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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. 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. PMID:20727005

  4. Microspectrofluorometry and fluorescence imaging in the study of human cytopathology.

    PubMed

    Kohen, E; Gatt, S; Schachtschabel, A; Schachtschabel, D O; Kohen, C; Agmon, V; Hirschberg, J G; Monti, M

    2000-12-01

    The study of energy pools and dynamics of specific pathways in living cells by microspectrofluorometry and fluorescence imaging produces spectral and topographic images characterizing structural and functional changes associated with cytopathology. Microspectro-fluorometry and fluorescence imaging have been applied, together with organelle morphometry to a number of cells mimicking certain cytopathologies, including melanoma cells, long-term malignant cells, and gene-defective cells. These investigations of cellular pathology indicate that there is a convergence of various physiopathological processes. Cellular states that have similarities include senescence, detoxification, and transformation. While the NAD(P)H metabolic transients have been studied before, our emphasis in this article is on very rapidly scanned fluorescence images related to organelle integration and photoinduced cellular senescence. PMID:11074618

  5. 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. PMID:26093936

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

    PubMed Central

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

    2015-01-01

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

  7. 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. PMID:27460234

  8. Applications of Imaging Flow Cytometry for Microalgae.

    PubMed

    Hildebrand, Mark; Davis, Aubrey; Abbriano, Raffaela; Pugsley, Haley R; Traller, Jesse C; Smith, Sarah R; Shrestha, Roshan P; Cook, Orna; Sánchez-Alvarez, Eva L; Manandhar-Shrestha, Kalpana; Alderete, Benjamin

    2016-01-01

    The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression. PMID:27460237

  9. 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.

  10. Infrared imaging of small molecules in living cells: from in vitro metabolic analysis to cytopathology.

    PubMed

    Quaroni, Luca; Zlateva, Theodora; Wehbe, Katia; Cinque, Gianfelice

    2016-06-23

    A major topic in InfraRed (IR) spectroscopic studies of living cells is the complexity of the vibrational spectra, involving hundreds of overlapping absorption bands from all the cellular components present at detectable concentrations. We focus on the relative contribution of both small-molecule metabolites and macromolecules, while defining the spectroscopic properties of cells and tissue in the middle IR (midIR) region. As a consequence, we show the limitations of current interpretative schemes that rely on a small number of macromolecules for IR band assignment. The discussion is framed specifically around the glycolytic metabolism of cancer cells because of the potential pharmacological applications. Several metabolites involved in glycolysis by A549 lung cancer cells can be identified by this approach, which we refer to as Correlated Cellular Spectro-Microscopy (CSM). It is noteworthy that the rate of formation or consumption of specific molecules could be quantitatively assessed by this approach. We now extend this analysis to the two-dimensional case by performing IR imaging on single cells and cell clusters, detecting variations of metabolite concentration in time and space across the sample. The molecular detail obtained from this analysis allows its use in evaluating the pharmacological effect of inhibitors of glycolytic enzymes with potential consequences for in vitro drug testing. Finally we highlight the implications of the spectral contribution from cellular metabolites on applications in IR spectral cytopathology (SCP). PMID:27049435

  11. Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations.

    PubMed

    Kuksin, Dmitry; Kuksin, Christina Arieta; Qiu, Jean; Chan, Leo Li-Ying

    2016-06-15

    Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples. PMID:27033005

  12. Visualization of DNA replicons by image cytometry

    SciTech Connect

    Gratzner, H.G. )

    1993-01-01

    Replication of DNA in eukaryotic organisms proceeds bidirectionally along the double helix in replicon substructures. The process can be visualized by autoradiography of spreads of genomic DNA from lysed, whole cells which have been incubated with radioactive DNA precursors. The objective of the present study was to develop techniques to measure DNA strand initiation and elongation using immunofluorescence and image cytometry. Peripheral blood lymphocytes were cultured for 3 days with PHA and then pulsed for 5 or 15 minutes with iododeoxyuridine. Chromatin spreads were then produced on microscope slides by lysing with detergent and slides were immunofluorescently stained by an indirect anti-BrdU technique. Individual replicons of mammalian cells were visualized by immunofluorescence at high resolution and digital images were analyzed to determine the rates of elongation as well as initiation parameters. Elongation rates by the method were approximately 1 [mu]M/min. The methods are applicable to visualization and quantitation of the effects of radiation or other agents on DNA damage or repair.

  13. 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.

  14. 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

  15. Fluorescent In Situ Hybridization in Suspension by Imaging Flow Cytometry.

    PubMed

    Maguire, Orla; Wallace, Paul K; Minderman, Hans

    2016-01-01

    The emergence of imaging flow cytometry (IFC) has brought novel applications exploiting its advantages over conventional flow cytometry and microscopy. One of the new applications is fluorescence in situ hybridization in suspension (FISH-IS). Conventional FISH is a slide-based approach in which the spotlike imagery resulting from hybridization with fluorescently tagged probes is evaluated by fluorescence microscopy. The FISH-IS approach evaluated by IFC enables the evaluation of tens to hundreds of thousands of cells in suspension and the analysis can be automated and standardized diminishing operator bias from the analysis. The high cell number throughput of FISH-IS improves the detection of rare events compared to conventional FISH. The applicability of FISH-IS is currently limited to detection of abnormal quantitative differences of hybridization targets such as occur in numerical chromosome abnormalities, deletions and amplifications.Here, we describe a protocol for FISH-IS using chromosome enumeration probes as an example. PMID:27460240

  16. 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.

  17. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.

    PubMed

    Giesen, Charlotte; Wang, Hao A O; Schapiro, Denis; Zivanovic, Nevena; Jacobs, Andrea; Hattendorf, Bodo; Schüffler, Peter J; Grolimund, Daniel; Buhmann, Joachim M; Brandt, Simone; Varga, Zsuzsanna; Wild, Peter J; Günther, Detlef; Bodenmiller, Bernd

    2014-04-01

    Mass cytometry enables high-dimensional, single-cell analysis of cell type and state. In mass cytometry, rare earth metals are used as reporters on antibodies. Analysis of metal abundances using the mass cytometer allows determination of marker expression in individual cells. Mass cytometry has previously been applied only to cell suspensions. To gain spatial information, we have coupled immunohistochemical and immunocytochemical methods with high-resolution laser ablation to CyTOF mass cytometry. This approach enables the simultaneous imaging of 32 proteins and protein modifications at subcellular resolution; with the availability of additional isotopes, measurement of over 100 markers will be possible. We applied imaging mass cytometry to human breast cancer samples, allowing delineation of cell subpopulations and cell-cell interactions and highlighting tumor heterogeneity. Imaging mass cytometry complements existing imaging approaches. It will enable basic studies of tissue heterogeneity and function and support the transition of medicine toward individualized molecularly targeted diagnosis and therapies. PMID:24584193

  18. [A plea for cytopathology].

    PubMed

    Dalquen, P

    2012-07-01

    Compared to other European and non-European countries the benefits of cytopathology for the diagnosis of many tumors is still underestimated in Germany for traditional reasons. Cytological methods provide excellent material from many organs for morphological, immunochemical and molecular examinations so that a definitive diagnosis is cytologically possible in many cases and the number of exploratory surgical operations could therefore be reduced. Improvements in this deplorable situation will only be possible if a standardized training period in cytology is consistently included in the training of general pathologists. This requires organizational and infrastructural changes within the institutes of pathology. In this respect, the university institutes as important training institutions should lead the way. PMID:22711371

  19. Flow cytometry using Brillouin imaging and sensing via time-resolved optical (BISTRO) measurements.

    PubMed

    Meng, Zhaokai; Petrov, Georgi I; Yakovlev, Vladislav V

    2015-11-01

    A novel concept of Brillouin imaging and sensing via time-resolved optical (BISTRO) measurements is introduced for flow cytometry applications. The system affords robust, maintenance-free and high-speed elasticity-sensitive measurements. PMID:26347908

  20. Biospecimen repositories and cytopathology.

    PubMed

    Krishnamurthy, Savitri

    2015-03-01

    Biospecimen repositories are important for the advancement of biomedical research. Literature on the potential for biobanking of fine-needle aspiration, gynecologic, and nongynecologic cytology specimens is very limited. The potential for biobanking of these specimens as valuable additional resources to surgically excised tissues appears to be excellent. The cervicovaginal specimens that can be used for biobanking include Papanicolaou-stained monolayer preparations and residual material from liquid-based cytology preparations. Different types of specimen preparations of fine-needle aspiration and nongynecologic specimens, including Papanicolaou-stained and Diff-Quik-stained smears, cell blocks. and dedicated passes/residual material from fine-needle aspiration stored frozen in a variety of solutions, can be used for biobanking. Because of several gaps in knowledge regarding the standard of operative procedures for the procurement, storage, and quality assessment of cytology specimens, further studies as well as national conferences and workshops are needed not only to create awareness but also to facilitate the use of cytopathology specimens for biobanking. PMID:25524469

  1. 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.

  2. Quantitative tissue cytometry (Tissomics): multimodal slide-based cytometry, confocal imaging, and volume rendering is the key

    NASA Astrophysics Data System (ADS)

    Tarnok, Attila; Mittag, Anja; Kuska, Jens-Peer; Braumann, Ulf-Dietrich; Mosch, Birgit; Arendt, Thomas

    2007-02-01

    Multiplexed high-content cytometric analysis of cells is a prerequisite for Cytomics and Systems Biology. Slide Based Cytometry (SBC) analysis yields quantitative cell related data on various cell constituents. It allows to measure and identify in high-throughput hundred-thousands of objects and obtain cytometric data on light absorption, scatter and fluorescence signals. Selected cells of interest can be rescanned and morphologically evaluated. To be cytometric SBC measurement needs high focal depth in order to acquire the fluorescence of the whole cell. For tissue analysis section thickness of >30μm is needed to reduce cell sectioning leading in multiple labelled specimens to an overestimation of multiple stained cells due to stereology, mimicking co-expression or elevated expression that is in fact due to coincidences in the z-axis direction. By confocal sectioning and 3D-reconstruction these overlays could be eliminated but confocal 3D imaging is slow and the resulting data are not cytometric. To overcome this obstacle, we combined SBC analysis with confocal imaging using a Laser Scanning Cytometer (iCys, Compucyte Corp., MA). Single to triple labelled 30-120μm thick human brain sections were scanned cytometrically (up to three laser 405nm, 488nm, 633nm) and double and triple labeled cells were identified. In the second step these objects were relocated, scanned confocally and 3D-reconstructed (Mathematica®, MathGL3d). This combination of high-throughput SBC and high-resolution confocal imaging enables for unequivocal identification of multiple labelled objects and is a prerequisite for Cytomic tissue analysis, Tissomics. (Support: HBFG 036/379-1)

  3. Multimodality Molecular Imaging (FDG-PET/CT, US Elastography, and DWI-MRI) as Complimentary Adjunct for Enhancing Diagnostic Confidence in Reported Intermediate Risk Category Thyroid Nodules on Bethesda Thyroid Cytopathology Reporting System

    PubMed Central

    Basu, Sandip; Mahajan, Abhishek; Arya, Supreeta

    2016-01-01

    The potential complimentary role of various molecular imaging modalities [fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT), ultrasound (US)-elastography, and diffusion weighted imaging-magnetic resonance imaging (DWI-MRI)] in characterizing thyroid nodules, which have been designated as “intermediate risk category” on the Bethesda thyroid cytopathology reporting system (BTCRS), is illustrated in this communication. The clinical cases described (category III thyroid nodules on BTCRS) show the imaging features and the final diagnostic impressions rendered by the interpreting physicians with the modalities that have been independently compared in a tabular format at the end; of particular note is the high negative predictive value of these (specifically FDG-PET/CT), which could aid in enhancing the diagnostic confidence in the reported “intermediate risk category” thyroid nodules, a “gray zone” from the patient management viewpoint. PMID:27134564

  4. Label-free cell cycle analysis for high-throughput imaging flow cytometry

    PubMed Central

    Blasi, Thomas; Hennig, Holger; Summers, Huw D.; Theis, Fabian J.; Cerveira, Joana; Patterson, James O.; Davies, Derek; Filby, Andrew; Carpenter, Anne E.; Rees, Paul

    2016-01-01

    Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types. PMID:26739115

  5. Image cytometry of estrogen receptors in breast carcinomas.

    PubMed

    Cohen, O; Brugal, G; Seigneurin, D; Demongeot, J

    1988-11-01

    A significant level of estrogen receptors (ER) in breast cancer cells is an indication of tumor differentiation and suggests that a homeostatic control of cell growth may persist in these cancers. In medical practice, the Dextran-coated charcoal assays (DCCA) are still the most frequently used test to characterize patients having ER-positive malignant breast tumors and for whom hormonal therapy is justified. Nevertheless, this routine biochemical technique is not satisfactory because it is a broad method unsuitable for revealing receptor tissue heterogeneity. However, immunocytochemical labeling, such as the ER-ICA method, which involves a monoclonal antibody linked to peroxidase, is a specific reaction for this purpose but which until now was not quantitative. The present study uses an original cell preparation technique combining the PAP reaction with toluidine blue counterstain for image analysis on the SAMBA system. Special software has been developed for the quantitative analysis of immunocytochemistry in cancers. Results obtained showed a high correlation between the DCCA values and the score derived from the mean ER concentration per positive tumor cell and the labeling index. In addition, intracell and intratumor heterogeneity can be displayed according to several parameters and were shown to vary according to tumor and to antiestrogen (Tamoxifen) presurgical therapy. PMID:2463134

  6. Image cytometry of duct cells from benign and malignant breast disease

    SciTech Connect

    Mayall, B.H.; Gadenne, C.; King, E.B.; Chew, K.L.; Duarte, L.A.; Petrakis, N.L.

    1987-01-22

    We used image cytometry to classify benign and malignant breast lesions, to identify parameters that classify premalignant lesions, and to assess these parameters for a biological association that may be linked with malignancy. Results suggest that quantitative image analysis is able to discriminate ''normal'' from ''abnormal'' breast lesions. Thus image cytometry may provide an objective approach for early detection of malignant changes in the breast. The parameters selected by stepwise discriminant and regression analyses as being most useful for discriminating among breast lesions relate to nuclear size, shape and DNA content. These parameters are robust and easily measured and are similar to those used by others for discriminating benign from malignant lesions. Significantly, no parameter based on nuclear chromatin distribution ranked as highly in this study, unlike our experience with studies of other premalignant lesions. 9 refs., 1 fig., 3 tabs.

  7. Cutting-edge analysis of extracellular microparticles using ImageStream(X) imaging flow cytometry.

    PubMed

    Headland, Sarah E; Jones, Hefin R; D'Sa, Adelina S V; Perretti, Mauro; Norling, Lucy V

    2014-01-01

    Interest in extracellular vesicle biology has exploded in the past decade, since these microstructures seem endowed with multiple roles, from blood coagulation to inter-cellular communication in pathophysiology. In order for microparticle research to evolve as a preclinical and clinical tool, accurate quantification of microparticle levels is a fundamental requirement, but their size and the complexity of sample fluids present major technical challenges. Flow cytometry is commonly used, but suffers from low sensitivity and accuracy. Use of Amnis ImageStream(X) Mk II imaging flow cytometer afforded accurate analysis of calibration beads ranging from 1 μm to 20 nm; and microparticles, which could be observed and quantified in whole blood, platelet-rich and platelet-free plasma and in leukocyte supernatants. Another advantage was the minimal sample preparation and volume required. Use of this high throughput analyzer allowed simultaneous phenotypic definition of the parent cells and offspring microparticles along with real time microparticle generation kinetics. With the current paucity of reliable techniques for the analysis of microparticles, we propose that the ImageStream(X) could be used effectively to advance this scientific field. PMID:24913598

  8. Cutting-Edge Analysis of Extracellular Microparticles using ImageStreamX Imaging Flow Cytometry

    PubMed Central

    Headland, Sarah E.; Jones, Hefin R.; D'Sa, Adelina S. V.; Perretti, Mauro; Norling, Lucy V.

    2014-01-01

    Interest in extracellular vesicle biology has exploded in the past decade, since these microstructures seem endowed with multiple roles, from blood coagulation to inter-cellular communication in pathophysiology. In order for microparticle research to evolve as a preclinical and clinical tool, accurate quantification of microparticle levels is a fundamental requirement, but their size and the complexity of sample fluids present major technical challenges. Flow cytometry is commonly used, but suffers from low sensitivity and accuracy. Use of Amnis ImageStreamX Mk II imaging flow cytometer afforded accurate analysis of calibration beads ranging from 1 μm to 20 nm; and microparticles, which could be observed and quantified in whole blood, platelet-rich and platelet-free plasma and in leukocyte supernatants. Another advantage was the minimal sample preparation and volume required. Use of this high throughput analyzer allowed simultaneous phenotypic definition of the parent cells and offspring microparticles along with real time microparticle generation kinetics. With the current paucity of reliable techniques for the analysis of microparticles, we propose that the ImageStreamX could be used effectively to advance this scientific field. PMID:24913598

  9. An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry

    PubMed Central

    Smirnov, Asya; Solga, Michael D; Lannigan, Joanne; Criss, Alison K

    2015-01-01

    Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type. PMID:25967947

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

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2013-01-01

    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. PMID:23603893

  12. Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

    PubMed Central

    Zhu, Hongying; Ozcan, Aydogan

    2013-01-01

    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 mm2. 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. PMID:23603893

  13. Cytopathology of Follicular Cell Nodules.

    PubMed

    Damiani, Domenico; Suciu, Voichita; Vielh, Philippe

    2015-12-01

    This article corresponds to a lecture delivered during the Endocrine Pathology Society symposium held in Boston on 21 March 2015 (104th USCAP meeting, March 21-27). It focuses on the importance of cytopathology in endocrine thyroid pathology and the limits and pitfalls of diagnosis in follicular cell lesions. Lights and shadows are present in each diagnostic technique: Fine needle aspiration has imposed itself as a gold standard in thyroid nodules thanks to its easiness of execution and high cost-effectiveness ratio. A milestone in this field is represented by the National Cancer Institute (NCI) Thyroid Fine Needle Aspiration (FNA) State of the State of the Science Conference hosted in October 22-23, 2007 by the NCI, followed by a series of documents published in Diagnostic Cytopathology and Cytojournal (2008) as well as in an atlas entitled: The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC): terminology and criteria (2010, Springer). "Gray" zones still remain, causing difficulties and anxiety to the cytopathologist when facing challenging cases. Each diagnostic category of TBSRTC is analyzed and discussed in a concise fashion with special emphasis on challenging cases such as atypia of undetermined significance (AUS), suspicion for follicular neoplasms (SFNs), diagnoses of papillary thyroid carcinoma (PTC) in Hashimoto thyroiditis and follicular variant of papillary carcinoma (FVPTC). Our aim was to better define and clarify the spectrum of follicular cell lesions in thyroid nodule samplings and to underline the diagnostic limits in order to avoid pitfalls. New emerging molecular biology techniques may represent useful tools in selected morphological challenging cases and lead to new therapeutic approaches in line with drug-tailored therapy and personalized medicine. PMID:26227345

  14. A comparative study of quantitative stains for DNA in image cytometry.

    PubMed

    Mikel, U V; Becker, R L

    1991-08-01

    In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide. PMID:1718295

  15. A Telepathology Based Virtual Reference and Certification Centre for DNA Image Cytometry

    PubMed Central

    Haroske, G.; Giroud, Francoise; 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. PMID:11339562

  16. 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. PMID:11339562

  17. 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

  18. 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.

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

    PubMed Central

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

    2008-01-01

    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. PMID:19049958

  20. 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}.

  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). PMID:26429450

  2. 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.

  3. High-throughput detection of DNA double-strand breaks using image cytometry

    PubMed Central

    Fowler, Tyler L.; Bailey, Alison M.; Bednarz, Bryan P.; Kimple, Randall J.

    2015-01-01

    Assessment of γH2AX expression for studying DNA double-strand break formation is often performed by manual counting of foci using immunofluorescence microscopy, an approach that is laborious and subject to significant foci selection bias. Here we present a novel high-throughput method for detecting DNA double-strand breaks using automated image cytometry assessment of cell average γH2AX immunofluorescence. Our technique provides an expedient, high-throughput, objective, and cost-effective method for γH2AX analysis. PMID:25605579

  4. High-throughput detection of DNA double-strand breaks using image cytometry.

    PubMed

    Fowler, Tyler L; Bailey, Alison M; Bednarz, Bryan P; Kimple, Randall J

    2015-01-01

    Assessment of γH2AX expression for studying DNA double-strand break formation is often performed by manual counting of foci using immunofluorescence microscopy, an approach that is laborious and subject to significant foci selection bias. Here we present a novel high-throughput method for detecting DNA double-strand breaks using automated image cytometry assessment of cell average γH2AX immunofluorescence. Our technique provides an expedient, high-throughput, objective, and cost-effective method for γH2AX analysis. PMID:25605579

  5. Application of image cytometry to characterize heterologous lipid flippases in yeast.

    PubMed

    Jensen, Maria S; Costa, Sara R; Theorin, Lisa; Christensen, Jan Pravsgaard; Pomorski, Thomas Günther; López-Marqués, Rosa L

    2016-07-01

    Lipid flippases are integral membrane proteins that play a central role in moving lipids across cellular membranes. Some of these transporters are ATPases that couple lipid translocation to ATP hydrolysis, whereas others function without any discernible metabolic energy input. A growing number of lipid flippases has been identified but key features of their activity remain to be elucidated. A well-established method to characterize ATP-driven flippases is based on their heterologous expression in yeast, followed by incubation of the cells with fluorescent lipids. Internalization of these probes is typically monitored by flow cytometry, a costly and maintenance-intensive method. Here, we have optimized a protocol to use an automated image-based cell counter to accurately measure lipid uptake by heterologous lipid flippases expressed in yeast. The method was validated by comparison with the classical flow cytometric evaluation of lipid-labeled cells. In addition, we demonstrated that expression of fluorescently tagged flippase complexes can be directly co-related with fluorescent lipid uptake using the image-based cell counter system. The method extends the number of techniques available for characterization of lipid flippase activity, and should be readily adaptable to analyze a variety of other transport systems in yeast, parasites, and mammalian cells. © 2016 International Society for Advancement of Cytometry. PMID:27272389

  6. FlowCam: Quantification and Classification of Phytoplankton by Imaging Flow Cytometry.

    PubMed

    Poulton, Nicole J

    2016-01-01

    The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments. PMID:27460250

  7. 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

  8. High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

    Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

  9. 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

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

    PubMed

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

    2016-07-01

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

  11. Quantification of heterotypic granule fusion in human neutrophils by imaging flow cytometry

    PubMed Central

    Björnsdottir, Halla; Welin, Amanda; Dahlgren, Claes; Karlsson, Anna; Bylund, Johan

    2015-01-01

    Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules. To date, at least four distinct granule/vesicle subsets have been identified. These organelles may secrete their content extracellularly following mobilization to and fusion with the plasma membrane, but some of them may also fuse with internal membrane-enclosed organelles, typically a plasma membrane-derived phagosome. There are also instances where different granules appear to fuse with one another, a process that would enable mixing of their matrix and membrane components. Such granule fusion enables e.g., myeloperoxidase-processing of intragranular oxygen radicals, a key event in the formation of neutrophil extracellular traps (Björnsdottir et al., 2015) [1]. Described herein are data that show the quantification of such heterotypic granule–granule fusion by the use of imaging flow cytometry, a technique that combines flow cytometry with microscopy. The analysis described is based on immunofluorescent staining of established granule markers (lactoferrin and/or NGAL for one granule subset; the specific granules, and CD63 for another granule subset, the azurophil granules) and calculation of a colocalization score for resting and PMA-stimulated neutrophils. PMID:26862586

  12. The Analysis of Cell Cycle, Proliferation, and Asymmetric Cell Division by Imaging Flow Cytometry.

    PubMed

    Filby, Andrew; Day, William; Purewal, Sukhveer; Martinez-Martin, Nuria

    2016-01-01

    Measuring cellular DNA content by conventional flow cytometry (CFC) and fluorescent DNA-binding dyes is a highly robust method for analysing cell cycle distributions within heterogeneous populations. However, any conclusions drawn from single-parameter DNA analysis alone can often be confounded by the asynchronous nature of cell proliferation. We have shown that by combining fluorescent DNA stains with proliferation tracking dyes and antigenic staining for mitotic cells one can elucidate the division history and cell cycle position of any cell within an asynchronously dividing population. Furthermore if one applies this panel to an imaging flow cytometry (IFC) system then the spatial information allows resolution of the four main mitotic phases and the ability to study molecular distributions within these populations. We have employed such an approach to study the prevalence of asymmetric cell division (ACD) within activated immune cells by measuring the distribution of key fate determining molecules across the plane of cytokinesis in a high-throughput, objective, and internally controlled manner. Moreover the ability to perform high-resolution, temporal dissection of the cell division process lends itself perfectly to investigating the influence chemotherapeutic agents exert on the proliferative capacity of transformed cell lines. Here we describe the method in detail and its application to both ACD and general cell cycle analysis. PMID:27460238

  13. 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. PMID:26841315

  14. Quantitating MHC class II trafficking in primary dendritic cells using imaging flow cytometry

    PubMed Central

    Hennies, Cassandra M.; Lehn, Maria A.; Janssen, Edith M.

    2015-01-01

    Presentation of antigenic peptides in MHC class II (MHCII) on dendritic cells (DCs) is the first step in the activation of antigen-specific CD4+T cells. The expression of surface MHCII-peptide complexes is tightly regulated as the frequency of MHCII-peptide complexes can affect the magnitude, as well as the phenotype of the ensuing CD4+T cell response. The surface MHCII-peptide levels are determined by the balance between expression of newly generated complexes, complex internalization, and their subsequent re-emergence or degradation. However, the molecular mechanisms that underpin these processes are still poorly understood. Here we describe a multispectral imaging flow cytometry assay to visualize MHCII trafficking that can be used as a tool to dissect the molecular mechanisms that regulate MHCII homeostasis in primary mouse and human DCs. PMID:25967952

  15. 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

  16. Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry.

    PubMed

    Lau, Andy K S; Shum, Ho Cheung; Wong, Kenneth K Y; Tsia, Kevin K

    2016-05-10

    Optical imaging is arguably the most effective tool to visualize living cells with high spatiotemporal resolution and in a nearly noninvasive manner. Driven by this capability, state-of-the-art cellular assay techniques have increasingly been adopting optical imaging for classifying different cell types/stages, and thus dissecting the respective cellular functions. However, it is still a daunting task to image and characterize cell-to-cell variability within an enormous and heterogeneous population - an unmet need in single-cell analysis, which is now widely advocated in modern biology and clinical diagnostics. The challenge stems from the fact that current optical imaging technologies still lack the practical speed and sensitivity for measuring thousands to millions of cells down to the single-cell precision. Adopting the wisdom in high-speed fiber-optics communication, optical time-stretch imaging has emerged as a completely new optical imaging concept which is now proven for ultrahigh-throughput optofluidic single-cell imaging, at least 1-2 orders-of-magnitude higher (up to ∼100 000 cells per second) compared to the existing imaging flow cytometers. It also uniquely enables quantification of intrinsic biophysical markers of individual cells - a largely unexploited class of single-cell signatures that is known to be correlated with the overwhelmingly investigated biochemical markers. With the aim of reaching a wider spectrum of experts specializing in cellular assay developments and applications, this paper highlights the essential basics of optical time-stretch imaging, followed by reviewing the recent developments and applications of optofluidic time-stretch imaging. We will also discuss the current challenges of this technology, in terms of providing new insights in basic biology and enriching the clinical diagnostic toolsets. PMID:27099993

  17. Population-based study of DNA image cytometry as screening method for esophageal cancer

    PubMed Central

    Zhao, Lin; Wei, Wen-Qiang; Zhao, De-Li; Hao, Chang-Qing; Lin, Dong-Mei; Pan, Qin-Jing; Li, Xin-Qing; Lei, Fu-Hua; Wang, Jin-Wu; Wang, Guo-Qing; Shang, Qi; Qiao, You-Lin

    2012-01-01

    AIM: To explore the DNA image cytometry (DNA-ICM) technique as a primary screening method for esophageal squamous precancerous lesions. METHODS: This study was designed as a population-based screening study. A total of 582 local residents aged 40 years-69 years were recruited from Linzhou in Henan and Feicheng in Shandong. However, only 452 subjects had results of liquid-based cytology, DNA-ICM and pathology. The sensitivity and specificity of DNA-ICM were calculated and compared with liquid-based cytology in moderate dysplasia or worse. RESULTS: Sensitivities of DNA-ICM ranging from at least 1 to 4 aneuploid cells were 90.91%, 86.36%, 79.55% and 77.27%, respectively, which were better than that of liquid-based cytology (75%). Specificities of DNA-ICM were 70.83%, 84.07%, 92.65% and 96.81%, but the specificity of liquid-based cytology was 91.91%. The sensitivity and specificity of a combination of liquid-based cytology and DNA-ICM were 84.09% and 85.78%, respectively. CONCLUSION: It is possible to use DNA-ICM technique as a primary screening method for esophageal squamous precancerous lesions. PMID:22294844

  18. Automated enumeration and viability measurement of canine stromal vascular fraction cells using fluorescence-based image cytometry method.

    PubMed

    Chan, Leo Li-Ying; Cohen, Donald A; Kuksin, Dmitry; Paradis, Benjamin D; Qiu, Jean

    2014-07-01

    In recent years, the lipoaspirate collected from adipose tissue has been seen as a valuable source of adipose-derived mesenchymal stem cells for autologous cellular therapy. For multiple applications, adipose-derived mesenchymal stem cells are isolated from the stromal vascular fraction (SVF) of adipose tissue. Because the fresh stromal vascular fraction typically contains a heterogeneous mixture of cells, determining cell concentration and viability is a crucial step in preparing fraction samples for downstream processing. Due to a large amount of cellular debris contained in the SVF sample, as well as counting irregularities standard manual counting can lead to inconsistent results. Advancements in imaging and optics technologies have significantly improved the image-based cytometric analysis method. In this work, we validated the use of fluorescence-based image cytometry for SVF concentration and viability measurement, by comparing to standard flow cytometry and manual hemocytometer. The concentration and viability of freshly collected canine SVF samples are analyzed, and the results highly correlated between all three methods, which validated the image cytometry method for canine SVF analysis, and potentially for SVF from other species. PMID:24740550

  19. Measurement of Low-Abundance Intracellular mRNA Using Amplified FISH Staining and Image-Based Flow Cytometry.

    PubMed

    Henning, Andrea L; Sampson, Jill N Best; McFarlin, Brian Keith

    2016-01-01

    Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image-based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR-gamma in peripheral blood monocytes, which have been exposed in vitro to modified LDL, is described. The process of PPAR-gamma activation following uptake of modified LDL is believed to play a role in the development of atherogenesis. PPAR-gamma mRNA measurement was made possible using an amplified FISH technique (PrimeFlow RNA Assay) that allowed for detection of low-abundant intracellular mRNA expression. This protocol represents a continued effort by the authors' laboratory to establish and validate new techniques to assess the role of the immune system in chronic disease. © 2016 by John Wiley & Sons, Inc. PMID:27037579

  20. 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

  1. Hyperspectral cytometry.

    PubMed

    Grégori, Gérald; Rajwa, Bartek; Patsekin, Valery; Jones, James; Furuki, Motohiro; Yamamoto, Masanobu; Paul Robinson, J

    2014-01-01

    Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches. PMID:24271566

  2. High-throughput detection and quantification of mitochondrial fusion through imaging flow cytometry.

    PubMed

    Nascimento, Aldo; Lannigan, Joanne; Kashatus, David

    2016-08-01

    Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to

  3. QUANTITATIVE IMAGE CYTOMETRY OF HEPATOCYTES EXPRESSING GAMMA-GLUTAMYL TRANSPEPTIDASE AND GLUTATHIONE S-TRANSFERASE IN DIETHYLNITROSAMINE-INITIATED RATS TREATED WITH PHENOBARBITAL AND/OR PHTHALATE ESTERS

    EPA Science Inventory

    Image cytometry was used to quantify the volume of liver tissue expressing two widely accepted biochemical markers of neoplasia, gammaglutamyl transpeptidase (GGT) and the placental isozyme of glutathione s-transferase (GST-P). ats were treated with hepatocarcinogen, diethylnitro...

  4. 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.

  5. Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

    PubMed Central

    Konstantinidis, Diamantis G.; Pushkaran, Suvarnamala; Giger, Katie; Manganaris, Stefanos; Zheng, Yi; Kalfa, Theodosia A.

    2014-01-01

    Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis. We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells. PMID:24962543

  6. 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.

  7. 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.

  8. 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. PMID:24585634

  9. 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

  10. 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. PMID:23543297

  11. 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. © 2015 International Society for Advancement of Cytometry. PMID:26013098

  12. An Automated Cytopathology System in an Integrated Hospital Information System

    PubMed Central

    Shafarman, M.; Miller, T.; Simborg, D.W.

    1983-01-01

    At University of California, San Francisco, an automated cytopathology system has been developed to meet two main objectives: the information processing needs of the cytopathology department, and the integration of the cytopathology system into both the surgical pathology system, and the hospital information system. The cytopathology system has been in operation since March 1,1982. Benefits to the department include automatic SNOMED coding of diagnoses, online retrieval of diagnoses, automatic billing, faster turnaround between accession and signout, improved management, and reduced paperflow. Current interactions with the hospital information system include access to the centralized patient demographic file, access to medical data from other systems such as the clinical lab, medical records, radiology, and surgical pathology. Planned extensions include online signout of cases, and transmittal of cytology diagnoses to other clinical systems.

  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. 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

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

    PubMed

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

    2015-12-01

    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 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 bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal 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, noninvasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes. PMID:26424696

  16. Localization and relative quantification of carbon nanotubes in cells with multispectral imaging flow cytometry.

    PubMed

    Marangon, Iris; Boggetto, Nicole; Ménard-Moyon, Cécilia; Luciani, Nathalie; Wilhelm, Claire; Bianco, Alberto; Gazeau, Florence

    2013-01-01

    Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization. This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light. PMID:24378540

  17. 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.

  18. 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.

  19. Screening for Drugs Against the Plasmodium falciparum Digestive Vacuole by Imaging Flow Cytometry.

    PubMed

    Lee, Yan Quan; Hall, Brian E; Tan, Kevin S W

    2016-01-01

    Phenotypic assays are increasingly employed to provide clues about drug mechanisms. In antimalarial drug screening, however, the majority of assays are designed to only measure parasite-killing activity. We describe here a high-content assay to detect drug-mediated perturbation of the digestive vacuole integrity in the trophozoite stage of Plasmodium falciparum, using the ImageStream imaging flow cytometer. PMID:27460247

  20. 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.

  1. 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. PMID:26132783

  2. Review of HIV-Related Cytopathology

    PubMed Central

    Lang, Tee U.; Khalbuss, Walid E.; Monaco, Sara E.; Michelow, Pam; Pantanowitz, Liron

    2011-01-01

    Exfoliative and aspiration cytologies play a major role in the management of patients with human immunodeficiency virus infection. Common cytology samples include cervicovaginal and anal Papanicolaou tests, fine needle aspirations, respiratory specimens, body fluids, Tzanck preparations, and touch preparations from brain specimens. While the cytopathologists need to be aware of specific infections and neoplasms likely to be encountered in this setting, they should be aware of the current shift in the pattern of human immunodeficiency virus-related diseases, as human immunodeficiency virus patients are living longer with highly active antiretroviral therapy and suffering fewer opportunistic infections with better antimicrobial prophylaxis. There is a rise in nonhuman immunodeficiency virus-defining cancers (e.g., anal cancer, Hodgkin's lymphoma) and entities (e.g., gynecomastia) from drug-related side effects. Given that fine needle aspiration is a valuable, noninvasive, and cost-effective tool, it is frequently employed in the evaluation and diagnosis of human immunodeficiency virus-related diseases. Anal Papanicolaou tests are also increasing as a result of enhanced screening of human immunodeficiency virus-positive patients for cancer. This paper covers the broad spectrum of disease entities likely to be encountered with human immunodeficiency virus-related cytopathology. PMID:21559199

  3. Hyperchromatic laser scanning cytometry

    NASA Astrophysics Data System (ADS)

    Tárnok, Attila; Mittag, Anja

    2007-02-01

    In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

  4. MEMS-based flow cytometry: microfluidics-based cell identification system by fluorescent imaging.

    PubMed

    Wu, W K; Liang, C K; Huang, J Z

    2004-01-01

    This study utilizes MEMS technology to realize a novel low-cost microfluidics-based biochip system for flow-type cell handling. Powered by vacuum pump, the microfluidic driving system enables cells to move in order one by one in the biochip by an effect of sheath flow prefocus. Then, cells are guided to a fluorescent inspection region where two detection tasks such as cell image identification and cell counting are conducted. Currently, the glass-based biochip has been manufactured and all the related devices have been well set up in our laboratory. With this proposed prototype system, typical results about cell separation of yeast cell and PC-3 cell are available and their separated images are also presented, respectively. PMID:17270801

  5. Flow cytometry

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.

    1984-09-01

    Flow cytometry instrumentation developed from early efforts to count cells and particles in liquid suspension as they passed through a sensing device. Since the mid-1960's sophisticated instruments have been designed for analyzing cells based on various cytological, biochemical, and functional properties. These instruments have revolutionized automated cell analysis methods in that measurements are made at high speed, multiparameter data is correlated on each cell, statistical precision is high, and cells are separated in high purity from heterogeneous mixtures for identification and functional analysis. Advanced instruments capable of measuring cell volume, surface area, multicolor fluorescence, fluorescence polarization, light scatter within various angular regions, and axial light loss (extinction) at different wavelengths are being used in biomedical research for analyzing and sorting normal and abnormal cell populations. This article reviews the development of flow cytometers, the conceptual basis of flow measurements, and discusses some of the numerous applications of the technology in biology and medicine.

  6. 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

    2016-09-01

    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. PMID:27444383

  7. Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

    PubMed Central

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

    2014-01-01

    Granulocytes play a key role in the body’s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm). PMID:25591001

  8. In situ label-free static cytometry by monitoring spatiotemporal fluctuations of image gray values

    NASA Astrophysics Data System (ADS)

    Wohl, Ishay; Zurgil, Naomi; Hakuk, Yaron; Sobolev, Maria; Galmidi, Moti; Deutsch, Mordechai

    2015-10-01

    Spatiotemporal fluctuation of homogeneity and randomness of gray values within an image was explored and utilized as a label-free means for cell examination. This was done by utilizing a user-friendly combination of simple bright field microscope and Cytocapture dish, wherein cells are individually held, each within a picoliter optical chamber, forming an array of cells to be repeatedly measured over time and biomanipulated in situ at single-cell resolution. First, the measured gray level information entropy (GLIE) was used and, based on the fact that living cells are not in a state of thermodynamic equilibrium but rather in a metastable state, two fluctuation-sensitive measures were proposed and examined: ASDE-the spatial average of temporal standard deviation (SD) of GLIE, and AA-the average time autocorrelation of GLIE. System performance was validated on cell-free solutions. This was followed by examining the performance of the measures AGLIE, ASDE, and AA to distinguish among individual live-still, dead and live cells from various cell lines, as well as between cells which were and were not induced to differentiate. Results, which were obtained on four types of cells, indicate advantages of the proposed measures which are believed to be significant additions to the microscope-based probe-free toolbox.

  9. 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. PMID:25858228

  10. Cytopathological Analysis of Cyst Fluid Enhances Diagnostic Accuracy of Mucinous Pancreatic Cystic Neoplasms

    PubMed Central

    Utomo, Wesley K.; Braat, Henri; Bruno, Marco J.; van Eijck, Casper H.J.; Koerkamp, Bas Groot; Krak, Nanda C.; van de Vreede, Adriaan; Fuhler, Gwenny M.; Peppelenbosch, Maikel P.; Biermann, Katharina

    2015-01-01

    Abstract Widespread use of cross-sectional imaging and increasing age of the general population has increased the number of detected pancreatic cystic lesions. However, several pathological entities with a variety in malignant potential have to be discriminated to allow clinical decision making. Discrimination between mucinous pancreatic cystic neoplasms (PCNs) and nonmucinous pancreatic lesions is the primary step in the clinical work-up, as malignant transformation is mostly associated with mucinous PCN. We performed a retrospective analysis of all resected PCN in our tertiary center from 2000 to 2014, to evaluate preoperative diagnostic performance and the results of implementation of the consensus guidelines over time. This was followed by a prospective cohort study of patients with an undefined pancreatic cyst, where the added value of cytopathological mucin evaluation to carcinoembryonic antigen (CEA) in cyst fluid for the discrimination of mucinous PCN and nonmucinous cysts was investigated. Retrospective analysis showed 115 patients operated for a PCN, with a correct preoperative classification in 96.2% of the patients. High-grade dysplasia or invasive carcinoma was observed in only 32.3% of mucinous PCN. In our prospective cohort (n = 71), 57.7% of patients were classified as having a mucinous PCN. CEA ≥192 ng/mL had an accuracy of 63.4%, and cytopathological mucin evaluation an accuracy of 73.0%. Combining these 2 tests further improved diagnostic accuracy of a mucinous PCN to 76.8%. CEA level and mucin evaluation were not predictive of the degree of dysplasia. These findings show that adding cytopathology to cyst fluid biochemistry improves discrimination between mucinous PCN and nonmucinous cysts.

  11. 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.

  12. Quantifying nuclear p65 as a parameter for NF-κB activation: Correlation between ImageStream cytometry, microscopy, and Western blot.

    PubMed

    Maguire, Orla; Collins, Christine; O'Loughlin, Kieran; Miecznikowski, Jeffrey; Minderman, Hans

    2011-06-01

    The nuclear factor kappa B (NF-κB) pathway, which regulates many cellular processes including proliferation, apoptosis, and survival, has emerged as an important therapeutic target in cancer. Activation of the NF-κB transcription factor is associated with nuclear translocation of the p65 component of the complex. Conventional methods employed to determine nuclear translocation of NF-κB either lack statistical robustness (microscopy) or the ability to discern heterogeneity within the sampled populations (Western blotting and Gel Shift assays). The ImageStream platform combines the high image content information of microscopy with the high throughput and multiparameter analysis of flow cytometry which overcomes the aforementioned limitations of conventional assays. It is demonstrated that ImageStream assessment of receptor-mediated (TNFα) and drug (Daunorubicin, DNR)-induced NF-κB translocation in leukemic cell lines correlates well with microscopy analysis and Western blot analysis. It is further demonstrated that ImageStream cytometry enables quantitative assessment of p65 translocation in immunophenotypically defined subpopulations; and that this assessment is highly reproducible. It is also demonstrated that, quantitatively, the DNR-induced nuclear translocation of NF-κB correlates well with a biological response (apoptosis). We conclude that the ImageStream has the potential to be a powerful tool to evaluate NF-κB /p65 activity as a determinant of response to therapies designed to target aberrant NF-κB signaling activities. PMID:21520400

  13. Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

    PubMed

    Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

    2015-05-01

    The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods. PMID:24643390

  14. Archival Fine-Needle Aspiration Cytopathology (FNAC) Samples

    PubMed Central

    Killian, J. Keith; Walker, Robert L.; Suuriniemi, Miia; Jones, Laura; Scurci, Stephanie; Singh, Parvati; Cornelison, Robert; Harmon, Shannon; Boisvert, Nichole; Zhu, Jack; Wang, Yonghong; Bilke, Sven; Davis, Sean; Giaccone, Giuseppe; Smith, William I.; Meltzer, Paul S.

    2010-01-01

    Microarray technologies provide high-resolution maps of chromosome imbalances and epigenomic aberrations in the cancer cell genome. Such assays are often sensitive to sample DNA integrity, voiding the utility of many archival pathology specimens and necessitating the special handling of prospective clinical specimens. We have identified the remarkable preservation of higher-molecular weight DNA in archival fine-needle aspiration cytopathology specimens from patients greater than 10 years of age. We further demonstrate the outstanding technical performance of 57 fine-needle aspiration cytopathology samples for aberration detection on high-resolution comparative genomic hybridization array, DNA methylation, and single nucleotide polymorphism genotyping platforms. Forty-four of 46 malignant aspirates in this study manifested unequivocal genomic aberrations. Importantly, matched Papanicolaou and Diff-Quik fine-needle aspiration cytopathology samples showed critical differences in DNA preservation and DNA integrity. Overall, this study identifies a largely untapped reserve of human pathology specimens for molecular profiling studies, with ramifications for the prospective collection of clinical biospecimens. PMID:20959611

  15. Diagnostic value of AgNOR method in thyroid cytopathology: correlation with morphometric measurements.

    PubMed

    Solymosi, T; Tóth, V; Sápi, Z; Bodó, M; Gál, I; Csanádi, L

    1996-03-01

    A silver staining technique was applied to 51 thyroid smears. The numbers of silver-stained nucleolar organizer regions (AgNORs) were counted, and the mean AgNOR and nuclear area per cell were determined with an image analyzer. The mean AgNOR count per cell was significantly higher in malignant than in benign lesions, but there was a considerable overlap. The mean AgNOR area and the mean SD of the AgNOR area per cell were significantly higher in carcinomas than in benign lesions (P = 10-9 and P = 5 x 10-10, respectively) and there were only two and one benign cases, respectively, of overlap. A strong correlation was observed between the mean AgNOR area and the mean nuclear area (r = 0.88), the former being a better discriminator between benign and malignant lesions. The AgNOR technique may contribute to routine thyroid cytopathology. PMID:8964170

  16. Outcomes of cytopathology studies presented at national pathology meetings.

    PubMed

    Ciesla, M C; Wojcik, E M

    2001-10-01

    The purpose of this study was to determine what factors influence the final publication status of cytopathology studies presented at national meetings. Abstracts involving cytopathology material were obtained from the following journals: Modern Pathology (volume 11, 1998), Acta Cytologica (volume 42, 1998), and the American Journal of Clinical Pathology (volumes 109 and 110, 1998). Using the National Library of Medicine Website, each abstract was searched by author and topic to determine if the study was published as a peer-reviewed article. The following parameters were evaluated: meeting where the abstract was presented, type of institution where the research was based, type of material used in the study, and application of ancillary techniques used in the study. The subsequent published articles were evaluated for journal and time to publication. Out of 257 studies presented in 1998, 85 (33%) were published in peer-reviewed journals by May 2000. The majority of papers were published in Diagnostic Cytopathology (n = 21), Acta Cytologica (n = 15), and Cancer (n = 18). The mean time for publication was 12.8 mo. The highest percentage of published studies was presented at the United States and Canadian Academy of Pathology (USCAP) meeting (50% of presented abstracts), followed by American Society of Cytopathology (ASC) (28%) and American Society of Clinical Pathologists (ASCP) (17%) meetings. Ancillary techniques were applied in 40 of 85 (47%) published studies, 27 of 85 (32%) articles focused on morphology, and 18 of 85 (21%) papers covered other topics (e.g., quality assurance (QA), cost, and role of cytology). In nonpublished studies (n = 172), special techniques were the main focus in 40%, morphology in 25%, and other topics in 35% of abstracts. The great majority (97%) of published studies were from academic institutions. Gynecological and nongynecological material were roughly equally covered in published and nonpublished studies. Only a relatively small

  17. 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

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

    PubMed

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

    2016-02-16

    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

  19. 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. PMID:8778332

  20. 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. PMID:26957591

  1. 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.

  2. 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

  3. In vivo micro-vascular imaging and flow cytometry in zebrafish using two-photon excited endogenous fluorescence

    PubMed Central

    Zeng, Yan; Yan, Bo; Sun, Qiqi; He, Sicong; Jiang, Jun; Wen, Zilong; Qu, Jianan Y.

    2014-01-01

    Zebrafish has rapidly evolved as a powerful vertebrate model organism for studying human diseases. Here we first demonstrate a new label-free approach for in vivo imaging of microvasculature, based on the recent discovery and detailed characterization of the two-photon excited endogenous fluorescence in the blood plasma of zebrafish. In particular, three-dimensional reconstruction of the microvascular networks was achieved with the depth-resolved two-photon excitation fluorescence (TPEF) imaging. Secondly, the blood flow images, obtained by perpendicularly scanning the focal point across the blood vessel, provided accurate information for characterizing the hemodynamics of the circulatory system. The endogenous fluorescent signals of reduced nicotinamide adenine dinucleotide (NADH) enabled visualization of the circulating granulocytes (neutrophils) in the blood vessel. The development of acute sterile inflammation could be detected by the quantitative counting of circulating neutrophils. Finally, we found that by utilizing a short wavelength excitation at 650 nm, the commonly used fluorescent proteins, such as GFP and DsRed, could be efficiently excited together with the endogenous fluorophores to achieve four-color TPEF imaging of the vascular structures and blood cells. The results demonstrated that the multi-color imaging could potentially yield multiple view angles of important processes in living biological systems. PMID:24688803

  4. 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

  5. Diagnostic Thyroidectomy May Be Preferable in Patients With Suspicious Ultrasonography Features After Cytopathology Diagnosis of AUS/FLUS in the Bethesda System.

    PubMed

    Lee, Yong Sang; Kim, Hyeung Kyoo; Chang, Hojin; Kim, Seok Mo; Kim, Bup-Woo; Chang, Hang-Seok; Park, Cheong Soo

    2015-12-01

    Atypia/follicular lesion of undetermined significance (AUS/FLUS) is a new category in the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) for which repeat fine-needle aspiration cytology (FNAC) is recommended. The aim of this study was to identify specific ultrasonography and clinical predictors of malignancy in a subset of thyroid nodules associated with cytology diagnoses of AUS/FLUS.Between January 2011 and December 2102, 5440 patients underwent thyroid surgery at our institution. Of these, 213 patients were diagnosed AUS/FLUS at the preoperative cytopathology diagnosis. The frequency of FNAC and ultrasonography images was compared between patients with cancerous and benign tumors based on their final pathology.Of the 213 patients, 158 (74.2%) were diagnosed with thyroid carcinoma in their final pathology reports. In univariate and multivariate analyses, the frequency of FNAC was not significantly correlated with the cancer diagnosis. Hypoechogenicity (odds ratio 2.521, P = 0.007) and microcalcification (odds ratio 3.247, P = 0.005) were statistically correlated with cancer risk.Although AUS/FLUS in cytopathology is recommended for repeating FNAC in BSRTC, we proposed that thyroid nodules with ultrasonography findings that suggest the possibility of cancer should undergo thyroidectomy with diagnostic intent. PMID:26705204

  6. 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

    2011-01-01

    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. PMID:21060932

  7. High throughput measurement of Ca²⁺ dynamics for drug risk assessment in human stem cell-derived cardiomyocytes by kinetic image cytometry.

    PubMed

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

    2012-01-01

    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

  8. 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

  9. Cytopathological Features of a Severe Type of Corneal Intraepithelial Neoplasia

    PubMed Central

    Fukuoka, Hideki; Kawasaki, Satoshi; Yokoi, Norihiko; Yamasaki, Kenta; Kinoshita, Shigeru

    2016-01-01

    Purpose To report the cytopathological features of corneal intraepithelial neoplasia (CIN) through the investigation of cytokeratin expression pattern, keratinization, cell proliferation, apoptosis, and epithelial mesenchymal transition. Patient and Methods Corneal tissue excised from a CIN patient was examined in this study. Cryosections of the excised CIN epithelial tissue were examined by immunostaining analysis using antibodies against cytokeratins, keratinization-related proteins, Ki-67, human telomerase reverse transcriptase (hTERT), and epithelial mesenchymal transition (EMT)-related proteins. Subcellular localization of F-actin was also analyzed using phalloidin. For the detection of apoptotic cells, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed. Real-time polymerase chain reaction was performed to quantify the expression level of hTERT in the CIN epithelium. Results The CIN epithelium exhibited a significantly altered cytokeratin expression pattern compared to normal corneas with an upregulated expression of keratinization-related proteins. The CIN epithelium also demonstrated an increased number of Ki-67-positive cells with an upregulated expression of hTERT, while exhibiting an increased number of apoptotic cells. EMT did not occur in the CIN epithelium. Conclusion CIN epithelium seems to be slightly dedifferentiated from the corneal epithelial lineage. The status of cell proliferation and apoptosis in the CIN epithelium was significantly altered from that of normal corneal epithelium, but its malignancy level does not appear to be as high as that of metastasis-competent malignant cancers. PMID:27462252

  10. 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. PMID:19942954

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. Flow cytometry-based apoptosis detection

    PubMed Central

    Wlodkowic, Donald; Skommer, Joanna; Darzynkiewicz, Zbigniew

    2013-01-01

    An apoptosing cell demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the stimuli and cell type. The gross majority of classical apoptotic hallmarks can be rapidly examined by flow and image cytometry. Cytometry thus became a technology of choice in diverse studies of cellular demise. A large variety of cytometric methods designed to identify apoptotic cells and probe mechanisms associated with this mode of cell demise have been developed during the past two decades. In the present chapter we outline a handful of commonly used methods that are based on the assessment of: mitochondrial transmembrane potential, activation of caspases, plasma membrane alterations and DNA fragmentation. PMID:19609746

  17. 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.

  18. LETTUCE INFECTIOUS YELLOWS VIRUS-ENCODED P26 INDUCES PLASMALEMMA DEPOSIT CYTOPATHOLOGY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 re...

  19. The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages.

    PubMed

    Libby, S J; Lesnick, M; Hasegawa, P; Weidenhammer, E; Guiney, D G

    2000-02-01

    The pathogenesis of serious systemic Salmonella infections is characterized by survival and proliferation of bacteria inside macrophages. Infection of human monocyte-derived macrophages in vitro with S. typhimurium or S. dublin produces cytopathology characterized by detachment of cells that contain large numbers of proliferating bacteria. This cytopathology is dependent on the expression of the bacterial spv genes, a virulence locus previously shown to markedly enhance the ability of Salmonella to produce systemic disease. After 24 h of infection, macrophage cultures contain two populations of bacteria: (i) proliferating organisms present in a detached cell fraction; and (ii) a static bacterial population in macrophages remaining attached to the culture well. Mutations in either the essential transcriptional activator SpvR or the key SpvB protein markedly reduce the cytopathic effect of Salmonella infection. The spv-dependent cytopathology in macrophages exhibits characteristics of apoptosis, with release of nucleosomes into the cytoplasm, nuclear condensation and DNA fragmentation. The current findings suggest that the mechanism of the spv effect is through induction of increased cytopathology in host macrophages. PMID:11207562

  20. 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.

  1. Flow cytometry: an introduction.

    PubMed

    Givan, Alice L

    2011-01-01

    A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chapter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts into the field. Other chapters in this book will provide more details, more references, and even an intriguing view of the future of cytometry. PMID:21116976

  2. 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.

  3. 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. PMID:27114371

  4. Triage of patients with AUS/FLUS on thyroid cytopathology: effectiveness of the multimodal diagnostic techniques.

    PubMed

    Kim, Tae Hyuk; Jeong, Dae Joon; Hahn, Soo Yeon; Shin, Jung Hee; Oh, Young Lyun; Ki, Chang-Seok; Kim, Jong-Won; Jang, Ju Young; Cho, Yoon Young; Chung, Jae Hoon; Kim, Sun Wook

    2016-05-01

    The management of patients with thyroid cytopathologic diagnosis of atypia (or follicular lesion) of undetermined significance (AUS/FLUS) is a complex clinical problem. The purpose of this study was to develop a practical triage scheme based on multiple diagnostic tests in general use. We performed a retrospective cohort study involving 15,335 consecutive patients with a referral diagnosis of thyroid nodule between April 2011 and March 2015 using an institutional database. We obtained 904 patients with an initial cytopathologic diagnosis of AUS/FLUS who underwent repeat fine-needle aspiration or core needle biopsy, 388 of whom had a corresponding histopathological diagnosis for excised index lesions. The diagnostic performance of ultrasound (US) findings, repeat biopsy, and BRAF(V) (600E) mutation in cytopathologic specimens were evaluated individually or as a set. Of the 388 resected AUS/FLUS cases, 338 (87.1%) were thyroid cancer. The positive likelihood ratios (LRs) for BRAF(V) (600E) mutation and repeat biopsy result of suspicious for malignant cell (SMC) or worse were 11.6 (95% CI = 1.7-77.8) and 13.7 (95% CI = 4.6-41.0), respectively. The absence of suspicious findings on US combined with cytologic result of less than SMC or negative BRAF(V) (600E) mutation produced negative LRs ranging from 0.06 to 0.15, corresponding to negative predictive values of over 90% in both primary and referral settings. For patients with AUS/FLUS cytopathology, clinical decision making can be guided by a simple triage scheme based on US findings, repeat biopsy, or BRAF(V) (600E) mutation. PMID:26775803

  5. Archival fine-needle aspiration cytopathology (FNAC) samples: untapped resource for clinical molecular profiling.

    PubMed

    Killian, J Keith; Walker, Robert L; Suuriniemi, Miia; Jones, Laura; Scurci, Stephanie; Singh, Parvati; Cornelison, Robert; Harmon, Shannon; Boisvert, Nichole; Zhu, Jack; Wang, Yonghong; Bilke, Sven; Davis, Sean; Giaccone, Giuseppe; Smith, William I; Meltzer, Paul S

    2010-11-01

    Microarray technologies provide high-resolution maps of chromosome imbalances and epigenomic aberrations in the cancer cell genome. Such assays are often sensitive to sample DNA integrity, voiding the utility of many archival pathology specimens and necessitating the special handling of prospective clinical specimens. We have identified the remarkable preservation of higher-molecular weight DNA in archival fine-needle aspiration cytopathology specimens from patients greater than 10 years of age. We further demonstrate the outstanding technical performance of 57 fine-needle aspiration cytopathology samples for aberration detection on high-resolution comparative genomic hybridization array, DNA methylation, and single nucleotide polymorphism genotyping platforms. Forty-four of 46 malignant aspirates in this study manifested unequivocal genomic aberrations. Importantly, matched Papanicolaou and Diff-Quik fine-needle aspiration cytopathology samples showed critical differences in DNA preservation and DNA integrity. Overall, this study identifies a largely untapped reserve of human pathology specimens for molecular profiling studies, with ramifications for the prospective collection of clinical biospecimens. PMID:20959611

  6. Chemical Cytometry: Fluorescence-Based Single-Cell Analysis

    NASA Astrophysics Data System (ADS)

    Cohen, Daniella; Dickerson, Jane A.; Whitmore, Colin D.; Turner, Emily H.; Palcic, Monica M.; Hindsgaul, Ole; Dovichi, Norman J.

    2008-07-01

    Cytometry deals with the analysis of the composition of single cells. Flow and image cytometry employ antibody-based stains to characterize a handful of components in single cells. Chemical cytometry, in contrast, employs a suite of powerful analytical tools to characterize a large number of components. Tools have been developed to characterize nucleic acids, proteins, and metabolites in single cells. Whereas nucleic acid analysis employs powerful polymerase chain reaction-based amplification techniques, protein and metabolite analysis tends to employ capillary electrophoresis separation and ultrasensitive laser-induced fluorescence detection. It is now possible to detect yoctomole amounts of many analytes in single cells.

  7. 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

  8. 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.

  9. Sputum cytopathology: use and potential in monitoring the workplace environment by screening for biological effects of exposure

    SciTech Connect

    Frost, J.K.; Ball, W.C. Jr.; Levin, M.L.; Tockman, M.S.; Erozan, Y.S.; Gupta, P.K.; Eggleston, J.C.; Pressman, N.J.; Donithan, M.P.; Kimball, A.W. Jr.

    1986-08-01

    Sputum cytopathologic monitoring detects squamous cell lung cancers at an extremely early stage (x-ray negative). It holds further potential for preventing disease by detecting epithelial alterations which reflect environmental hazards. The addition of sputum cytology screening to screening by chest x-ray film does not significantly reduce mortality from all types of lung cancer, but preliminary analysis of Johns Hopkins Lung Project data suggests that mortality from squamous cell carcinoma is reduced. Quantitative automated cytopathology systems and biochemical/immunological cell markers enhance understanding of these precursors and offer great promise for increasing capacity, accuracy, and usefulness in cytopathology screening of workers. Cytological specimens collected over years of screening workers considered at risk may be important to eventually understanding development and prevention of major occupational diseases.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Management of Thyroid Nodular Disease: Current Cytopathology Classifications and Genetic Testing.

    PubMed

    Kuo, Lindsay E; Kelz, Rachel R

    2016-01-01

    Preoperative diagnosis and operative planning for patients with thyroid nodules has improved over the last decade. The Bethesda criteria for cytopathologic classification of thyroid nodule aspirate has enhanced communication between pathologists and clinicians. Multiple genetic tests, including molecular markers and the Afirma gene expression classifier, have been developed and validated. The tests, along with clinical and radiologic information, are most useful in the setting of indeterminate cytology. The development of an updated diagnostic and treatment algorithm incorporating all available tests will help standardize the management of patients with nodular thyroid disease and reduce variation and inefficiencies in care. PMID:26610771

  12. 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. PMID:26421636

  13. The Papanicolaou Society of Cytopathology classification for pulmonary specimens: an overview.

    PubMed

    Layfield, L J

    2016-06-01

    The Papanicolaou Society of Cytopathology has formulated guidelines for respiratory cytology which include a classification scheme. The recommended classification scheme is based on the expertise of the committee members, extensive review of the literature and feedback from presentations at national and international meetings. Each category of the classification system is closely defined and link to a known risk for malignancy. The classification contains six categories designated as: 1) Non-diagnostic; 2) Negative for Malignancy; 3) Atypical; 4) Neoplastic (Benign and low grade cancer); 5) Suspicious for Malignancy; and 6) Malignant. PMID:27221749

  14. 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. PMID:27581017

  15. Continuous automated imaging-in-flow cytometry for detection and early warning of Karenia brevis blooms in the Gulf of Mexico.

    PubMed

    Campbell, Lisa; Henrichs, Darren W; Olson, Robert J; Sosik, Heidi M

    2013-10-01

    Monitoring programs for harmful algal blooms (HABs) typically rely on time-consuming manual methods for identification and enumeration of phytoplankton, which make it difficult to obtain results with sufficient temporal resolution for early warning. Continuous automated imaging-in-flow by the Imaging FlowCytobot (IFCB) deployed at Port Aransas, TX has provided early warnings of six HAB events. Here we describe the progress in automating this early warning system for blooms of Karenia brevis. In 2009, manual inspection of IFCB images in mid-August 2009 provided early warning for a Karenia bloom that developed in mid-September. Images from 2009 were used to develop an automated classifier that was employed in 2011. Successful implementation of automated file downloading, processing and image classification allowed results to be available within 4 h after collection and to be sent to state agency representatives by email for early warning of HABs. No human illness (neurotoxic shellfish poisoning) has resulted from these events. In contrast to the common assumption that Karenia blooms are near monospecific, post-bloom analysis of the time series revealed that Karenia cells comprised at most 60-75 % of the total microplankton. PMID:23307076

  16. 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

  17. Flow cytometry and cell sorting.

    PubMed

    Ibrahim, Sherrif F; van den Engh, Ger

    2007-01-01

    Flow cytometry and cell sorting are well-established technologies in clinical diagnostics and biomedical research. Heterogeneous mixtures of cells are placed in suspension and passed single file across one or more laser interrogation points. Light signals emitted from the particles are collected and correlated to entities such as cell morphology, surface and intracellular protein expression, gene expression, and cellular physiology. Based on user-defined parameters, individual cells can then be diverted from the fluid stream and collected into viable, homogeneous fractions at exceptionally high speeds and a purity that approaches 100%. As such, the cell sorter becomes the launching point for numerous downstream studies. Flow cytometry is a cornerstone in clinical diagnostics, and cheaper, more versatile machines are finding their way into widespread and varied uses. In addition, advances in computing and optics have led to a new generation of flow cytometers capable of processing cells at orders of magnitudes faster than their predecessors, and with staggering degrees of complexity, making the cytometer a powerful discovery tool in biotechnology. This chapter will begin with a discussion of basic principles of flow cytometry and cell sorting, including a technical description of factors that contribute to the performance of these instruments. The remaining sections will then be divided into clinical- and research-based applications of flow cytometry and cell sorting, highlighting salient studies that illustrate the versatility of this indispensable technology. PMID:17728993

  18. 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. PMID:26243567

  19. 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

  20. 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

    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 also have

  1. 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

  2. Two-photon flow cytometry

    NASA Astrophysics Data System (ADS)

    Zhong, Cheng F.; Ye, Jing Yong; Myc, Andrzej; Thomas, Thommey P.; Bielinska, Anna; Baker, James R., Jr.; Norris, Theodore B.

    2005-03-01

    Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantization 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 (T3FC) 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.

  3. Brevipalpus-transmitted plant virus and virus-like diseases: cytopathology and some recent cases.

    PubMed

    Kitajima, E W; Chagas, C M; Rodrigues, J C V

    2003-01-01

    An increasing number of diseases transmitted by Brevipalpus mite species (Acari: Tenuipalpidae) is being identified that affect economically important plants such as citrus, coffee, passion fruit, orchids, and several ornamentals. All of these diseases are characterized by localized lesions (chlorotic, green spots, or ringspots) on leaves, stems, and fruits. Virus or virus-like agents are considered to be the causal agents, possibly transmitted in a circulative-propagative manner by Brevipalpus mites. The virus or virus-like particles are short, rod-like, or bacilliform, that induce two characteristic types of cell alteration: (1) 'Nuclear type'--nuclei of parenchyma and epidermal cells in the lesions often contain a large electron lucent inclusion. Short, naked, rod-like (40-50 nm x 100-110 nm) particles may be seen in the viroplasm or nucleoplasm and in the cytoplasm. These particles are commonly arranged perpendicularly on the membranes of the nuclear envelope or endoplasmic reticulum (ER). In a very few instances, they were found to be membrane-bound, within the ER cavities. (2) 'Cytoplasmic type'--short bacilliform particles (60-70 nm x 110-120 nm) are present within the cisternae of the ER and often have electron dense viroplasm of varied shapes present in the cytoplasm. Bacilliform particles may be seen budding into the ER lumen near the viroplasm. These particles resemble those of members of the Rhabdoviridae, but are shorter. The only sequenced virus of this group, orchid fleck virus (OFV), has a negative sense (bipartite) type ssRNA genome, but its organization is similar to known rhabdoviruses, which are monopartite. Both types of cytopathological effects have been found associated with citrus leprosis. In orchids, OFV has a 'nuclear type' of cytopathology, but in some species the 'cytoplasmic type' has been found associated with ringspot symptoms. In Hibiscus and Clerodendron, green spot symptoms have been associated with the cytoplasmic type of cell

  4. 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

  5. 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. PMID:27410612

  6. 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

    2015-07-01

    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. PMID:26058598

  7. 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.

  8. 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

  9. The Bethesda System for Reporting Thyroid Cytopathology is applicable to frozen section diagnosis in children.

    PubMed

    Arnold, Michael A; Nicol, Kathleen K

    2015-01-01

    The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) offers standardized and widely understood diagnostic categories for reporting thyroid cytology diagnoses. We compared the utility of TBSRTC categories in pediatric cytology diagnoses and pediatric intraoperative frozen section diagnoses. We examined the experience of our primary and referral care center over a 20-year period. This included 182 thyroidectomy patients who underwent 64 preoperative fine-needle aspirations and 91 intraoperative frozen section evaluations, including 38 patients evaluated sequentially by each method. All diagnoses were retrospectively reclassified into TBSRTC categories and correlated with the final thyroidectomy diagnoses. For each sampling method, malignant final diagnoses were observed at similar frequencies to rates predicted by TBSRTC. Malignant final diagnoses following fine-needle aspiration or frozen section diagnoses in TBSRTC categories other than malignant or suspicious for malignancy most often resulted from difficulty in detecting papillary carcinoma, including difficulty detecting the nuclear characteristics of papillary carcinoma in frozen sections. The limitations of needle biopsy and frozen section evaluations differ, yet serial utilization of these procedures was rarely informative. Based on the experience of our institution, classification of cytology and frozen section diagnosis by TBSRTC predicts a risk of malignancy similar to the guidance offered by TBSRTC. We recommend including a TBSRTC category when reporting either thyroid cytology or frozen section diagnoses in children. PMID:25625563

  10. Update in salivary gland cytopathology: Recent molecular advances and diagnostic applications.

    PubMed

    Pusztaszeri, Marc P; Faquin, William C

    2015-07-01

    Salivary gland tumors (SGT) are notorious for their extraordinary diversity and for the morphological overlap that exists between many of these entities. Fine-needle aspiration biopsy (FNAB) has a well-established role in the evaluation of patients with a salivary gland lesion, helping to guide clinical management. However, salivary gland FNAB has several limitations and does not allow for a specific diagnosis in some cases. For these reasons, salivary gland FNAB is considered one of the most challenging areas in cytopathology. Over the last decade, new salivary gland entities have been recognized, enlarging SGT diversity and complexity even more. In addition, a subset of SGT, including common entities such as pleomorphic adenoma and uncommon new entities such as mammary analog secretory carcinoma, have been characterized cytogenetically by the presence of specific translocations. The molecular consequences of these translocations and their potential prognostic and therapeutic values are not yet well characterized. However, these translocations and their resulting fusion oncogenes and oncoproteins can be used as diagnostic clues in salivary gland FNAB material in order to overcome the limitations of cytomorphological evaluation alone. In this review, we focus on SGTs currently known to harbor translocations and fusion genes, including uncommon and recently recognized entities, and discuss their potential application to salivary gland FNAB. PMID:25613003

  11. Cytopathologic characteristics of SMARCB1 (INI-1) deficient sinonasal carcinoma: A potential diagnostic pitfall.

    PubMed

    Allison, Derek B; Bishop, Justin A; Ali, Syed Z

    2016-08-01

    Tumors of the head and neck are extremely diverse and a subset are poorly differentiated and difficult to classify. Recently, a new entity has been described with rhabdoid and/or plasmacytoid cytologic features and a characteristic genetic signature-inactivation of the SMARCB1 (INI-1) tumor suppressor gene. To date, only 16 cases of SMARCB1 (INI-1) deficient sinonasal carcinoma have been described, and there are currently no reports of the cytopathologic features by fine needle aspiration (FNA) cytology. A case of a 77-year-old man who presented with a posterior ethmoid sinus lesion with invasion into the skull base and bone was reported. FNA cytology of a right retropharyngeal lymph node revealed relatively monomorphic, loosely cohesive clusters of plasmacytoid cells with occasional nucleoli, rare intranuclear cytoplasmic inclusions, and mitotic figures in a background of necrosis and absence of overt squamous or glandular differentiation. A diagnosis of metastatic myoepithelial carcinoma was made; however, retrospectively, the surgical excision showed loss of the SMARCB1 (INI-1) tumor suppressor gene by immunohistochemistry. In summary, the cytomorphologic features of SMARCB1 (INI-1) deficient sinonasal carcinoma are relatively nonspecific and overlap with other regional tumors, including myoepithelial neoplasms. As a result, this entity should be considered in the differential diagnosis for a plasmacytoid tumor arising in the sinonasal tract by FNA cytology. Diagn. Cytopathol. 2016;44:700-703. © 2016 Wiley Periodicals, Inc. PMID:27177850

  12. 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. PMID:19619150

  13. Mass Cytometry: Single Cells, Many Features.

    PubMed

    Spitzer, Matthew H; Nolan, Garry P

    2016-05-01

    Technology development in biological research often aims to either increase the number of cellular features that can be surveyed simultaneously or enhance the resolution at which such observations are possible. For decades, flow cytometry has balanced these goals to fill a critical need by enabling the measurement of multiple features in single cells, commonly to examine complex or hierarchical cellular systems. Recently, a format for flow cytometry has been developed that leverages the precision of mass spectrometry. This fusion of the two technologies, termed mass cytometry, provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes. In this Primer, we review the current state of mass cytometry, providing an overview of the instrumentation, its present capabilities, and methods of data analysis, as well as thoughts on future developments and applications. PMID:27153492

  14. 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.

  15. GenePattern flow cytometry suite

    PubMed Central

    2013-01-01

    Background Traditional flow cytometry data analysis is largely based on interactive and time consuming analysis of series two dimensional representations of up to 20 dimensional data. Recent technological advances have increased the amount of data generated by the technology and outpaced the development of data analysis approaches. While there are advanced tools available, including many R/BioConductor packages, these are only accessible programmatically and therefore out of reach for most experimentalists. GenePattern is a powerful genomic analysis platform with over 200 tools for analysis of gene expression, proteomics, and other data. A web-based interface provides easy access to these tools and allows the creation of automated analysis pipelines enabling reproducible research. Results In order to bring advanced flow cytometry data analysis tools to experimentalists without programmatic skills, we developed the GenePattern Flow Cytometry Suite. It contains 34 open source GenePattern flow cytometry modules covering methods from basic processing of flow cytometry standard (i.e., FCS) files to advanced algorithms for automated identification of cell populations, normalization and quality assessment. Internally, these modules leverage from functionality developed in R/BioConductor. Using the GenePattern web-based interface, they can be connected to build analytical pipelines. Conclusions GenePattern Flow Cytometry Suite brings advanced flow cytometry data analysis capabilities to users with minimal computer skills. Functionality previously available only to skilled bioinformaticians is now easily accessible from a web browser. PMID:23822732

  16. 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

  17. 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. PMID:25805180

  18. Use of a laboratory information system driven tool for pre-signout quality assurance of random cytopathology reports

    PubMed Central

    Kamat, Sonal; Parwani, Anil V.; Khalbuss, Walid E.; Monaco, Sara E.; Kelly, Susan M.; Wiehagen, Luke T.; Piccoli, Anthony L.; Lassige, Karen M.; Pantanowitz, Liron

    2011-01-01

    Background: Quality assurance (QA) programs in cytopathology laboratories in the USA currently primarily involve the review of Pap tests per clinical laboratory improvement amendments of 1988 federal regulations. A pre-signout quality assurance tool (PQAT) at our institution allows the laboratory information system (LIS) to also automatically and randomly select an adjustable percentage of non-gynecological cytopathology cases for review before release of the final report. The aim of this study was to review our experience and the effectiveness of this novel PQAT tool in cytology. Materials and Methods: Software modifications in the existing LIS application (CoPathPlus, Cerner) allow for the random QA of 8% of cases prior to signout. Selected cases are assigned to a second QA cytopathologist for review and all agreement and disagreements tracked. Detected errors are rectified before the case is signed out. Data from cases selected for PQAT over an 18-month period were collected and analyzed. Results: The total number of non-gynecological cases selected for QA review was 1339 (7.45%) out of 17,967 cases signed out during this time period. Most (1304) cases (97.4%) had an agreement in diagnosis. In 2.6% of cases, there were disagreements, including 34 minor and only 1 major disagreement. Average turnaround time of cases selected for review was not significantly altered. Conclusion: The PQAT provides a prospective QA mechanism in non-gynecological cytopathology to prevent diagnostic errors from occurring. This LIS-driven tool allows for peer review and corrective action to be taken prior to reporting without delaying turnaround time, thereby improving patient safety. PMID:21969923

  19. Endoscopic ultrasound-guided fine-needle aspiration with on-site cytopathology versus core biopsy: a comparison of both techniques performed at the same endoscopic session

    PubMed Central

    Lin, Michael; Hair, Clark D.; Green, Linda K.; Vela, Stacie A.; Patel, Kalpesh K.; Qureshi, Waqar A.; Shaib, Yasser H.

    2014-01-01

    Background: Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) with bedside cytopathology is the gold standard for assessment of pancreatic, subepithelial, and other lesions in close proximity to the gastrointestinal tract, but it is time-consuming, has certain diagnostic limitations, and bedside cytopathology is not widely available. Aims: The goal of this study is to compare the diagnostic yield of EUS-guided FNA with on-site cytopathology and EUS-guided core biopsy. Methods: Twenty-six patients with gastrointestinal mass lesions requiring biopsy at a tertiary medical center were included in this retrospective analysis of a prospective cohort. Two core biopsies were taken using a 22 gauge needle followed by FNA guided by a bedside cytopathologist at the same endoscopic session. The diagnostic yield and test characteristics of EUS core biopsy and EUS FNA with bedside cytopathology were examined. Results: The mean number of passes was 3.2 for FNA, and the mean procedure time was 39.4 minutes. The final diagnosis was malignant in 92.3 %. Sensitivity and specificity were 83 % and 100 %, respectively, for FNA, and 91.7 % and 100 %, respectively, for core biopsy. Diagnostic accuracy was 92.3 % for FNA and 84.6 % for core biopsy. The two approaches were in agreement in 88.4 % with a kappa statistic of 0.66 (95 % confidence interval 0.33 – 0.99). Conclusions: An approach using two passes with a core biopsy needle is comparable to the current gold standard of FNA with bedside cytopathology. The performance of two core biopsies is time-efficient and could represent a good alternative to FNA with bedside cytopathology. PMID:26135096

  20. 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.

  1. 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.

  2. Cell blocks in cytopathology: a review of preparative methods, utility in diagnosis and role in ancillary studies.

    PubMed

    Jain, D; Mathur, S R; Iyer, V K

    2014-12-01

    The cell block (CB) is a routine procedure in cytopathology that has gained importance because of its pivotal role in diagnosis and ancillary studies. There is no precise review in the published literature that deals with the various methods of preparation of CB, its utility in diagnosis, immunocytochemistry (ICC) or molecular testing, and its drawbacks. An extensive literature search on CB in cytology using internet search engines was performed for this review employing the following keywords: cell block, cytoblock, cytology, cytopathology, methods, preparation, fixatives, diagnostic yield, ancillary and molecular studies. Ever since its introduction more than a century ago, the CB technique has undergone numerous modifications to improve the quality of the procedure; however, the overall principle remains the same in each method. CBs can be prepared from virtually all varieties of cytological samples. In today's era of personalized medicine, cytological specimens, including CBs, augment the utility of cytological samples in analysing the molecular alterations as effectively as surgical biopsies or resection specimens. With the availability of molecular targeted therapy for many cancers, a large number of recent studies have used cytological material or CBs for molecular characterization. The various techniques of CB preparation with different fixatives, their advantages and limitations, and issues of diagnostic yield are discussed in this review. PMID:25113785

  3. 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

  4. New flow cytometry approaches in equine andrology.

    PubMed

    Peña, Fernando J; Ortega Ferrusola, Cristina; Martín Muñoz, Patricia

    2016-07-01

    Flow cytometry is currently recognized as a robust tool for the evaluation of sperm quality and function. However, within equine reproduction, this technique has not reached the sophistication of other areas of biology and medicine. In recent years, more sophisticated flow cytometers have been introduced in andrology laboratories, and the number of tests that can be potentially used in the evaluation of sperm physiology has increased accordingly. In this review, recent advances in the evaluation of stallion spermatozoa will be discussed. These new techniques in flow cytometry are able to simultaneously measure damage to different sperm regions and/or changes in functionality. PMID:27160445

  5. 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.

  6. 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

    2016-06-01

    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. PMID:27216436

  7. In vitro confirmation of newly established lung cancer cell lines using flow cytometry and multicellular tumor spheroids.

    PubMed

    Inoue, S; Takaoka, K; Endo, T; Mizuno, S; Ogawa, Y; Yoshida, M; Ohnuma, T

    1997-05-01

    We report on a simplified method of cytomorphological in vitro confirmation of newly established lung cancer cell lines by using multicellular tumor spheroids (MTS) and flow cytometry (FCM). Eleven cell lines were established from 11 patients with lung cancer. The MTS were produced by culturing cells in agar-coated dish. Cytomorphological studies were made using smears of crushed MTS and frozen sections of MTS. The MTS were fixed doubly with paraformaldehyde and osmic acid for scanning and transmission electron microscopy. Bivariate fluorescence of fluorescein isothyocyanate (FITC, tumor associated antigen, TAA) and propidium iodide (DNA) were measured by FCM. The MTS grew anchorage-independently. Cytopathological and electron microscopic findings of MTS were similar to those of the original clinical specimens. The DNA index and TAA were useful in evaluating the presence or absence of contamination by cells of non-tumor origin. The new cell lines satisfied a minimum of four conditions to confirm their establishment: (a) they originated from humans, (b) they were cytomorphologically identified with specimens from primary lesions, (c) they showed tumorigenicity, and (d) they were free from contamination by cells of different origin. From these findings, the establishment of new cell lines can be confirmed in vitro by using MTS and FCM. PMID:9194029

  8. 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. PMID:21812528

  9. Assaying Cell Cycle Status Using Flow Cytometry.

    PubMed

    Kim, Kang Ho; Sederstrom, Joel M

    2015-01-01

    In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation-specific marker (Ki-67) and cellular DNA content, which discriminate resting/quiescent cell populations (G0 cell) and quantify cell cycle distribution (G1, S, or G2/M), respectively. The second is based on differential staining of DNA and RNA through co-staining of Hoechst 33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, two additional methods for cell proliferation assays with recent updates of newly developed fluorophores, which allow multiplex analysis of cell cycle status, cell proliferation, and a gene of interest using flow cytometry, are outlined. PMID:26131851

  10. A clinical flow cytometry data analysis assistant

    SciTech Connect

    Salzman, G.C. ); Stewart, C.C. ); Duque, R.E. ); Braylan, R.C. . Coll. of Medicine)

    1990-01-01

    A rule-based expert system is being developed to assist clinicians in the analysis of multivariate flow cytometry data for patients with leukemias or lymphomas. The cells are stained with fluorescently labeled monoclonal antibodies and the cell fluorescence is measured with a flow cytometer. Cluster analysis is used to isolate subpopulations in the data on which the clinical decisions are made. Symbolic facts for the expert system are instantiated using these numerical data and the knowledge of the clinicians and experts in flow cytometry. The first prototype used a decision tree and rigid rules. Is successfully classified only nine of eleven leukemia cases. A second prototype incorporating certainty factors into the rules is now being developed that should remove the need for a rigid decision tree. 9 refs.

  11. 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. PMID:24668857

  12. 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

  13. Coaxial-Flow System for Chemical Cytometry

    PubMed Central

    Marc, Paul J.; Sims, Christopher E.; Allbritton, Nancy L.

    2008-01-01

    Over the past decade, chemical cytometry performed by capillary electrophoresis (CE) has become increasingly valuable as a bio-analytical tool to quantify analytes from single cells. However, extensive use of CE-based chemical cytometry has been hindered by the relatively low throughput for the analysis of single adherent cells. In order to overcome the low throughput of CE-based analysis of adherent cells and increase its utility in evaluating cellular attributes, new higher throughput methods are needed. Integration of a coaxial buffer exchange system with CE-based chemical cytometry increased the rate of serial analyses of cells. In the designed system, fluid flow through a tube coaxial to the separation capillary was used to supply electrophoretic buffer to the capillary. This sheath or coaxial fluid was turned off between analysis of cells and on during cell sampling and electrophoresis. Thus, living cells were not exposed to the nonphysiologic electrophoretic buffer prior to lysis. Key parameters of the system such as the relative capillary-sheath positions, buffer flow velocities, and the cell chamber design were optimized. To demonstrate the utility of the system, rat basophilic leukemic cells loaded with Oregon Green and fluorescein were serially lysed and loaded into a capillary. Separation of the contents of 20 cells at a rate of 0.5 cells/min was demonstrated. PMID:17979298

  14. 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...

  15. In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop.

    PubMed Central

    Donaldson, Y K; Bell, J E; Holmes, E C; Hughes, E S; Brown, H K; Simmonds, P

    1994-01-01

    The distribution, cell tropism, and cytopathology in vivo of human immunodeficiency virus (HIV) was investigated in postmortem tissue samples from a series of HIV-infected individuals who died either of complications associated with AIDS or for unrelated reasons while they were asymptomatic. Proviral sequences were detected at a high copy number in lymphoid tissue of both presymptomatic patients and patients with AIDS, whereas significant infection of nonlymphoid tissue such as that from brains, spinal cords, and lungs were confined to those with AIDS. V3 loop sequences from both groups showed highly restricted sequence variability and a low overall positive charge of the encoded amino acid sequence compared with those of standard laboratory isolates of HIV type 1 (HIV-1). The low charge and the restriction in sequence variability were comparable to those observed with isolates showing a non-syncytium-inducing (NSI) and macrophage-tropic phenotype in vitro. All patients were either exclusively infected (six of seven cases) or predominantly infected (one case) with variants with a predicted NSI/macrophage-tropic phenotype, irrespective of the degree of disease progression. p24 antigen was detected by immunocytochemical staining of paraffin-fixed sections in the germinal centers within lymphoid tissue, although little or no antigen was found in areas of lymph node or spleen containing T lymphocytes from either presymptomatic patients or patients with AIDS. The predominant p24 antigen-expressing cells in the lungs and brains of the patients with AIDS were macrophages and microglia (in brains), frequently forming multinucleated giant cells (syncytia) even though the V3 loop sequences of these variants resembled those of NSI isolates in vitro. These studies indicate that lack of syncytium-forming ability in established T-cell lines does not necessarily predict syncytium-forming ability in primary target cells in vivo. Furthermore, variants of HIV with V3 sequences

  16. 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

  17. Optimized flow cytometry isolation of murine spermatocytes.

    PubMed

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

    2014-06-01

    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 mouse 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 MPI. PMID:24664803

  18. CYTOMETRY OF APOPTOSIS. HISTORICAL PERSPECTIVE AND NEW ADVANCES

    PubMed Central

    Wlodkowic, D.; Skommer, J.; Darzynkiewicz, Z.

    2012-01-01

    Characteristic changes in cell morphology paralleled by the appearance of a multitude of molecular and biochemical markers occur during apoptosis. These changes vary depending on the cell type, mechanism of induction of apoptosis, and the time-window at which the process of apoptosis is analyzed. By virtue of the capability of rapid measurement of individual cells the flow- and imaging-cytometry become preferred technologies to detect, identify and record incidence of apoptosis in large cell populations. It also provided a valuable tool to investigate molecular mechanisms in field of necrobiology. This review outlines the progress in development of the most commonly used cytometric methods probing cells death based on analysis of fragmentation of DNA, activation of caspases, analysis of mitochondrial potential, alterations in plasma membrane structure and other features that characterize programmed cell death. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”. PMID:23070010

  19. 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.

  20. 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

  1. 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

  2. Cosmetic fat augmentation following breast reconstruction: sonographic appearance with cytopathologic correlation.

    PubMed

    Arleo, Elizabeth Kagan; Saleh, Marwa; Schwartz, Mark H; Eisen, Carolyn Sharyn

    2014-01-01

    This series presents the history and imaging of patients who had cosmetic fat augmentation following mastectomy and reconstruction. The cases provide the useful reminder that a complete surgical history is essential when assessing the imaging of a post-operative breast patient and that speaking directly with patients can be a critical step in putting together a complete clinical picture and adding value to their care. PMID:25128090

  3. 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.

  4. Optical clearing in photoacoustic flow cytometry

    PubMed Central

    Menyaev, Yulian A.; Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Juratli, Mazen A.; Galanzha, Ekaterina I.; Tuchin, Valery V.; Zharov, Vladimir P.

    2013-01-01

    Clinical applications of photoacoustic (PA) flow cytometry (PAFC) for detection of circulating tumor cells in deep blood vessels are hindered by laser beam scattering, that result in loss of PAFC sensitivity and resolution. We demonstrate biocompatible and rapid optical clearing (OC) of skin to minimize light scattering and thus, increase optical resolution and sensitivity of PAFC. OC effect was achieved in 20 min by sequent skin cleaning, microdermabrasion, and glycerol application enhanced by massage and sonophoresis. Using 0.8 mm mouse skin layer over a blood vessel in vitro phantom we demonstrated 1.6-fold decrease in laser spot blurring accompanied by 1.6-fold increase in PA signal amplitude from blood background. As a result, peak rate for B16F10 melanoma cells in blood flow increased 1.7-fold. By using OC we also demonstrated the feasibility of PA contrast improvement for human hand veins. PMID:24409398

  5. Optical clearing in photoacoustic flow cytometry.

    PubMed

    Menyaev, Yulian A; Nedosekin, Dmitry A; Sarimollaoglu, Mustafa; Juratli, Mazen A; Galanzha, Ekaterina I; Tuchin, Valery V; Zharov, Vladimir P

    2013-01-01

    Clinical applications of photoacoustic (PA) flow cytometry (PAFC) for detection of circulating tumor cells in deep blood vessels are hindered by laser beam scattering, that result in loss of PAFC sensitivity and resolution. We demonstrate biocompatible and rapid optical clearing (OC) of skin to minimize light scattering and thus, increase optical resolution and sensitivity of PAFC. OC effect was achieved in 20 min by sequent skin cleaning, microdermabrasion, and glycerol application enhanced by massage and sonophoresis. Using 0.8 mm mouse skin layer over a blood vessel in vitro phantom we demonstrated 1.6-fold decrease in laser spot blurring accompanied by 1.6-fold increase in PA signal amplitude from blood background. As a result, peak rate for B16F10 melanoma cells in blood flow increased 1.7-fold. By using OC we also demonstrated the feasibility of PA contrast improvement for human hand veins. PMID:24409398

  6. 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.

  7. Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry.

    PubMed

    Leipold, Michael D; Newell, Evan W; Maecker, Holden T

    2015-01-01

    The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays. PMID:26420710

  8. Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry

    PubMed Central

    Leipold, Michael D.; Newell, Evan W.; Maecker, Holden T.

    2016-01-01

    The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays. PMID:26420710

  9. Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the methyl green-pyronin technique with the gallocyanin chromalum and Feulgen procedures using image cytometry.

    PubMed

    Schulte, E K; Lyon, H O; Hoyer, P E

    1992-06-01

    For simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA. The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen reaction. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm. After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum. We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine. PMID:1378824

  10. 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

  11. Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry.

    PubMed

    Wang, Lili; Abbasi, Fatima; Ornatsky, Olga; Cole, Kenneth D; Misakian, Martin; Gaigalas, Adolfas K; He, Hua-Jun; Marti, Gerald E; Tanner, Scott; Stebbings, Richard

    2012-07-01

    To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach. PMID:22539147

  12. Spectrum of cytopathologic features of epithelioid sarcoma in a series of 7 uncommon cases with immunohistochemical results, including loss of INI1/SMARCB1 in two test cases.

    PubMed

    Rekhi, Bharat; Singh, Neha

    2016-07-01

    Diagnosis of an epithelioid sarcoma (ES) is challenging on fine needle aspiration cytology (FNAC) smears. There are few documented series describing cytopathologic features and immunostaining results of ESs. The present study describes cytopathologic features of seven cases of ES. All seven tumors occurred in males within age-range of 22-61 years; in sites, such as forearm (n = 3), hand (n = 2), thigh (n = 1), and inguinal region (n = 1). FNAC was performed for metastatic lesions (n = 5), recurrent lesions (n = 4), as well as for a primary diagnosis (n = 1). FNAC smears in most cases were moderate to hypercellular, composed of polygonal cells(seven cases) and spindle cells(three cases), arranged in loosely cohesive groups, non-overlapping clusters, and scattered singly, containing moderate to abundant cytoplasm, defined cell borders, vesicular nuclei, and discernible nucleoli. Variable cytopathologic features identified in certain cases were "rhabdoid-like" intracytoplasmic inclusions (n = 5), giant cells (n = 3), and interspersed scanty, metachromatic stroma (n = 4). Histopathologic examination revealed two cases of conventional-type ES, three of proximal/large cell-type ES, and two cases of mixed-type ES, displaying features of conventional and proximal subtypes. By immunohistochemistry (IHC), tumor cells were positive for cytokeratin (CK)(4/5), epithelial membrane antigen (EMA) (6/6), panCK (1/1), vimentin (3/3), and CD34 (7/7). Tumor cells were completely negative for INI1/SMARCB1 (0/2) and CD31 (0/5). In our settings, FNAC was mostly performed in recurrent and/or metastatic cases of ES, and rarely for a primary diagnosis of ES. Important cytopathologic features of ESs include loosely cohesive, non-overlapping clusters of polygonal cells with variable "rhabdoid-like" and spindle cells. Optimal diagnostic IHC markers in such cases include CK, EMA, AE1AE3, CD34, and INI1/SMARCB1. Clinical correlation is imperative in all

  13. Use of CCD sensors in flow cytometry for nonimaging applications

    NASA Astrophysics Data System (ADS)

    Beisker, Wolfgang

    1997-05-01

    The use of charge coupled devices (CCDs) as non-imaging sensors in flow cytometric systems to replace the classical photomultplier tubes (PMTs) is very advantageous: the quantum efficiency of the CCDs is about 5 to 10 times higher as for PMTs, the charge storage capability of CCDs avoids analogue processing of the fluorescence signals, the dynamic range is up to 18 bits and the fluorescence intensity at different wavelengths can be recorded on the same chip. In this report a full frame CCD imager is used in a thermoelectrically cooled environment. The output signal for the CCD is digitized with a 12-bit ADC and the data are sorted as list-mode data typically used in flow cytometric work. The performance of the system is demonstrated with DNA staining of mammalian cells with acridine-orange, propidium iodide and ethidium bromide. DNA histograms comparable with standard flow cytometry are recorded. From the same data set pulse-widths histograms can be processed and used for doublet discrimination. The high quantum efficiency of the CCD sensors is of special interest for fluorescing dyes in the dark red or near IR wavelength range.

  14. Evolution of flow cytometry standard, FCS3.0, into a DICOM-compatible format

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.; Leif, Suzanne B.

    1997-05-01

    The International Society for Analytical Cytology, ISAC, has developed a flow cytometry standard (FCS) to permit data interchange, ISAC will soon replace FCS 2.0 with FCS 3.0. Unfortunately, the proposed FCS 3.0 is still fraught with problems, which are of sufficient magnitude as to warrant its early replacement. The most reasonable replacement is as a supplement to the digital imaging and communications in medicine, DICOM 3.0, standard. The recent digital microscopy extension of DICOM can be extended and modified to include flow cytometry data. DICOM includes: image graphics objects, specifications for describing: studies, reports, the acquisition of the data and the individuals involved, physician, patient, etc. Storing the present FCS data in a database, which has already been accomplished with the QC tracker software, will facilitate the transition of FCS to DICOM.

  15. 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.

  16. Bioaerosol characterization by flow cytometry with fluorochrome.

    PubMed

    Chen, Pei-Shih; Li, Chih-Shan

    2005-10-01

    Traditional culture and microscopy methods for evaluation of bioaerosols are slow, tedious, and rather imprecise. In this study, the application of flow cytometry that was combined with a fluorescent technique (FCM/FL) was evaluated as a technique to quickly and accurately determine and quantify the total concentration and viability of bioaerosols. The optimal conditions of five fluorescent dyes [acridine orange (AO), SYTO-13, propidium iodide (PI), YOPRO-1, and 5-cyano-2,3-ditolytetrazolium chloride (CTC)] used in FCM/FL were determined for laboratory samples of bacterial aerosols (Escherichia coli, and endospores of Bacillus subtilis) and fungal aerosols (Candida famata and Penicillium citrinum spores). Based on the measured cell concentration, fluorescence intensity, and staining efficiency as indicators for dye performance evaluation, SYTO-13 was found to be the most suitable fluorescent dye for determining the total concentration of the bioaerosols, as well as YOPRO-1 was the most suitable for determining viability. Moreover, the established optimal FCM/FL with dyes was validated for characterizing microorganism profiles from both air and water samples from the aeration tank of hospital wastewater treatment plant. In conclusion, the FCM/FL successfully assessed the total concentration and viability for bacterial and fungal microorganisms in environmental field samples. PMID:16193165

  17. [Flow cytometry: applications in transfusion medicine].

    PubMed

    Boval, B

    2000-06-01

    In transfusion medicine, flow cytometry (FCM) is a methodology combining laser radiation, optics and a computerized treatment of numerous results. We can measure size, cellularity and fluorescence intensity of cells or particles in suspension after the binding of appropriate fluorescent antibodies or fluorescent dyes. The main utilisation of FCM in transfusion medicine is for quality control of the process of leukocyte reduction in red cell concentrates or in platelet units, using commercial kits. In addition, it is used for the enumeration of CD 34 positive cells before bone marrow transplantation and for control of platelet function in platelet units. For clinical investigations, FCM may be used for red cell phenotyping, essentially to detect minor populations (chimerism), for the estimation of red cell survival, or for the detection of fetal erythrocytes. In the field of platelet immunology, FCM is an essential tool for detecting platelet antibodies (auto or allo), for platelet phenotyping or for cross-matching. In the future perhaps, FCM will permit us to detect bacterial contamination or prion protein in transfused blood cells. PMID:10919227

  18. 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

  19. New Horizons in Platelets Flow Cytometry

    PubMed Central

    Saboor, Muhammad; Moinuddin, Moinuddin; Ilyas, Samina

    2013-01-01

    Platelet flow cytometry is an emerging tool in diagnostic and therapeutic hematology. It is eminently suited to study the expression of platelet surface receptors both qualitatively as well as quantitatively. It can serve as a useful marker for the documentation of in vivo platelet activation, and thus, fore-warn the risk of thromboembolism in patients with diabetes mellitus, coronary syndromes, peripheral vascular diseases, and pre-eclampsia. This technique can also be extended to study and compare the effect of various antiplatelet drugs on the level of activation of platelets and to establish any dose-effect relationship of these drugs. Topographical localization of platelet granules and study of platelet-platelet and platelet-leukocyte interaction is also possible by this procedure. All these parameters serve as pointers towards the presence of activated platelets in the circulation with its thromboembolic consequences. This is a simple reliable and cost effective technique which has a wide application in the diagnosis of various inherited and acquired platelet disorders. Study of platelet cluster of differentiation (CD) markers in various inherited disorders i.e. Bernard Soulier’s disease, von Willebrand disease, Glanzman’s disease, and Grey platelet syndrome may help categories the molecular lesions in these oft under-studied disorders. PMID:23983579

  20. Flow-cytometry techniques in radiation biology

    SciTech Connect

    McCarthy, K.F.; Hale, M.L.

    1988-01-01

    Considerable evidence exists that all blood cells are derived from HSC. These cells are of interest to radiobiologists because they are highly sensitive to low doses of ionizing radiation. Hematopoietic stem cells (HSC) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC. Because of the importance of HSC in the post-irradiation syndrome, the authors developed a new rapid method based on flow cytometry not only to assay but also to purify and characterize HSC. This new method makes extensive use of non-clonal antibodies conjugated to fluorescent phycobiliproteins through the sulfhydryls of the hinge region of the IgG molecule. An optical bench arrangement with a dye laser and an argon laser was used for dual excitation of the phycobiliprotein-monoclonal antibody conjugates and various cellular and DNA probes. Using 4', 6-diamidino 2-phenylindole dihydrochloride (DAP) exclusion to identify viable cells, it was possible to follow regeneration of post-irradiated rat marrow HSC.

  1. 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

  2. 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

  3. Metastatic mixed acinar-neuroendocrine carcinoma of the pancreas to the liver: a cytopathology case report with review of the literature.

    PubMed

    Lee, Lili; Bajor-Dattilo, Ewa B; Das, Kasturi

    2013-02-01

    A case of metastatic mixed acinar-neuroendocrine carcinoma (MANEC) of the pancreas to the liver is reported. A diagnostic percutaneous US-guided FNA and core biopsy of a liver nodule was performed. The FNA smears were cellular and showed neoplastic cells in clusters with acinar formation, isolated single cells, and scattered naked nuclei. The cytoplasm was finely granular. The nuclei were relatively uniform, some with speckled chromatin and prominent nucleoli. The immunohistochemistry performed on the cell block showed strong positivity for cytokeratin AE1/AE3, chromogranin, and synaptophysin. Furthermore, the tumor cells were weakly positive for α1-antichymotrypsin. The Ki-67 mitotic index was up to 50%. Based on the morphology and supporting immunohistochemical stains, the final cytopathologic diagnosis rendered was "Positive for malignant cells. Carcinoma with mixed acinar and endocrine features." To our knowledge, this is the first report of a metastatic MANEC to the liver diagnosed based on cytology with confirmatory histology. The difficulties in the cytopathologic diagnosis and differential diagnosis of MANEC are discussed in this article. PMID:22903971

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

    SciTech Connect

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

    1989-11-01

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

  5. Enveloped double-stranded DNA insect virus with novel structure and cytopathology

    PubMed Central

    Federici, Brian A.

    1983-01-01

    An unusual type of virus has been isolated from larvae of the cabbage looper, Trichoplusia ni (Lepidoptera; Noctuidae). The virus infects a variety of tissues, including fat body, epidermis, and tracheal matrix, causing a chronic, fatal disease. Viral replication begins in the nucleus and is accompanied by invagination of the nuclear envelope and extensive nuclear and cellular hypertrophy. The nuclear envelope eventually ruptures and fragments, after which viral-induced membranes are assembled along planes through the cell and around its periphery. Subsequently, these membranes coalesce, partitioning most of the cell, including viroplasms and virions in various stages of assembly, among a cluster of vesicles. The vesicles dissociate and are liberated into the hemolymph where they accumulate in large numbers (>108 vesicles per ml), causing the blood to become opaque white. The virus has been isolated from T. ni and transmitted per os and by injection to T. ni and several other species of the family Noctuidae. The virions produced by this virus are large (ca. 130 × 400 nm), enveloped, and allantoid in shape with complex symmetry and contain apparently linear, double-stranded DNA of Mr of ≈ 1.00 × 108. The envelope contains subunits arranged in a hexagonal pattern that impart a distinctive reticular appearance to virions in negatively stained preparations. The unique structural and developmental properties of this virus indicate that it is a member of a group of enveloped, double-stranded DNA viruses not observed previously. Images PMID:16593397

  6. Deep profiling of multitube flow cytometry data

    PubMed Central

    O’Neill, Kieran; Aghaeepour, Nima; Parker, Jeremy; Hogge, Donna; Karsan, Aly; Dalal, Bakul; Brinkman, Ryan R.

    2015-01-01

    Motivation: Deep profiling the phenotypic landscape of tissues using high-throughput flow cytometry (FCM) can provide important new insights into the interplay of cells in both healthy and diseased tissue. But often, especially in clinical settings, the cytometer cannot measure all the desired markers in a single aliquot. In these cases, tissue is separated into independently analysed samples, leaving a need to electronically recombine these to increase dimensionality. Nearest-neighbour (NN) based imputation fulfils this need but can produce artificial subpopulations. Clustering-based NNs can reduce these, but requires prior domain knowledge to be able to parameterize the clustering, so is unsuited to discovery settings. Results: We present flowBin, a parameterization-free method for combining multitube FCM data into a higher-dimensional form suitable for deep profiling and discovery. FlowBin allocates cells to bins defined by the common markers across tubes in a multitube experiment, then computes aggregate expression for each bin within each tube, to create a matrix of expression of all markers assayed in each tube. We show, using simulated multitube data, that flowType analysis of flowBin output reproduces the results of that same analysis on the original data for cell types of >10% abundance. We used flowBin in conjunction with classifiers to distinguish normal from cancerous cells. We used flowBin together with flowType and RchyOptimyx to profile the immunophenotypic landscape of NPM1-mutated acute myeloid leukemia, and present a series of novel cell types associated with that mutation. Availability and implementation: FlowBin is available in Bioconductor under the Artistic 2.0 free open source license. All data used are available in FlowRepository under accessions: FR-FCM-ZZYA, FR-FCM-ZZZK and FR-FCM-ZZES. Contact: rbrinkman@bccrc.ca. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25600947

  7. Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death

    PubMed Central

    Wlodkowic, Donald; Telford, William; Skommer, Joanna; Darzynkiewicz, Zbigniew

    2012-01-01

    A cell undergoing apoptosis demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the inducer of apoptosis, cell type and the “time window” at which the process of apoptosis is observed. Because the gross majority of apoptotic hallmarks can be revealed by flow and image cytometry, the cytometric methods become a technology of choice in diverse studies of cellular demise. Variety of cytometric methods designed to identify apoptotic cells, detect particular events of apoptosis and probe mechanisms associated with this mode of cell death have been developed during the past two decades. In the present review, we outline commonly used methods that are based on the assessment of mitochondrial transmembrane potential, activation of caspases, DNA fragmentation, and plasma membrane alterations. We also present novel developments in the field such as the use of cyanine SYTO and TO-PRO family of probes. Strategies of selecting the optimal multiparameter approaches, as well as potential difficulties in the experimental procedures, are thoroughly summarized. PMID:21722800

  8. 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.

  9. Improving the signal analysis for in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Niu, Zhenyu; Yang, Ping; Wei, Dan; Tang, Shuo; Wei, Xunbin

    2015-03-01

    At early stage of cancer, a small number of circulating tumor cells (CTCs) appear in the blood circulation. Thus, early detection of malignant circulating tumor cells has great significance for timely treatment to reduce the cancer death rate. We have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of CTCs and record the signals from target cells. Information of target cells which is helpful to the early therapy would be obtained through analyzing and processing the signals. 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 PAFC technique can detect signals from circulating tumor cells or other particles. The processing methods have a great potential for analyzing signals accurately and rapidly.

  10. Peripancreatic paraganglioma: a potential diagnostic challenge in cytopathology and surgical pathology.

    PubMed

    Singhi, Aatur D; Hruban, Ralph H; Fabre, Monique; Imura, Johji; Schulick, Richard; Wolfgang, Christopher; Ali, Syed Z

    2011-10-01

    Paragangliomas are rare neuroendocrine neoplasms arising in extra-adrenal chromaffin cells of the autonomic nervous system. In rare instances, paragangliomas present around and involve the pancreas, thereby mimicking one of the more common primary pancreatic lesions. These neoplasms present considerable diagnostic difficulty not only for the clinician and radiologist but also for the pathologist. We have collected a series of 9 peripancreatic paragangliomas clinically simulating a primary pancreatic lesion. The paragangliomas were diagnosed in 4 men and 5 women with an age range of 37 to 78 years (mean, 50 y). Patients presented clinically either with diffuse epigastric and abdominal pain (7 of 9, 78%) or with an incidental mass (2 of 9, 22%) discovered on routine radiographic imaging. All patients were found to have mass lesions suspicious for a primary pancreatic neoplasm on radiographic examination. The lesions were predominantly located in the body of the pancreas (5 of 9, 56%) and ranged in size from 5.5 to 17.0 cm (mean, 10.0 cm). Five of 9 (56%) neoplasms also demonstrated cystic change. Fine-needle aspiration (FNA) was performed on 6 cases; however, the diagnostic accuracy was low, with 3 of 6 (50%) neoplasms misdiagnosed as pancreatic neuroendocrine tumor (PanNET) (n=1), spindle cell neoplasm (n=1), or pseudocyst (n=1). In addition, 2 of 8 (25%) surgically resected tumors were misdiagnosed by the referring pathologist as a PanNET. Immunohistochemistry was performed on all cases, confirming the characteristic 2-cell populations: chief cells (synaptophysin positive and chromogranin A positive) and sustentacular cells (S-100 protein positive). Follow-up information was available for all patients and ranged from 2 months to 11.6 years (mean, 2.7 y). Three of 9 (33%) patients developed metastatic disease, and 2 of these 3 died of their disease at 2.8 and 4.6 years after diagnosis. In summary, in unsuspected cases, interpretation of FNA and surgical pathology

  11. 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.

  12. New fluorogenic dyes for analysis of cellular processes by flow cytometry and confocal microscopy.

    PubMed

    Nikolova, Kalina; Kaloyanova, Stefka; Mihaylova, Nikolina; Stoitsova, Stoyanka; Chausheva, Stela; Vasilev, Aleksey; Lesev, Nedyalko; Dimitrova, Petya; Deligeorgiev, Todor; Tchorbanov, Andrey

    2013-12-01

    Fluorescent microscopy and fluorescent imaging by flow cytometry are two of the fastest growing areas in the medical and biological research. Innovations in fluorescent chemistry and synthesis of new dye probes are closely related to the development of service equipment such as light sources, and detection techniques. Among compounds known as fluorescent labels, the cyanine-based dyes have become widely used since they have high excitation coefficients, narrow emission bands and high fluorescence upon binding to nucleic acids. The key methods for evaluation of apoptosis and cell cycle allow measuring DNA content by several flow cytometric techniques. We have synthesized new monomethine cyanine dyes and have characterized their applicability for staining of live and/or apoptotic cells. Imaging experiments by flow cytometry and confocal laser scanning microscopy (CLSM) have been also performed. Two of the dyes have shown high-affinity binding to the nuclei at high dilutions, up to 10(-9)M. Flow cytometry and CLSM have confirmed that these dyes labeled selectively non-living, e.g. ethanol-fixed cells that makes them appropriate for estimations of cell viability and apoptosis. The novel structures proved to be appropriate also for analysis of the cell cycle. PMID:24231377

  13. 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

  14. 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

  15. 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.

  16. 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

  17. The application of flow cytometry to histocompatibility testing.

    PubMed

    Horsburgh, T; Martin, S; Robson, A J

    2000-03-01

    Flow cytometry is a powerful technique that enables the sensitive and quantitative detection of both cellular antigens and bound biological moieties. This article reviews how flow cytometry is increasingly being used as histocompatibility laboratories for the analysis of antibody specificity and HLA antigen expression. A basic description of flow cytometry principles and standardisation is given, together with an outline of clinical application in the areas of pre-transplant cross-matching, antibody screening, post-transplant antibody monitoring and HLA-B27 detection. It is concluded that flow cytometry is a useful multi-parametric analytical tool, yielding clinical benefit especially in the identification of patients at risk of early transplant rejection. PMID:10834606

  18. 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. PMID:27068992

  19. 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

  20. Postbrushing and fine-needle aspiration biopsy follow-up and treatment options for patients with pancreatobiliary lesions: the Papanicolaou Society of Cytopathology guidelines.

    PubMed

    Kurtycz, Daniel; Tabatabai, Z Laura; Michaels, Claire; Young, Nancy; Schmidt, C Max; Farrell, James; Gopal, Deepak; Simeone, Diane; Merchant, Nipun B; Field, Andrew; Pitman, Martha Bishop

    2014-04-01

    The papanicolaou society of cytopathology (PSC) 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 for pancreatobiliary cytology, ancillary testing, and postprocedure 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 PSC web site [www.papsociety.org]. This document selectively presents the results of these discussions and focuses on the follow-up and treatment options for patients after procedures performed for obtaining cytology samples for the evaluation of biliary strictures and solid and cystic masses in the pancreas. These recommendations follow the six-tiered terminology and nomenclature scheme proposed by Committee III. PMID:24639399

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

    PubMed

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

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. PMID:26908592

  2. Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states.

    PubMed

    Samadder, Partha; Weng, Ning; Doetschman, Thomas; Heimark, Ronald L; Galbraith, David W

    2016-05-01

    The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis. © 2016 International Society for Advancement of Cytometry. PMID:27003621

  3. Algorithmic tools for mining high-dimensional cytometry data

    PubMed Central

    Chester, Cariad; Maecker, Holden T.

    2015-01-01

    The advent of mass cytometry has lead to an unprecedented increase in the number of analytes measured in individual cells, thereby increasing the complexity and information content of cytometric data. While this technology is ideally suited to detailed examination of the immune system, the applicability of the different methods for analyzing such complex data are less clear. Conventional data analysis by ‘manual’ gating of cells in biaxial dotplots 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 recently applied to mass cytometry data. Herein, 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 identify suitable algorithmic tools for their particular projects. PMID:26188071

  4. Application of Flow Cytometry in the Evaluation of Primary Immunodeficiencies.

    PubMed

    Fleisher, Thomas A; Madkaikar, Manisha; Rosenzweig, Sergio D

    2016-05-01

    Primary immunodeficiency disorders (PIDDs) are a heterogeneous group of inherited disorders of the immune system. Currently more than 250 different PIDDs with a known genetic defect have been recognized. The diagnosis of many of these disorders is supported strongly by a wide variety of flow cytometry applications. Flow cytometry offers a rapid and sensitive tool for diagnosis and classification of PIDDs. It is applicable in the initial workup and subsequent management of several primary immunodeficiency diseases. As our understanding of the pathogenesis and management of these diseases increases, the majority of these tests can be easily established in the diagnostic laboratory. Thus, the focus of this article is on the application of flow cytometry in the diagnosis and/or evaluation of PIDDs. PMID:26865168

  5. Flow cytometry measurements of human chromosome kinetochore labeling

    SciTech Connect

    Fantes, J.A.; Green, D.K.; Malloy, P.; Sumner, A.T.

    1989-03-01

    A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.

  6. 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

  7. Deep Profiling Human T Cell Heterogeneity by Mass Cytometry.

    PubMed

    Cheng, Y; Newell, E W

    2016-01-01

    Advances of mass cytometry and high-dimensional single-cell data analysis have brought cellular immunological research into a new generation. By coupling these two powerful technology platforms, immunologists now have more tools to resolve the tremendous diversity of immune cell subsets, and their heterogeneous functionality. Since the first introduction of mass cytometry, many reports have been published using this novel technology to study a range of cell types. At the outset, studies of human hematopoietic stem cell and peripheral CD8(+) T cells using mass cytometry have shad the light of future experimental approach in interrogating immune cell phenotypic and functional diversity. Here, we briefly revisit the past and present understanding of T cell heterogeneity, and the technologies that facilitate this knowledge. In addition, we review the current progress of mass cytometry and high-dimensional cytometric analysis, including the methodology, panel design, experimental procedure, and choice of computational algorithms with a special focus on their utility in exploration of human T cell immunology. PMID:27235682

  8. Per-channel basis normalization methods for flow cytometry data

    PubMed Central

    Hahne, Florian; Khodabakhshi, Alireza Hadj; Bashashati, Ali; Wong, Chao-Jen; Gascoyne, Randy D.; Weng, Andrew P.; Seifert-Margolis, Vicky; Bourcier, Katarzyna; Asare, Adam; Lumley, Thomas; Gentleman, Robert; Brinkman, Ryan R.

    2013-01-01

    Between-sample variation in high throughput flow cytometry data poses a significant challenge for analysis of large scale data sets, such as those derived from multi-center clinical trials. It is often hard to match biologically relevant cell populations across samples due to technical variation in sample acquisition and instrumentation differences. Thus normalization of data is a critical step prior to analysis, particularly in large-scale data sets from clinical trials, where group specific differences may be subtle and patient-to-patient variation common. We have developed two normalization methods that remove technical between-sample variation by aligning prominent features (landmarks) in the raw data on a per-channel basis. These algorithms were tested on two independent flow cytometry data sets by comparing manually gated data, either individually for each sample or using static gating templates, before and after normalization. Our results show a marked improvement in the overlap between manual and static gating when the data are normalized, thereby facilitating the use of automated analyses on large flow cytometry data sets. Such automated analyses are essential for high throughput flow cytometry. PMID:19899135

  9. 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…

  10. Platinum-conjugated antibodies for application in mass cytometry.

    PubMed

    Mei, Henrik E; Leipold, Michael D; Maecker, Holden T

    2016-03-01

    Mass cytometry has overcome limitations of fluorescent single cell cytometry by allowing for the measurement of up to currently ∼40 different parameters on a single cell level. However, the cellular proteome comprises many more potential analytes, and current mass cytometry instrumentation allows for theoretically up to 121 different mass detection channels. The labeling of specific probes with appropriate metal ions is a significant hurdle for exploiting more of mass cytometry's analytical capacity. To this end, we here describe the labeling of antibody with natural abundance or isotopically purified platinum as formulated in cisplatin and circumventing the use of chelator-loaded polymers. We confirm the utility of cisplatin-antibody-conjugates for surface, intracellular, and phosphoepitope-specific immunophenotyping, as well as for application in cell surface CD45-based barcoding. Cisplatin-labeling of antibody increases the analytical capacity of the CyTOF(®) platform by two channels based on available reagents, and has the potential to add a total of six channels for detection of specific probes, thus helping to better extend the analytical mass range of mass cytometers. PMID:26355391

  11. 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

  12. 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

  13. Multiparametric cytometry for exploration of complex cellular dynamics.

    PubMed

    Gondois-Rey, Françoise; Granjeaud, Samuel; Kieu, Suong Le Thi; Herrera, Diana; Hirsch, Ivan; Olive, Daniel

    2012-04-01

    The development of polychromatic cytometry has contributed to significant progress in the field of human immunology. Although numerous functional studies of rare cell populations have been performed using this technology, here we used polychromatic cytometry to explore the dynamics of complex cellular systems implicated in innate immunity. We used PBMC stimulated with live influenza virus as an experimental model. We studied the time course of activation of PBMC, which contain DC, monocytes, and NK cells, all of which are, in addition to their innate immune properties, susceptible to Flu infection. We developed 12 color panels to investigate intracellular expression of IFN-α, TNF-α, IL-12, IL-6, IFN-γ, CD107, and influenza virus nucleoprotein simultaneously in these cell populations. These panels allowed reproducible determination of activation markers induced in DC after their direct exposure to various stimulations or in NK cells by indirect DC-mediated activation within the complex cellular environment. The ability to use a low number of cells and reduced quantities of reagents permitted us to perform kinetic experiments. The power of polychromatic cytometry associated with bioinformatic tools allowed us to analyze the multiple functional data generated as dynamic clustering maps. These maps present a readily understandable view of activation events induced in different populations of PBMC. In addition, it reveals new information on the coordination of the complex pathways induced and on the cellular interactions that sustained indirect DC-mediated NK cell activation. Our work shows that polychromatic cytometry is a tool for discoveries in unexplored complex cell systems, at the crossroads of immunology and virology. © 2012 International Society for Advancement of Cytometry. PMID:22278900

  14. Nanocrystal-based biomimetic system for quantitative flow cytometry

    NASA Astrophysics Data System (ADS)

    Yim, Peter; Dobrovolskaia, Marina; Kang, HyeongGon; Clarke, Matthew; Patri, Anil K.; Hwang, Jeeseong

    2007-02-01

    Flow cytometry has been instrumental in rapid analysis of single cells since the 1970s. One of the common approaches is the immunofluorescence study involving labeling of cells with antibodies conjugated to organic fluorophores. More recently, as the application of flow cytometry extended from simple cell detection to single-cell proteomic analysis, the need of determining the actual number of antigens in a single cell has driven the flow cytomery technique towards a quantitative methodology. However, organic fluorophores are challenging to use as probes for quantitative detection due to the lack of photostability and of quantitative fluorescence standards. National Institute of Standards and Technologies (NIST) provides a set of fluorescein isothiocyanate (FITC) labeled beads, RM 8640, which is the only nationally recognized fluorescent particle standard. On the other hand, optical characteristics of semiconductor nanocrystals or quantum dots or QDs are superior to traditional dye molecules for the use as tags for biological and chemical fluorescent sensors and detectors. Compelling advantages of QDs include long photostability, broad spectral coverage, easy excitation, and suitability for multiplexed sensing. Recently, novel surface coatings have been developed to render QDs water soluble and bio-conjugation ready, leading to their use as fluorescent tags and sensors for a variety of biological applications including immunolabeling of cells. Here, we describe our approach of using fluorescent semiconductor QDs as a novel tool for quantitative flow cytometry detection. Our strategy involves the development of immuno-labeled QD-conjugated silica beads as "biomimetic cells." In addition to flow cytometry, the QD-conjugated silica beads were characterized by fluorescence microscopy to quantitate the number of QDs attached to a single silica bead. Our approach enables flow cytometry analysis to be highly sensitive, quantitative, and encompass a wide dynamic range of

  15. 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.

  16. 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

  17. Preparation of cells from paraffin-embedded tissue for cytometry and cytomorphologic evaluation.

    PubMed

    van Driel-Kulker, A M; Mesker, W E; van der Burg, M J; Ploem, J S

    1987-06-01

    A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells. PMID:3304329

  18. Barrett's esophagus. Correlation between mucin histochemistry, flow cytometry, and histologic diagnosis for predicting increased cancer risk.

    PubMed Central

    Haggitt, R. C.; Reid, B. J.; Rabinovitch, P. S.; Rubin, C. E.

    1988-01-01

    A predominance of sulfated mucin in the nongoblet columnar cells of Barrett's specialized metaplastic epithelium has been postulated to be a form of mild dysplasia and to indicate an increased risk of adenocarcinoma. Flow cytometry for the analysis of nuclear DNA content and cell cycle parameters has also been postulated to be an objective aid in the diagnosis of dysplasia and carcinoma in Barrett's esophagus. The authors investigated the relationship among sulfated mucin, flow cytometric data, and histologic diagnosis in each of 152 biopsies from 42 patients who had Barrett's specialized metaplastic epithelium. Sulfated mucin, as detected by the high iron diamine-Alcian blue stain, was present in biopsies from 8 of 11 (73%) patients with the histologic diagnosis of dysplasia or carcinoma, in 7 of 9 (78%) patients whose biopsies were indefinite for dysplasia, and in 12 of 22 (55%) patients whose biopsies were negative for dysplasia (P = 0.37). Sulfated mucins predominated in 9%, 22%, and 9% of the patients, respectively (P = 0.56). Abnormal flow cytometry (aneuploidy or increased G2/tetraploid fraction) was found in all patients with the histologic diagnosis of dysplasia or carcinoma, in 3 of 9 (33%) indefinite for dysplasia, and in 1 of 22 (5%) negative for dysplasia (P = less than 0.0001). Neither the presence nor the predominance of sulfated mucin in the specialized metaplastic epithelium of Barrett's esophagus has sufficiently high sensitivity or specificity for dysplasia or carcinoma to be of value in managing patients. Abnormal flow cytometry shows excellent correlation with the histologic diagnosis of dysplasia and carcinoma; it detects a subset of patients whose biopsies are histologically indefinite or negative for dysplasia, but who have flow cytometric abnormalities similar to those otherwise seen only in dysplasia and carcinoma. Images Figure 1 Figure 2 Figure 3 PMID:3354644

  19. 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.

  20. Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments.

    PubMed

    Rahman, Adeeb H; Tordesillas, Leticia; Berin, M Cecilia

    2016-06-01

    The analysis of heterogeneous cell samples by mass cytometry (CyTOF) relies on the assumption that metal labeled antibodies accurately bind to their target antigens. We report a previously unappreciated experimental artifact of non-specific antibody binding by eosinophils during intracellular CyTOF analysis of human whole blood samples. We hypothesized that this non-specific binding results from a charge-based interaction between the metal-labeled antibodies and highly cationic proteins found in eosinophillic granules and found that this non-specific staining artifact could be reduced to background levels with a simple blocking protocol using heparin as a competing anionic protein. This protocol eliminates a potential source of erroneous data interpretation in all experiments involving intracellular staining of human whole blood samples, and allows accurate assessment of dynamic changes in intracellular proteins in eosinophils by CyTOF. © 2016 International Society for Advancement of Cytometry. PMID:27061608

  1. 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

  2. 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. PMID:23396282

  3. Analyzing Schizosaccharomyces pombe DNA Content by Flow Cytometry.

    PubMed

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

    2016-01-01

    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. PMID:27250946

  4. 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

  5. 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.

  6. 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

  7. Flow cytometry applications in the study of immunological lung disorders.

    PubMed

    Mortaz, Esmaeil; Gudarzi, Hoda; Tabarsi, Payam; M Adcock, Ian; Masjedi, Mohamad Reza; Jamaati, Hamid Reza; Garssen, Johan; Velayati, Ali Akbar; A Redegeld, Frank

    2015-02-01

    The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. Flow cytometric analysis provides a rapid qualitative and quantitative description of multiple characteristics of individual cells. For example, it is possible to detect the cell size and granularity, aspects of DNA and RNA content and the presence of cell surface and nuclear markers which are used to characterize the phenotype of single cells. Flow cytometry has been used for the immunophenotyping of a variety of specimens including whole blood, bone marrow, serous cavity fluids, (cerebrospinal fluid) CSF, urine and all types of body fluids. The technique has also been applied to human bronchoalveolar lavage (BAL) fluid, peritoneal fluids and blood. In this review, we describe the current status of the application of flow cytometry as a diagnostic tool in various lung diseases. We focus on the analysis of BAL cell composition in chronic obstructive lung disease (COPD), asthma, lung cancer, sarcoidosis, tuberculosis and idiopathic eosinophilic pneumonia (IEP). PMID:25530134

  8. Discriminating cellular heterogeneity using microwell-based RNA cytometry

    PubMed Central

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

    2014-01-01

    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. Moreover, we use Monte-Carlo simulation and RNA-cytometry data to calculate the minimum number of cells required for detecting heterogeneity. We applied this system to characterize the RNA distributions of aging related genes in a highly purified mouse hematopoietic stem cell population. We identified genes that reveal novel heterogeneity of these cells. We also show that changes in expression of genes such as Birc6 during aging can be attributed to the shift of relative portions of cells in the high-expressing subgroup versus low-expressing subgroup. PMID:24667995

  9. Does DNA cytometry have a place in the clinical laboratory

    SciTech Connect

    Mayall, B. ); Waldman, F.; Chew, K.; Christov, K.; Goodson, W.; Ljung, B.M. . Lab. for Cell Analysis); Smith, H.S. )

    1990-01-24

    We are investigating the potential utility of cellular markers, including cellular proliferation and DNA cytometry, as independent diagnostic and prognostic markers in human breast cancer. However, as the clinical laboratory is responsible for providing physicians with data relevant to the patient, it is essential first to establish the validity of such markers before their use is recommended. Prospective validation is time-consuming and costly for tests of human malignancies, such as breast cancer, which may follow a lengthy and indolent course requiring patients to be followed for a decade or more before their clinical outcome is known. Therefore, retrospective studies on archival material are used whenever possible. Cell proliferation is recognized as an important diagnostic and prognostic marker for human breast cancer and a tritiated thymidine DNA labeling index greater than 5% is associated with a markedly less favorable outcome. Incorporation of bromodeoxyuridine (BrdUrd) into the DNA of S phase cells gives a similar labeling index. Unfortunately, paraffin-embedded archival material is rarely pre-labeled, and so DNA cytometry of either whole nuclei disaggregated from thick sections or partial nuclei in thin sections must be used as an indirect approach to estimate cellular proliferative activity. We are particularly interested in validating the DNA cytometry of thin sections and in relating the DNA histogram to in vivo BrdUrd labeling index, which is our standard for cellular proliferation. 6 refs., 1 fig.

  10. Barcoding of live human PBMC for multiplexed mass cytometry*

    PubMed Central

    Mei, Henrik E.; Leipold, Michael D.; Schulz, Axel Ronald; Chester, Cariad; Maecker, Holden T.

    2014-01-01

    Mass cytometry is developing as a means of multiparametric single cell analysis. Here, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a CyTOF® instrument. Using six different anti-CD45 antibody (Ab) conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and reduces wet work and antibody consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45-barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and should be applicable to fluorescence flow cytometry as well. PMID:25609839