; Vymazal, Ondřej; Frade, José María; Táborská, Eva

    2016-01-01

    Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. In consideration of the results

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. The BSCC code of practice--exfoliative cytopathology (excluding gynaecological cytopathology).

    PubMed

    Chandra, A; Cross, P; Denton, K; Giles, T; Hemming, D; Payne, C; Wilson, A; Wilson, P

    2009-08-01

    Exfoliative cytopathology (often referred to as non-gynaecological cytology) is an important part of the workload of all diagnostic pathology departments. It clearly has a role in the diagnosis of neoplastic disease but its role in establishing non-neoplastic diagnoses should also be recognised. Ancillary tests may be required to establish a definitive diagnosis. Clinical and scientific teamwork is essential to establish an effective cytology service and staffing levels should be sufficient to support preparation, prescreening, on-site adequacy assessment and reporting of samples as appropriate. Routine clinical audit and histology/cytology correlation should be in place as quality control of a cytology service. Cytology staff should be involved in multidisciplinary meetings and appropriate professional networks. Laboratories should have an effective quality management system conforming to the requirements of a recognised accreditation scheme such as Clinical Pathology Accreditation (UK) Ltd. Consultant pathologists should sign out the majority of exfoliative cytology cases. Where specimens are reported by experienced biomedical scientists (BMS), referred to as cytotechnologists outside the UK, this must only be when adequate training has been given and be defined in agreed written local protocols. An educational basis for formalising the role of the BMS in exfoliative cytopathology is provided by the Diploma of Expert Practice in Non-gynaecological Cytology offered by the Institute of Biomedical Science (IBMS). The reliability of cytological diagnoses is dependent on the quality of the specimen provided and the quality of the preparations produced. The laboratory should provide feedback and written guidance on specimen procurement. Specimen processing should be by appropriately trained, competent staff with appropriate quality control. Microscopic examination of preparations by BMS should be encouraged wherever possible. Specific guidance is provided on the

  10. Cytopathologic features of NUT midline carcinoma: A series of 26 specimens from 13 patients.

    PubMed

    Bishop, Justin A; French, Christopher A; Ali, Syed Z

    2016-12-01

    NUT midline carcinoma (NMC) is an increasingly recognized neoplasm defined by rearrangements of the nuclear protein in testis (NUT) gene (also known as NUTM1). NMC is important to diagnose for prognostic and diagnostic reasons, but to date, only a small case series and rare case reports of the cytopathologic features of NMC have been published. All NMC specimens (confirmed by molecular testing and/or NUT immunoreactivity) with cytopathologic material available were identified at 2 academic centers. All smears were reviewed, and the cytologic characteristics were described. Twenty-six cytopathologic specimens of NMC were identified from 13 patients: 8 men and 5 women ranging in age from 16 to 68 years (mean, 35 years). The NMCs arose in the mediastinum (n = 4), sinonasal tract (n = 4), neck (n = 2), lung (n = 1), lung and mediastinum (n = 1), and kidney (n = 1). Cytologic specimens included serous cavity effusions (n = 13), fine-needle aspirates (n = 9), bronchial brushings (n = 2), bronchial lavage (n = 1), and bronchial washings (n = 1). Ancillary studies were performed on cell blocks for only 6 samples from 4 patients: immunohistochemistry (n = 6) and flow cytometry (n = 1). All 13 NMCs had corresponding surgical pathology material. The NUT rearrangement status was known in 10 cases, and in 3 cases, the diagnosis was established by immunoreactivity for NUT. On cytologic smears, the NMCs were mostly hypercellular with monotonous, small to midsize, primitive-appearing cells largely distributed singly in a discohesive pattern. The tumor cells had round to oval nuclei that appeared mostly naked and devoid of cytoplasm. The nuclei varied in chromatin density from mostly pale, open chromatin to a hyperchromatic, neuroendocrine-type appearance, often with focal cell-to-cell molding, and most examples had a distinct, small nucleolus. NMC is a recently recognized tumor that should be considered in the differential

  11. Flow cytometry and laser scanning cytometry, a comparison of techniques.

    PubMed

    Mach, William J; Thimmesch, Amanda R; Orr, James A; Slusser, Joyce G; Pierce, Janet D

    2010-08-01

    Flow and laser scanning cytometry are used extensively in research and clinical settings. These techniques provide clinicians and scientists information about cell functioning in a variety of health and disease states. An in-depth knowledge and understanding of cytometry techniques can enhance interpretation of current research findings. Our goal with this review is to reacquaint clinicians and scientists with information concerning differences between flow and laser scanning cytometry by comparing their capabilities and applications. A Pubmed abstract search was conducted for articles on research, reviews and current texts relating to origins and use of flow and laser scanning cytometry. Attention was given to studies describing application of these techniques in the clinical setting. Both techniques exploit interactions between the physical properties of light. Data are immediately and automatically acquired; they are distinctly different. Flow cytometry provides valuable rapid information about a wide variety of cellular or particle characteristics. This technique does not provide the scanned high resolution image analysis needed for investigators to localize areas of interest within the cell for quantification. Flow cytometry requires that the sample contain a large amount disaggregated, single, suspended cells. Laser scanning cytometry is slide-based and does not require as large of a sample. The tissue sample is affixed to a slide allowing repeated sample analyses. These cytometry techniques are used in the clinical setting to understand pathophysiological derangements associated with many diseases; cardiovascular disease, diabetes, acute lung injury, hemorrhagic shock, surgery, cancer and Alzheimer's disease. Understanding the differences between FCM and LSCM can assist investigators in planning and design of their research or clinical testing. Researchers and clinicians optimize these technique capabilities with the cellular characteristics they wish to

  12. 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.

  13. A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high-content high-resolution multiparameter image-cytometry.

    PubMed

    Furia, Laura; Pelicci, Pier Giuseppe; Faretta, Mario

    2013-04-01

    Dissection of complex molecular-networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high-resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image-analysis of data from confocal or widefield robotized microscopes. We have applied this image-cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell-cycle profile and active DNA-replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (γH2AX) and 53BP1, as markers of DNA-damage response (DDR); and (iii) p53 and p21, as checkpoint-activation markers. Our data suggest the existence of cell-cycle modulated mechanisms involving different functions of γH2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell-cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance.

  14. 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.

  15. 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.

  16. Sensitivity of cytopathological examination in the diagnosis of feline sporotrichosis.

    PubMed

    Pereira, Sandro A; Menezes, Rodrigo C; Gremião, Isabella D F; Silva, Jéssica N; Honse, Carla de O; Figueiredo, Fabiano B; da Silva, Denise T; Kitada, Amanda A Braga; dos Reis, Erica G; Schubach, Tânia M P

    2011-04-01

    Sporotrichosis is caused by Sporothrix schenckii. The cat is the animal species most affected by this mycosis and plays an important role in the zoonotic transmission of this disease. The definitive diagnosis is made by isolation of the fungus in culture; however, cytopathological examination is frequently used in cats. Medical records from cats treated at Instituto de Pesquisa Clínica Evandro Chagas/Fiocruz, Rio de Janeiro, Brazil, between 2004 and 2006 were reviewed. Criteria for inclusion were a diagnosis by isolation of S schenckii in culture and cytopathological examination of the same lesion as that used for collection of the culture material. Eight hundred and six cats were included in the study. Yeast-like structures suggestive of S schenckii were observed in 636 cases. The sensitivity of the method was 78.9%. Its practicality, low cost and sensitivity validate cytopathology as a safe technique for the presumptive diagnosis of sporotrichosis in cats. Copyright © 2010 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

  17. Comparison of glass slides and various digital-slide modalities for cytopathology screening and interpretation.

    PubMed

    Hanna, Matthew G; Monaco, Sara E; Cuda, Jacqueline; Xing, Juan; Ahmed, Ishtiaque; Pantanowitz, Liron

    2017-09-01

    Whole-slide imaging in cytology is limited when glass slides are digitized without z-stacks for focusing. Different vendors have started to provide z-stacking solutions to overcome this limitation. The Panoptiq imaging system allows users to create digital files combining low-magnification panoramic images with regions of interest (ROIs) that are imaged with high-magnification z-stacks. The aim of this study was to compare such panoramic images with conventional whole-slide images and glass slides for the tasks of screening and interpretation in cytopathology. Thirty glass slides, including 10 ThinPrep Papanicolaou tests and 20 nongynecologic cytology cases, were digitized with an Olympus BX45 integrated microscope with an attached Prosilica GT camera. ViewsIQ software was used for image acquisition and viewing. These glass slides were also scanned on an Aperio ScanScope XT at ×40 (0.25 μm/pixel) with 1 z-plane and were viewed with ImageScope software. Digital and glass sides were screened and dotted/annotated by a cytotechnologist and were subsequently reviewed by 3 cytopathologists. For panoramic images, the cytotechnologist manually created digital maps and selected representative ROIs to generate z-stacks at a higher magnification. After 3-week washout periods, panoramic images were compared with Aperio digital slides and glass slides. The Panoptiq system permitted fine focusing of thick smears and cell clusters. In comparison with glass slides, the average screening times were 5.5 and 1.8 times longer with Panoptiq and Aperio images, respectively, but this improved with user experience. There was no statistical difference in diagnostic concordance between all 3 modalities. Users' diagnostic confidence was also similar for all modalities. The Aperio whole-slide scanner with 1 z-plane scanning and the Panoptiq imaging system with z-stacking are both suitable for cytopathology screening and interpretation. However, ROI z-stacks do offer a superior mechanism

  18. 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.

  19. 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.

  20. Is there an advantage to be gained from adding digital image cytometry of brush cytology to a standard biopsy protocol in patients with Barrett's esophagus?

    PubMed

    Borovicka, J; Schönegg, R; Hell, M; Kradolfer, D; Bauerfeind, P; Dorta, G; Netzer, P; Binek, J; Meyenberger, C; Fischer, J E; Spieler, P

    2009-05-01

    The current gold standard in Barrett's esophagus monitoring consists of four-quadrant biopsies every 1-2 cm in accordance with the Seattle protocol. Adding brush cytology processed by digital image cytometry (DICM) may further increase the detection of patients with Barrett's esophagus who are at risk of neoplasia. The aim of the present study was to assess the additional diagnostic value and accuracy of DICM when added to the standard histological analysis in a cross-sectional multicenter study of patients with Barrett's esophagus in Switzerland. One hundred sixty-four patients with Barrett's esophagus underwent 239 endoscopies with biopsy and brush cytology. DICM was carried out on 239 cytology specimens. Measures of the test accuracy of DICM (relative risk, sensitivity, specificity, likelihood ratios) were obtained by dichotomizing the histopathology results (high-grade dysplasia or adenocarcinoma vs. all others) and DICM results (aneuploidy/intermediate pattern vs. diploidy). DICM revealed diploidy in 83% of 239 endoscopies, an intermediate pattern in 8.8%, and aneuploidy in 8.4%. An intermediate DICM result carried a relative risk (RR) of 12 and aneuploidy a RR of 27 for high-grade dysplasia/adenocarcinoma. Adding DICM to the standard biopsy protocol, a pathological cytometry result (aneuploid or intermediate) was found in 25 of 239 endoscopies (11%; 18 patients) with low-risk histology (no high-grade dysplasia or adenocarcinoma). During follow-up of 14 of these 18 patients, histological deterioration was seen in 3 (21%). DICM from brush cytology may add important information to a standard biopsy protocol by identifying a subgroup of BE-patients with high-risk cellular abnormalities.

  1. 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.

  2. [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.

  3. Fine social aspiration: Twitter as a voice for cytopathology.

    PubMed

    Lepe, M; Gardner, J M

    2017-08-01

    Social media is an influential tool that has the power to transform cytopathology. Twitter is being used more and more to share cutting-edge updates from pathology meetings ("live-tweeting"). Modern smartphones can now take high resolution microscopic photographs and easily transmit them worldwide via Twitter, Facebook, and other social media, allowing cytopathologists to share educational pearls and discuss difficult cases on a global scale like never before. Social media also allows cytopathologists to share a behind-the-scenes look at their subspecialty with other physicians and even the non-medical public, helping them to better understand the crucial importance of cytopathology in modern medicine. This could positively impact rapport with other specialties, influence policy making, and possibly even improve delivery of patient care. Rare disease patient communities are being formed by patients on Facebook. By joining and volunteering with these patient groups, cytopathologists would have further opportunity to interact directly with patients and their family members, explaining the role of cytopathology in patient care and helping patients to better understand their own diseases. Social media enables cytopathologists and their colleagues in other pathology subspecialties to easily and rapidly form a broad and diverse worldwide network with one another. The authors believe that this is the key to a bright future for our specialty, a strong unified global community of pathologists all working together for education, patient advocacy, and outstanding patient care. Social media can allow us to build that community, strengthen its bonds, and harness its power like never before in history. Diagn. Cytopathol. 2017;45:705-713. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. 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.

  5. CytometryML and other data formats

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.

    2006-02-01

    Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many

  6. 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.

  7. 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.

  8. Microfluidic CARS cytometry

    PubMed Central

    Wang, Han-Wei; Bao, Ning; Le, Thuc T.; Lu, Chang; Cheng, Ji-Xin

    2009-01-01

    Coherent anti-stokes Raman scattering (CARS) flow cytometry was demonstrated by combining a laser-scanning CARS microscope with a polydimethylsiloxane (PDMS) based microfluidic device. Line-scanning across the hydrodynamically focused core stream was performed for detection of flowing objects. Parameters were optimized by utilizing polystyrene beads as flowing particles. Population measurements of adipocytes isolated from mouse fat tissues demonstrated the viability of microfluidic CARS cytometry for quantitation of adipocyte size distribution. CARS cytometry could be a new modality for quantitative analysis with vibrational selectivity. PMID:18542688

  9. 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

  10. Diagnostic digital cytopathology: Are we ready yet?

    PubMed

    House, Jarret C; Henderson-Jackson, Evita B; Johnson, Joseph O; Lloyd, Mark C; Dhillon, Jasreman; Ahmad, Nazeel; Hakam, Ardeshir; Khalbuss, Walid E; Leon, Marino E; Chhieng, David; Zhang, Xiaohui; Centeno, Barbara A; Bui, Marilyn M

    2013-01-01

    The cytology literature relating to diagnostic accuracy using whole slide imaging is scarce. We studied the diagnostic concordance between glass and digital slides among diagnosticians with different profiles to assess the readiness of adopting digital cytology in routine practice. This cohort consisted of 22 de-identified previously screened and diagnosed cases, including non-gynecological and gynecological slides using standard preparations. Glass slides were digitalized using Aperio ScanScope XT (×20 and ×40). Cytopathologists with (3) and without (3) digital experience, cytotechnologists (4) and senior pathology residents (2) diagnosed the digital slides independently first and recorded the results. Glass slides were read and recorded separately 1-3 days later. Accuracy of diagnosis, time to diagnosis and diagnostician's profile were analyzed. Among 22 case pairs and four study groups, correct diagnosis (93% vs. 86%) was established using glass versus digital slides. Both methods more (>95%) accurately diagnosed positive cases than negatives. Cytopathologists with no digital experience were the most accurate in digital diagnosis, even the senior members. Cytotechnologists had the fastest diagnosis time (3 min/digital vs. 1.7 min/glass), but not the best accuracy. Digital time was 1.5 min longer than glass-slide time/per case for cytopathologists and cytotechnologists. Senior pathology residents were slower and less accurate with both methods. Cytopathologists with digital experience ranked 2(nd) fastest in time, yet last in accuracy for digital slides. There was good overall diagnostic agreement between the digital whole-slide images and glass slides. Although glass slide diagnosis was more accurate and faster, the results of technologists and pathologists with no digital cytology experience suggest that solid diagnostic ability is a strong indicator for readiness of digital adoption.

  11. Diagnostic digital cytopathology: Are we ready yet?

    PubMed Central

    House, Jarret C.; Henderson-Jackson, Evita B.; Johnson, Joseph O.; Lloyd, Mark C.; Dhillon, Jasreman; Ahmad, Nazeel; Hakam, Ardeshir; Khalbuss, Walid E.; Leon, Marino E.; Chhieng, David; Zhang, Xiaohui; Centeno, Barbara A.; Bui, Marilyn M.

    2013-01-01

    Background: The cytology literature relating to diagnostic accuracy using whole slide imaging is scarce. We studied the diagnostic concordance between glass and digital slides among diagnosticians with different profiles to assess the readiness of adopting digital cytology in routine practice. Materials and Methods: This cohort consisted of 22 de-identified previously screened and diagnosed cases, including non-gynecological and gynecological slides using standard preparations. Glass slides were digitalized using Aperio ScanScope XT (×20 and ×40). Cytopathologists with (3) and without (3) digital experience, cytotechnologists (4) and senior pathology residents (2) diagnosed the digital slides independently first and recorded the results. Glass slides were read and recorded separately 1-3 days later. Accuracy of diagnosis, time to diagnosis and diagnostician's profile were analyzed. Results: Among 22 case pairs and four study groups, correct diagnosis (93% vs. 86%) was established using glass versus digital slides. Both methods more (>95%) accurately diagnosed positive cases than negatives. Cytopathologists with no digital experience were the most accurate in digital diagnosis, even the senior members. Cytotechnologists had the fastest diagnosis time (3 min/digital vs. 1.7 min/glass), but not the best accuracy. Digital time was 1.5 min longer than glass-slide time/per case for cytopathologists and cytotechnologists. Senior pathology residents were slower and less accurate with both methods. Cytopathologists with digital experience ranked 2nd fastest in time, yet last in accuracy for digital slides. Conclusions: There was good overall diagnostic agreement between the digital whole-slide images and glass slides. Although glass slide diagnosis was more accurate and faster, the results of technologists and pathologists with no digital cytology experience suggest that solid diagnostic ability is a strong indicator for readiness of digital adoption. PMID:24392242

  12. THE EFFECT OF AN OPTIMIZED IMAGING FLOW CYTOMETRY ANALYSIS TEMPLATE ON SAMPLE THROUGHPUT IN THE REDUCED CULTURE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY.

    PubMed

    Rodrigues, M A; Probst, C E; Beaton-Green, L A; Wilkins, R C

    2016-12-01

    In cases of overexposure to ionizing radiation, the cytokinesis-block micronucleus (CBMN) assay can be performed in order to estimate the dose of radiation to an exposed individual. However, in the event of a large-scale radiation accident with many potentially exposed casualties, the assay must be able to generate accurate dose estimates to within ±0.5 Gy as quickly as possible. The assay has been adapted to, validated and optimized on the ImageStream(X) imaging flow cytometer. The ease of running this automated version of the CBMN assay allowed investigation into the accuracy of dose estimates after reducing the volume of whole blood cultured to 200 µl and reducing the culture time to 48 h. The data analysis template used to identify binucleated lymphocyte cells (BNCs) and micronuclei (MN) has since been optimized to improve the sensitivity and specificity of BNC and MN detection. This paper presents a re-analysis of existing data using this optimized analysis template to demonstrate that dose estimations from blinded samples can be obtained to the same level of accuracy in a shorter data collection time. Here, we show that dose estimates from blinded samples were obtained to within ±0.5 Gy of the delivered dose when data collection time was reduced by 30 min at standard culture conditions and by 15 min at reduced culture conditions. Reducing data collection time while retaining the same level of accuracy in our imaging flow cytometry-based version of the CBMN assay results in higher throughput and further increases the relevancy of the CBMN assay as a radiation biodosimeter. © Crown copyright 2016.

  13. 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

  14. Application of the tissue transfer technique in veterinary cytopathology.

    PubMed

    Stone, Brett M; Gan, David

    2014-06-01

    Limited availability of diagnostic cytopathologic material may preclude additional diagnostic techniques. Tissue transfer allows for preparation of additional slides from a single original slide. Information pertaining to the application of the tissue transfer technique in veterinary cytopathology is lacking. The objectives were to evaluate the application of the tissue transfer technique on Quick Dip-stained veterinary cytologic smears and to assess if a selection of histochemical and immunocytochemical stains, and PCR analyses could be performed on transferred material. Archived Quick Dip-stained canine lymph node aspirate smears from previously diagnosed lymphoma cases were utilized to validate and optimize the tissue transfer technique. In this technique, diagnostic material is lifted from the original stained slide, is divided and transferred to multiple new slides. Histochemical stains such as Gram, periodic acid Schiff, Congo red, and Ziehl-Neelson, immunohistochemistry for CD3 and PAX5, and PCR for cryptococcal and mycobacterial organisms were selectively performed on transferred material. The tissue transfer technique was simple, and transferred Quick Dip-stained material retained cellular morphology. Histochemical and immunohistochemical stains, and PCR analysis yielded reliable results when performed on the additional smears produced by this technique. The tissue transfer technique was simple and easy to perform on previously Quick Dip-stained cytology smears. Cellular detail was preserved and multiple additional ancillary diagnostic techniques were facilitated, such as histochemical and immunohistochemical stains, and PCR analysis. © 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

  15. Herpes simplex virus: isolation, cytopathological characterization and antiviral sensitivity*

    PubMed Central

    Nozawa, Carlos; Hattori, Lilian Yumi; Galhardi, Ligia Carla Faccin; Lopes, Nayara; Bomfim, Wesley Andrade; de Cândido, Ligyana Korki; de Azevedo, Elbens Marcos Minoreli; Gon, Airton dos Santos; Linhares, Rosa Elisa Carvalho

    2014-01-01

    BACKGROUND Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually self-limiting, though they can be serious, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resistant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensitivity are important in the establishment of an antivirogram for controlling the infection. OBJECTIVE This study sought to isolate and examine the cytopathological characteristics of circulating strains of the Herpes simplex virus, from clinical specimens and their sensitivity to commercially available antiherpesvirus drugs, acyclovir, phosphonophormic acid and trifluridine. METHODS Herpes simplex virus isolation, cytopathological features and antiviral sensitivity assays were performed in cell culture by tissue culture infectious dose or plaque forming unit assay. RESULTS From twenty-two clinical specimens, we isolated and adapted nine strains. Overall, the cytopathic effect was detected 24 h post-infection (p.i.) and the presence of syncytia was remarkable 48 h p.i., observed after cell staining. Out of eight isolates, four developed plaques of varying sizes. All the isolates were sensitive to acyclovir, phosphonophormic and trifluridine, with the percentage of virus inhibition (%VI) ranging from 49.7-100%. CONCLUSIONS The methodology for HSV isolation and characterization is a straightforward approach, but the drug sensitivity test, regarded as being of great practical importance, needs to be better understood. PMID:24937819

  16. 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.

  17. DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

    PubMed Central

    Mendonça, Maria Andréia Corrêa; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo

    2010-01-01

    Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus “Leghorn” nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229–235, 2010) PMID:19875846

  18. 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

  19. 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.

  20. Diagnostic accuracy of histopathologic and cytopathologic examination of Aspergillus species.

    PubMed

    Shah, Akeesha A; Hazen, Kevin C

    2013-01-01

    To assess the diagnostic accuracy of histopatho-logic and cytopathologic examination (HCE) of Aspergillus species (spp), we performed an 11-year retrospective review to correlate surgical/cytology cases with a diagnosis of Aspergillus spp with their concurrent fungal culture results. Diagnostic accuracy was defined as the percentage of cases with culture-proven Aspergillus spp divided by the number of cases diagnosed as Aspergillus spp on HCE that had growth on fungal culture. Ninety surgical/cytology cases with concurrent fungal culture were reviewed, 58 of which grew a fungal organism. Of these 58 cases, 45 grew an Aspergillus spp, whereas 13 grew an organism other than Aspergillus spp, including both common (Scedosporium, Fusarium, and Paecilomyces spp) and uncommon mimickers (Trichosporon loubieri), resulting in a diagnostic accuracy of 78%. The low diagnostic accuracy indicates that several fungal organisms can morphologically mimic Aspergillus spp and can only be distinguished by fungal culture and DNA sequencing.

  1. Cytopathology of pulmonary alveolar proteinosis complicating lung transplantation.

    PubMed

    Gal, Anthony A; Bryan, John A; Kanter, Kirk R; Lawrence, E Clinton

    2004-01-01

    Pulmonary alveolar proteinosis is a disorder of unknown origin that occurs rarely after lung transplantation. We identified a patient with pulmonary alveolar proteinosis 66 days after undergoing single lung transplantation for idiopathic pulmonary fibrosis. We based the diagnosis on the presence of amorphous clumps or globules of acellular and finely granular material in bronchoalveolar lavage fluid (BALF). This material persisted for an 18.5-month period and was present in 9 of 14 lavage specimens. However, despite its presence in the native lung at autopsy, the material was seen in only 1 of 14 transbronchial lung biopsy specimens. Although uncommon, pulmonary alveolar proteinosis can be diagnosed readily in BALF by its distinctive cytopathologic features and should be considered in the differential diagnosis of pulmonary disease in lung transplant recipients.

  2. Small cell osteosarcoma: cytopathologic characteristics and differential diagnosis.

    PubMed

    Bishop, Justin A; Shum, Chung H; Sheth, Sheila; Wakely, Paul E; Ali, Syed Z

    2010-05-01

    Small cell osteosarcoma may present a challenging primary diagnosis on cytologic assessment owing to its rarity and its morphologic similarity to other small round blue cell tumors. Five cases of small cell osteosarcoma from our cytopathology archives were identified and reviewed and cytologic features elaborated. Three cases were fine-needle aspirations from bony lesions in the classic location for osteosarcoma (2 distal femur and 1 proximal tibia), and 2 aspirations were from metastases. Common cytomorphologic features included relatively small to intermediate cell size, high nuclear/cytoplasmic ratios, round nuclei, minimal anisonucleosis, finely granular nuclear chromatin, fine cytoplasmic vacuoles, and only rare osteoid. Small cell osteosarcoma shares many of the well-described cytomorphologic features of classic osteosarcoma, but the relatively small cells, round hyperchromatic nuclei, and scant osteoid constitute the common denominator. Correlation with radiographic findings and ancillary tests can aid in definitive diagnosis.

  3. 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

  4. 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.

  5. National Flow Cytometry Resource

    SciTech Connect

    Bell-Prince, C.; Dickson, J.A.; Jett, J.H.; Stevenson, A.P.; Sklar, L.A. )

    1993-01-01

    thee National Flow Cytometry and Sorting Resource (NFCR) was established in 1982 to develop advanced flow cytometric instrumentation and methodology, to provide facilities for using the fruits of the NFCR developments in collaborative projects and to disseminate the results to the cytometry community at large. Achievements of the NFCR for 1992 include: (1) preliminary studies of DNA inactivation in preparation for the development of an optical chromosome sorter; (2) modeling of real-time cytometry data using th ISML software package on a Cray supercomputer; (3) execution of proof-of-principle experiments on a phase sensitive flow cytometer in which cellular fluorescence lifetimes were determined; (4) continued development of the DiDAC data acquisition system to include bit mapped sorting and multi-laser capabilities; (5) development of new display modalities for flow cytometric data using the high level graphics language IDL; (6) development and testing of new approaches to clustering of multivariate data; (7) novel applications of Fourier transform flow cytometry to questions of cell activation and molecular structure.

  6. 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.

  7. Multiphoton flow cytometry strategies and applications.

    PubMed

    Tkaczyk, Eric R; Tkaczyk, Alan H

    2011-10-01

    A handful of research teams around the world have recently begun to utilize multiphoton techniques in cytometry, especially for in vivo applications. These approaches offer similar enhancements to flow cytometry as the multiphoton phenomenon brought to the field of microscopy at the turn of the 20th century, with at least six advantages over single-photon excitation. Here, we review the published literature on multiphoton cytometry in vivo or in vitro from the initial experiments in 1999 to present. Multiphoton cytometry instrumentation set-ups vary from adapted multiphoton microscopy to a dedicated system, with laser pulse power and repetition rate serving as important variables. Two-beam geometry enables quantitation of cell size. Labeling strategies include conjugated fluorophore targeting, with folate and/or dendrimer platforms. With two-color measurement, ratiometric labeling is also possible, where one dye serves as a trigger to indicate the amount of excitation a cell receives, and another informs of cellular function. With two-color labeling, geometric fluorophore distribution proves important in theory and experiment for detection sensitivity curves and detected event intensity correlation. The main biological achievements to date using this young technology are reviewed, with emphasis on real-time monitoring of minute-by-minute and long-term cell dynamics as well as the clinically significant surveillance of circulating tumor cells. For this goal, minimally invasive two-photon flow cytometry with a fiber probe may overcome the primary issue of sample volume. The technique of multicolor, multiphoton flow cytometry greatly enhances the capabilities of flow cytometry to investigate the dynamics of circulating cells in cancer and other important diseases, and may in the future benefit from advances in microscopy such as super-resolution imaging, coherent control, and bioluminescence. Copyright © 2011 International Society for Advancement of Cytometry.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. Connected component masking accurately identifies the ratio of phagocytosed and cell-bound particles in individual cells by imaging flow cytometry.

    PubMed

    Fei, Chenjie; Lillico, Dustin M E; Hall, Brian; Rieger, Aja M; Stafford, James L

    2017-04-01

    Innate immune cell-mediated recognition, capture, and engulfment of large particulate targets such as bacteria is known as phagocytosis. This highly dynamic cellular process involves a series of steps including receptor-mediated target binding, phagocytic cup formation, pseudopod extension, and phagosome closure, which depend on distinct actin polymerization events. Using flow cytometry, precise determination of target locations relative to cell membranes (i.e., surface-bound vs. fully engulfed/internalized) during the phagocytic process is difficult to quantify. Here, we describe the application of new analysis features within the IDEAS® software to distinguish internalized and surface-bound particles on individual cells with a high degree of accuracy and reproducibility. Through the use of connected component masks, the accurate discrimination of surface-bound beads versus those internalized is clearly demonstrated. In addition, we were able to further analyze the ratio of beads that had been surface-bound or internalized within individual cells. This novel method of analyzing the phagocytic process provides more accurate determination of target-cell interactions that will assist in examination of the signalling events that occur during the various stages of phagocytosis. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  14. 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

  15. CytometryML: a markup language for analytical cytology

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.; Leif, Stephanie H.; Leif, Suzanne B.

    2003-06-01

    Cytometry Markup Language, CytometryML, is a proposed new analytical cytology data standard. CytometryML is a set of XML schemas for encoding both flow cytometry and digital microscopy text based data types. CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. These schemas provide representations for the keywords in FCS 3.0 and will soon include DICOM microscopic image data. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. A preliminary version of a list mode binary data type, which does not presently exist in DICOM, has been designed. This binary type is required to enhance the storage and transmission of flow cytometry and digital microscopy data. Index files based on Waveform indices will be used to rapidly locate the cells present in individual subsets. DICOM has the advantage of employing standard file types, TIF and JPEG, for Digital Microscopy. Using an XML schema based representation means that standard commercial software packages such as Excel and MathCad can be used to analyze, display, and store analytical cytometry data. Furthermore, by providing one standard for both DICOM data and analytical cytology data, it eliminates the need to create and maintain special purpose interfaces for analytical cytology data thereby integrating the data into the larger DICOM and other clinical communities. A draft version of CytometryML is available at www.newportinstruments.com.

  16. 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.

  17. Application of low vacuum scanning electron microscopy for Papanicolaou-stained slides for cytopathology examinations.

    PubMed

    Yano, Tetsuya; Soejima, Yurie; Sawabe, Motoji

    2016-06-01

    Papanicolaou (Pap)-stained slides are usually observed using a transmitted light microscope for cytopathology. However, progress in pathological examinations has created a need for new diagnostic tools, because cytopathological preparations do not allow additional examinations without a loss of specimen, unlike histopathology. Low-vacuum scanning electron microscopy (LVSEM) can reveal the surface topography at an ultrastructual resolution without metal coating. The aim of this study was to determine the conditions required for observing Pap-stained slides of oral smears using LVSEM without any loss of specimen and to reexamine the same slides again using light microscopy, while preserving the cytopathological information. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. 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.

  19. 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

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

    PubMed

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

    2016-01-01

    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. 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. 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. 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.

  1. Pineal gland lesions: a cytopathologic study of 20 specimens.

    PubMed

    Parwani, Anil V; Baisden, Blaire L; Erozan, Yener S; Burger, Peter C; Ali, Syed Z

    2005-04-25

    Pineal gland lesions are rare, with only a few cytologic descriptions occurring in the literature, according to the authors' knowledge. The current article describes the cytopathologic characteristics of 20 such lesions with discussion of differential diagnoses. Cytologic material was obtained either by fine-needle aspiration biopsy (FNAB) under stearotactic radiologic guidance or by touch imprinting (TI) at the time of frozen sectioning. The 20 specimens include pineoblastoma (five specimens), pineocytoma (four specimens), astrocytoma (three specimens), germ cell tumor (three specimens), meningioma (one specimen), epidermoid cyst (three specimens), and pineal cyst (one specimen). Smears were stained with Diff-Quik and with Papanicolaou and hematoxylin and eosin stains. In selected specimens, immunoperoxidase (IPOX) stains were performed on cell block sections using synaptophysin, neuron-specific enolase, placental alkaline phosphatase, glial fibrillary acidic protein, leukocyte common antigen, cytokeratins, and human chorionic gonadotropin antibodies. Several cytomorphologic characteristics unique to each lesional category with occasional overlapping features were observed. The unique features included the following: small, hyperchromatic, round to oval cells with frequent rosetting (pineocytoma), with a few specimens in addition showing hypercellularity, crowding, mitoses, and necrosis (pineoblastoma); pleomorphic round cells in a fibrillary background (astrocytoma); large polygonal cells with prominent nucleoli and clear cytoplasm (germ cell tumor); spindled fibroblastic cells (meningioma); anucleate squames and mature squamous cells (epidermoid cyst); and small uniform polygonal cells (pineal cyst). When necessary, IPOX studies supported the morphologic diagnoses. FNAB and TI cytology were found to provide a rapid and reliable diagnosis of pineal lesions. This is particularly important when dealing with minute amounts of tissue material. Both techniques

  2. 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.

  3. Flow cytometry using spectrally encoded confocal microscopy.

    PubMed

    Golan, Lior; Yelin, Dvir

    2010-07-01

    Flow cytometry techniques often rely on detecting fluorescence from single cells flowing through the cross section of a laser beam, providing invaluable information on vast numbers of cells. Such techniques, however, are often limited in their ability to resolve clusters of cells or parallel cell flow through large vessels. We present a confocal imaging technique that images unstained cells flowing in parallel through a wide channel, using spectrally encoded reflectance confocal microscopy that does not require mechanical scanning. Images of red blood cells from our system are compared to conventional transmission microscopy, and imaging of flowing red blood cells in vitro is experimentally demonstrated.

  4. INFECTIOUS BOVINE RHINOTRACHEITIS VIRUS REPLICATION, CYTOPATHOLOGY, AND PLAQUE FORMATION IN THE PRESENCE AND ABSENCE OF NUCLEIC ACID ANALOGUES

    PubMed Central

    Stevens, Jack G.; Groman, Neal B.

    1964-01-01

    Stevens, Jack G. (University of Washington, Seattle), and Neal B. Groman. Infectious bovine rhinotracheitis virus replication, cytopathology, and plaque formation in the presence and absence of nucleic acid analogues. J. Bacteriol. 87:446–453. 1964.—Cytopathology induced by infectious bovine rhinotracheitis (IBR) virus was correlated with the one-step growth cycle. Nuclear alterations, including the development of inclusion bodies, preceded the appearance of virus. It was found that similar effects occurred in the presence of 5-bromodeoxyuridine (BUDR) and 5-fluorodeoxyuridine (FUDR), compounds which depress the yield of “standard” virus from a range of 116 to 500 to less than 0.5 plaque-forming units per cell. As with known members of the herpesvirus group, IBR virus plaques developed and enlarged indefinitely in the presence of specific antibody. An analysis of the mechanism operative in this process was undertaken. The evidence suggested that neither viral nor subviral particles capable of replicating “standard” virus passed between cells during the first 8 hr of infection. This is the time preceding the release of extracellular virus from initially infected cells. With BUDR and FUDR, it was shown that plaques also developed in this system in the virtual absence of production of “standard” infectious virus. However, a class of analogue-dependent virus was found which may have been at least partly responsible for plaque formation in the analogue-treated system. The relative contributions of subviral particles or of a self-sustaining molecular disorganization to the process have not been completely assessed as yet. Images PMID:14151070

  5. Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry.

    PubMed

    Elmendorf, S; McSharry, J; Laffin, J; Fogleman, D; Lehman, J M

    1988-05-01

    An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two-color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell-cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque-forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.

  6. 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.

  7. 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.

  8. Basics of flow cytometry.

    PubMed

    Radcliff, G; Jaroszeski, M J

    1998-01-01

    In summary, a beginner requires fundamental knowledge about flow cytometric instrumentation in order to effectively use this technology. It is important to remember that flow cytometers are very complex instruments that are composed of four closely related systems. The fluidic system transports particles from a suspension through the cytometer for interrogation by an illumination system. The resulting light scattering and fluorescence is collected, filtered, and converted into electrical signals by the optical and electronics system. The data storage and computer control system saves acquired data and is also the user interface for controlling most instrument functions. These four systems provide a very unique and powerful analytical tool for researchers and clinicians. This is because they analyze the properties of individual particles, and thousands of particles can be analyzed in a matter of seconds. Thus, data for a flow cytometric sample are a collection of many measurements instead of a single bulk measurement. Basic knowledge of instrumentation is a tremendous aid to designing experiments that can be successfully analyzed using flow cytometry. For example, it is important to know the emission wavelength of the laser in the instrument that will be used for analysis. This wavelength is critical knowledge for selecting probes. It is also important to understand that a different range of wavelengths is detected for each fluorescent channel. This will aid selection of probes that are compatible with the flow cytometer. Understanding the complication that emission spectra overlap contributes to detection can be used to guide fluorochrome selections for multicolor analysis. All of these experiment design considerations that rely on knowledge of how flow cytometers work are a very practical and effective means of avoiding wasted time, energy, and costly reagents. Data analysis is a paramount issue in flow cytometry. Analysis includes interpreting as well as

  9. Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes.

    PubMed

    Durdik, Matus; Kosik, Pavol; Gursky, Jan; Vokalova, Lenka; Markova, Eva; Belyaev, Igor

    2015-12-01

    Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. γH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, γH2AX foci and their co-localization induced by γ-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of γH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses ≥ 5 cGy when analyzed by IFC. Analysis of the co-localizing foci by ImageStream and Metafer showed statistical significance at doses ≥ 2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of γH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating γH2AX foci positive (with 1 or more γH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ≥ 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF. © 2015 International Society for Advancement of Cytometry.

  10. 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.

  11. 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.

  12. [On-site fine-needle aspiration cytology of thyroid nodules. Quality assurance of the Bethesda System for Reporting Thyroid Cytopathology (2008)].

    PubMed

    Bak, Mihály; Péter, Ilona; Nyári, Tibor; Simon, Péter; Újlaky, Mátyás; Boér, András; Kásler, Miklós

    2015-10-11

    The methods available for the diagnosis of thyroid nodules include physical examination, imaging, laboratory and fine-needle aspiration cytology tests. The aim of this study was to determine the quality assurance of fine-needle aspiration cytology of thyroid nodules. Cytology results were rated to 6 categories according to the Bethesda System for Reporting Thyroid Cytopathology (2008) (I. nondiagnostic; II. benign; III. atypia of undetermined significance; IV. follicular neoplasia; V. suspicious for malignancy; VI. malignant). All cytology reports were compared with the final histology diagnosis. A total of 1384 patient with thyroid nodule underwent fine-needle aspiration biopsy cytology. Smears were classified I. inadequate in 214 (15.9%); II. benign 986; III. atypical 56; IV. follicular neoplasm 41; V. suspicious for malignancy 18; VI. malignant 33 cases. Two hundred and twenty seven (16.8%) of the cases were operated and histologically verified. The positive predictive value in the benign category was 98.25% and in the malignant 88.46%. The sensitivity of the follicular neoplasm was 66.67%. The results suggest that fine-needle aspiration cytology of thyroid nodules using the Bethesda System for Reporting Thyroid Cytopathology has a high diagnostic accuracy. The auditing values of the results meet the proposed threshold values.

  13. College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference on good laboratory practices in gynecologic cytology: background, rationale, and organization.

    PubMed

    Tworek, Joseph A; Henry, Michael R; Blond, Barbara; Jones, Bruce Allen

    2013-02-01

    Gynecologic cytopathology is a heavily regulated field, with Clinical Laboratory Improvement Amendments of 1988 mandating the collection of many quality metrics. There is a lack of consensus regarding methods to collect, monitor, and benchmark these data and how these data should be used in a quality assurance program. Furthermore, the introduction of human papilloma virus testing and proficiency testing has provided more data to monitor. To determine good laboratory practices in quality assurance of gynecologic cytopathology. Data were collected through a written survey consisting of 98 questions submitted to 1245 Clinical Laboratory Improvement Amendments-licensed or Department of Defense laboratories. There were 541 usable responses. Additional input was sought through a Web posting of results and questions on the College of American Pathologists Web site. Four senior authors who authored the survey and 28 cytopathologists and cytotechnologists were assigned to 5 working groups to analyze data and present statements on good laboratory practices in gynecologic cytopathology at the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference. Ninety-eight attendees at the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference discussed and voted on good laboratory practice statements to obtain consensus. This paper describes the rationale, background, process, and strengths and limitations of a series of papers that summarize good laboratory practice statements in quality assurance in gynecologic cytopathology.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  15. Quantitative asymmetric-detection time-stretch optical microscopy (Q-ATOM) for ultrafast quantitative phase imaging flow cytometry (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Lau, Andy K. S.; Tang, Anson H. L.; Chung, Bob M. F.; Tsang, Kwok Yeung; Chan, Antony C. S.; Wei, Xiaoming; Wong, Kenneth K.; Lam, Edmund Y.; Cheah, Kathryn S. E.; Shum, Anderson H. C.; Tsia, Kevin K.

    2016-03-01

    Based on the interferometric or holographic approaches, recent QPM techniques provide quantitative-phase information, e.g cell volume, dry mass and optical scattering properties for label-free cellular physical phenotyping. These approaches generally rely on iterative phase-retrieval algorithms to obtain quantitative-phase information, which are computationally intensive. Moreover, current QPM techniques can only offer limited image acquisition rate by using CMOS/CCD image sensors, these two limitations hinder QPM for high-throughput quantitative image-based single-cell analysis in real-time. To this end, we demonstrate an interferometry-free quantitative phase microscopy developed on a new generation of time-stretch microscopy, asymmetric-detection time-stretch optical microscopy (ATOM), which is coined quantitative ATOM (Q-ATOM) - featuring an unprecedented cell measurement throughput together with the assorted intrinsic optical phenotypes (e.g. angular light scattering profile) and the derived physical properties of the cells (e.g. cell size, dry mass density etc.). Based on a similar concept to Schlieren imaging, Q-ATOM retrieves quantitative-phase information through multiple off-axis light-beam detection at a line-scan rate of <10 MHz - a speed unachievable by any existing QPM techniques. Phase retrieval in Q-ATOM relies on a non-iterative method, significantly reducing the computational complexity of the technique. It is a particularly important feature which facilitates real-time continuous label-free single-cell analysis in Q-ATOM. With the use of a non-interferometric configuration, we demonstrate ultrafast Q-ATOM of mouse chondrocytes and hypertrophic chondrocytes in ultrafast microfluidic flow with sub-cellular resolution at an imaging throughput equivalent to ~100,000 cells/sec without image blur. This technique shows a great potential for ultrahigh throughput label-free image-based single-cell biophysical phentotyping.

  16. 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.

  17. Infrared micro-spectroscopy for cyto-pathological classification of esophageal cells.

    PubMed

    Townsend, Douglas; Miljković, Miloš; Bird, Benjamin; Lenau, Kathleen; Old, Oliver; Almond, Max; Kendall, Catherine; Lloyd, Gavin; Shepherd, Neil; Barr, Hugh; Stone, Nick; Diem, Max

    2015-04-07

    We report results from a study utilizing infrared spectral cytopathology (SCP) to detect abnormalities in exfoliated esophageal cells. SCP has been developed over the past decade as an ancillary tool to classical cytopathology. In SCP, the biochemical composition of individual cells is probed by collecting infrared absorption spectra from each individual, unstained cell, and correlating the observed spectral patterns, and the variations therein, against classical diagnostic methods to obtain an objective, machine-based classification of cells. In the past, SCP has been applied to the analysis and classification of cells exfoliated from the cervix and the oral cavity. In these studies, it was established that SCP can distinguish normal and abnormal cell types. Furthermore, SCP can differentiate between truly normal cells, and cells with normal morphology from the vicinity of abnormalities. Thus, SCP may be a valuable tool for the screening of early stages of dysplasia and pre-cancer.

  18. 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.

  19. Techniques for cytologic sampling of pancreatic and bile duct lesions: The Papanicolaou Society of Cytopathology Guidelines.

    PubMed

    Brugge, William R; De Witt, John; Klapman, Jason B; Ashfaq, Raheela; Shidham, Vinod; Chhieng, David; Kwon, Richard; Baloch, Zubair; Zarka, Matthew; Staerkel, Gregg

    2014-01-01

    The Papanicolaou Society of Cytopathology has developed a set of guidelines for pancreatobiliary cytology, including indications for endoscopic ultrasound guided fine-needle aspiration biopsy, techniques of the endoscopic retrograde cholangiopancreatography, terminology and nomenclature of pancreatobiliary disease, ancillary testing, and postbiopsy management. All documents are based on the expertise of the authors, a review of 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 website [www.papsociety.org]. This document presents the results of these discussions regarding the use of sampling techniques in the cytological diagnosis of biliary and pancreatic lesions. This document summarizes the current state of the art for techniques in acquiring cytology specimens from the biliary tree as well as solid and cystic lesions of the pancreas.

  20. Cytopathologic evaluation of patients submitted to radiotherapy for uterine cervix cancer.

    PubMed

    Padilha, Cátia Martins Leite; Araújo, Mário Lúcio Cordeiro; Souza, Sergio Augusto Lopes de

    2017-04-01

    Cervical cancer is an important public health problem. Pap smear is the leading strategy of screening programs for cervical cancer worldwide. However, delayed diagnosis leads to more aggressive and less effective treatments. Patients with uterine cervix malignancies who are referred for radiotherapy have advanced-stage disease, which results in high rates of locoregional recurrence. The use of radiotherapy as a treatment for cervical cancer causes morphological changes in neoplastic and non-neoplastic epithelial cells, as well as in stromal cells, which make it difficult to diagnose the residual lesion, resulting in a dilemma in cytopathological routine. Based on the difficulties of cytopathologic evaluation for the follow-up of patients treated with radiotherapy for cervical cancer, our objective was to describe the actinic cytopathic effects. Our paper was based on a structured review including the period from June 2015 to April 2016, aiming at an exploratory-descriptive study. Bibliographic investigations were carried out through selection and analysis of articles, list of authors and keywords, selection of new articles focused on the analysis of bibliographic references to previously selected documents, as well as textbooks of recognized merit. The most incident actinic cytopathological alterations as described in the literature are: cellular gigantism, nuclear and cytoplasmic vacuolization, dyskeratosis, bi- and multinucleated (B/M) cells, macro and multiple nucleoli, anisokaryosis, anisonucleolosis and nuclear pyknosis. To date, a protocol has not been established that can precisely differentiate the morphological characteristics between benign cells with actinic effects from recurrent malignant cells on post-radiotherapy smears.

  1. 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.

  2. 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.

  3. 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

  4. 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.

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

    PubMed Central

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

    2013-01-01

    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

  6. 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

  7. Engineered viral nanoparticles for flow cytometry and fluorescence microscopy applications.

    PubMed

    Robertson, Kelly L; Liu, Jinny L

    2012-01-01

    Viral nanoparticles (VNPs) are attractive platforms for use in the biotechnology and biomedical fields because of their biological nature. A wide variety of these particles, labeled with fluorescent reporters, have been characterized using flow cytometry and cellular imaging techniques. Fluorescence microscopy allows the direct observation of VNPs on the cell surface or inside the membrane as well as the cellular localization of the nanoparticles while flow cytometry allows the statistical quantification of nanoparticle uptake and targeting specificity. These techniques are essential when characterizing the properties of VNPs and provide information toward the use of VNPs for targeting, imaging, and/or cargo delivery. Copyright © 2012 Wiley Periodicals, Inc.

  8. 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

  9. Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III

    PubMed Central

    Konečný, Petr; Frydrych, Ivo; Koberna, Karel

    2017-01-01

    The approach for the detection of replicational activity in cells using 5-bromo-2′-deoxyuridine, a low concentration of hydrochloric acid and exonuclease III is presented in the study. The described method was optimised with the aim to provide a fast and robust tool for the detection of DNA synthesis with minimal impact on the cellular structures using image and flow cytometry. The approach is based on the introduction of breaks into the DNA by the low concentration of hydrochloric acid followed by the subsequent enzymatic extension of these breaks using exonuclease III. Our data showed that the method has only a minimal effect on the tested protein localisations and is applicable both for formaldehyde- and ethanol-fixed cells. The approach partially also preserves the fluorescence of the fluorescent proteins in the HeLa cells expressing Fluorescent Ubiquitin Cell Cycle Indicator. In the case of the short labelling pulses that disabled the use of 5-ethynyl-2′-deoxyuridine because of the low specific signal, the described method provided a bright signal enabling reliable recognition of replicating cells. The optimized protocol was also successfully tested for the detection of trifluridine, the nucleoside used as an antiviral drug and in combination with tipiracil also for the treatment of some types of cancer. PMID:28426799

  10. Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2'-deoxyuridine, low concentration of hydrochloric acid and exonuclease III.

    PubMed

    Ligasová, Anna; Konečný, Petr; Frydrych, Ivo; Koberna, Karel

    2017-01-01

    The approach for the detection of replicational activity in cells using 5-bromo-2'-deoxyuridine, a low concentration of hydrochloric acid and exonuclease III is presented in the study. The described method was optimised with the aim to provide a fast and robust tool for the detection of DNA synthesis with minimal impact on the cellular structures using image and flow cytometry. The approach is based on the introduction of breaks into the DNA by the low concentration of hydrochloric acid followed by the subsequent enzymatic extension of these breaks using exonuclease III. Our data showed that the method has only a minimal effect on the tested protein localisations and is applicable both for formaldehyde- and ethanol-fixed cells. The approach partially also preserves the fluorescence of the fluorescent proteins in the HeLa cells expressing Fluorescent Ubiquitin Cell Cycle Indicator. In the case of the short labelling pulses that disabled the use of 5-ethynyl-2'-deoxyuridine because of the low specific signal, the described method provided a bright signal enabling reliable recognition of replicating cells. The optimized protocol was also successfully tested for the detection of trifluridine, the nucleoside used as an antiviral drug and in combination with tipiracil also for the treatment of some types of cancer.

  11. Post-fine-needle aspiration biopsy communication and the integrated and standardized cytopathology report.

    PubMed

    Pitman, Martha B; Black-Schaffer, W Stephen

    2017-06-01

    Communication between cytopathologists and patients and their care team is a critical component of accurate and timely patient management. The most important single means of communication for the cytopathologist is through the cytopathology report. Implementation of standardized terminology schemes and structured, templated reporting facilitates the ability of the cytopathologist to provide a comprehensive and integrated report. Cytopathology has been among the pathology subspecialties that have led the way in developing standardized reporting, beginning with the 1954 Papanicolaou classification scheme for cervical-vaginal cytology and continuing through the Bethesda systems for gynecological cytology and several nongynecological cytology systems. The effective reporting of cytopathology necessarily becomes more complex as it addresses increasingly sophisticated management options, requiring the integration of information from a broader range of sources. In addition to the complexity of information inputs, a wider spectrum of consumers of these reports is emerging, from patients themselves to primary care providers to subspecialized disease management experts. Both these factors require that the reporting cytopathologist provide the integration and interpretation necessary to translate diverse forms of information into meaningful and actionable reports that will inform the care team while enabling the patient to meaningfully participate in his or her own care. To achieve such broad and focused communications will require first the development of standardized and integrated reports and ultimately the involvement of cytopathologists in the development of the clinical informatics needed to treat all these items of information as structured data elements with flexible reporting operators to address the full range of patient and patient care needs. Cancer Cytopathol 2017;125(6 suppl):486-93. © 2017 American Cancer Society. © 2017 American Cancer Society.

  12. The Milan System for Reporting Salivary Gland Cytopathology: Analysis and suggestions of initial survey.

    PubMed

    Rossi, Esther Diana; Faquin, William C; Baloch, Zubair; Barkan, Güliz A; Foschini, Maria Pia; Pusztaszeri, Marc; Vielh, Philippe; Kurtycz, Daniel F I

    2017-07-14

    An international panel of experts in the field of salivary gland cytology (SGC), supported by the American Society of Cytopathology (ASC) and the International Academy of Cytology, conducted a survey to seek evidence and practice patterns regarding SGC. Results were used to provide focus for the proposed Milan System for Reporting Salivary Gland Cytopathology. The study group, formed during the 2015 European Congress of Cytology held in Milan, Italy, generated a survey that included 49 specific questions related to the taxonomies, practices, and diagnostic entities of salivary cytology. Qualtrics software was used as the study platform. Software and server support were provided by the division of information technology at the University of Wisconsin. The survey was available online from November 2015 until February 2016. Participants were invited through the Web sites of the ASC, the International Academy of Cytology, and the Papanicolaou Society of Cytopathology as well as by the ASC e-mail "ListServe"; responses were evaluated by the Milan System editors. Responses from a total of 515 participants were collected and reviewed. A total of 347 participants provided demographic data information. Responses revealed variations in diagnostic practice and subsequent management. Participants believed that the acceptable rate for nondiagnostic samples should not be higher than 10%. There were varied opinions regarding the approach to neoplastic lesions of uncertain malignant potential, those that may or may have not local invasion and distant spread. Results of the survey demonstrated strong support for the development of a unified system for reporting SGC. Cancer Cytopathol 2017. © 2017 American Cancer Society. © 2017 American Cancer Society.

  13. 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.

  14. 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.

  15. Cancer screening via infrared spectral cytopathology (SCP): results for the upper respiratory and digestive tracts.

    PubMed

    Diem, Max; Miljković, Miloš; Bird, Benjamin; Mazur, Antonella I; Schubert, Jen M; Townsend, Douglas; Laver, Nora; Almond, Max; Old, Oliver

    2016-01-21

    Instrumental advances in infrared micro-spectroscopy have made possible the observation of individual human cells and even subcellular structures. The observed spectra represent a snapshot of the biochemical composition of a cell; this composition varies subtly but reproducibly with cellular effects such as progression through the cell cycle, cell maturation and differentiation, and disease. The aim of this summary is to provide a synopsis of the progress achieved in infrared spectral cytopathology (SCP) - the combination of infrared micro-spectroscopy and multivariate methods of analysis - for the detection of abnormalities in exfoliated human cells of the upper respiratory and digestive tract, namely the oral and nasopharyngeal cavities, and the esophagus.

  16. 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.

  17. Levitational Image Cytometry with Temporal Resolution.

    PubMed

    Tasoglu, Savas; Khoory, Joseph A; Tekin, Huseyin C; Thomas, Clemence; Karnoub, Antoine E; Ghiran, Ionita C; Demirci, Utkan

    2015-07-08

    A simple, yet powerful magnetic-levitation-based device is reported for real-time, label-free separation, as well as high-resolution monitoring of cell populations based on their unique magnetic and density signatures. This method allows a wide variety of cellular processes to be studied, accompanied by transient or permanent changes in cells' fundamental characteristics as a biological material. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. 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.

  19. Phasor plotting with frequency-domain flow cytometry

    PubMed Central

    Cao, Ruofan; Jenkins, Patrick; Peria, William; Sands, Bryan; Naivar, Mark; Brent, Roger; Houston, Jessica P.

    2016-01-01

    Interest in time resolved flow cytometry is growing. In this paper, we collect time-resolved flow cytometry data and use it to create polar plots showing distributions that are a function of measured fluorescence decay rates from individual fluorescently-labeled cells and fluorescent microspheres. Phasor, or polar, graphics are commonly used in fluorescence lifetime imaging microscopy (FLIM). In FLIM measurements, the plotted points on a phasor graph represent the phase-shift and demodulation of the frequency-domain fluorescence signal collected by the imaging system for each image pixel. Here, we take a flow cytometry cell counting system, introduce into it frequency-domain optoelectronics, and process the data so that each point on a phasor plot represents the phase shift and demodulation of an individual cell or particle. In order to demonstrate the value of this technique, we show that phasor graphs can be used to discriminate among populations of (i) fluorescent microspheres, which are labeled with one fluorophore type; (ii) Chinese hamster ovary (CHO) cells labeled with one and two different fluorophore types; and (iii) Saccharomyces cerevisiae cells that express combinations of fluorescent proteins with different fluorescence lifetimes. The resulting phasor plots reveal differences in the fluorescence lifetimes within each sample and provide a distribution from which we can infer the number of cells expressing unique single or dual fluorescence lifetimes. These methods should facilitate analysis time resolved flow cytometry data to reveal complex fluorescence decay kinetics. PMID:27410612

  20. Phasor plotting with frequency-domain flow cytometry.

    PubMed

    Cao, Ruofan; Jenkins, Patrick; Peria, William; Sands, Bryan; Naivar, Mark; Brent, Roger; Houston, Jessica P

    2016-06-27

    Interest in time resolved flow cytometry is growing. In this paper, we collect time-resolved flow cytometry data and use it to create polar plots showing distributions that are a function of measured fluorescence decay rates from individual fluorescently-labeled cells and fluorescent microspheres. Phasor, or polar, graphics are commonly used in fluorescence lifetime imaging microscopy (FLIM). In FLIM measurements, the plotted points on a phasor graph represent the phase-shift and demodulation of the frequency-domain fluorescence signal collected by the imaging system for each image pixel. Here, we take a flow cytometry cell counting system, introduce into it frequency-domain optoelectronics, and process the data so that each point on a phasor plot represents the phase shift and demodulation of an individual cell or particle. In order to demonstrate the value of this technique, we show that phasor graphs can be used to discriminate among populations of (i) fluorescent microspheres, which are labeled with one fluorophore type; (ii) Chinese hamster ovary (CHO) cells labeled with one and two different fluorophore types; and (iii) Saccharomyces cerevisiae cells that express combinations of fluorescent proteins with different fluorescence lifetimes. The resulting phasor plots reveal differences in the fluorescence lifetimes within each sample and provide a distribution from which we can infer the number of cells expressing unique single or dual fluorescence lifetimes. These methods should facilitate analysis time resolved flow cytometry data to reveal complex fluorescence decay kinetics.

  1. 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

  2. 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.

  3. 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.

  4. Cellular analysis by open-source software for affordable cytometry.

    PubMed

    Mittag, Anja; Pinto, Fernanda E; Endringer, Denise C; Tarnok, Attila; Lenz, Dominik

    2011-01-01

    Image cytometry is an important technique in affordable healthcare and cellular research. Some efforts toward establishing a personal, low-cost cytometer have been described in the literature. However, a self-assembled fluorescence microscope requires software for cytometric analysis. There are some open-source image-based software analysis applications available. However, for a quantitative analysis of images, software that can generate data comparable to those of previously evaluated cytometric analyses programs is required. Hence, the aim of this study is to compare results of a commercially available image cytometry program to data obtained using the open-source software CellProfiler (CP). Leukocytes and fluorescent bead images obtained using a Laser Scanning Cytometer were analyzed by CP and the results compared with those of conventional cytometric analyses' programs. Algorithms were developed enabling the analysis of leukocytes and beads by CP. CP provided similar results to those obtained by the cytometer software. Hallmark parameters, including cell count and fluorescence intensity, revealed a high correlation in the analysis of both programs. Therefore, CP is appropriate for cellular analysis on a self-assembled microscope, thereby enabling affordable cytometry. Copyright © 2011 Wiley Periodicals, Inc.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. Data Standards for Flow Cytometry

    PubMed Central

    SPIDLEN, JOSEF; GENTLEMAN, ROBERT C.; HAALAND, PERRY D.; LANGILLE, MORGAN; MEUR, NOLWENN LE; OCHS, MICHAEL F.; SCHMITT, CHARLES; SMITH, CLAYTON A.; TREISTER, ADAM S.; BRINKMAN, RYAN R.

    2009-01-01

    Flow cytometry (FCM) is an analytical tool widely used for cancer and HIV/AIDS research, and treatment, stem cell manipulation and detecting microorganisms in environmental samples. Current data standards do not capture the full scope of FCM experiments and there is a demand for software tools that can assist in the exploration and analysis of large FCM datasets. We are implementing a standardized approach to capturing, analyzing, and disseminating FCM data that will facilitate both more complex analyses and analysis of datasets that could not previously be efficiently studied. Initial work has focused on developing a community-based guideline for recording and reporting the details of FCM experiments. Open source software tools that implement this standard are being created, with an emphasis on facilitating reproducible and extensible data analyses. As well, tools for electronic collaboration will assist the integrated access and comprehension of experiments to empower users to collaborate on FCM analyses. This coordinated, joint development of bioinformatics standards and software tools for FCM data analysis has the potential to greatly facilitate both basic and clinical research—impacting a notably diverse range of medical and environmental research areas. PMID:16901228

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. CytometryML: a data standard which has been designed to interface with other standards

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.

    2007-02-01

    Because of the differences in the requirements, needs, and past histories including existing standards of the creating organizations, a single encompassing cytology-pathology standard will not, in the near future, replace the multiple existing or under development standards. Except for DICOM and FCS, these standardization efforts are all based on XML. CytometryML is a collection of XML schemas, which are based on the Digital Imaging and Communications in Medicine (DICOM) and Flow Cytometry Standard (FCS) datatypes. The CytometryML schemas contain attributes that link them to the DICOM standard and FCS. Interoperability with DICOM has been facilitated by, wherever reasonable, limiting the difference between CytometryML and the previous standards to syntax. In order to permit the Resource Description Framework, RDF, to reference the CytometryML datatypes, id attributes have been added to many CytometryML elements. The Laboratory Digital Imaging Project (LDIP) Data Exchange Specification and the Flowcyt standards development effort employ RDF syntax. Documentation from DICOM has been reused in CytometryML. The unity of analytical cytology was demonstrated by deriving a microscope type and a flow cytometer type from a generic cytometry instrument type. The feasibility of incorporating the Flowcyt gating schemas into CytometryML has been demonstrated. CytometryML is being extended to include many of the new DICOM Working Group 26 datatypes, which describe patients, specimens, and analytes. In situations where multiple standards are being created, interoperability can be facilitated by employing datatypes based on a common set of semantics and building in links to standards that employ different syntax.

  18. Flow Cytometry: Impact on Early Drug Discovery.

    PubMed

    Edwards, Bruce S; Sklar, Larry A

    2015-07-01

    Modern flow cytometers can make optical measurements of 10 or more parameters per cell at tens of thousands of cells per second and more than 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 multiparameter 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. © 2015 Society for Laboratory Automation and Screening.

  19. 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

  20. 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.

  1. PREDICTIVE VALUE OF SOMATIC MUTATIONS FOR THE DEVELOPMENT OF MALIGNANCY IN THYROID NODULES BY CYTOPATHOLOGY.

    PubMed

    Halászlaki, Csaba; Tóbiás, Bálint; Balla, Bernadett; Kósa, János P; Horányi, János; Bölöny, Eszter; Nagy, Zsolt; Speer, Gábor; Járay, Balázs; Székely, Eszter; Istók, Roland; Székely, Tamás; Putz, Zsuzsanna; Dank, Magdolna; Lakatos, Péter; Takács, István

    2016-09-01

    The purpose of our prospective longitudinal study was to evaluate the predictive efficacy of genetic testing for malignancies in fine-needle aspiration biopsy samples that are cytologically benign at the time of biopsy. A total of 779 aspirated cytological samples collected from thyroid nodules of 626 patients were included in a 3-year follow-up study. Consecutive patients with cytologically benign thyroid nodules by the Bethesda System for Reporting Thyroid Cytopathology were enrolled in the study. At enrollment, somatic 1-point nucleotide polymorphisms of BRAF and RAS family genes were tested by melting-point analysis, while RET/PTC and PAX8/PPAR-gamma rearrangements were examined by real-time polymerase chain reaction. The genetic test was considered to be positive if a somatic mutation was found. Malignant cytopathologic diagnoses were confirmed by histopathology. In samples collected from 779 thyroid nodules, there were 39 BRAF, 33 RAS mutations, and 1 RET/PTC rearrangements found at the beginning of the study. No PAX8/PPAR-gamma rearrangement was identified. There were 52 malignant thyroid tumors removed during follow-up, out of which 24 contained a somatic mutation. The specificity of the presence of somatic mutations for malignancies was as high as 93.3%, and sensitivity was 46.2%. The negative predictive value of genetic testing reached 96.0%. Our results show that our set of genetic tests can predict the appearance of malignancy in benign thyroid nodules (at the beginning of follow-up) with high specificity and strong negative predictive value. BRAF = v-raf murine sarcoma viral oncogene homolog B1 FLUS = follicular lesion of undetermined significance FNAB = fine-needle aspiration biopsy FTC = follicular thyroid carcinoma HRAS = homologous to the oncogene from the Harvey rat sarcoma virus KRAS = homologous to the oncogene from the Kirsten rat sarcoma virus NRAS = first isolated from a human neuroblastoma/neuroblastoma RAS = viral oncogene homolog PAX8

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Cytopathological findings of primary pulmonary Ewing family of tumors with EWSR1 translocation: A case report.

    PubMed

    Mizuguchi, Keishi; Minato, Hiroshi; Onishi, Hitomi; Mitani, Yuki; Kawai, Jun

    2016-09-01

    Primary pulmonary neoplasms of the Ewing family of tumors (EFT) are extremely rare and usually occur in adolescents or young adults. Only about 40 cases of pulmonary EFT have been reported in English literature, and no cytological studies have been documented. In this report, we describe the cytopathological findings of a primary pulmonary EFT in an elderly patient. A 70-year-old man sought care because of a progressing cough and dyspnea. Chest computed tomography revealed a circumscribed mass of 6 cm in the left upper lobe. Fine needle aspiration cytology and core needle biopsy revealed uniform round cell proliferation. The predominant population consisted of cells with thickened nuclear membranes, finely dispersed chromatin, single distinct nucleoli, and indistinct cytoplasm. The other population consisted of smaller cells with darker chromatin. The cytoplasm stained positive for periodic acid-Schiff stain and was digested by diastase. Immunohistochemistry showed positivity for MIC2 (CD99), and focal positivity for neuron specific enolase, synaptophysin, and chromogranin A. Fluorescence in situ hybridization (FISH) revealed EWSR1 translocation. Although rare, pulmonary EFT cannot be disregarded, regardless of age. When two populations of uniform, round cells are observed, immunohistochemistry with MIC2 (CD99) and cytogenetic analysis by reverse transcription polymerase chain reaction or FISH should be considered. Cytological diagnosis may play an important role in the early diagnosis and treatment of pulmonary EFT. © 2016 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  7. Identification of AgNORs and cytopathological changes in oral lichen planus lesions.

    PubMed

    Ferreira, Stefânia Jeronimo; Machado, Maria Ângela Naval; de Lima, Antônio Adilson Soares; Johann, Aline Cristina Batista Rodrigues; Grégio, Ana Maria Trindade; Azevedo-Alanis, Luciana Reis

    2017-01-01

    To evaluate cytopathological changes in epithelial cells of the oral mucosa of patients with oral lichen planus (OLP) compared with patients without OLP. Swabs were collected from the oral mucosa of 20 patients with OLP (case group) and 20 patients without OLP (control group) using liquid-based cytology. After Papanicolaou staining, the smears were characterized based on Papanicolaou classification and degree of maturation. Nuclear area (NA) measurements, cytoplasmic area (CA) measurements, and the NA/CA ratio were determined from 50 epithelial cells per slide. For quantification of argyrophilic nucleolar organizer regions (AgNORs), the smears were stained with silver nitrate, and the number of AgNORs was counted in 100 cells. In both groups, there was a predominance of Papanicolaou Class I nucleated cells in the superficial layer. The average values of NA (p>0.05) and CA (p=0.000) were greater in the case group (NA=521.6, CA=22,750.3) compared with the control group (NA=518.9, CA=18,348.0). The NA/CA ratio was 0.025 for the case group and 0.031 for the control group (p=0.004). There was no significant difference between the mean AgNORs values of both groups (p>0.05). The oral mucosa of patients with OLP exhibited significant cytomorphometric changes. However, there was no evidence of malignancy. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. GABAergic neuroaxonal dystrophy and other cytopathological alterations in feline Niemann-Pick disease type C.

    PubMed

    March, P A; Thrall, M A; Brown, D E; Mitchell, T W; Lowenthal, A C; Walkley, S U

    1997-08-01

    Feline Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disease which shares many of the clinical, biochemical and pathological features of the corresponding human disorder. Cytopathological alterations in distinct neuronal cell populations were investigated in this animal model to gain a better understanding of the pathogenesis of brain dysfunction. Golgi and immunocytochemical methods were employed to characterize the cell architectural changes occurring in neuronal somata, dendrites and axons at different stages of disease progression. Cortical pyramidal neurons in laminae II, III, and V exhibited various degrees of meganeurite and/or swollen axon hillock formation with or without ectopic dendritogenesis. Enlarged axon hillock regions with neuritic processes and spines were recognized early in the progression of feline NPC but were less prevalent in mid to late stages of the disease. Glutamic acid decarboxylase (GAD) immunocytochemistry demonstrated immunoreactive spheroids in numerous GABAergic axons in neocortex, subcortical areas, and cerebellum. Parvalbumin-immunoreactive axonal spheroid distribution in brain closely mirrored results from the GAD studies, whereas calbindin D-28k-immunoreactive spheroids were conspicuously absent in most cortical and subcortical areas examined. Purkinje cell axonal spheroid formation progressed in a distal to proximal direction, with eventual involvement of recurrent axon collaterals. Purkinje cell death and a concomitant decrease in the numbers of spheroids in the cerebellum were observed late in the disease course. Clinical neurological signs in feline NPC occur in parallel with neuronal structural alterations and suggest that GABAergic neuroaxonal dystrophy is a contributor to brain dysfunction in this disease.

  9. 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.

  10. 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

  11. Thyroid cytopathology with an emphasis on the 'atypical cells of uncertain significance' category: a 3-year audit with cytohistologic correlation.

    PubMed

    Fatman, Luvo; Michelow, Pamela

    2015-01-01

    The National Cancer Institute meeting of 2007 resulted in the reporting terminology for thyroid cytopathology. The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) aims to standardise thyroid cytopathology reporting for cytology centres and clinicians alike. To compare our laboratory's performance against TBSRTC. The second aim was to determine our laboratory's atypia of undetermined significance/follicular cells of undetermined significance (AUS/FLUS) reporting rate and malignant outcomes. Our laboratory subclassifies the AUS/FLUS category into AUS/FLUS not otherwise specified (NOS) and AUS/FLUS cannot exclude malignancy. All thyroid reports were retrieved from our computerised database for the period of January 1, 2008 to March 31, 2011. Histologic correlation was obtained where available, and cases were classified according to their original diagnosis into 1 of the 6 categories of TBSRTC. A total of 1,767 cases were retrieved. The categories were as follows: inadequate (n=415; 23%), benign (n=1,063; 60%), AUS/FLUS (n=141; 8%) [NOS (n=93; 5%) and cannot exclude malignancy (n=48; 3%)] suspicious for follicular/Hürthle cell neoplasm (n=68; 4%), suspicious for malignancy (n=37; 2%) and malignant (n=43; 2%). The malignant rates for the categories were as follows: -6 (26%), 0 (0%), 8 (40%), 9 (38%), 11 (42%), 15 (62.5%), and 15 (94%), respectively. We have shown that the AUS category carries a higher malignant rate than that of the AUS category in TBSRTC of 5-15%. We conclude that subclassifying the AUS/FLUS category into NOS and cannot exclude malignancy helps to better identify patients with an increased risk of malignancy in the AUS/FLUS cannot exclude malignancy category. © 2015 S. Karger AG, Basel.

  12. Flipping The Practice Based Pathology Laboratory-Can It Support Development Of Practitioner Capability For Trainee Pathologists in Gynaecological Cytopathology?

    PubMed

    Smith, Sara; Ganesan, Raji; Martin, Jan

    2017-09-01

    This study investigated the role of 'flipping', the practice-based pathology laboratory and classroom to support the development of trainee pathologist practitioners' in the field of gynaecological cytopathology, addressing development of their knowledge and practical application in the clinical setting. Content-rich courses traditionally involve lecture led delivery which restricts tutors from adopting approaches that support greater student engagement in the topic area and application of knowledge to practice. We investigated the role of 'flipping', the practice-based pathology laboratory and classroom where 'virtual lectures' were accessed outside of 'class time' allowing more time for students to engage in active learning under the supervision of a consultant histopathologist. 'Flipping' was used to support two gynaecological cytopathology training courses with cohorts of eight trainee pathologists on the first course and six on the second. Lectures were made available to the trainees to watch before attending the workshops. The workshops consisted of group activities and individual practical exercises allowing trainees to review and report on patient practice cases with the support of their peers and tutors. Focus group sessions were held after each course, allowing trainee pathologists to reflect on their experiences. Discussions were transcribed and thematic analysis was used to capture key themes discussed by the trainees. Trainees' identified that 'flipping' provided them with more time during face-to-face sessions, enabling a greater depth of questioning and engagement with the consultant histopathologists. Having already watched the lectures, trainees were able to attend the sessions having identified areas in which they needed additional support and development. Trainee pathologists reported they had more time to concentrate on developing their skills and practise under the guidance of the consultant histopathologists so developing their capability in

  13. 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.

  14. Assessment of 100% Rapid Review as an Effective Tool for Internal Quality Control in Cytopathological Services.

    PubMed

    Queiroz Filho, José; de Oliveira Crispim Freitas, Janaina Cristiana; Caldas Pessoa, Daliana; Eleutério Júnior, José; Giraldo, Paulo César; Gonçalves, Ana Katherine

    2017-01-01

    The aim of this study was to evaluate the 100% rapid review (100%-RR) as an effective tool for internal quality control (IQC) in gynecological cytopathology services. A total of 8,677 swabs were analyzed; the negative results were submitted to 100%-RR. Divergent cases were discussed in a consensus meeting to reach a conclusion on the final diagnosis. The data were entered into SAS statistical software, and the agreement of the 100%-RR results with the final diagnosis was tested with the weighted kappa statistic. Of the 8,155 smears characterized as negative, 255 (3.13%) were abnormal smears, and 552 (6.77%) unsatisfactory smears were deemed negative. Regarding the results on the 8,155 smears subjected to 100%-RR when compared with the final diagnosis, there was agreement in 7,063 (86.60%) of them, and there were 1,092 (13.40%) discordant results (65.6%, unsatisfactory; 5.47%, atypical squamous cells of undetermined significance [ASC-US]). The κ index had an agreement of 0.867, with κ = 0.734 (p < 0.0001). Compared with the final diagnosis, the sensitivity of 100%-RR was 99.91% and its specificity was 99.4% for severe abnormalities. The sensitivity for high-grade squamous intraepithelial lesions was 88.2%, with a specificity of 100.00%. For abnormalities considered borderline, such as ASC-US, the sensitivity was 94.50% and the specificity was 99.5%. The 100%-RR was considered efficient when used as an IQC method. © 2017 S. Karger AG, Basel.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. Thyroid nodules with KRAS mutations are different from nodules with NRAS and HRAS mutations with regard to cytopathologic and histopathologic outcome characteristics.

    PubMed

    Radkay, Lisa A; Chiosea, Simion I; Seethala, Raja R; Hodak, Steven P; LeBeau, Shane O; Yip, Linwah; McCoy, Kelly L; Carty, Sally E; Schoedel, Karen E; Nikiforova, Marina N; Nikiforov, Yuri E; Ohori, N Paul

    2014-12-01

    Mutations in the RAS gene in the thyroid gland result in the activation of signaling pathways and are associated with a follicular growth pattern and the probability of a carcinoma outcome ranging from 74% to 87%. In the current study, the authors investigated the cytopathologic and histopathologic features of common RAS mutation subtypes. Malignant, indeterminate, and selected benign thyroid cytology cases were tested prospectively for the presence of NRAS61, HRAS61, and KRAS12/13 mutations. For each case, the Bethesda System for thyroid cytopathology diagnosis, additional cytologic descriptors, and surgical pathology outcomes were documented. The Fisher exact test and Wilcoxon 2-sample test were used for statistical comparison between the groups. A total of 204 thyroid fine-needle aspiration cases with RAS mutations (93.6% of which were associated with indeterminate cytopathology diagnoses) and corresponding surgical pathology resection specimens were identified. The KRAS12/13 mutation was associated with a significantly lower carcinoma outcome (41.7%) when compared with HRAS61 (95.5%) and NRAS61 (86.8%) mutations (P<.0001). Furthermore, oncocytic change was observed in a significantly higher percentage of cytology and resection specimens with KRAS12/13 mutations (66.7% and 75.0%, respectively) in comparison with those with HRAS61 (4.5% and 4.5%, respectively) and NRAS61 (15.4% and 14.7%, respectively) mutations (P<.0001). RAS mutations also were identified in cases of poorly differentiated carcinoma (NRAS61), anaplastic carcinoma (HRAS61), and medullary thyroid carcinoma (HRAS61 and KRAS12/13). Subclassification of RAS mutations in conjunction with cytopathologic evaluation improves presurgical risk stratification, provides better insight into lesional characteristics, and may influence patient management. In particular, KRAS12/13-mutated thyroid nodules were found to be different from HRAS61-mutated and NRAS61-mutated nodules with regard to cytopathologic and

  20. 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

  1. 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.

  2. Fluorescence lifetime excitation cytometry by kinetic dithering

    PubMed Central

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

    2014-01-01

    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. PMID:24668857

  3. 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

  4. Multinode acoustic focusing for parallel flow cytometry

    PubMed Central

    Piyasena, Menake E.; Suthanthiraraj, Pearlson P. Austin; Applegate, Robert W.; Goumas, Andrew M.; Woods, Travis A.; López, Gabriel P.; Graves, Steven W.

    2012-01-01

    Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry. PMID:22239072

  5. 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.

  6. 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. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. [Why does the prevalence of cytopathological results of cervical cancer screening can vary significantly between two regions of Brazil?].

    PubMed

    Discacciati, Michelle Garcia; Barboza, Bárbara Maria Santos; Zeferino, Luiz Carlos

    2014-05-01

    To analyze the prevalence of cervical cytopathological results for the screening of cervical cancer with regard to women's age and time since the last examination in Maceió and Rio de Janeiro, Brazil, among those assisted by the Brazilian Unified Health System. Cervical cytopathological results available in the Information System of Cervical Cancer Screening for the year 2011 were analyzed, corresponding to 206,550 for Rio de Janeiro and 45,243 for Maceió. In Rio de Janeiro, examination at one and two year intervals predominated, while in Maceió examination at one and three year intervals had a higher predominance. Women who underwent cervical smear screening in Maceió were older than those in Rio de Janeiro. The prevalence of invasive squamous cell carcinoma was similar for the two cities, but all the other results presented a higher prevalence in Rio de Janeiro: ASCUS (PR=5.32; 95%CI 4.66-6.07); ASCH (PR=4.27; 95%CI 3.15-5.78); atypical glandular cells (PR=10.02; 95%CI 5.66-17.76); low-grade squamous intraepithelial lesions (PR=6.10; 95%CI 5.27-7.07); high-grade squamous intraepithelial lesions (PR=8.90; 95%CI 6.50-12.18) and adenocarcinoma (PR=3.00; 95%CI 1.21-7.44). The rate of unsatisfactory cervical samples was two times higher in Maceió and that of rejected samples for analysis was five times higher in Maceió when compared to Rio de Janeiro. The prevalence rates of altered cervical cytopathological results was significantly higher in Rio de Janeiro than in Maceió. There is no objective information that may justify this difference. One hypothesis is that there may be a difference in the diagnostic performance of the cervical cancer screening, which could be related to the quality of the Pap smear. Thus, these findings suggest that it would be necessary to perform this evaluation at national level, with emphasis on the performance of cervical cancer screening in order to improve the effectiveness of cervical cancer control.

  8. Recent advances in cytometry applications: preclinical, clinical, and cell biology.

    PubMed

    Mittag, Anja; Tarnok, Attila

    2011-01-01

    The acceptance of flow cytometry (FCM) in clinical laboratory medicine is a major stepping stone towards development new cell analyses, improvement of accuracy, and finally a new range of diagnostic tests. Applications range from differential blood count determination to the identification of fluorescence-labeled subpopulations of disease-specific cell types in cell suspensions. Even new disease patterns can be identified by FCM. However, FCM is not only applicable for making a diagnosis but also for disease monitoring and routine check-ups. It is often used in oncology-related analyses, such as for leukemia and lymphoma patients. Here, not only cell numbers are relevant but also the degree of antigen expression which can be determined in a standardized way. Next to FCM also image cytometry has entered clinical applications although manual review by pathologists is still standard. In general, the multicolor approach and hence the ability for multiparametric analyses has led FCM to a central cornerstone in cell biology research. This review is intended to present an overview of cytometric applications which have entered clinical practice and led to deeper understanding in biological processes. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Photothermal and photoacoustic Raman cytometry in vitro and in vivo

    PubMed Central

    Shashkov, Evgeny V.; Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2010-01-01

    An integrated Raman-based cytometry was developed with photothermal (PT) and photoacoustic (PA) detection of Raman-induced thermal and acoustic signals in biological samples with Raman-active vibrational modes. The two-frequency, spatially and temporally overlapping pump–Stokes excitation in counterpropagating geometry was provided by a nanosecond tunable (420–2300 nm) optical parametric oscillator and a Raman shifter (639 nm) pumped by a double-pulsed Q-switched Nd:YAG laser using microscopic and fiberoptic delivery of laser radiation. The PA and PT Raman detection and imaging technique was tested in vitro with benzene, acetone, olive oil, carbon nanotubes, chylomicron phantom, and cancer cells, and in vivo in single adipocytes in mouse mesentery model. The integration of linear and nonlinear PA and PT Raman scanning and flow cytometry has the potential to enhance its chemical specificity and sensitivity including nanobubble-based amplification (up to 10- fold) for detection of absorbing and nonabsorbing targets that are important for both basic and clinically relevant studies of lymph and blood biochemistry, cancer, and fat distribution at the single-cell level. PMID:20389713

  10. 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...

  11. 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...

  12. Targeting tyrosine kinases in cancer: the converging roles of cytopathology and molecular pathology in the era of genomic medicine.

    PubMed

    Dumur, Catherine I; Idowu, Michael O; Powers, Celeste N

    2013-02-01

    Because of knowledge gained in the field of cancer biology, clinicians are currently witnessing an explosion of molecular tests as companion diagnostics to targeted therapies against growth factor receptors and their signaling pathways. Such tests are being applied increasingly to cytology specimens as essential components of genomic medicine, because less invasive diagnostic procedures are becoming the norm. The objective of this review was to present an overview of the current and future role of cytopathology in molecular diagnostics, including the adequacy of cytology specimens for such studies. The authors also discuss the critical methodologic aspects of the molecular assays used for the selection of tyrosine kinase treatment for oncology patients. Copyright © 2012 American Cancer Society.

  13. Internal quality control indicators of cervical cytopathology exams performed in laboratories monitored by the External Quality Control Laboratory.

    PubMed

    Ázara, Cinara Zago Silveira; Manrique, Edna Joana Cláudio; Tavares, Suelene Brito do Nascimento; de Souza, Nadja Lindany Alves; Amaral, Rita Goreti

    2014-09-01

    To evaluate the impact of continued education provided by an external quality control laboratory on the indicators of internal quality control of cytopathology exams. The internal quality assurance indicators for cytopathology exams from 12 laboratories monitored by the External Quality Control Laboratory were evaluated. Overall, 185,194 exams were included, 98,133 of which referred to the period preceding implementation of a continued education program, while 87,061 referred to the period following this intervention. Data were obtained from the Cervical Cancer Database of the Brazilian National Health Service. Following implementation of the continued education program, the positivity index (PI) remained within recommended limits in four laboratories. In another four laboratories, the PI progressed from below the limits to within the recommended standards. In one laboratory, the PI remained low, in two laboratories, it remained very low, and in one, it increased from very low to low. The percentage of exams compatible with a high-grade squamous intraepithelial lesion (HSIL) remained within the recommended limits in five laboratories, while in three laboratories it progressed from below the recommended levels to >0.4% of the total number of satisfactory exams, and in four laboratories it remained below the standard limit. Both the percentage of atypical squamous cells of undetermined significance (ASC-US) in relation to abnormal exams, and the ratio between ASC-US and intraepithelial lesions remained within recommended levels in all the laboratories investigated. An improvement was found in the indicators represented by the positivity index and the percentage of exams compatible with a high-grade squamous intraepithelial lesion, showing that the role played by the external quality control laboratory in providing continued education contributed towards improving laboratory staff skills in detecting cervical cancer precursor lesions.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. 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.

  1. Microfluidics in flow cytometry and related techniques.

    PubMed

    Béné, M C

    2017-05-01

    Technological advances in laboratory automation are now well understood and applied as they considerably improved the speed and robustness of haematological laboratory data, in the companion fields of blood analyzers and flow cytometry. Still rather confidential is the field of microfluidics, mostly confined so far to academic settings and research laboratories. The literature in the field of microfluidics is growing and applications in hematology range from cell counting to flow cytometry, cell sorting, or ex vivo testing. A literature search allows to identify many innovative solutions developed to master the specific physics of fluid movements in microchips. Miniaturization also dwells on findings that have emerged from different areas such as electronics and nanoengineering. This review proposes an overview of the major principles guiding developments in microfluidics and describes a necessarily limited and nonexhaustive series of specific applications. Readers are strongly encouraged to consult the documents referred to in the references section to learn more about this world knocking at our door and possibly liable to revolutionize our profession of hematology biologists in a not so far future. © 2017 John Wiley & Sons Ltd.

  2. 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

  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. 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.

  5. Detection of metal induced cytopathological alterations and DNA damage in the gills and hepatopancreas of green mussel Perna viridis from Ennore Estuary, Chennai, India.

    PubMed

    Vasanthi, Lourduraj A; Revathi, Peranandam; Babu Rajendran, Ramaswamy; Munuswamy, Natesan

    2017-04-15

    This study report the impact of heavy metals on cytopathology and DNA damage in the gills and hepatopancreas of Perna viridis collected from Ennore estuary and the Kovalam coastal waters. Principal Component Analysis (PCA) showed significant differences among all variables at the scale of plots. The ultrastructural alterations such as lack of microvilli, distorted mitochondria, electron dense particles and the presence of large mucous droplets were common in the gill and hepatopancreatic cells of mussels from Ennore estuary. However, the gill and hepatopancreatic cells of P. viridis from Kovalam revealed normal compartmentalization of cells. The percentage of tail DNA in the mussels from Ennore estuary was recorded as 12.44 and 10.14% in the gills and hepatopancreas respectively. Overall, it has been demonstrated that the Comet and cytopathological assays are useful biomarkers to assess the level of pollution and it provide reliable information on ecotoxicology and genotoxicology of coastal waters. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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. © 2013 International Society for Advancement of Cytometry.

  13. Guidelines for cytopathologic diagnosis of epithelioid and mixed type malignant mesothelioma. Complementary statement from the International Mesothelioma Interest Group, also endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology

    PubMed Central

    Hjerpe, Anders; Ascoli, Valeria; Bedrossian, Carlos; Boon, Mathilde; Creaney, Jenette; Davidson, Ben; Dejmek, Annika; Dobra, Katalin; Fassina, Ambrogio; Field, Andrew; Firat, Pinar; Kamei, Toshiaki; Kobayashi, Tadao; Michael, Claire W.; Önder, Sevgen; Segal, Amanda; Vielh, Philippe

    2015-01-01

    To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma (MM). Cytopathologists involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC), who have an interest in the field contributed to this update. Reference material includes peer-reviewed publications and textbooks. This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists, who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in cape town. During the previous IMIG biennial meetings, thorough discussions have resulted in published guidelines for the pathologic diagnosis of MM. However, previous recommendations have stated that the diagnosis of MM should be based on histological material only.[12] Accumulating evidence now indicates that the cytological diagnosis of MM supported by ancillary techniques is as reliable as that based on histopathology, although the sensitivity with cytology may be somewhat lower.[345] Recognizing that noninvasive diagnostic modalities benefit both the patient and the health system, future recommendations should include cytology as an accepted method for the diagnosis of this malignancy.[67] The article describes the consensus of opinions of the authors on how cytology together with ancillary testing can be used to establish a reliable diagnosis of MM. PMID:26681974

  14. Diagnostic Frequency Ratios Are Insufficient to Measure Laboratory Precision with The Bethesda System for Reporting Thyroid Cytopathology.

    PubMed

    Fazeli, Roghayeh; Schneider, Eric B; Ali, Syed Z; Zeiger, Martha A; Olson, Matthew T

    2015-01-01

    Diagnostic frequency ratios such as the atypia of undetermined significance (AUS):malignant ratio are touted to be useful for laboratory precision benchmarking. We therefore sought to examine their reproducibility and usefulness at a tertiary hospital. We reviewed thyroid fine-needle aspirates (FNA) submitted to our institution from outside laboratories and evaluated the ability of diagnostic frequency ratios to capture the complexity of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). Specifically, we evaluated the ability of the AUS:malignant ratio to describe the frequencies of the other TBSRTC diagnoses. A total of 2,784 cases from 19 laboratories were included. The use of the AUS category varied the most. There was insufficient reflection of the non-AUS nonmalignant TBSRTC diagnostic frequencies in our analysis, and these results do not appear to arise from observer variability in the outside laboratories. Diagnostic frequency ratios are not reproducible in our experience and fail to describe the other TBSRTC categories. As such, they are unlikely to prove sufficient for benchmarking laboratory precision with TBSRTC. © 2015 S. Karger AG, Basel.

  15. Tularemia: potential role of cytopathology in differential diagnosis of cervical lymphadenitis: multicenter experience in 53 cases and literature review.

    PubMed

    Tuncer, Ersin; Onal, Binnur; Simsek, Gulcin; Elagoz, Sahande; Sahpaz, Ahmet; Kilic, Selcuk; Altuntas, Emine Elif; Ulu Kilic, Aysegul

    2014-03-01

    Tularemia is a zoonosis caused by Francisella tularensis. Tularemia outbreaks occurred in Central Anatolia during 2009 and 2011. We evaluated the clinical characteristics and cytomorphologies of fine needle aspirations (FNAs) from cervical lymph nodes in serologically confirmed tularemia cases. To our knowledge, this is the first large series concerning FNA morphology of Tularemia. FNA smears of 53 patients of the 290, diagnosed by microagglutination tests and PCR, were evaluated at three Pathology centers. FNAs were performed by cytopathologists or ear-nose-throat surgeons. Of all patients, 17 had also lymph node resections. FNAs showed the presence of suppuration and abscess. Rare epithelioid histiocytes and granulomas, seldom phagocytosed bacilli-like microorganisms were observed. On histopathology; granulomas, necrosis, and suppurative inflammation extending extracapsular areas were seen. Tularemia is endemic in certain areas of the Northern Hemisphere. The benefit from cytopathology is limited and cytological suspicion should be confirmed by serology. However FNA cytology is helpful in differential diagnosis of tularemia and other diseases presented with suppurative, granulomatous cervical lymphadenitis. It is also useful in providing the material for PCR and culture in early phase when the serology is negative and the treatment is more effective. © 2013 APMIS. Published by John Wiley & Sons Ltd.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. DNA Detection by Flow Cytometry using PNA-Modified Metal-Organic Framework Particles.

    PubMed

    Mejia-Ariza, Raquel; Rosselli, Jessica; Breukers, Christian; Manicardi, Alex; Terstappen, Leon W M M; Corradini, Roberto; Huskens, Jurriaan

    2017-03-23

    A DNA-sensing platform is developed by exploiting the easy surface functionalization of metal-organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL-88A, using either covalent or non-covalent interactions, resulting in alkyne-modified and biotin-modified MIL-88A, respectively. Covalent surface coupling of an azide-dye and the alkyne-MIL-88A was achieved by means of a click reaction. Non-covalent streptavidin-biotin interactions were employed to link biotin-PNA to biotin-MIL-88A particles mediated by streptavidin. Characterization by confocal imaging and flow cytometry demonstrated that DNA can be bound selectively to the MOF surface. Flow cytometry provided quantitative data of the interaction with DNA. Making use of the large numbers of particles that can be simultaneously processed by flow cytometry, this MOF platform was able to discriminate between fully complementary, single-base mismatched, and randomized DNA targets. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  3. Real-time three-dimensional counting and shape measurement of RBCs using digital holographic cytometry

    NASA Astrophysics Data System (ADS)

    Funamizu, Hideki; Sonoda, Kotaro; Goto, Ryoji; Aizu, Yoshihisa

    2017-04-01

    Digital holography is a useful technique for recording and reconstruction of the complex amplitude of an optical field. In this technique, an interference pattern of two waves is detected by an image sensor, and digital holograms are acquired in computer. The wavefront is reconstructed by a numerical calculation. In this study, we present the real-time threedimensional counting and shape measurement of RBCs using flow cytometry with digital holographic microscopy.

  4. 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.

  5. 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.

  6. 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

  7. Toward lower contrast computer vision in vivo flow cytometry.

    PubMed

    Markovic, Stacey; Siyuan Li; Tianxue Zhang; Niedre, Mark

    2014-01-01

    There are many applications in biomedical research where detection and enumeration of circulating cells (CCs) is important. Existing techniques involve drawing and enriching blood samples and analyzing them ex vivo. More recently, small animal "in vivo flow cytometry" (IVFC) techniques have been developed, where fluorescently-labeled cells flowing through small arterioles (ear, retina) are detected and counted. We recently developed a new high-sensitivity IVFC technique termed "Computer Vision(CV)-IVFC". Here, large circulating blood volumes were monitored in the ears of mice with a wide-field video-rate near-infrared (NIR) fluorescent camera. Cells were labeled with a membrane dye and were detected and tracked in noisy image sequences. This technique allowed enumeration of CCs in vivo with overall sensitivity better than 10 cells/mL. However, an ongoing area of interest in our lab is optimization of the system for lower-contrast imaging conditions, e.g. when CCs are weakly labeled, or in the case higher background autofluorescence with visible dyes. To this end, we developed a new optical flow phantom model to control autofluorescence intensity and physical structure to better mimic conditions observed in mice. We acquired image sequences from a series of phantoms with varying levels of contrast and analyzed the distribution of pixel intensities, and showed that we could generate similar conditions to those in vivo. We characterized the performance of our CV-IVFC algorithm in these phantoms with respect to sensitivity and false-alarm rates. Use of this phantom model in optimization of the instrument and algorithm under lower-contrast conditions is the subject of ongoing work in our lab.

  8. 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

  9. CD4/CD8 Ratio in Mediastinal Lymph Nodes Involved by Sarcoidosis: Analysis of Flow Cytometry Data Obtained by Endobronchial Ultrasound-guided Transbronchial Needle Aspiration.

    PubMed

    Ruiz, Sory J; Zhang, Yaxia; Mukhopadhyay, Sanjay

    2016-10-01

    Despite mixed results in the literature, some clinicians continue to consider an elevated CD4/CD8 ratio in bronchoalveolar lavage (BAL) fluid to be supportive of a diagnosis of sarcoidosis. However, the CD4/CD8 ratio in mediastinal lymph nodes involved by sarcoidosis has not been extensively studied. The primary aim of this study was to evaluate the utility of the CD4/CD8 ratio in mediastinal lymph node aspirates obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for diagnosing sarcoidosis. Our archives were searched for EBUS-TBNAs in which mediastinal lymph node aspirates had been submitted for flow cytometry (n=160). Clinical and pathologic findings in these cases were reviewed retrospectively. Cases were included in the study if they had (1) a clinical diagnosis of sarcoidosis supported by cytopathologic confirmation of non-necrotizing granulomas in EBUS-TBNA-derived lymph node aspirates (23 cases), or (2) a pathologically confirmed non-neoplastic diagnosis other than sarcoidosis (7 cases). Cases that did not fulfil these criteria were excluded (130 cases). The CD4/CD8 ratios in mediastinal lymph nodes and BAL fluid were compared. The CD4/CD8 ratio was elevated in mediastinal lymph nodes in 12/23 (52%) cases of sarcoidosis and 3/7 (43%) pathologically confirmed nonsarcoid cases. BAL fluid had been concurrently submitted for flow cytometry in 20/23 cases of sarcoidosis and 5/7 nonsarcoid cases. CD4/CD8 was elevated in BAL fluid in 9/20 (45%) cases of sarcoidosis and 2/5 (40%) nonsarcoid cases. As in BAL fluid, the CD4/CD8 ratio in mediastinal lymph nodes involved by sarcoid granulomas is highly variable and does not reliably confirm or exclude sarcoidosis.

  10. 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.

  11. Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.

    PubMed

    Clarke, Scott T; Calderon, Veronica; Bradford, Jolene A

    2017-10-02

    The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio-orthogonal to most living systems and does not non-specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  12. Laser scanning cytometry: principles and applications-an update.

    PubMed

    Pozarowski, Piotr; Holden, Elena; Darzynkiewicz, Zbigniew

    2013-01-01

    Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.

  13. 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.

  14. 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

    2017-07-01

    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. Copyright © 2017 by the American Society of Nephrology.

  15. The impact of category, cytopathology and cytogenetics on development and progression of clonal and malignant myeloid transformation in inherited bone marrow failure syndromes

    PubMed Central

    Cada, Michaela; Segbefia, Catherin I.; Klaassen, Robert; Fernandez, Conrad V.; Yanofsky, Rochelle A.; Wu, John; Pastore, Yves; Silva, Mariana; Lipton, Jeffrey H.; Brossard, Josee; Michon, Bruno; Abish, Sharon; Steele, MacGregor; Sinha, Roona; Belletrutti, Mark; Breakey, Vicky; Jardine, Lawrence; Goodyear, Lisa; Sung, Lillian; Shago, Mary; Beyene, Joseph; Sharma, Preeti; Zlateska, Bozana; Dror, Yigal

    2015-01-01

    Inherited bone marrow failure syndromes are a group of rare, heterogeneous genetic disorders with a risk of clonal and malignant myeloid transformation including clonal marrow cytogenetic abnormalities, myelodysplastic syndrome and acute myeloid leukemia. The clinical characteristics, risk classification, prognostic factors and outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes are largely unknown. The aims of this study were to determine the impact of category, cytopathology and cytogenetics, the three components of the “Category Cytology Cytogenetics” classification of pediatric myelodysplastic syndrome, on the outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure. We used data from the Canadian Inherited Marrow Failure Registry. Among 327 patients with inherited bone marrow failure syndrome enrolled in the registry, the estimated risk of clonal and malignant myeloid transformation by the age of 18 years was 37%. The risk of clonal and malignant myeloid transformation varied according to the type of inherited bone marrow failure syndrome but was highest in Fanconi anemia. The development of clonal and malignant myeloid transformation significantly affected overall survival. Mortality varied based on cytopathological group. The largest group of patients had refractory cytopenia. Clonal marrow cytogenetic abnormalities were identified in 87% of patients with clonal and malignant myeloid transformation, and different cytogenetic groups had different impacts on disease progression. We conclude that category, cytopathology and cytogenetics in cases of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes have an important impact on outcome and that the classification of such cases should incorporate these factors. PMID:25682607

  16. Prevention of total thyroidectomy in noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) based on combined interpretation of ultrasonographic and cytopathologic results.

    PubMed

    You, Sung-Hye; Lee, Kyu Eun; Yoo, Roh-Eul; Choi, Hye Jeong; Jung, Kyeong Cheon; Won, Jae-Kyung; Kang, Koung Mi; Yoon, Tae Jin; Choi, Seung Hong; Sohn, Chul-Ho; Kim, Ji-Hoon

    2017-09-12

    To explore the potential preoperative ultrasonography (US) and cytopathological features to avoid total thyroidectomy in NIFTP. Recently, it has been proposed that that noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) be classified as tumours, rather than cancer. A total of 142 surgically proven follicular variant papillary thyroid carcinomas (FVPTCs; 45 NIFTP, 97 non-NIFTP; mean size: 20.4±11.0 mm, range: 10.0-65.0 mm) from 142 patients were included in this study. Three preoperative features of thyroid nodules (each US finding, US and Bethesda category) were compared in NIFTP and non-NIFTP groups. The preoperative decision-making process to avoid total thyroidectomy in NIFTP was evaluated based on combination of those features. In each US finding, there was only significantly less macrocalcification in the NIFTP group than in the non-NIFTP group (8.8% [4/45] vs 32.0% [31/97], P = .006). In US category, all of the NIFTP nodules were a low or intermediate suspicion (100% [45/45]). In Bethesda category, 26.7% [12/45] of the NIFTP was diagnosed as either suspicious malignancy or malignant, which increased the risk of a total thyroidectomy. In our study, a total thyroidectomy might be avoided in all of the NIFTP cases if lobectomy was selected for the nodules classified as a low or intermediate suspicion in US, despite being classified as a suspicious malignancy or malignant by cytopathology. Combining the US and cytopathological results could sensitively reduce total thyroidectomy in cases of NIFTP. © 2017 John Wiley & Sons Ltd.

  17. 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.

  18. Atrophic vaginitis: concordance and interpretation of slides in the College of American Pathologists Cervicovaginal Interlaboratory Comparison Program in Gynecologic Cytopathology.

    PubMed

    Crothers, Barbara A; Booth, Christine N; Darragh, Teresa M; Means, Marilee M; Souers, Rhona J; Thomas, Nicole; Moriarty, Ann T

    2012-11-01

    Atrophic vaginitis is a commonly reported subset of Papanicolaou test results that are negative for intraepithelial lesion or malignancy, but interpretive criteria overlap with atrophic changes and other entities, hindering concordance among observers. To report on the participant concordance from 2000 to 2009 in the College of American Pathologists Interlaboratory Comparison Program in Gynecologic Cytopathology, with a reference interpretation of atrophic vaginitis, and to investigate cytologic features of good and poorly performing slides to identify criteria useful in the interpretation of atrophic vaginitis. We summarized 18 302 responses from the program for slides with a reference interpretation of atrophic vaginitis. We randomly selected 18 Papanicolaou test results (3 conventional, 4 SurePath, and 11 ThinPrep) from good and poor performers for prospective, blinded criteria scoring for the following features: abundance of neutrophils, more than 100 degenerating parabasal cells, more than 25% necrotic background, more than 100 pseudoparakeratotic cells, and the presence of stripped or streaked nuclei, histiocytes, and superficial or intermediate squamous cells. Most Papanicolaou test results (>90%) with a specific reference interpretation of atrophic vaginitis were categorized as negative. Cytotechnologists are more likely than pathologists are to label it negative for intraepithelial lesion or malignancy (NILM) and are equally likely to mistake it for a high-grade lesion. Degenerating parabasal cells, pseudoparakeratosis, and necrotic background are associated with atrophic vaginitis (P  =  .001) on Papanicolaou. Abundant neutrophils (>100 per ×400 field) are also significantly correlated (P  =  .01). Exact concordance to atrophic vaginitis is less than 90%. Most of the discrepancies are negative results for intraepithelial lesion or malignancy. Advanced atrophic features are as significant as neutrophils are to the interpretation of atrophic

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Web-Based Analysis and Publication of Flow Cytometry Experiments

    PubMed Central

    Kotecha, Nikesh; Krutzik, Peter O.; Irish, Jonathan M.

    2014-01-01

    Cytobank is a web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permissions from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at www.cytobank.org PMID:20578106

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. Cytometry and DNA ploidy: clinical uses and molecular perspective in gastric and lung cancer.

    PubMed

    D'Urso, Vittorio; Collodoro, Angelo; Mattioli, Eliseo; Giordano, Antonio; Bagella, Luigi

    2010-03-01

    Flow cytometry is one of the most powerful and specific methods used for the integrated study of the molecular and morphological events occurring during cell proliferation. Many methods have been described for investigating this process. Several cell cycle regulators controlling the correct entry and progression through the cell cycle are altered in tumors. In fact, in most, if not all, human cancers there is a deregulated control of G1 phase progression, the period when cells decide if they will start proliferation or stay quiescent. Cytometry (flow and image) is able to analyze DNA content thanks to the use of the same "molecule" conjugates with a fluorochrome that permits to identify DNA content of single cell in a sample. Most important results of studies on DNA ploidy have been reviewed during the last years and as a result the analyses of DNA ploidy in cancer may provide clinically useful information on diagnostic, therapeutic and prognostic aspects. In fact, aneuploid cancer has a high proliferative activity and a metastatic or invasive potential, markers of a poor prognosis. Multiparametric flow cytometry should allow the simultaneous determination of morphology, phenotype, intracellular protein expression, and status of chromatin and DNA. Evaluating if a particular protein is responsible for the aggressiveness of cancer, or the alteration of DNA content, or if the activation of its state is the cause of rapid growth of cancer cells, is very important and it can facilitate the clinical treatment of patients.

  9. Multiscale cytometry and regulation of 3D cell cultures on a chip.

    PubMed

    Sart, Sébastien; Tomasi, Raphaël F-X; Amselem, Gabriel; Baroud, Charles N

    2017-09-07

    Three-dimensional cell culture is emerging as a more relevant alternative to the traditional two-dimensional format. Yet the ability to perform cytometry at the single cell level on intact three-dimensional spheroids or together with temporal regulation of the cell microenvironment remains limited. Here we describe a microfluidic platform to perform high-density three-dimensional culture, controlled stimulation, and observation in a single chip. The method extends the capabilities of droplet microfluidics for performing long-term culture of adherent cells. Using arrays of 500 spheroids per chip, in situ immunocytochemistry and image analysis provide multiscale cytometry that we demonstrate at the population scale, on 10(4) single spheroids, and over 10(5) single cells, correlating functionality with cellular location within the spheroids. Also, an individual spheroid can be extracted for further analysis or culturing. This will enable a shift towards quantitative studies on three-dimensional cultures, under dynamic conditions, with implications for stem cells, organs-on-chips, or cancer research.3D cell culture is more relevant than the two-dimensional format, but methods for parallel analysis and temporal regulation of the microenvironment are limited. Here the authors develop a droplet microfluidics system to perform long-term culture of 3D spheroids, enabling multiscale cytometry of individual cells within the spheroid.

  10. 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

  11. Scalable clustering algorithms for continuous environmental flow cytometry.

    PubMed

    Hyrkas, Jeremy; Clayton, Sophie; Ribalet, Francois; Halperin, Daniel; Armbrust, E Virginia; Howe, Bill

    2016-02-01

    Recent technological innovations in flow cytometry now allow oceanographers to collect high-frequency flow cytometry data from particles in aquatic environments on a scale far surpassing conventional flow cytometers. The SeaFlow cytometer continuously profiles microbial phytoplankton populations across thousands of kilometers of the surface ocean. The data streams produced by instruments such as SeaFlow challenge the traditional sample-by-sample approach in cytometric analysis and highlight the need for scalable clustering algorithms to extract population information from these large-scale, high-frequency flow cytometers. We explore how available algorithms commonly used for medical applications perform at classification of such a large-scale, environmental flow cytometry data. We apply large-scale Gaussian mixture models to massive datasets using Hadoop. This approach outperforms current state-of-the-art cytometry classification algorithms in accuracy and can be coupled with manual or automatic partitioning of data into homogeneous sections for further classification gains. We propose the Gaussian mixture model with partitioning approach for classification of large-scale, high-frequency flow cytometry data. Source code available for download at https://github.com/jhyrkas/seaflow_cluster, implemented in Java for use with Hadoop. hyrkas@cs.washington.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Algorithmic Tools for Mining High-Dimensional Cytometry Data.

    PubMed

    Chester, Cariad; Maecker, Holden T

    2015-08-01

    The advent of mass cytometry has led to an unprecedented increase in the number of analytes measured in individual cells, thereby increasing the complexity and information content of cytometric data. Although this technology is ideally suited to the detailed examination of the immune system, the applicability of the different methods for analyzing such complex data is less clear. Conventional data analysis by manual gating of cells in biaxial dot plots is often subjective, time consuming, and neglectful of much of the information contained in a highly dimensional cytometric dataset. Algorithmic data mining has the promise to eliminate these concerns, and several such tools have been applied recently to mass cytometry data. We review computational data mining tools that have been used to analyze mass cytometry data, outline their differences, and comment on their strengths and limitations. This review will help immunologists to identify suitable algorithmic tools for their particular projects. Copyright © 2015 by The American Association of Immunologists, Inc.

  13. 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

  14. Human chromosome karyotyping and molecular biology by flow cytometry

    SciTech Connect

    Yu, L.C.; Gray, J.W.; Langlois, R.; Van Dilla, M.A.; Carrano, A.V.

    1982-03-22

    Flow cytometry is a sensitive analytical tool for rapdily measuring the biological, chemical and physical properties of cells and cellular components, such as chromosomes and has become a promising system for automating human chromosomal karyotyping. Unlike traditional approaches based upon chromosomal length, centromeric index, and banding patterns, it is based on the measurement of chromosomal DNA content and base composition and can classify chromosomes more objectively. In this paper we describe flow karyotyping and chromosome sorting and compare flow cytometry with the use of cell hybrids for gene mapping and the construction of chromosome-specific genomic libraries.

  15. Associations between oral HPV16 infection and cytopathology: evaluation of an oropharyngeal “Pap-test equivalent” in high-risk populations

    PubMed Central

    Fakhry, Carole; Rosenthal, Barbara T.; Clark, Douglas P.; Gillison, Maura L.

    2012-01-01

    Human papillomavirus (HPV) is responsible for the rising incidence of oropharyngeal squamous cell cancers (OSCC) in the United States (U.S.), and yet, no screening strategies have been evaluated. Secondary prevention by means of HPV detection and cervical cytology has led to a decline in cervical cancer incidence in the U.S. Here, we explored an analogous strategy by evaluating associations between HPV16 infection, cytopathology and histopathology in two populations at elevated risk for OSCC. In the first, a cross-sectional study population (PAP1), cytology specimens were collected by means of brush biopsy from patients presenting with oropharyngeal abnormalities. In the second (PAP2), a nested case-control study, bilateral tonsillar cytology samples were collected at 12-month intervals from HIV-infected individuals. The presence of cytopathological abnormality in HPV16-positive tonsil brush biopsies (cases) was compared to HPV16-negative samples (controls) matched on age and gender. HPV16 was detected in samples by consensus primer PCR and/or type-specific PCR. Univariate logistic regression was used to evaluate associations. In PAP1, HPV16 alone (OR 6.1, 95%CI 1.6–22.7) or in combination with abnormal cytology (OR 20, 95%CI 4.2–95.4) was associated with OSCC. In PAP2, 4.7% (72 of 1524) of tonsillar cytology specimens from HIV-infected individuals without oropharyngeal abnormalities were HPV16-positive. Tonsillar HPV16 infection was not associated with atypical squamous cells of unknown significance (ASCUS), the only cytological abnormality identified. Therefore, HPV16 was associated with OSCC among individuals with accessible oropharyngeal lesions, but not with cytological evidence of dysplasia among high-risk individuals without such lesions. An oropharyngeal Pap-test equivalent may not be feasible, likely due to limitations in sampling the relevant tonsillar crypt epithelium. PMID:21836021

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

    PubMed Central

    Frelinger, Jacob; Kepler, Thomas B; Chan, Cliburn

    2008-01-01

    Flow is an open source software application for clinical and experimental researchers to perform exploratory data analysis, clustering and annotation of flow cytometric data. Flow is an extensible system that offers the ease of use commonly found in commercial flow cytometry software packages and the statistical power of academic packages like the R BioConductor project. PMID:18559108

  17. 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.

  18. 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…

  19. 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

  20. 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…

  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. Cluster stability in the analysis of mass cytometry data.

    PubMed

    Melchiotti, Rossella; Gracio, Filipe; Kordasti, Shahram; Todd, Alan K; de Rinaldis, Emanuele

    2017-01-01

    Manual gating has been traditionally applied to cytometry data sets to identify cells based on protein expression. The advent of mass cytometry allows for a higher number of proteins to be simultaneously measured on cells, therefore providing a means to define cell clusters in a high dimensional expression space. This enhancement, whilst opening unprecedented opportunities for single cell-level analyses, makes the incremental replacement of manual gating with automated clustering a compelling need. To this aim many methods have been implemented and their successful applications demonstrated in different settings. However, the reproducibility of automatically generated clusters is proving challenging and an analytical framework to distinguish spurious clusters from more stable entities, and presumably more biologically relevant ones, is still missing. One way to estimate cell clusters' stability is the evaluation of their consistent re-occurrence within- and between-algorithms, a metric that is commonly used to evaluate results from gene expression. Herein we report the usage and importance of cluster stability evaluations, when applied to results generated from three popular clustering algorithms - SPADE, FLOCK and PhenoGraph - run on four different data sets. These algorithms were shown to generate clusters with various degrees of statistical stability, many of them being unstable. By comparing the results of automated clustering with manually gated populations, we illustrate how information on cluster stability can assist towards a more rigorous and informed interpretation of clustering results. We also explore the relationships between statistical stability and other properties such as clusters' compactness and isolation, demonstrating that whilst cluster stability is linked to other properties it cannot be reliably predicted by any of them. Our study proposes the introduction of cluster stability as a necessary checkpoint for cluster interpretation and

  3. 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.

  4. 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

  5. Microfluidic impedance flow cytometry enabling high-throughput single-cell electrical property characterization.

    PubMed

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

    2015-04-29

    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.

  6. 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

  7. 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.

  8. 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.

  9. Impact of standardization on clinical cell analysis by flow cytometry.

    PubMed

    Keeney, M; Barnett, D; Gratama, J W

    2004-01-01

    The evolution of flow cytometry from a research tool to a pivotal technology for clinical diagnostic purposes has required significant efforts to standardize methods. The great advantage of flow cytometry is that it's applications are highly amenable to standardization. Here, we review the efforts that have been made for flow cytometric applications in four major fields of clinical cell analysis: CD4+ T-cell enumeration, CD34+ hematopoietic stem and progenitor cell enumeration, screening for the HLA-B27 antigen and leukemia/lymphoma immunophenotyping. These standardization efforts have been parallelled by the establishment of external quality assessment (EQA) schemes in many countries worldwide. The goal of these EQA exercises has been primarily educa-tional, but their results will increasingly serve as a basis for laboratory accreditation. This important development requires that the EQA schemes, in particular the quality of the distributed samples and the procedures for evaluating the results, meet the highest standards.

  10. 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.

  11. 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.

  12. Data Analysis in Flow Cytometry: The Future Just Started

    PubMed Central

    Lugli, Enrico; Roederer, Mario; Cossarizza, Andrea

    2010-01-01

    In the last 10 years, a tremendous progress characterized flow cytometry in its different aspects. In particular, major advances have been conducted regarding the hardware/instrumentation and reagent development, thus allowing fine cell analysis up to 20 parameters. As a result, this technology generates very complex data sets that demand for the development of optimal tools of analysis. Recently, many independent research groups approached the problem by using both supervised and unsupervised methods. In this paper, we will review the new developments concerning the use of bioinformatics for polychromatic flow cytometry and propose what should be done in order to unravel the enormous heterogeneity of the cells we interrogate each day. PMID:20583274

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  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. Guide to Red Fluorescent Proteins and Biosensors for Flow Cytometry

    PubMed Central

    Piatkevich, Kiryl D.; Verkhusha, Vladislav V.

    2014-01-01

    Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. PMID:21704849

  5. [Flow cytometry in datecting lymph node micrometastasis in colorectal cancer].

    PubMed

    Sun, Q; Ding, Y; Zhang, J

    2001-01-25

    To study the methodology and significance of flow cytometry in detecting lymph node micrometastasis of colorectal cancer. One hundred sixty-two cellular suspensions were prepared with lymph nodes which were resected radically on 25 patients with colorectal cancer and in which no cancer cells were found by HE staining. Different concentrations of cultured Lovo colorectal cancer cells were added into the celular suspension prepared from lymph node tissue of persons without colorectal cancer in order to prepare a control model. Dual staining with CK/FTTC and PI was made to the sedimetns from those 2 kinds of suspension. Flow cytometry was used to detect cancer cells. An ideal correlation was obtained between the detection value and the theoretical value of cancer cells in the specimen suspensions and control models (r = 0.097 6) with a sensitivity rate of 10/10(5). Cancer cells were detected from 7 out of the 25 patients and 30 of the 162 cellular suspensions. The detection rate was correlated with the size and infiltrating depth of the cancer. Flow cytometry is a reliable, rapid, and quantitative method for detecting lymph node micrometastasis in colorectal cancer.

  6. Discriminating cellular heterogeneity using microwell-based RNA cytometry.

    PubMed

    Dimov, Ivan K; Lu, Rong; Lee, Eric P; Seita, Jun; Sahoo, Debashis; Park, Seung-min; Weissman, Irving L; Lee, Luke P

    2014-03-25

    Discriminating cellular heterogeneity is important for understanding cellular physiology. However, it is limited by the technical difficulties of single-cell measurements. Here we develop a two-stage system to determine cellular heterogeneity. In the first stage, we perform multiplex single-cell RNA cytometry in a microwell array containing over 60,000 reaction chambers. In the second stage, we use the RNA cytometry data to determine cellular heterogeneity by providing a heterogeneity likelihood score (HLS). Moreover, we use Monte-Carlo simulation and RNA cytometry data to calculate the minimum number of cells required for detecting heterogeneity. We apply this system to characterize the RNA distributions of ageing-related genes in a highly purified mouse haematopoietic stem cell population. We identify genes that reveal novel heterogeneity of these cells. We also show that changes in expression of genes such as Birc6 during ageing can be attributed to the shift of relative portions of cells in the high-expressing subgroup versus low-expressing subgroup.

  7. Minimal residual disease studies by flow cytometry in acute leukemia.

    PubMed

    Campana, Dario; Coustan-Smith, Elaine

    2004-01-01

    Minimal residual disease (MRD) assays are increasingly important in the clinical management of patients with acute leukemia. Among the methods available for monitoring MRD, flow cytometry holds great promise for clinical application because of its simplicity and wide availability. Several studies have demonstrated strong correlations between MRD levels by flow cytometry during clinical remission and treatment outcome, lending support to the reliability of this approach. Flow-cytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. Its sensitivity depends on the specificity of the immunophenotypes used to track leukemic cells and on the number of cells available for study. Immunophenotypes that allow detection of 1 leukemic cell in 10,000 normal cells can be identified in at least 90% of patients with acute lymphoblastic leukemia; immunophenotypes that allow detection of 1 leukemic cell in 1,000-10,000 normal cells can be identified in at least 85% of patients with acute myeloid leukemia. Identification of new markers of leukemia by gene array technology should lead to the design of simple and reliable antibody panels for universal monitoring of MRD. Here we review the relative advantages and disadvantages of flow cytometry for MRD studies, as well as results obtained in correlative studies with treatment outcome. Copyright 2004 S. Karger AG, Basel

  8. Normalization of mass cytometry data with bead standards

    PubMed Central

    Finck, Rachel; Simonds, Erin F.; Jager, Astraea; Krishnaswamy, Smita; Sachs, Karen; Fantl, Wendy; Pe’er, Dana; Nolan, Garry P.; Bendall, Sean C.

    2013-01-01

    Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold. PMID:23512433

  9. Cytometry: The Journal of the International Society for Analytical Cytology, Supplement 6, 1993: Abstracts

    SciTech Connect

    Mayall, B.H.; Landay, A.L.; Shapiro, H.M.; Visser, J.W.M.

    1993-12-31

    This contains the 465 presentation and poster abstracts for the XVI Congress of the International Society for Analytical Cytology, March 1993. Plenary Sessions included the following: Industrial Cytometry; Clinical Issues (in Cytology); Molecular Pathology; biotechnology; new biology; temporal cytometry.

  10. 78 FR 5186 - Clinical Flow Cytometry in Hematologic Malignancies; Public Workshop; Request for Comments

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-24

    ... workshop include: (1) Overview of Quality control and standardization issues associated with Clinical Flow... monoclonal antibodies and flow cytometry contributed to the automation of lymphocyte subset analysis. Flow... cytometry system devices included flow cytometers, reagents, controls, and associated software for...

  11. Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

    PubMed Central

    Telford, William G.; Shcherbakova, Daria M.; Buschke, David; Hawley, Teresa S.; Verkhusha, Vladislav V.

    2015-01-01

    Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo. PMID:25811854

  12. Microfluidic Single Cell Array Cytometry for the Analysis of Tumour Apoptosis

    PubMed Central

    Wlodkowic, Donald; Faley, Shannon; Zagnoni, Michele; Wikswo, John P.; Cooper, Jonathan M.

    2013-01-01

    Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anti-cancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual non-adherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of non-adherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anti-cancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anti-cancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (~300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15,000 – 30,000 cells. PMID:19514700

  13. Measuring sickle cell morphology in flow using spectrally encoded flow cytometry (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kviatkovsky, Inna; Zeidan, Adel; Yeheskely-Hayon, Daniella; Dann, Eldad J.; Yelin, Dvir

    2017-02-01

    During a sickle cell crisis in sickle cell anemia patients, deoxygenated red blood cells may change their mechanical properties and block small blood vessels, causing pain, local tissue damage and even organ failure. Measuring these cellular structural and morphological changes is important for understanding the factors contributing to vessel blockage and developing an effective treatment. In this work, we use spectrally encoded flow cytometry for confocal, high-resolution imaging of flowing blood cells from sickle cell anemia patients. A wide variety of cell morphologies were observed by analyzing the interference patterns resulting from reflections from the front and back faces of the cells' membrane. Using numerical simulation for calculating the two-dimensional reflection pattern from the cells, we propose an analytical expression for the three-dimensional shape of a characteristic sickle cell and compare it to a previous from the literature. In vitro spectrally encoded flow cytometry offers new means for analyzing the morphology of sickle cells in stress-free environment, and could provide an effective tool for studying the unique physiological properties of these cells.

  14. In vivo flow cytometry of circulating clots using negative phototothermal and photoacoustic contrasts

    PubMed Central

    Galanzha, Ekaterina I.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Keyrouz, Salah G.; Mehta, Jawahar L.; Zharov, Vladimir P.

    2012-01-01

    Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of severe thromboembolisms including ischemia at strokes and myocardial dysfunction at heart attack. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 µm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting them by negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. PMID:21976458

  15. 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.

  16. 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...

  17. 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...

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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. Copyright © 2016 John Wiley & Sons, Inc.

  11. 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.

  12. Neutrophil aggregation measured in whole blood by flow cytometry

    SciTech Connect

    Simon, S.I.; Sklar, L.A. Los Alamos National Lab., NM )

    1991-03-15

    Flow cytometry has enabled measurement of the kinetics of formyl peptide stimulated neutrophil aggregation and its dependence on the CD11b/CD18 adhesion molecule. The authors are currently measuring aggregation of neutrophils in whole blood using flow cytometry. Fresh whole blood samples were kept at 4C and stained with LDS-751 a vital nucleic stain. Cytometric detection of neutrophil aggregation in whole blood at 37C was achieved by thresholding on LDS-751 fluorescence and then gating on forward and right angle light scatter. Aggregation was up to 10 times more efficient in whole blood than in purified cells, despite the fact that the number of CD11b/CD18 sites was upregulated 5-10 fold in elutriated neutrophil preparations. The time course of whole blood aggregation was often irreversible as compared to elutriated cells. Aggregation was only partially blocked by preincubation with concentrations of antibody to the CD18 integrin effective in blocking aggregation in elutriated cells. Further study is needed to distinguish between the contributions of other cell types, as well as the activity and number of CD11b/CD18 adhesive sites on the kinetics and efficiency of neutrophil collisions in whole blood.

  13. 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.

  14. 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.

  15. 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

  16. Detection of Significant Bacteriuria by Automated Urinalysis Using Flow Cytometry

    PubMed Central

    Okada, Hiroshi; Sakai, Yutaka; Miyazaki, Shigenori; Arakawa, Soichi; Hamaguchi, Yukio; Kamidono, Sadao

    2000-01-01

    A new flow cytometry-based automated urine analyzer, the UF-50, was evaluated for its ability to screen urine samples for significant bacteriuria. One hundred eighty-six urine specimens from patients attending an outpatient clinic of a university-based hospital were examined. The results obtained with the UF-50 were compared with those obtained by conventional quantitative urine culture. The UF-50 detected significant bacteriuria with a sensitivity of 83.1%, a specificity of 76.4%, a positive predictive value of 62.0%, a negative predictive value of 90.7%, and an accuracy of 78.5%. These results are comparable to those obtained by previously reported screening procedures. Besides detecting significant bacteriuria, the UF-50 can also perform routine urinalysis, including measurement of concentrations of red blood cells, white blood cells, epithelial cells, and casts, within 70 s. This capability renders this new flow cytometry-based urine analyzer superior to previously reported rapid screening methods. PMID:10921941

  17. 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.

  18. 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.

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

    PubMed Central

    Abdelrahman, Ahmed I.; Ornatsky, Olga; Bandura, Dmitry; Kinach, Robert; Dai, Sheng; Thickett, Stuart C.; Tanner, Scott

    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. PMID:20390041

  20. Thrombocytopenia: diagnosis with flow cytometry and antiplatelet antibodies.

    PubMed

    Guerra, João Carlos de Campos; Kanayama, Ruth Hissae; Nozawa, Sonia Tsukasa; Ioshida, Márcia Regina; Takiri, Irina Yoko; Lazaro, Robson José; Hamerschlak, Nelson; Rosenfeld, Luiz Gastão Mange; Guerra, Celso Carlos de Campos; Bacal, Nydia Strachman

    2011-06-01

    To identify antiplatelet antibodies by flow cytometry (direct method) in patients with thrombocytopenia. Between January 1997 and March 2004 a total of 15100 patients were referred to the Centro de Hematologia de São Paulo for hematological investigation of several diagnoses (anemia, leukopenia, thrombocytopenia, coagulation abnormalities, adenomegaly, leukemia and others). Of those, 1057 were referred because of thrombocytopenia and were divided into two groups: Group Idiopathic thrombocytopenic purpura, with no identifiable cause; and Group Other thrombocytopenia, which included low normal platelet counts cause to be established, hepatitis C and HIV infection, hypersplenism, EDTA-induced artifacts, laboratory error, and other causes. Flow cytometry immunophenotyping was done in 115 cases to identify platelet autoantibodies (direct method). Of the total number of patients, 1057 (7%) presented low platelet counts, 670 were females (63.4%) and age range of one to 75 years. Of the 115 cases (9.7%) submitted to immunophenotyping, the results were positive in 40% and the test was inconclusive in 5%. Idiopathic thrombocytopenic purpura was found in 52% of patients, more often in women. Hepatitis C virus infection was found in 7% and HIV infection in 1%. Low normal platelet counts were found in 17%, laboratory errors in 6%, and laboratory artifacts in 1% of cases. Platelet autoantibodies were found in 76.9% of all idiopathic thrombocytopenic purpura cases. It was negative in 83.3% of the low normal counts. antiplatelet autoantibodies when present help to diagnose idiopathic thrombocytopenic purpura. When absent, suggest other causes of thrombocytopenia.

  1. 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.

  2. 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.

  3. Bleaching response of Symbiodinium (zooxanthellae): determination by flow cytometry.

    PubMed

    Lee, Co Sin; Yeo, Yin Sheng Wilson; Sin, Tsai Min

    2012-10-01

    Coral bleaching is of increasing concern to reef management and stakeholders. Thus far, quantification of coral bleaching tends to be heavily reliant on the enumeration of zooxanthellae, with less emphasis on assessment of photosynthetic or physiological condition, these being often assessed separately by techniques such as liquid chromatography. Traditional methods of enumeration using microscopy are time consuming, subjected to low precision and great observer error. In this study, we presented a method for the distinction of physoiological condition and rapid enumeration of zooxanthellae using flow cytometry (FCM). Microscopy verified that healthy looking/live versus damaged/dead zooxanthellae could be reliably and objectively distinguished and counted by FCM on the basis of red and green fluorescence and light scatter. Excellent correlations were also determined between FCM and microscopy estimates of cell concentrations of fresh zooxanthellae isolates from Pocillopora damicornis. The relative intensities of chlorophyll and β-carotene fluorescences were shown to be important in understanding the results of increased cell counts in freshly isolated zooxanthellae experimentally exposed to high temperatures (34, 36, and 38°C) over 24 h, with ambient temperature (29°C) used as controls. The ability to simultaneously identify and enumerate subpopulations of different physiological states in the same sample provides an enormous advantage in not just determining bleaching responses, but elucidating adaptive response and mechanisms for tolerance. Therefore, this approach might provide a rapid, convenient, and reproducible methodology for climate change studies and reef management programs. Copyright © 2012 International Society for Advancement of Cytometry.

  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. A rapid method for quantifying cytoplasmic versus nuclear localization in endogenous peripheral blood leukocytes by conventional flow cytometry.

    PubMed

    Brittain, George C; Gulnik, Sergei

    2017-04-01

    A biochemical system and method have been developed to enable the quantitative measurement of cytoplasmic versus nuclear localization within cells in whole blood. Compared with the analyses of nuclear localization by western blot or fluorescence microscopy, this system saves a lot of time and resources by eliminating the necessity of purification and culturing steps, and generates data that are free from the errors and artifacts associated with using tumor cell lines or calculating nuclear signals from 2D images. This user-friendly system enables the analysis of cell signaling within peripheral blood cells in their endogenous environment, including measuring the kinetics of nuclear translocation for transcription factors without requiring protein modifications. We first demonstrated the efficiency and specificity of this system for targeting nuclear epitopes, and verified the results by fluorescence microscopy. Next, the power of the technique to analyze LPS-induced signaling in peripheral blood monocytes was demonstrated. Finally, both FoxP3 localization and IL-2-induced STAT5 signaling in regulatory T cells were analyzed. We conclude that this system can be a useful tool for enabling multidimensional molecular-biological analyses of cell signaling within endogenous peripheral blood cells by conventional flow cytometry. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  7. Identification and characterization of neutrophil extracellular trap shapes in flow cytometry

    NASA Astrophysics Data System (ADS)

    Ginley, Brandon; Emmons, Tiffany; Sasankan, Prabhu; Urban, Constantin; Segal, Brahm H.; Sarder, Pinaki

    2017-03-01

    Neutrophil extracellular trap (NET) formation is an alternate immunologic weapon used mainly by neutrophils. Chromatin backbones fused with proteins derived from granules are shot like projectiles onto foreign invaders. It is thought that this mechanism is highly anti-microbial, aids in preventing bacterial dissemination, is used to break down structures several sizes larger than neutrophils themselves, and may have several more uses yet unknown. NETs have been implied to be involved in a wide array of systemic host immune defenses, including sepsis, autoimmune diseases, and cancer. Existing methods used to visually quantify NETotic versus non-NETotic shapes are extremely time-consuming and subject to user bias. These limitations are obstacles to developing NETs as prognostic biomarkers and therapeutic targets. We propose an automated pipeline for quantitatively detecting neutrophil and NET shapes captured using a flow cytometry-imaging system. Our method uses contrast limited adaptive histogram equalization to improve signal intensity in dimly illuminated NETs. From the contrast improved image, fixed value thresholding is applied to convert the image to binary. Feature extraction is performed on the resulting binary image, by calculating region properties of the resulting foreground structures. Classification of the resulting features is performed using Support Vector Machine. Our method classifies NETs from neutrophils without traps at 0.97/0.96 sensitivity/specificity on n = 387 images, and is 1500X faster than manual classification, per sample. Our method can be extended to rapidly analyze whole-slide immunofluorescence tissue images for NET classification, and has potential to streamline the quantification of NETs for patients with diseases associated with cancer and autoimmunity.

  8. 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

  9. Quantitative absorption cytometry for measuring red blood cell hemoglobin mass and volume.

    PubMed

    Schonbrun, Ethan; Malka, Roy; Di Caprio, Giuseppe; Schaak, Diane; Higgins, John M

    2014-04-01

    We present an optical system, called the quantitative absorption cytometer (QAC), to measure the volume and hemoglobin mass of red blood cells flowing through a microfluidic channel. In contrast to clinical hematology analyzers, where cells are sphered in order for both volume and hemoglobin to be measured accurately, the QAC measures cells in their normal physiological shape. Human red blood cells are suspended in a refractive index-matching absorbing buffer, driven through a microfluidic channel, and imaged using a transmission light microscope onto a color camera. A red and a blue LED illuminate cells and images at each color are used to independently retrieve cell volume and hemoglobin mass. This system shows good agreement with red blood cell indices retrieved by a clinical hematology analyzer and in fact measures a smaller coefficient of variation of hemoglobin concentration. In addition to cell indices, the QAC returns height and mass maps of each measured cell. These quantitative images are valuable for analyzing the detailed morphology of individual cells as well as statistical outliers found in the data. We also measured red blood cells in hypertonic and hypotonic buffers to quantify the correlation between volume and hemoglobin mass under osmotic stress. Because this method is invariant to cell shape, even extremely nonspherical cells in hypertonic buffers can be measured accurately. © 2014 International Society for Advancement of Cytometry.

  10. Chromosome classification and purification using flow cytometry and sorting

    SciTech Connect

    Gray, J.W.; Langlois, R.G. )

    1986-01-01

    The authors review here both the techniques and the applications of flow karyotyping and sorting, hereafter called flow cytogenetics. Included in our review of the techniques are the procedures for chromosome isolation and staining, the principles of flow cytometry and sorting, and the analytical procedures for classification of the chromosomes from flow cytometric data. Applications reviewed include classification of normal human chromosomes, identification of disease-linked structural and numerical aberrations, quantification of the frequency of structurally aberrant chromosomes as a measure of the extent of induced genetic damage, and purification of chromosomes of a single type to facilitate interpretation of flow karyotypes, gene mapping, and production of recombinant DNA libraries from chromosomes of a single type.

  11. Quantitative karyotyping of human chromosomes by dual beam flow cytometry

    SciTech Connect

    Langlois, R.G.; Yu, L.C.; Gray, J.W.; Carrano, A.V.

    1982-12-01

    Dual beam flow cytometry of chromosomes stained with Hoechst 33258 and chromomycin A3 has been proposed as a method for quantitative classification of human chromosomes (bivariate flow karotyping). In this paper we investigate the sources and magnitudes of variability in the mean fluorescence intensities of each chromosome group resolved in bivariate flow karyotypes and study the impact of this variablity on chromosome classification. Replicate bivariate flow karyotypes of chromosomes isolated from lymphocyctes from 10 individuals demonstrated that person-to-person variability was significantly greater than run-to-run variability. The total variability was sufficiently small that it did not interfere with classification of normal chromosome types except chromosomes 9 through 12 and chromosomes 14 and 15. Furthermore, the variability was generally smaller than 1/600th of the mitotic genome, so that one-band rearrangements should be detectable in bivariate flow karoyotypes.

  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. 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.

  15. 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.

  16. 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.

  17. Multiparametric flow cytometry profiling of neoplastic plasma cells in multiple myeloma.

    PubMed

    Johnsen, Hans E; Bøgsted, Martin; Klausen, Tobias W; Gimsing, Peter; Schmitz, Alexander; Kjaersgaard, Erik; Damgaard, Tina; Voss, Pia; Knudsen, Lene M; Mylin, Anne K; Nielsen, Johan Lanng; Björkstrand, Bo; Gruber, Astrid; Lenhoff, Stig; Remes, Kari; Dahl, Inger Marie; Fogd, Kirsten; Dybkaer, Karen

    2010-09-01

    The clinical impact of multiparametric flow cytometry (MFC) in multiple myeloma (MM) is still unclear and under evaluation. Further progress relies on multiparametric profiling of the neoplastic plasma cell (PC) compartment to provide an accurate image of the stage of differentiation. The primary aim of this study was to perform global analysis of CD expression on the PC compartment and subsequently to evaluate the prognostic impact. Secondary aims were to study the diagnostic and predictive impact. The design included a retrospective analysis of MFC data generated from diagnostic bone marrow (BM) samples of 109 Nordic patients included in clinical trials within NMSG. Whole marrow were analyzed by MFC for identification of end-stage CD45(-) /CD38(++) neoplastic PC and registered the relative numbers of events and mean fluorescence intensity (MFI) staining for CD19, CD20, CD27, CD28, CD38, CD44, CD45, CD56, and isotypes for cluster analysis. The median MFC-PC number was 15%, and the median light microscopy (LM)-PC number was 35%. However, the numbers were significant correlated and the prognostic value with an increased relative risk (95% CI) of 3.1 (1.7-5.5) and 2.9 (1.4-6.2), P < 0.0003 and P < 0.004 of MFC-PC and LM-PC counts, respectively. Unsupervised clustering based on global MFI assessment on PC revealed two clusters based on CD expression profiling. Cluster I with high intensity for CD56, CD38, CD45, right-angle light-scatter signal (SSC), forward-angle light-scatter signal (FSC), and low for CD28, CD19, and a Cluster II, with low intensity of CD56, CD38, CD45, SSC, FSC, and high for CD28, CD19 with a median survival of 39 months and 19 months, respectively (P = 0.02). The MFC analysis of MM BM samples produces diagnostic, prognostic, and predictive information useful in clinical practice, which will be prospectively validated within the European Myeloma Network (EMN). © 2010 International Clinical Cytometry Society. Copyright © 2010 International

  18. AutoGate: automating analysis of flow cytometry data

    PubMed Central

    Meehan, Stephen; Walther, Guenther; Moore, Wayne; Orlova, Darya; Meehan, Connor; Parks, David; Ghosn, Eliver; Philips, Megan; Mitsunaga, Erin; Waters, Jeffrey; Kantor, Aaron; Okamura, Ross; Owumi, Solomon; Yang, Yang; Herzenberg, Leonard A.

    2015-01-01

    Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis “trees” that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this “early adopter” phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users. PMID:24825775

  19. 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

  20. [Immunophenotype in multiple myeloma cells detected by multiparameter flow cytometry].

    PubMed

    Cao, Fang-Fang; Chen, Fang; Hu, Yan-Ping; Zhang, Ji-Hong

    2012-06-01

    This study was purposed to investigate the immunophenotypic characteristics in multiple myeloma (MM) cells and their significance. Thirty three cases of MM and 12 cases of reactive plasmacytosis (as control group) were enrolled in the study. The expressions of surface antigens in MM cells were detected with flow cytometry by using direct immunofluorescent technique and gating method of CD38/SSC and were confirmed with morphologic observation of myeloma cells. The results indicated that the proportion of myeloma cells detected by morphologic examination was 6.0% - 76.0%. With CD38/SSC gating method, a cluster of CD38 bright positive cells could be detected in their scatter plot, the proportion ranged from 0.99% to 57.54%. Most phenotype of MM was 38(st+)CD138(+)CD19(-)CD56(+) (78.8%). While the expressions of CD20, CD33, CD117, HLA-DR were seen in some MM patients, the positive rates were 12.1%, 15.2%, 30.3%, 9.1%, respectively; the expression of other antigens was negative. cκ or cλ monoclonal restriction was detected in 27 cases (81.8%) of MM, both cκ and cλ in the remaining cases of MM was negative. It is concluded that detecting the immunophenotype of MM patients by flow cytometry with CD38/SSC gating method and basing on the heterogeneity of cell antigens can discriminate myeloma cells from normal plasma cells, which provides evidence for targeted therapy and prognosis evaluation.

  1. Determination of DNA Content of Aquatic Bacteria by Flow Cytometry

    PubMed Central

    Button, D. K.; Robertson, Betsy R.

    2001-01-01

    The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell−1 was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell−1. PMID:11282616

  2. 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.

  3. 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.

  4. 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.

  5. DNA methylation analysis for the diagnosis of thyroid nodules - a pilot study with reference to BRAF(V) (600E) mutation and cytopathology results.

    PubMed

    Chang, H; Shin, B K; Kim, A; Kim, H K; Kim, B H

    2016-04-01

    Promoter hypermethylation and the BRAF(V) (600E) mutation are both involved in thyroid tumorigenesis. We conducted a pilot study on the diagnosis of thyroid nodules by analysis of promoter hypermethylation status with reference to BRAF(V) (600E) mutation and cytopathology results using formalin-fixed, paraffin-embedded (FFPE) tissues and liquid-based preparation (LBP) thyroid fine needle aspiration (FNA) samples to predict more reliably the possibility of papillary carcinoma. We initially performed MethyLight analysis for 30 genes that are known to be hypermethylated in malignancies using 164 papillary carcinomas and 77 benign tissue samples. Five genes selected from the tissue analysis were subsequently analysed in 75 surgically proven benign and 66 surgically proven papillary carcinoma LBP FNA samples. Samples that showed two or more positive results among the five genes were classified as methylation positive. We also analysed the BRAF(V) (600E) mutation status of the FNA samples. We identified five genes that were significantly hypermethylated in malignant tissues: PTGS2, HOXA1, TMEFF2, p16 and PTEN. With respect to diagnostic potential, results obtained using the BRAF(V) (600E) mutation test combined with cytological examination were not significantly different from those obtained with cytological examination only. Combining methylation analyses with cytological examination or performing all three tests for diagnoses did not improve significantly the negative predictive values and sensitivity, but a significant decrease in positive predictive value and specificity was observed. Further studies are needed on larger samples to assess the potential value of methylation analysis of thyroid FNA. © 2015 John Wiley & Sons Ltd.

  6. The clinical impact of immediate on-site cytopathology evaluation during endoscopic ultrasound-guided fine needle aspiration of pancreatic masses: a prospective multicenter randomized controlled trial.

    PubMed

    Wani, Sachin; Mullady, Daniel; Early, Dayna S; Rastogi, Amit; Collins, Brian; Wang, Jeff F; Marshall, Carrie; Sams, Sharon B; Yen, Roy; Rizeq, Mona; Romanas, Maria; Ulusarac, Ozlem; Brauer, Brian; Attwell, Augustin; Gaddam, Srinivas; Hollander, Thomas G; Hosford, Lindsay; Johnson, Sydney; Kushnir, Vladimir; Amateau, Stuart K; Kohlmeier, Cara; Azar, Riad R; Vargo, John; Fukami, Norio; Shah, Raj J; Das, Ananya; Edmundowicz, Steven A

    2015-10-01

    Observational data on the impact of on-site cytopathology evaluation (OCE) during endoscopic ultrasonography-guided fine needle aspiration (EUS-FNA) of pancreatic masses have reported conflicting results. We aimed to compare the diagnostic yield of malignancy and proportion of inadequate specimens between patients undergoing EUS-FNA of pancreatic masses with and without OCE. In this multicenter randomized controlled trial, consecutive patients with solid pancreatic mass underwent randomization for EUS-FNA with or without OCE. The number of FNA passes in the OCE+ arm was dictated by the on-site cytopathologist, whereas seven passes were performed in OCE- arm. EUS-FNA protocol was standardized, and slides were reviewed by cytopathologists using standardized criteria for cytologic characteristics and diagnosis. A total of 241 patients (121 OCE+, 120 OCE-) were included. There was no difference between the two groups in diagnostic yield of malignancy (OCE+ 75.2% vs. OCE- 71.6%, P=0.45) and proportion of inadequate specimens (9.8 vs. 13.3%, P=0.31). Procedures in OCE+ group required fewer EUS-FNA passes (median, OCE+ 4 vs. OCE- 7, P<0.0001). There was no significant difference between the two groups with regard to overall procedure time, adverse events, number of repeat procedures, costs (based on baseline cost-minimization analysis), and accuracy (using predefined criteria for final diagnosis of malignancy). There was no difference between the two groups with respect to cytologic characteristics of cellularity, bloodiness, number of cells/slide, and contamination. Results of this study demonstrated no significant difference in the diagnostic yield of malignancy, proportion of inadequate specimens, and accuracy in patients with pancreatic mass undergoing EUS-FNA with or without OCE.

  7. Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP): Implications for the risk of malignancy (ROM) in the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC).

    PubMed

    Zhou, Haijun; Baloch, Zubair W; Nayar, Ritu; Bizzarro, Tommaso; Fadda, Guido; Adhikari-Guragain, Deepti; Hatem, Joseph; Larocca, Luigi M; Samolczyk, Julia; Slade, Jamie; Rossi, Esther Diana

    2017-09-20

    The introduction of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) affects the risk of malignancy (ROM) mostly in the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) categories. In this multi-institutional, retrospective study, the authors investigated variations in the impact of an NIFTP diagnosis on the associated ROM for each TBSRTC category with an emphasis on the influence of pathologist and institutional diagnostic thresholds on the ROM. Baseline data on cytology and histology diagnostic categories were collected over a 3-year period at 3 academic center hospitals (institutions A, B, and C). Histology slides for all cases diagnosed as follicular variant of papillary thyroid carcinoma (FVPTC) were re-reviewed at each institution, and those that qualifying as NIFTP were separated from other PTCs. The collective case cohort from the 3 institutions included 15,973 thyroid fine-needle aspiration cytology (FNAC) specimens and 5090 thyroid surgical resection specimens. Significant differences in baseline cytology and histology data were noted among the 3 institutions. The number of cases classified as NIFTP compared with FVPTC was highly variable (institution A, 14%; institution B, 39%; and institution C, 12%). For 3250 resected thyroid nodules with a previous FNAC diagnosis, the average decrease in ROM after the exclusion of NIFTP for all TBSRTC categories was as follows: institution A, 9.8%; institution B, 3.9%; and institution C, 1.3%. The institutional frequency of NIFTP histopathology diagnosis and cytology baseline data will impact the ROM associated with specific FNAC diagnoses, especially among the indeterminate TBSRTC categories. The range of ROM for each TBSRTC diagnostic category is reflective of the inherent diagnostic thresholds and interobserver and interinstitutional variability in the diagnosis of thyroid lesions. Cancer Cytopathol 2017. © 2017 American Cancer Society. © 2017 American Cancer

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput Förster resonance energy transfer screening.

    PubMed

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

    2015-02-01

    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 fluorescence

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow Cytometry