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Sample records for cytopathology image cytometry

  1. Combined analysis of cervical smears. Cytopathology, image cytometry and in situ hybridization.

    PubMed

    Multhaupt, H; Bruder, E; Elit, L; Rothblat, I; Warhol, M

    1993-01-01

    This study was an attempt to correlate the Bethesda System of Papanicolaou smear classification with DNA content by image analysis and the presence of human papillomavirus (HPV) as determined by in situ hybridization. DNA histograms were classified as normal diploid, diploid proliferative, polyploid and aneuploid. HPV in situ hybridization was performed with a cocktail of probes specific to HPV types 6, 11, 16 and 18. There was a good correlation between normal cytology and normal DNA histograms. Cytologically normal smears with bacterial or fungal infections showed a high proliferation index. HPV infection correlated with DNA polyploidy but was seen in 15 of 29 smears classified as cytologically normal. Morphologically abnormal Papanicolaou smears correlated with aneuploid DNA content. Smears classified as intraepithelial neoplasia correlated with aneuploid DNA content in all 12 cases. Four of five cases cytologically suspicious for HPV infection had HPV by in situ hybridization.

  2. Digital spectral imaging for histopathology and cytopathology

    NASA Astrophysics Data System (ADS)

    Levenson, Richard M.; Farkas, Daniel L.

    1997-05-01

    The process by which diseases, particularly neoplastic diseases, are diagnosed by pathologists using microscopic evaluation of tissue has changed little over the last several decades despite the advent of molecular medicine. Cells or tissues, stained with complex and sometimes poorly characterized organic dyes, are examined using a subjective, pattern-matching approach whose accuracy and reproducibility has increasingly been challenged. Furthermore, the ability of pathologists to deliver accurate prognoses using histological and clinical parameters remains limited, leading to both over- and under-treatment. While molecular techniques hold out great promise as diagnostic and prognostic tools, they are currently still largely investigational, and costly. A complementary approach is proposed, whereby more information is obtained by improved analysis of conventionally prepared histological and cytological samples. Using inverse Fourier transform multi- pixel spectroscopy, a new instrument has been developed which can display a complete transmittance or emission spectrum at every pixel of an image, providing much more color information than can be appreciated by eye or by conventional red-green-blue color cameras. Since spectra variations in staining behavior correlate with alterations in subcellular macromolecular composition it seems likely that they may also correlate with diagnosis and clinical behavior for a number of disease states. Examples of how this approach may prove useful in clinical practice are provided.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Temporal Heterogeneity in Apoptosis Determined by Imaging Flow Cytometry.

    PubMed

    Vorobjev, Ivan A; Barteneva, Natasha S

    2016-01-01

    Apoptotic process is highly heterogeneous, and a long-standing question is how many parameters define time and reversibility of the apoptotic response at a population and single-cell levels. Cell death analysis applications have greatly expanded since the introduction of flow cytometry. Classical approach for evaluation of apoptosis is en masse analysis of cells treated with different stimuli, but these methods cannot demonstrate heterogeneity in the population. Single-cell heterogeneity is now usually assessed by multicolor fluorescence microscopy; however obtaining reasonable statistics is time consuming and laborious. Therefore we combined flow cytometry, imaging flow cytometry, and fluorescent microscopy to characterize at a single-cell and population level sequence of apoptotic events induced by a variety of treatments (Vorobjev, Barteneva, J Histochem Cytochem 63:494-510, 2015). We show that simultaneous use of membrane potential dye TMRE, caspases 3/7 sensor, Annexin V and nuclear staining along with morphological parameters demonstrate heterogeneity of the whole process and is a valuable method for quantitative study of the apoptosis execution. Imaging flow cytometry allowed us to analyze correlation between TMRE, caspases 3/7, and Annexin V staining and morphological characteristics providing valuable information on the process of apoptotic execution. Importantly, comparisons of different data sets obtained by three methods allowed us to achieve temporal resolution of the whole process superior to that had been obtained by only one method. PMID:27460249

  12. Temporal Heterogeneity in Apoptosis Determined by Imaging Flow Cytometry.

    PubMed

    Vorobjev, Ivan A; Barteneva, Natasha S

    2016-01-01

    Apoptotic process is highly heterogeneous, and a long-standing question is how many parameters define time and reversibility of the apoptotic response at a population and single-cell levels. Cell death analysis applications have greatly expanded since the introduction of flow cytometry. Classical approach for evaluation of apoptosis is en masse analysis of cells treated with different stimuli, but these methods cannot demonstrate heterogeneity in the population. Single-cell heterogeneity is now usually assessed by multicolor fluorescence microscopy; however obtaining reasonable statistics is time consuming and laborious. Therefore we combined flow cytometry, imaging flow cytometry, and fluorescent microscopy to characterize at a single-cell and population level sequence of apoptotic events induced by a variety of treatments (Vorobjev, Barteneva, J Histochem Cytochem 63:494-510, 2015). We show that simultaneous use of membrane potential dye TMRE, caspases 3/7 sensor, Annexin V and nuclear staining along with morphological parameters demonstrate heterogeneity of the whole process and is a valuable method for quantitative study of the apoptosis execution. Imaging flow cytometry allowed us to analyze correlation between TMRE, caspases 3/7, and Annexin V staining and morphological characteristics providing valuable information on the process of apoptotic execution. Importantly, comparisons of different data sets obtained by three methods allowed us to achieve temporal resolution of the whole process superior to that had been obtained by only one method.

  13. DNA image cytometry in acquired immune deficiency syndrome (AIDS).

    PubMed

    Auffermann, W; Krueger, G R; Böcking, A

    1986-03-01

    In nine cases with the acquired immune deficiency syndrome (AIDS), including four stage I cases, three stage II cases and two stage III cases, DNA image cytometry was performed on Feulgen-stained lymph node imprint smears. Diploidy was found in three cases, tetraploidy in three cases and octoploidy in two cases. Aneuploid DNA distribution patterns were not seen. The lymphoid cells showed an enormously increased proliferation rate. Two cases in stage I revealed characteristic intranuclear DNA inclusions in lymphoid cells. These results indicate that DNA image cytometry may be useful as an adjunct to surgical pathology in certain cases to assist in the differential diagnosis between AIDS and benign conditions of the lymphoid system as well as between AIDS and malignant lymphomas, which usually have aneuploid DNA patterns.

  14. Optofluidic fluorescent imaging cytometry on a cell phone.

    PubMed

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

    2011-09-01

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

  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. The century for cytopathology.

    PubMed

    Naylor, B

    2000-01-01

    By the end of the 19th century, exfoliated cancer cells had been described in all of the types of specimen in which we find them today. However, it was not until Drs. Papanicolaou and Traut published their account of the diagnosis of uterine cancer from exfoliated cells (1941 and 1943) that cytopathology acquired the momentum to develop into the powerful presence that it has in human medicine today. These and the subsequent publications by Papanicolaou stimulated the development and application of cytopathology worldwide, resulting in abundant literature on the subject and a galaxy of outstanding practitioners. The 1980s saw the development and widespread use of aspiration cytology. This was followed in the 1990s by the development of automated screening systems, marking the latest stage in the evolution of cytopathology. These and other events and achievements in cytopathology, from its meager beginnings in the early 20th century to its worldwide use and acceptance today, mark this century as the "century for cytopathology."

  17. Label-free high-throughput imaging flow cytometry

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  18. Diagnostic and prognostic value of DNA image cytometry in myelodysplasia.

    PubMed Central

    Auffermann, W; Fohlmeister, I; Böcking, A

    1988-01-01

    The DNA content of erythropoietic cells from 10 patients with refractory anaemia (RA) with megaloblastic changes, who subsequently developed acute non-lymphoblastic leukaemia (ANL), and from seven patients with megaloblastic marrow aspirates due to pernicious anaemia were compared by DNA image cytometry. The DNA distribution, the rate of aneuploid cells exceeding 5c (5cER), and the square deviation index of DNA values from the normal 2c-peak (2cDI) were recorded. Both variables were of diagnostic and prognostic importance for epithelial tumours, malignant lymphomas, and dysplastic lesions. A rate of 5cER greater than 0 was found in eight of 10 myelodysplastic, but in none of seven control cases. Hypodiploidy was equally pronounced in both groups of patients. The 5cE had the highest discriminative value of all variables calculated. The 2cDI was not significantly different in either group. In pernicious anaemia the 2cDI depended mainly on the percentage of S cells, reflecting the defect of DNA synthesis. In RA with megaloblastosis the 2cDI correlated with the percentage of G2 cells, reflecting G2 arrest. In the myelodysplastic group the 2cDI correlated positively with the length of time until ANL developed, indicating the prognostic relevance of 2cDI. Our findings show that in megaloblastic anaemia DNA image cytometry can distinguish myelodysplasia from pernicious anaemia and that it also provides prognostic information. PMID:3384994

  19. 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.

  20. Image Cytometry Data From Breast Lesions Analyzed using Hybrid Networks.

    PubMed

    Mat Sakim, H A; Mat Isa, N A; G Naguib, Raouf; Sherbet, Gajanan

    2005-01-01

    The treatment and therapy to be administered on breast cancer patients are dependent on the stage of the disease at time of diagnosis. It is therefore crucial to determine the stage at the earliest time possible. Tumor dissemination to axillary lymph nodes has been regarded as an indication of tumor aggression, thus the stage of the disease. Neural networks have been employed in many applications including breast cancer prognosis. The performance of the networks have often been quoted based on accuracy and mean squared error. In this paper, the performance of hybrid networks based on Multilayer Perceptron and Radial Basis Function networks to predict axillary lymph node involvement have been investigated. A measurement of how confident the networks are with respect to the results produced is also proposed. The input layer of the networks include four image cytometry features extracted from fine needle aspiration of breast lesions. The highest accuracy achieved by the hybrid networks was 69% only. However, most of the correctly predicted cases had a high confidence level.

  1. [Breast cytopathology bestiary].

    PubMed

    Bretz-Grenier, Marie-Françoise

    2006-10-01

    Breast cytopathology of tubular and lobular carcinomas may be very difficult. Indeed, the classical morphological signs of malignancy are often scarce or absent; the identification of special patterns of cell clusters may therefore be very useful to reach a diagnosis. Following the review of many cases of cytological smears with histological confirmation, a series of peculiar cytological aspects (often mimicking little animals such as butterfly, eagle, dog, lizard, fish, rabbit...) were identified and found helpful to report here from a diagnostic point of view.

  2. [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

  3. 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

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

    PubMed Central

    Samsel, Leigh; McCoy, J Philip

    2015-01-01

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

  5. Automated identification of circulating tumor cells by image cytometry.

    PubMed

    Scholtens, Tycho M; Schreuder, Frederik; Ligthart, Sjoerd T; Swennenhuis, Joost F; Greve, Jan; Terstappen, Leon W M M

    2012-02-01

    Presence of circulating tumor cells (CTC), as detected by the CellSearch System, in patients with metastatic carcinomas is associated with poor survival prospects. CellTracks TDI, a dedicated image cytometer, was developed to improve the enumeration of these rare CTC. The CellSearch System was used to enumerate CTC in 7.5 mL blood of 68 patients with cancer and 9 healthy controls. Cartridges containing the fluorescently labeled CTC from this system were reanalyzed using the image cytometer, which acquires images with a TDI camera using a 40×/0.6 NA objective and lasers as light source. Automated classification of events was performed by the Random Forest method using Matlab. An automated classifier was developed to classify events into CTC, apoptotic CTC, CTC debris, leukocytes, and debris not related to CTC. A high agreement in classification was obtained between the automated classifier and five expert reviewers. Comparison of images from the same events in CellTracks TDI and CellTracks Analyzer II shows improved resolution in fluorescence images and improved classification by adding bright-field images. Improved detection efficiency for CD45-APC avoids the classification of leukocytes nonspecifically binding to cytokeratin as CTC. The correlation between number of CTC detected in CellTracks TDI and CellTracks Analyzer II is good with a slope of 1.88 and a correlation coefficient of 0.87. Automated classification of events by CellTracks TDI eliminates the operator error in classification of events as CTC and permits quantitative assessment of parameters. The clinical relevance of various CTC definitions can now be investigated.

  6. Classification of retinopathic injury using image cytometry and vasculature complexity

    NASA Astrophysics Data System (ADS)

    Staniszewski, K.; Sepehr, R.; Sorenson, C. M.; Sheibani, N.; Ranji, M.

    2012-03-01

    Retinopathic injuries are a common symptom of many diseases. However, if detected early, much of the damage caused by these injuries can be prevented, or in some cases reversed. In this study, images of retinas were classified as normal or injured using the vascular cell count, vasculature coverage, and vessel caliber. To model retinal vasculopathies, retinal vasculature from mice with the BCL-2 gene either partially or completely knocked out were compared. The bcl-2 gene is a critical regulator of apoptosis and angiogenesis, and therefore its absence has a significant impact on the number of vascular cells and vasculature complexity. When the aforementioned features were extracted from the images, classification was performed using a majority vote between a linear classifier, k-nearest-neighbors classification, and a support vector machine. This resulted in a classification accuracy of 81% using the "leave one out" error determination method.

  7. 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

  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. 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

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

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

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

  11. 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.

  12. 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.

  13. 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}.

  14. An automated image cytometry system for monitoring DNA ploidy and other cell features of radiotherapy and chemotherapy patients.

    PubMed

    Zhang, Y; LeRiche, J C; Jackson, S M; Garner, D; Palcic, B

    1999-01-01

    DNA content and distribution in cell nuclei were studied in samples of fine-needle aspiration (FNA) from 27 locally advanced breast and head and neck cancers in two going randomized trials that compared accelerated fractionation to standard fractionation radiation in locally advanced breast cancer and head and neck cancer. Two image cytometry methods were compared: a new, fully automated DNA image cytometry system (AIC) and a conventional image cytometry (CIC) system with manual selection, focusing, and segmentation of cells. The results of both techniques were compared on the basis of DNA histogram parameters including DNA index (DI), mean DNA values (MDV), and Auer's DNA histogram patterns. An excellent correlation was achieved between the two imaging techniques in terms of DI (r=0.985, p<0.001) and MDV (r=0.951, p<0.001) as well as between Auer's histogram patterns, where both methods agreed completely. It was concluded in these analyses that the two image cytometry methods were equivalent. However, the AIC offered an advantage by scanning samples in a fully automated way, which represented significant time saving for cytopathologists working with the system, as well as a larger number of cells used in the automated analysis. With the automated image cytometer, 500 relevant cells were collected and analyzed in about 10 minutes, where with the interactive (manual) method, it took typically an hour to collect and analyze only about 250 cells. Seventeen samples were sufficient for flow analysis. Image cytometry and flow cytometry showed good agreement in DI determination; however, three cases reported as diploid by flow cytometry were found to be aneuploid by image cytometry techniques.

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

    PubMed Central

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

    2013-01-01

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

  16. 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

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

    PubMed

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

    2013-06-01

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

  18. 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

  19. Detection of DNA Strand Breaks in Apoptotic Cells by Flow- and Image-Cytometry

    PubMed Central

    Darzynkiewicz, Zbigniew; Zhao, Hong

    2010-01-01

    Extensive DNA fragmentation that generates a multitude of DNA double-strand breaks (DSBs) is a hallmark of apoptosis. A widely used approach to identify apoptotic cells relies on labeling DSBs in situ with fluorochromes. Flow or image cytometry is then used to detect and quantify apoptotic cells labeled this way. We developed several variants of the methodology that is based on the use of exogenous terminal deoxynucleotidyl transferase (TdT) to label 3′-OH ends of the DSBs with fluorochromes, defined as the TUNEL assay. This chapter describes the variant based on DSBs labeling using 5-Bromo-2′-deoxyuridine-5′-triphosphate (BrdUTP) as a TdT substrate and the incorporated BrdU is subsequently detected immunocytochemically with anti-BrdU antibody. We also describe modifications of the protocol that allow using other than BrdUTP deoxyribonucleotides to label DSBs. Concurrent differential staining of cellular DNA and multiparameter analysis of cells by flow- or image cytometry enables one to correlate the induction of apoptosis with the cell cycle phase. Examples of the detection of apoptotic cells in cultures of human leukemic cell lines treated with TNF-α and DNA topoisomerase I inhibitor topote-can are presented. The protocol can be applied to the cells growing in vitro, treated ex vivo with cytotoxic drugs as well as to clinical samples. PMID:21057923

  20. Cytopathological Diagnosis of an Unusual Cause of Malignant Hydrocele

    PubMed Central

    Jain, Ankur; Khadwal, Alka; Prakash, Gaurav; Gupta, Nalini; Varma, Subhash; Malhotra, Pankaj

    2016-01-01

    Testicular involvement in a case of acute lymphoblastic leukemia (ALL) is well reported, but occurrence of “isolated” malignant hydrocele is extremely uncommon. We herein report a case of a 22-year-old man who presented to our hematology clinic with fever and easy fatiguability of 2 weeks’ duration. Examination revealed pallor, cervical lymphadenopathy, and bilateral scrotal swellings. He was diagnosed as a case of Philadelphia-positive ALL (B-cell type) based on peripheral smear, bone marrow examination, and flow cytometry of the marrow aspirate. Ultrasonography of scrotum revealed bilateral hydrocele with normal testes. Cytopathological analysis of the hydrocele fluid showed the presence of lymphoblasts. The patient was treated with modified BFM-90 protocol along with imatinib mesylate (600 mg/day). He achieved complete remission with a minimal residual disease of <0.001% at the end of induction therapy. However, the hydrocele persisted and a repeat cytological examination of the aspirate did not reveal any lymphoblasts. The patient was treated with consolidation (high-dose methotrexate), bilateral testicular irradiation, and re-induction following which the hydrocele disappeared. The patient is currently on maintenance phase of BFM-90 protocol and is alive at one year of follow-up. Contiguous spread from the subclinical testicular involvement is hypothesized as the mechanism for development of hydrocele in the current case. The role of cytopathology in the early diagnosis of testicular involvement in ALL is emphasized here. PMID:27721665

  1. 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.

  2. Real-time Fourier transform spectrometry for fluorescence imaging and flow cytometry

    SciTech Connect

    Buican, T.N.

    1990-01-01

    We present a Fourier transform (FT) spectrometer that is suitable for real-time spectral analysis in fluorescence imaging and flow cytometry. The instrument consists of a novel type of interferometer that can be modulated at frequencies of up to 100 kHz and has a high light throughput; and a dedicated, parallel array processor for the real-time computation of spectral parameters. The data acquisition array processor can be programmed by a host computer to perform any desired linear transform on the interferogram and can thus separate contributions from multiple fluorescence microscopy. The integration of a flow cytometer and a spectral imaging fluorescence microscope is discussed, and the concepts of direct and reversed virtual sorting'' are introduced. 9 refs., 8 figs.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

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

    PubMed

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

    2012-09-19

    Imaging flow cytometry was used to analyze the self-assembly of DNA-conjugated polystyrene microspheres. This technique enables quantitative analysis of the assembly process and thereby enables detailed analysis of the effect of structural and process variables on the assembly yield. In a demonstration of the potential of this technique, the influence of DNA strand base pair (bp) length was examined, and it was found that 50 bp was sufficient to drive the assembly of microspheres efficiently, forming not only dimers but also chainlike structures. The effect of stoichiometry on the yield was also examined. The analysis demonstrated that self-assembly of 50 bp microspheres can be driven nearly to completion by stoichiometric excess in a manner similar to Le Chatelier's principle in common chemical equilibrium. PMID:22938015

  10. 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

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2016-10-01

    Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model. PMID:27577979

  13. 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

  14. Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

    PubMed

    Yason, John Anthony; Tan, Kevin Shyong Wei

    2015-01-01

    Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms. PMID:26618361

  15. Overestimation of heterotrophic bacteria in the Sargasso Sea: direct evidence by flow and imaging cytometry

    NASA Astrophysics Data System (ADS)

    Sieracki, Michael E.; Haugen, Elin M.; Cucci, Terry L.

    1995-08-01

    Accurate measurements of bacterial biomass in the ocean are needed for modeling marine microbial food webs and global biogeochemical cycling. We present direct evidence that previous estimates of heterotrophic bacteria biomass in the oligotrophic ocean are confounded by the presence of the abundant photosynthetic procaryote, Prochlorococcus. The chlorophyll autofluorescence of these photosynthetic bacterial cells is very faint and fades rapidly under epifluorescence microscopy. Detection and enumeration of these cells thus far has almost exclusively been by flow cytometry. Using a cooled, charge-coupled device (CCD) camera we were able to image these cells for direct biovolume measurements. A double-exposed image of DAPI-stained Prochlorococcus cells shows that they are indistinguishable from heterotrophic bacteria in standard slide preparations. At two Sargasso Sea stations Prochlorococcus could cause an overestimation of surface (top 150 m) integrated heterotrophic bacterial biovolume (biomass) of 18 and 22% determined by standard microscope methods. At the subsurface chlorophyll maximum Prochlorococcus was 33 and 43% of the heterotrophic bacterial biovolume (biomass) at these stations. Prochlorococcus cell size increased from 0.05 μm 3 in the surface mixed layer to about 0.2 μm 3 below 100 m, confirming previous interpretations of flow cytometric light scatter measurements. Shifting biomass from the heterotrophic bacteria pool to the primary producer compartment has significant implications for ecosystem structure and trophic transfer in marine food webs.

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

    PubMed

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

    2011-08-01

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

  17. Biological monitoring of chemical exposure in nickel workers by imaging cytometry (ICM) of nasal smears.

    PubMed

    Reith, A K; Reichborn-Kjennerud, S; Aubele, M; Jütting, U; Gais, P; Burger, G

    1994-01-01

    Highly sensitive and inexpensive methods that are not time consuming are desirable for monitoring the workplace environment for the detection of cytotoxic hazards, particularly cancerous risks. It is possible to detect precancerous and cancerous lesions in samples taken by brushing the nose, but the cytological diagnoses can be affected by an inability to obtain representative smears from the sometimes very small focal lesions, and uncertainties in the subjective interpretation of suspicious cells when these are small in number. In an attempt to improve diagnosis we applied imaging cytometry (ICM) and tested the concept of malignancy-associated changes (MAC) in routinely Papanicolaou-stained smears. Cells of non-goblet type that visually appeared normal were selected from nickel workers with and without dysplastic lesions of the nasal mucosa. A set of nuclear features was measured by ICM and used for discriminant analysis. We were able to differentiate between workers with non-dysplastic normal and suspicious mucosa smears and those with dysplastic lesions. Unexpectedly, it was found possible to distinguish between workers in the roasting/smelting and the electrolysis departments, who were exposed to different carcinogenic nickel compounds. A further surprising finding was the possibility to distinguish smokers and non-smokers among the nickel workers. PMID:8130134

  18. Biological monitoring of chemical exposure in nickel workers by imaging cytometry (ICM) of nasal smears.

    PubMed

    Reith, A K; Reichborn-Kjennerud, S; Aubele, M; Jütting, U; Gais, P; Burger, G

    1994-01-01

    Highly sensitive and inexpensive methods that are not time consuming are desirable for monitoring the workplace environment for the detection of cytotoxic hazards, particularly cancerous risks. It is possible to detect precancerous and cancerous lesions in samples taken by brushing the nose, but the cytological diagnoses can be affected by an inability to obtain representative smears from the sometimes very small focal lesions, and uncertainties in the subjective interpretation of suspicious cells when these are small in number. In an attempt to improve diagnosis we applied imaging cytometry (ICM) and tested the concept of malignancy-associated changes (MAC) in routinely Papanicolaou-stained smears. Cells of non-goblet type that visually appeared normal were selected from nickel workers with and without dysplastic lesions of the nasal mucosa. A set of nuclear features was measured by ICM and used for discriminant analysis. We were able to differentiate between workers with non-dysplastic normal and suspicious mucosa smears and those with dysplastic lesions. Unexpectedly, it was found possible to distinguish between workers in the roasting/smelting and the electrolysis departments, who were exposed to different carcinogenic nickel compounds. A further surprising finding was the possibility to distinguish smokers and non-smokers among the nickel workers.

  19. 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

  20. Cytopathologic diagnosis of liver mass lesions.

    PubMed

    Conrad, Rachel; Castelino-Prabhu, Shobha; Cobb, Camilla; Raza, Anwar

    2013-03-01

    The liver is a common site for metastatic malignancies, particularly from the gastrointestinal tract. It also may be involved by primary neoplasms, both benign and malignant. Cytopathologic examination of mass lesions of the liver with pertinent use of ancillary studies is a useful method of establishing a correct diagnosis for patient management. The authors reviewed the literature for articles pertaining to cytologic characteristics of specific tumor types, utility of immunohistochemical markers and pertinent molecular studies, differential diagnoses and pitfalls.

  1. 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

  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. 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.

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

    PubMed Central

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

    2011-01-01

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

  5. Imaging and Mapping of Tissue Constituents at the Single-Cell Level Using MALDI MSI and Quantitative Laser Scanning Cytometry.

    PubMed

    Rawlins, Catherine M; Salisbury, Joseph P; Feldman, Daniel R; Isim, Sinan; Agar, Nathalie Y R; Luther, Ed; Agar, Jeffery N

    2015-01-01

    For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells-giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods. PMID:26542720

  6. 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

  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. 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

  9. Differential effects of depleting agents on cytoplasmic and nuclear non-protein sulphydryls: a fluorescence image cytometry study.

    PubMed Central

    Thomas, M.; Nicklee, T.; Hedley, D. W.

    1995-01-01

    The intracellular distribution of glutathione (GSH) was measured by a quantitative image cytometry method, using the sulphydryl-reactive agent mercury orange. This readily forms fluorescent adducts with GSH and other non-protein sulphydryls (NPSH), but reacts much more slowly with protein sulphydryls. Under optimum staining conditions mean integrated mercury orange fluorescence per cell was closely correlated with a standard biochemical assay for GSH. Use of the DNA dye DAPI as a counterstain allowed measurement of nuclear NPSH. The mean nuclear-cytoplasmic ratio was 0.57 +/- 0.05. Isolation of nuclei under aqueous conditions resulted in the loss of approximately 90% of mercury orange fluorescence, compared with nuclear fluorescence from intact cells, suggesting that background labelling of protein sulphydryls or other macromolecules is low. Depletion of GSH with N-ethylmaleimide or diethylmaleate decreased mercury orange fluorescence in the nucleus and cytoplasm to a similar extent. In contrast, mercury orange fluorescence in the nucleus was much more resistant to DL-buthionine-S,R-sulphoximine (BSO) depletion than that in the cytoplasm. This finding is compatible with a distinct pool of GSH in the nucleus that is comparatively resistant to BSO depletion. Alternatively, the retention of fluorescence in the nucleus following GSH depletion by BSO treatment might be due to accumulation of cysteine. These findings have implications for cancer treatment since the level of NPSH in the nucleus might be a more important determinant of resistance to DNA-damaging agents than that in cytoplasm. The image cytometry method described here is quantitative, allows a measure of tumour cell heterogeneity and can be applied to small biopsy samples obtained by fine-needle aspiration. Thus it appears suitable for prospective clinical studies in cancer patients, and for monitoring the effects of GSH-depleting agents used as adjuncts to cancer chemotherapy or radiotherapy. Images Figure

  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. [AIDS in a woman having had sexual relations with a patient with hemophilia A. Characteristic findings in DNA image cytometry].

    PubMed

    Schaar, H; Auffermann, W; Böcking, A; Franke, P; Pusztai-Markos, Z; Reininghaus, A; Schmitt, H

    1986-12-19

    A 37-year-old female patient reported marked weight loss, prolonged alopecia, recurrent infections and watery diarrhoea. Examination revealed Salmonella infection, candidiasis and immunological signs of previous toxoplasmosis. Between 1978 and 1981, the patient had had close sexual relations to a patient with haemophilia A. Due to this fact, AIDS was suspected. Serological tests for HIV were not available at the time. The findings in DNA image cytometry (nuclear DNA inclusion bodies, polyploid lymphocyte nuclei and binuclear lymphocytes) suggested a viral infection of the lymphoid cells. Electron microscopy revealed in hepatocytes and cerebral cells intranuclear inclusion bodies whose size and contents were not compatible with an infection caused by cytomegalovirus, herpes virus or Epstein-Barr virus. In autopsy, infections of various organ systems such as pneumonia, tracheobronchitis, urocystitis, pyelonephritis, Candida oesophagitis and enteritis were found.

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

    PubMed

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

    2014-01-01

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

  13. Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

    PubMed Central

    2013-01-01

    Background The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Results Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. Conclusion The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs. PMID:23388071

  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. Rapid analysis of white blood cells with diffraction imaging flow cytometry

    NASA Astrophysics Data System (ADS)

    Hu, Xin-Hua; Lu, Jun Q.; Feng, Yuanming

    2011-03-01

    From a foundation of cell optics research program we have recently developed a diffraction imaging flow cytometer method which can be used to rapidly acquire and analyze diffraction image data from single flowing cells. Modeling and experimental investigation of light scattering by white blood cells (WBC) have been conducted which suggest a strong correlation between the fringe patterns related to the texture of the diffraction images and 3D morphological features of the cells. In particular, we present results of calculated and measured diffraction images obtained with three cell lines derived from leukemia patients to demonstrate that texture features of the diffraction images extracted with a gray-scale co-occurrence-matrix algorithm can be used for rapid cell classification.

  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. 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.

  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. 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

  2. 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

  3. 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.

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

    PubMed

    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. PMID:26506467

  5. 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.

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2015-06-01

    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.

  8. 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.

  9. Electron microscopy renders the diagnostic capabilities of cytopathology more precise: an approach to everyday practice.

    PubMed

    Turbat-Herrera, Elba A; Herrera, Guillermo A

    2005-01-01

    Cytology is a powerful diagnostic tool but to make definitive diagnoses, the use of ancillary techniques is imperative. By combining immunohistochemistry (IHC) and electron microscopy (EM), cytologic diagnoses can be as precise as those of surgical pathology. In the authors' daily practice of cytopathology they use all ancillary techniques available to them: histochemistry, IHC, EM, flow cytometry, and molecular pathology. IHC is frequently used as an ancillary technique in their daily practice but EM is many times their technique of choice. By the use of EM the authors can make specific final diagnoses, make the diagnosis more definitive, narrow the differential diagnosis, or determine the origin of a neoplasm with unknown primary site. Specimens obtained by fine-needle aspiration as well as all body fluids are suitable for EM. The limiting factor is to obtain the appropriate material with the diagnostic cells for ultrastructural examination. The common diagnostic dilemmas in the everyday practice of cytology are the following: mesothelioma vs. adenocarcinoma, neuroendocrine differentiation or not, the distinction of melanoma from adenocarcinoma and sarcoma, hepatocellular carcinoma vs. adenocarcinoma, and the origin of adenocarcinomas of unknown primary. The authors discuss how they approach these diagnostic problems in their everyday practice and how they incorporate EM in solving them.

  10. 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

  11. 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

  12. 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

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

    PubMed Central

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

    2014-01-01

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

  14. Cancer detection by quantitative fluorescence image analysis.

    PubMed

    Parry, W L; Hemstreet, G P

    1988-02-01

    Quantitative fluorescence image analysis is a rapidly evolving biophysical cytochemical technology with the potential for multiple clinical and basic research applications. We report the application of this technique for bladder cancer detection and discuss its potential usefulness as an adjunct to methods used currently by urologists for the diagnosis and management of bladder cancer. Quantitative fluorescence image analysis is a cytological method that incorporates 2 diagnostic techniques, quantitation of nuclear deoxyribonucleic acid and morphometric analysis, in a single semiautomated system to facilitate the identification of rare events, that is individual cancer cells. When compared to routine cytopathology for detection of bladder cancer in symptomatic patients, quantitative fluorescence image analysis demonstrated greater sensitivity (76 versus 33 per cent) for the detection of low grade transitional cell carcinoma. The specificity of quantitative fluorescence image analysis in a small control group was 94 per cent and with the manual method for quantitation of absolute nuclear fluorescence intensity in the screening of high risk asymptomatic subjects the specificity was 96.7 per cent. The more familiar flow cytometry is another fluorescence technique for measurement of nuclear deoxyribonucleic acid. However, rather than identifying individual cancer cells, flow cytometry identifies cellular pattern distributions, that is the ratio of normal to abnormal cells. Numerous studies by others have shown that flow cytometry is a sensitive method to monitor patients with diagnosed urological disease. Based upon results in separate quantitative fluorescence image analysis and flow cytometry studies, it appears that these 2 fluorescence techniques may be complementary tools for urological screening, diagnosis and management, and that they also may be useful separately or in combination to elucidate the oncogenic process, determine the biological potential of tumors

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

    PubMed

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

    2014-03-01

    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.

  16. Laser Scanning Cytometry

    PubMed Central

    Pozarowski, Piotr; Holden, Elena; Darzynkiewicz, Zbigniew

    2013-01-01

    Summary The laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of analytical capabilities. Multilaser-excited fluorescence emitted from individual cells is measured at several wavelength ranges, rapidly (up to 5000 cells/min), with high sensitivity and accuracy. The following applications of LSC are reviewed: (1) identification of cells that differ in degree of chromatin condensation (e.g., mitotic or apoptotic cells or lymphocytes vs granulocytes vs monocytes); (2) detection of translocation between cytoplasm vs nucleus or nucleoplasm vs nucleolus of regulatory molecules such as NF- κB, p53, or Bax; (3) semiautomatic scoring of micronuclei in mutagenicity assays; (4) analysis of fluorescence in situ hybridization; (5) enumeration and morphometry of nucleoli; (6) analysis of phenotype of progeny of individual cells in clonogenicity assay; (7) cell immunophenotyping; (8) visual examination, imaging, or sequential analysis of the cells measured earlier upon their relocation, using different probes; (9) in situ enzyme kinetics and other time-resolved processes; (10) analysis of tissue section architecture; (11) application for hypocellular samples (needle aspirate, spinal fluid, etc.); (12) other clinical applications. Advantages and limitations of LSC are discussed and compared with flow cytometry. PMID:16719355

  17. Malignant solitary fibrous tumor in the extremity: Cytopathologic findings.

    PubMed

    Khanchel, Fatma; Driss, Maha; Mrad, Karima; Romdhane, Khaled Ben

    2012-04-01

    Malignant solitary fibrous tumor (SFT) is an extremely rare neoplasm. There are only rare published accounts of the cytopathologic features of this tumor. We report a case of a 59-year-old woman presented with a 10-year history of a right thigh mass. A preoperative fine needle aspiration (FNA) was performed. Smears were hypercellular, with cohesive and crowded tissue fragments, haphazard cell arrangements and many single cells. The tumor cells were polymorphous, plump spindled or round with often indented or bare nuclei. A differential diagnosis of low grade sarcoma was favored. The diagnosis of malignant SFT is extremely difficult on FNA and must be included in the differential diagnosis of spindle cell neoplasms.

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2015-08-01

    Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity. PMID:25967950

  20. Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes.

    PubMed

    Gerner, Michael Y; Kastenmuller, Wolfgang; Ifrim, Ina; Kabat, Juraj; Germain, Ronald N

    2012-08-24

    Flow cytometry allows highly quantitative analysis of complex dissociated populations at the cost of neglecting their tissue localization. In contrast, conventional microscopy methods provide spatial information, but visualization and quantification of cellular subsets defined by complex phenotypic marker combinations is challenging. Here, we describe an analytical microscopy method, "histo-cytometry," for visualizing and quantifying phenotypically complex cell populations directly in tissue sections. This technology is based on multiplexed antibody staining, tiled high-resolution confocal microscopy, voxel gating, volumetric cell rendering, and quantitative analysis. We have tested this technology on various innate and adaptive immune populations in murine lymph nodes (LNs) and were able to identify complex cellular subsets and phenotypes, achieving quantitatively similar results to flow cytometry, while also gathering cellular positional information. Here, we employ histo-cytometry to describe the spatial segregation of resident and migratory dendritic cell subsets into specialized microanatomical domains, suggesting an unexpected LN demarcation into discrete functional compartments. PMID:22863836

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. DNA image cytometry test for primary screening of esophageal cancer: a population-based multi-center study in high-risk areas in China

    PubMed Central

    Wang, Meng; Hao, Changqing; Ma, Qing; Song, Guohui; Ma, Shanrui; Zhao, Deli; Zhao, Lin; Li, Xinqing; Wei, Wenqiang

    2016-01-01

    Objective To evaluate the feasibility of DNA image cytometry (DNA-ICM) as a primary screening method for esophageal squamous cell cancer (ESCC). Methods A total of 5,382 local residents aged 40–69 years from three high-risk areas in China (Linzhou in Henan province, Feicheng in Shandong province and Cixian in Hebei province) from 2008 to 2011 were recruited in this population-based screening study. And 2,526 subjects declined to receive endoscopic biopsy examination with Lugol’s iodine staining, while 9 and 815 subjects were excluded from liquid-based cytology and DNA-ICM test respectively due to slide quality. Finally, 2,856, 5,373 and 4,567 subjects were enrolled in the analysis for endoscopic biopsy examination, liquid-based cytology and DNA-ICM test, respectively. Sensitivity (SE), specificity (SP), negative predictive values (NPV) and positive predictive values (PPV) as well as their 95% confidence intervals (95% CI) for DNA-ICM, liquid-based cytology and the combination of the two methods were calculated. Receiver operating characteristic (ROC) curves were applied to determine the cutoff point of DNA-ICM for esophageal cancer. Results DNA-ICM results were significantly correlative with esophageal cancer and precancer lesions (χ2=18.016, P<0.001). The cutoff points were 5,802, 5,803 and 8,002 based on dissimilar pathological types of low grade intraepithelial neoplasia (LGIN), high grade intraepithelial neoplasia (HGIN), and ESCC, respectively, and 5,803 was chosen in this study considering the SE and SP. The SE, SP, PPV, NPV of DNA-ICM test (cutoff point 5,803) combined with liquid-based cytology [threshold atypical squamous cells of undetermined significance (ASCUS)] were separately 72.1% (95% CI: 70.3%-73.9%), 43.3% (95% CI: 41.3%-45.3%), 22.8% (95% CI: 21.1%-24.5%) and 87.0% (95% CI: 85.7%-88.3%) for LGIN, 85.7% (95% CI: 84.3%-87.1%), 41.3% (95% CI: 39.3%-43.3%), 4.6% (95% CI: 3.8%-5.4%) and 98.9% (95% CI: 98.5%-99.3%) for HGIN, and 96.0% (95% CI: 95

  8. DNA image cytometry test for primary screening of esophageal cancer: a population-based multi-center study in high-risk areas in China

    PubMed Central

    Wang, Meng; Hao, Changqing; Ma, Qing; Song, Guohui; Ma, Shanrui; Zhao, Deli; Zhao, Lin; Li, Xinqing; Wei, Wenqiang

    2016-01-01

    Objective To evaluate the feasibility of DNA image cytometry (DNA-ICM) as a primary screening method for esophageal squamous cell cancer (ESCC). Methods A total of 5,382 local residents aged 40–69 years from three high-risk areas in China (Linzhou in Henan province, Feicheng in Shandong province and Cixian in Hebei province) from 2008 to 2011 were recruited in this population-based screening study. And 2,526 subjects declined to receive endoscopic biopsy examination with Lugol’s iodine staining, while 9 and 815 subjects were excluded from liquid-based cytology and DNA-ICM test respectively due to slide quality. Finally, 2,856, 5,373 and 4,567 subjects were enrolled in the analysis for endoscopic biopsy examination, liquid-based cytology and DNA-ICM test, respectively. Sensitivity (SE), specificity (SP), negative predictive values (NPV) and positive predictive values (PPV) as well as their 95% confidence intervals (95% CI) for DNA-ICM, liquid-based cytology and the combination of the two methods were calculated. Receiver operating characteristic (ROC) curves were applied to determine the cutoff point of DNA-ICM for esophageal cancer. Results DNA-ICM results were significantly correlative with esophageal cancer and precancer lesions (χ2=18.016, P<0.001). The cutoff points were 5,802, 5,803 and 8,002 based on dissimilar pathological types of low grade intraepithelial neoplasia (LGIN), high grade intraepithelial neoplasia (HGIN), and ESCC, respectively, and 5,803 was chosen in this study considering the SE and SP. The SE, SP, PPV, NPV of DNA-ICM test (cutoff point 5,803) combined with liquid-based cytology [threshold atypical squamous cells of undetermined significance (ASCUS)] were separately 72.1% (95% CI: 70.3%-73.9%), 43.3% (95% CI: 41.3%-45.3%), 22.8% (95% CI: 21.1%-24.5%) and 87.0% (95% CI: 85.7%-88.3%) for LGIN, 85.7% (95% CI: 84.3%-87.1%), 41.3% (95% CI: 39.3%-43.3%), 4.6% (95% CI: 3.8%-5.4%) and 98.9% (95% CI: 98.5%-99.3%) for HGIN, and 96.0% (95% CI: 95

  9. Discrepancy analysis, communication, and feedback for cytotechnologist quality improvement of nongynecologic cytopathology.

    PubMed

    Whigham, Peggy; Ilario, Marius John Marc; Flanagan, Melina B; Mauser, Nancy; Raab, Stephen S; Ohori, N Paul

    2006-04-01

    The Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) detail the requirements for the cytotechnologist (CT) who evaluates gynecologic cytopathology specimens. However, the role of the CT in nongynecologic cytopathology is not clearly defined. Furthermore, non gynecologic cytopathology cases are diverse and the screening, interpretative, and diagnostic issues may be quite different from the gynecologic cases. At our institution, the CT and pathologist review nongynecologic cytopathology cases. Since CLIA '88 does not require the CT to screen nongynecologic cytopathology cases, there are few guidelines for quality assessment or quality improvement for the CT regarding nongynecologic cytopathology cases. To provide better understanding of the expectations of the CT and the needs of the pathologist, we developed a system comparing the CT's interpretation to the pathologist's interpretation as a means for enhanced communication and feedback. Using our Laboratory Information System (LIS), we generate a daily report that lists all cases with discrepancy in diagnoses between the CT and pathologist. The general supervisor reviews this report for diagnostic discrepancy in each case. To determine the degree of discrepancy, numerical values are assigned to each primary interpretation. Minor discrepancies are defined as differences less than +/-2.0. Major discrepancies are defined as differences greater than or equal to +/-2.0. For the entire laboratory, the overall percentage of concordant cases was consistently above 80% for each of the 6 mo analysis. Regarding the monthly discrepancies, the proportion of minor discrepancies ranged from 11.09% to 15.44% and the proportion of major discrepancies ranged from 1.40% to 3.56%. The frequency distribution of discrepancies by degree approximates a normal (Gaussian) curve and serves as baseline information that may be used for comparison when there are changes in practice or personnel. The CTs attend slide review sessions

  10. Uses of flow cytometry in virology.

    PubMed Central

    McSharry, J J

    1994-01-01

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

  11. Cytopathology of the pancreatobiliary tract-the agony, and sometimes, the ease of it.

    PubMed

    Conrad, Rachel; Castelino-Prabhu, Shobha; Cobb, Camilla; Raza, Anwar

    2013-06-01

    Pancreatic cytopathology is recognized as a rapid, reliable, safe and cost-beneficial modality of investigation of pancreatic mass lesions. Optimal cytodiagnosis depends on multiple factors including sample quality, and expertise of the cytopathologist and endoscopist. This article discusses key cytologic features of specific tumor types, specimen handling, differential diagnoses and pitfalls.

  12. 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.

  13. 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.

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

    PubMed

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

    2014-01-01

    The Papanicolaou Society of Cytopathology has developed a set of guidelines for pancreatobiliary cytology, including indications for endoscopic ultrasound guided fine-needle aspiration biopsy, techniques of the endoscopic retrograde cholangiopancreatography, terminology and nomenclature of pancreatobiliary disease, ancillary testing, and postbiopsy management. All documents are based on the expertise of the authors, a review of literature, discussions of the draft document at several national and international meetings over an 18 month period and synthesis of online comments of the draft document on the Papanicolaou Society of Cytopathology website [www.papsociety.org]. This document presents the results of these discussions regarding the use of sampling techniques in the cytological diagnosis of biliary and pancreatic lesions. This document summarizes the current state of the art for techniques in acquiring cytology specimens from the biliary tree as well as solid and cystic lesions of the pancreas.

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

    PubMed

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

    2015-04-01

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

  16. Guidelines for pancreaticobiliary cytology from the Papanicolaou Society of Cytopathology: A review.

    PubMed

    Pitman, Martha B; Layfield, Lester J

    2014-06-01

    The newest installment on state-of-the-art standards of practice in cytopathology from the Papanicolaou Society of Cytopathology (PSC) focuses on the pancreaticobiliary system. Similar to the National Cancer Institute recommendations for aspiration cytology of the thyroid, the PSC guidelines for pancreaticobiliary cytology addresses indications, techniques, terminology and nomenclature, ancillary studies, and postprocedure management. Each committee was composed of a multidisciplinary group of experts in diagnosing, managing, and treating patients with pancreaticobiliary disease. Draft documents were posted on an interactive Web-based forum hosted by the PSC Web site (www.papsociety.org) and the topics of terminology, ancillary testing, and management were presented at national and international meetings over an 18-month period for discussion and feedback from practicing pathologists around the world. This review provides a synopsis of these guidelines.

  17. Near infrared lasers in flow cytometry.

    PubMed

    Telford, William G

    2015-07-01

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

  18. 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 ...

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. A cytopathological approach to diagnosing intrathoracic lymphadenopathy using aspirates obtained by the transbronchial needle aspiration method.

    PubMed

    Özyalvaçlı, Gülzade; Yaşar, Zehra; Çetinkaya, Erdoğan

    2016-03-01

    Transbronchial needle aspiration (TBNA) is an effective, safe and cost-effective technique that allows for sampling of the mediastinal lymph node and peribronchial lesions. It is used in bronchogenic carcinoma staging, peribronchial and submucosal lesions, diagnosis of sarcoidosis and tuberculosis, differentiating submucosal invasion, and in diagnosing mediastinal masses. From our experience at the University of Abant Izzet Baysal and from a review of the literature, we discuss the adequacy and the differential diagnosis of aspiration material obtained by TBNA and cytopathological-histopathological evaluation in intrathoracic lymphadenopathies to increase the success rate of the TBNA method. PMID:27266286

  5. Small lasers in flow cytometry.

    PubMed

    Telford, William G

    2004-01-01

    Laser technology has made tremendous advances in recent years, particularly in the area of diode and diode-pumped solid state sources. Flow cytometry has been a direct beneficiary of these advances, as these small, low-maintenance, inexpensive lasers with reasonable power outputs are integrated into flow cytometers. In this chapter we review the contribution and potential of solid-state lasers to flow cytometry, and show several examples of these novel sources integrated into production flow cytometers. Technical details and critical parameters for successful application of these lasers for biomedical analysis are reviewed. PMID:14976380

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

    PubMed

    Meimaridou, Anastasia; Haasnoot, Willem; Shelver, Weilin L; Franek, Milan; Nielen, Michel W F

    2013-01-01

    Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays require a flow cytometer with sophisticated fluidics and optics. A new imaging super-paramagnetic SEMs-based alternative platform transports SEMs with considerably less fluid volume into a measuring chamber. Once there SEMs are held in a monolayer by a magnet. Light-emitting diodes (LEDs) are focused on the chamber to illuminate the SEMs - instead of lasers and they are imaged by a charge-coupled device (CCD) detector, offering a more compact sized, transportable and affordable system. The feasibility of utilising this system to develop a 3-plex SEMs-based imaging immunoassay (IMIA) for the screening of persistent organic pollutants (POPs) was studied. Moreover the performance characteristics of 3-plex IMIA were critically compared with the conventional 3-plex flow cytometric immunoassay (FCIA). Both SEM technologies have potential for the multiplex analysis of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) in buffer and fish extract with insignificant differences in assay sensitivities. Furthermore, we developed a faster and simpler, modified QuEChERS-like generic POPs extraction from tilapia fillet using sodium hydrogen carbonate as one of the salt additives and dispersive solid-phase extraction (dSPE) as a clean-up. Finally, a preliminary in-house validation using 40 different blank and spiked tilapia fillet samples was performed in both systems and the results obtained were critically compared. The lower-cost imaging SEMs-based system performed similarly to the original flow cytometer and, in combination with the new quicker QuEChERS-like extraction, it has high potential for future rapid screening of POPs in several other sample matrices such as other fish species, vegetable refined oils and environmental samples.

  7. 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

  8. Rise of the micromachines: microfluidics and the future of cytometry.

    PubMed

    Wlodkowic, Donald; Darzynkiewicz, Zbigniew

    2011-01-01

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

  9. 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

  10. The prognostic significance of determining DNA content in breast cancer by DNA image cytometry: the role of high grade aneuploidy in node negative breast cancer

    PubMed Central

    Yildirim‐Assaf, Selma; Coumbos, Alexandra; Hopfenmüller, Werner; Foss, Hans‐Dieter; Stein, Harald; Kühn, Wolfgang

    2007-01-01

    Aim To investigate the role of DNA aneuploidy, particularly in patients with node negative breast cancer, in order to identify the different risk profiles within the pool of heterogeneous breast cancers. Methods Imprint smears from 370 breast carcinomas were Feulgen‐stained and measured by DNA image analysis. DNA aneuploidy was graded by the amount of aneuploid cells (DNA content >5c) and highly aneuploid cells (DNA content >9c) in a breast tumour population. These results were correlated to the clinical long‐term follow‐up. A statistical cut‐off value of >10 aneuploid cells (>5c) and of >1 highly aneuploid cell (>9c) was evaluated as significant for disease‐free survival (DFS) and overall survival (OS). Results Subgroups among patients with breast cancer with aneuploid cells below the cut‐off value showed a significantly longer DFS and OS than those with aneuploid cells above this value. Patients with node negative breast cancer with >10 aneuploid cells (>5c) and >1 highly aneuploid cell (>9c) showed an unfavourable prognosis similar to patients with node positive breast cancer with <10 aneuploid cells (>5c) and <1 highly aneuploid tumour cell (>9c) in DFS and OS. Conclusion Nuclear DNA content, as an objective marker of tumour aggressiveness, provides prognostic information in patients with both node negative and node positive breast cancer. Based on DNA aneuploidy, the clinically inhomogeneous group of patients with node negative breast cancer can be stratified into low‐risk and high‐risk subgroups. Therefore, DNA ploidy analysis may identify high‐risk patients with lymph node negative breast cancer who might benefit from additional adjuvant therapy. PMID:17557867

  11. Teaching phagocytosis using flow cytometry.

    PubMed

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

    2004-05-01

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

  12. 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

  13. Measuring DNA content by flow cytometry in fission yeast.

    PubMed

    Sabatinos, Sarah A; Forsburg, Susan L

    2015-01-01

    Flow cytometry is an essential tool to monitor DNA content and determine cell cycle distribution. Its utility in fission yeast reflects the ease of sample preparation, the stochiometric binding of the most popular DNA dyes (propidium iodide and Sytox Green), and ability to monitor cell size. However, the study of DNA replication with multicolour flow analysis has lagged behind its use in mammalian cells. We present basic and advanced protocols for analysis of DNA replication in fission yeast by flow cytometry including whole cell, nuclear "ghosts," two-color imaging with BrdU, and estimates of DNA synthesis using EdU.

  14. Solitary fibrous tumor of the thyroid: cytopathologic findings and differential diagnosis.

    PubMed

    Parwani, Anil V; Galindo, Rene; Steinberg, David M; Zeiger, Martha A; Westra, William H; Ali, Syed Z

    2003-04-01

    Solitary fibrous tumor (SFT) is an uncommon mesenchymal neoplasm that arises primarily from the pleura. Extrapleural occurrences are rare. To our knowledge, there is no published account of this entity in the thyroid in the cytopathology literature. We report the case of a 61-yr-old man who was evaluated at The Johns Hopkins Hospital for a slow-growing thyroid mass that was present for 2 yr despite thyroid hormone suppression. Thyroid-stimulating hormone (TSH) was within normal limits. The patient underwent ultrasound-guided fine-needle aspiration (FNA), which showed predominantly discohesive slender spindle-shaped cells and fragments of collagenized stromal tissue. After the FNA diagnosis of "thyroid neoplasm" was made, the patient underwent a near-total thyroidectomy, which revealed a SFT. Differential diagnosis of spindle cell lesions in thyroid is also presented.

  15. Cytopathological features of villous adenoma of the urinary bladder in urine: A rare case report.

    PubMed

    Ishikawa, Ryou; Kadota, Kyuichi; Hayashi, Toshitetsu; Motoyama, Mutsumi; Matsunaga, Toru; Miyai, Yumi; Katsuki, Naomi; Kushida, Yoshio; Haba, Reiji

    2016-07-01

    Villous adenoma of the urinary bladder is a rare tumor that histologically mimics its enteric counterpart. Patients with an isolated villous adenoma have an excellent prognosis, but associated adenocarcinomas can frequently be identified in them as well. There is no literature that discusses the cytopathologic features of villous adenoma. Here we report a case which was diagnosed as villous adenoma histologically, which has been followed up with urine cytology. In urine cytology, many mucin producing cells are recognized. Few cell clusters show glandular formation or arrangement along the basement membrane. When glandular cells with columnar mucin-filled goblet cells are seen in urine cytology, the presence of a primary glandular lesion of the urinary bladder, such as villous adenoma, should be considered possible. Diagn. Cytopathol. 2016;44:632-635. © 2016 Wiley Periodicals, Inc.

  16. Cytopathological features of villous adenoma of the urinary bladder in urine: A rare case report.

    PubMed

    Ishikawa, Ryou; Kadota, Kyuichi; Hayashi, Toshitetsu; Motoyama, Mutsumi; Matsunaga, Toru; Miyai, Yumi; Katsuki, Naomi; Kushida, Yoshio; Haba, Reiji

    2016-07-01

    Villous adenoma of the urinary bladder is a rare tumor that histologically mimics its enteric counterpart. Patients with an isolated villous adenoma have an excellent prognosis, but associated adenocarcinomas can frequently be identified in them as well. There is no literature that discusses the cytopathologic features of villous adenoma. Here we report a case which was diagnosed as villous adenoma histologically, which has been followed up with urine cytology. In urine cytology, many mucin producing cells are recognized. Few cell clusters show glandular formation or arrangement along the basement membrane. When glandular cells with columnar mucin-filled goblet cells are seen in urine cytology, the presence of a primary glandular lesion of the urinary bladder, such as villous adenoma, should be considered possible. Diagn. Cytopathol. 2016;44:632-635. © 2016 Wiley Periodicals, Inc. PMID:27121034

  17. Nonsebaceous lymphadenoma of the parotid gland: cytopathologic findings and differential diagnosis.

    PubMed

    Castelino-Prabhu, Shobha; Li, Qing Kay; Ali, Syed Z

    2010-02-01

    Lymphadenomas (sebaceous and nonsebaceous types) of the salivary glands are extremely uncommon benign neoplasms. There are rare published reports of cytopathologic characteristics of "nonsebaceous lymphadenomas" of the parotid gland. We report herein, the case of an 80-year-old female who was evaluated at The Johns Hopkins Hospital for a 4.0 cm, nontender, mobile asymptomatic left parotid mass present for 3 months. An ultrasound-guided fine-needle aspiration revealed a uniform population of cohesive basaloid-type cells associated with scant myxoid stroma and was interpreted as "epithelioid neoplasm with basaloid features." Subsequently, a superficial parotidectomy was performed, which revealed a nonsebaceous type lymphadenoma. The rarity of this neoplasm and its superficial resemblance to more common salivary gland neoplasms may present diagnostic issues on FNA.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

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

    PubMed

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

    2013-08-15

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

  4. 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.

  5. 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

  6. 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.

  7. Capture of Fluorescence Decay Times by Flow Cytometry

    PubMed Central

    Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

    2012-01-01

    In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263

  8. Cytometry: today's technology and tomorrow's horizons.

    PubMed

    Chattopadhyay, Pratip K; Roederer, Mario

    2012-07-01

    Flow cytometry has been the premier tool for single cell analysis since its invention in the 1960s. It has maintained this position through steady advances in technology and applications, becoming the main force behind interrogating the complexities of the immune system. Technology development was a three-pronged effort, including the hardware, reagents, and analysis algorithms to allow measurement of as many as 20 independent parameters on each cell, at tens of thousands of cells per second. In the coming years, cytometry technology will integrate with other techniques, such as transcriptomics, metabolomics, and so forth. Ongoing efforts are aimed at algorithms to analyse these aggregated datasaets over large numbers of samples. Here we review the development efforts heralding the next stage of flow cytometry. PMID:22391486

  9. Spaceflight Flow Cytometry: Design Challenges and Applications

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  10. Rapid titration of viruses by flow cytometry.

    PubMed

    Drayman, Nir; Oppenheim, Ariella

    2011-06-01

    Traditionally, the most common methods used to titrate virus stocks are the plaque assay and the hemagglutination assay. The protocol presented here is based on the detection of viral-expressed proteins in infected cells by flow cytometry. It is simpler and more rapid than the traditional plaque-forming assay and it enables high-throughput analyses.

  11. Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

    PubMed Central

    Lee, Wing-Kee; Dittmar, Thomas

    2014-01-01

    A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin. PMID:25407650

  12. 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

  13. 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.

  14. Measurement of intracellular ions by flow cytometry.

    PubMed

    Posey, Avery D; Kawalekar, Omkar U; June, Carl H

    2015-01-01

    Using flow cytometry, single-cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation. PMID:25827486

  15. 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.

  16. 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...

  17. Detection of Kinase Translocation Using Microfluidic Electroporative Flow Cytometry

    NASA Astrophysics Data System (ADS)

    Lu, Chang; Wang, Jun; Bao, Ning; Paris, Leela; Wang, Hsiang-Yu; Geahlen, Robert

    2008-03-01

    Translocation of a protein between different subcellular compartments is a common event during signal transduction in living cells. Detection of these events has been largely carried out based on imaging of a low number of cells and subcellular fractionation/Western blotting. These conventional techniques either lack the high throughput desired for probing an entire cell population or provide only the average behaviors of cell populations without information from single cells. Here we demonstrate a new tool, referred to as microfluidic electroporative flow cytometry, to detect the translocation of an EGFP-tagged tyrosine kinase, Syk, to the plasma membrane in B cells at the level of the cell population. We combine electroporation with flow cytometry and observe the release of intracellular kinase out of the cells during electroporation. We found that the release of the kinase was strongly influenced by its subcellular localization. Cells stimulated through the antigen receptor have a fraction of the kinase at the plasma membrane and retain more kinase after electroporation than do cells without stimulation and translocation. This tool will have utility for kinase-related drug discovery and tumor diagnosis and staging.

  18. 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

  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. 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

  1. 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

  2. Honey Bee Hemocyte Profiling by Flow Cytometry

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-03-01

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

  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. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Advances in flow cytometry for sperm sexing.

    PubMed

    Sharpe, J C; Evans, K M

    2009-01-01

    This review presents the key technological developments that have been implemented in the 20 years since the first reports of successful measurement, sorting, insemination and live births using flow cytometry as a proven physical sperm separation technique. Since the first reports of sexed sperm, flow technology efforts have been largely focused on improving sample throughput by increasing the rate at which sperm are introduced to the sorter, and on improving measurement resolution, which has increased the proportion of cells that can be reliably measured and sorted. Today, routine high-purity sorting of X- or Y-chromosome-bearing sperm can be achieved at rates up to 8000 s(-1) for an input rate of 40,000 X- and Y-sperms(-1). With current protocols, straws of sex-sorted sperm intended for use in artificial insemination contain approximately 2 x 10(6)sperm. The sort rate of 8000 sperms(-1) mentioned above corresponds to a production capacity of approximately 14 straws of each sex per hour per instrument. PMID:18950849

  11. [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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Application of the Bethesda System for Reporting Thyroid Cytopathology in the Eastern Province of Saudi Arabia: phase I pilot retrospective analysis.

    PubMed

    Al-Abbadi, Mousa A; Shareef, Sameera Q; Ali, Jassim A; Yousef, Mohammad M

    2013-01-01

    We analyzed and evaluated our adequacy rate and the classification of our thyroid aspirates using the Bethesda System for Reporting Thyroid Cytopathology (BSRTC). All thyroid fine needle aspirates that were collected or referred to our institution were reviewed and reclassified according to the BSRTC. The results were tabulated and analyzed. Those with histological resection were correlated with our revised cytopathological evaluation using the BSRTC. A total of 205 thyroid aspirates from 186 patients were reviewed. There were 149 females (80%) and 37 males (20%) ranging in age from 23 to 81 (average age 48) years. All slides were reclassified using the BSRTC. The previous interpretations were not consistent with any apparent standards. The nondiagnostic rate was found to be 22%. Five cases were considered false negative and were upgraded to a more serious category with higher risk of malignancy. The high unsatisfactory rates can be reduced by an adequacy interpretation at the time of the procedure. The risk of malignancy in our cohort increased with each increase in the BSRTC category (I-VI). Communication about and awareness of the BSRTC and its implications by all our clinicians is a prime target of this study and is still work in progress. Hopefully, this study will increase the awareness of the BSRTC and its intended benefits in our region.

  19. 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.

  20. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1999-04-01

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

  2. Laser Scanning Cytometry: Principles and Applications—An Update

    PubMed Central

    Pozarowski, Piotr; Holden, Elena; Darzynkiewicz, Zbigniew

    2012-01-01

    Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM. PMID:23027005

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Cytopathologic observations of the lung of adult newts (Cynops pyrrhogaster) on-board the space shuttle, Columbia, during the Second International Microgravity Laboratory experiments.

    PubMed

    Pfeiffer, C J; Yamashita, M; Izumi-Kurotani, A; Koike, H; Asashima, M

    1995-10-01

    Four adult female Japanese newts, Cynops pyrrhogaster, were carried for 15 days aboard the orbiting space shuttle, Columbia, in July of 1994, as part of the Second International Microgravity Laboratory, IML-2 aquatic animal experiments. These previously fertilized newts, after stimulation with chorionic gonadotropin by a spaceflight adapted injection procedure, deposited numerous eggs for study of early development during weightlessness. The primitive saccular lungs of the two newts which survived the spaceflight revealed by TEM marked pulmonary cytopathologic changes including basal laminar separation, microvillar degeneration, and cytoplasmic granular changes in the primary granulated pneumocytes. Also, intracellular edema in the pulmonary collagenous matrix and vacuolar changes in the ciliated pulmonary lining cell type and in vascular endothelial cells were observed. These changes, triggered by the spaceflight, and not seen in controls also relying on respiration via the skin, may reflect a chronic mild hypoxia as it is known that newts undergoing oviposition are subject to increased oxygen demand.

  12. 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.

  13. 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

  14. 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

  15. 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

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

    PubMed

    Chester, Cariad; Maecker, Holden T

    2015-08-01

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

  17. 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.

  18. Microfluidic impedance cytometry of tumour cells in blood.

    PubMed

    Spencer, Daniel; Hollis, Veronica; Morgan, Hywel

    2014-11-01

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

  19. Coefficient of variation in flow cytometry of phagocytosis.

    PubMed

    Fujikawa-Yamamoto, K; Odashima, S

    1987-01-01

    Fluorescence histograms of V79 Chinese hamster lung cells containing phagocytized fluorescent microspheres were measured by flow cytometry. In the fluorescence histograms, the coefficient of variation (CV) of the peak for cells ingesting microspheres was not constant. Rather, it decreased with the number of microspheres ingested by the cells.

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

    PubMed

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

    2016-07-01

    In recent years, multispectral flow cytometry systems have come to attention. They differ from conventional flow cytometers in two key ways: a multispectral flow cytometer collects the full spectral information at the single cell level and the detector configuration is fixed and not explicitly tuned to a particular staining panel. This brings about clear hardware advantages, as a closed system should be highly stable, and ease-of-use should be improved if used in conjunction with custom unmixing software. An open question remains: what are the benefits of multispectral over conventional flow cytometry in terms of sensitivity and resolution? To probe this, we use Q (detection efficiency) and B (background) values and develop a novel "multivariate population overlap factor" to characterize the cytometer performance. To verify the usefulness of our factor, we perform representative experiments and compare our overlap factor to Q and B. Finally, we conclude that the increased light collection of multispectral flow cytometry does indeed lead to increased sensitivity, an improved detection limit, and a higher resolution. © 2016 International Society for Advancement of Cytometry. PMID:27295550

  1. 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…

  2. 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.

  3. 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

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

    PubMed

    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

    2016-01-14

    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 Ca(2+) and Mg(2+) 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 Ca(2+) or Mg(2+) 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.

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

    NASA Astrophysics Data System (ADS)

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

    2007-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. 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

  8. Applications of flow cytometry to artificial insemination: a review.

    PubMed

    Morrell, J M

    1991-10-26

    Flow cytometry is a technique in which sub-populations of cells can be analysed and separated according to the staining pattern seen with various fluorescent markers. This review describes some of the ways in which flow cytometry can be applied to the investigation of sperm populations, either as a means of quality control of semen or to examine the characteristics of different sub-populations of sperm within an ejaculate. These methods can replace or augment existing subjective assessments of semen characteristics. Using this technique it is possible to produce aliquots of sexed sperm for insemination or for in vitro fertilisation. An objective assessment can be made of the effects of environmental stress on male physiology by monitoring changes in semen quality. PMID:1720909

  9. XML-based Gating Descriptions in Flow Cytometry

    PubMed Central

    Spidlen, Josef; Leif, Robert; Moore, Wayne; Roederer, Mario; Brinkman, Ryan R.

    2008-01-01

    Background The lack of software interoperability with respect to gating due to lack of a standardized mechanism for data exchange has traditionally been a bottleneck preventing reproducibility of flow cytometry (FCM) data analysis and the usage of multiple analytical tools. Methods To facilitate interoperability among FCM data analysis tools, members of the International Society for the Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF) have developed an XML-based mechanism to formally describe gates (Gating-ML). Results Gating-ML, an open specification for encoding gating, data transformations and compensation, has been adopted by the ISAC DSTF as a Candidate Recommendation (CR). Conclusions Gating-ML can facilitate exchange of gating descriptions the same way that FCS facilitated for exchange of raw FCM data. Its adoption will open new collaborative opportunities as well as possibilities for advanced analyses and methods development. The ISAC DSTF is satisfied that the standard addresses the requirements for a gating exchange standard. PMID:18773465

  10. Antenatal screening for HPA-1a by flow cytometry.

    PubMed

    Lavu, E K; Nelson, M; Popp, H J; Gibson, J; Kronenberg, H; Pearson, H; Child, A

    1997-05-01

    Pregnant women who attended antenatal clinics at King George V Hospital, the Birth Centre or were referred by obstetricians from February 19 July, 1996 were screened for the platelet antigen HPA-1a by flow cytometry. Forty out of 2,300 (1.7%) were found to be negative for this antigen. Of the 28 women followed throughout their pregnancy, none developed antibody to HPA-1a. Platelet counts performed on samples from 17 babies born to 17 of these mothers were all normal. This study proves the simplicity and rapidity of flow cytometry for platelet antigen screening. The results were comparable with the Solid Phase Red Cell Adherence (SPRCA) method and with PCR. The lack of a plentiful supply of specific antibody and the rarity of fetomaternal alloimmune thrombocytopenia (FMAIT) argue against the introduction of routine screening for maternal HPA-1a status at the present time.

  11. High-throughput flow cytometry for drug discovery.

    PubMed

    Edwards, Bruce S; Young, Susan M; Saunders, Matthew J; Bologa, Cristian; Oprea, Tudor I; Ye, Richard D; Prossnitz, Eric R; Graves, Steven W; Sklar, Larry A

    2007-05-01

    High-throughput flow cytometry exploits a novel many-samples/one-file approach to dramatically speed data acquisition, limit aspirated sample volume to as little as 2 μl/well and produce multisample data sets that facilitate automated analysis of samples in groups as well as individually. It has been successfully applied to both cell- and microsphere-based bioassays in 96- and 384-well formats, to screen tens-of-thousands of compounds and identify novel bioactive structures. High-content multiparametric analysis capabilities have been exploited for assay multiplexing, allowing the assessment of biologic selectivity and specificity to be an integral component of primary screens. These and other advances in the last decade have contributed to the application of flow cytometry as a uniquely powerful tool for probing biologic and chemical diversity and complex systems biology.

  12. Immunoflow cytometry and cell block immunohistochemistry in the FNA diagnosis of lymphoma: a review of 73 consecutive cases

    PubMed Central

    Mayall, F.; Dray, M.; Stanley, D.; Harrison, B.; Allen, R.

    2000-01-01

    Aims—To review the results of 73 consecutive fine needle aspirations (FNAs) that were collected by a pathologist and analysed by immunoflow cytometry. Material for a cell block was also collected from some of these lesions. Methods—The setting was a large general hospital in rural New Zealand. The FNAs were performed by a pathologist, or a radiologist for image guided localisations. Material for immunoflow cytometry was collected into RPMI and, when required, material for a cell block was collected into formalin. Results—Of the 73 samples collected by FNA nine were inadequate. Light chain restriction could be demonstrated in most FNA samples from B cell lymphomas (28 of 30 adequate samples). The exceptions were two cases of T cell rich B cell lymphoma. Artefactual light chain restriction was seen occasionally in T cell lymphomas, presumably as a result of autoantibodies binding to the cell surfaces. It was possible to subtype most (18 of 30 adequate samples) B cell lymphomas as chronic lymphocytic leukaemia (CLL), follicle centre cell lymphoma (FCCL), or mantle cell lymphoma. The CD4 to CD8 ratio was not usually restricted in T cell lymphomas and coexpression of CD4 and CD8 was not usually found. Loss of pan-T cell antigens was seen in some T cell lymphomas. Four of the six T cell lymphomas and three of the four non-lymphoid malignancies were diagnosed with the aid of cell block immunohistochemistry. Only one of the four cases of Hodgkin's lymphoma showed Reed-Sternberg cells in the FNA smears. Conclusions—It is not always possible to characterise lymphomas as fully with FNA and immunoflow cytometry as is possible with biopsy histology and a full battery of modern investigations. Nevertheless, in the setting of a large rural general hospital immunoflow cytometry on FNA samples is a highly effective method of diagnosing and typing B cell lymphomas. Immunoflow cytometry is of little use for T cell lymphomas or Hodgkin's lymphomas. We advocate the use of cell

  13. 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

  14. 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.

  15. An efficient method for enumerating oral spirochetes using flow cytometry.

    PubMed

    Orth, Rebecca; O'Brien-Simpson, Neil; Dashper, Stuart; Walsh, Katrina; Reynolds, Eric

    2010-02-01

    Spirochetes, such as Treponema denticola, are thin walled, helical, motile bacteria. They are notoriously difficult to enumerate due to their thinness and the difficulties associated with culturing them. Here we have developed a modified oral bacterial growth medium (OBGM) that significantly improves the cultivation of T. denticola compared with a previously published growth medium. Three methods for the enumeration of T. denticola, semi-solid growth medium colony-forming unit (CFU) counts, DNA analysis and flow cytometry, are described and compared. Enumeration of T. denticola using the semi-solid agar method resulted in a positive linear relationship with absorbance of the culture (R(2)=0.9423). However, the semi-solid agar method was found to consistently underestimate (by 50 fold) the T. denticola cell density compared to previously published data. DNA analysis of T. denticola cultures reliably and consistently resulted in a positive linear relationship with absorbance (R(2)=0.9360), giving a calculated cell density of 6.9 x 10(8)cells/mL at an absorbance of 0.2 at 650 nm. Flow cytometry was also found to result in a positive linear relationship with absorbance (R(2)=0.9874), giving a calculated cell density of 6.6 x 10(8)cells/mL at an absorbance of 0.2 at 650 nm. In comparing all of these enumeration methods, the flow cytometry method was found to have distinct advantages, as it is accurate, rapid, and could distinguish between live and dead bacteria. Thus flow cytometry is a recommended means for the rapid and reliable enumeration of viable spirochetes from culture.

  16. 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

  17. 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

  18. Guide to Red Fluorescent Proteins and Biosensors for Flow Cytometry

    PubMed Central

    Piatkevich, Kiryl D.; Verkhusha, Vladislav V.

    2014-01-01

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

  19. Overcoming limitations of microparticle measurement by flow cytometry.

    PubMed

    Lacroix, Romaric; Robert, Stephane; Poncelet, Philippe; Dignat-George, Françoise

    2010-11-01

    Circulating microparticles are submicron vesicles released from cell membranes in response to activation or apoptosis. Acknowledgment of their role both as markers and pathogenic effectors in thrombosis, inflammation, and the spread of cancer has increased the interest of their measurement in clinical practice. However, assessment of their clinical use is impeded by technological issues. Among the different methodologies available, flow cytometry is the most commonly used technique. This review addresses flow cytometry limitations in microparticle measurement that may be subdivided into three domains: sizing, probing, and counting. This article also covers the various standardization strategies and technological improvements that have been proposed to overcome these limitations. New tools using size-calibrated beads and recent progress in instrumentation have opened new avenues to improve detection of microparticle populations of smaller sizes. Significant optimization in microparticle detection is also expected from the use of new fluorescent dyes and the establishment of practical recommendations. Finally, absolute counting of microparticles will also benefit from adapted bead-based strategies or, alternatively, from the generalized availability of volumetric systems. Overall, recent technological improvements maintain flow cytometry as a highly competitive analytical method to measure microparticles. Challenging these evolutions in pathological situations is a mandatory step to validate their real impact in clinical practice.

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

    NASA Astrophysics Data System (ADS)

    Markovic, Stacey; Li, Siyuan; Niedre, Mark

    2015-03-01

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

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

    EPA Science Inventory

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

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Misty Mountain clustering: application to fast unsupervised flow cytometry gating

    PubMed Central

    2010-01-01

    Background There are many important clustering questions in computational biology for which no satisfactory method exists. Automated clustering algorithms, when applied to large, multidimensional datasets, such as flow cytometry data, prove unsatisfactory in terms of speed, problems with local minima or cluster shape bias. Model-based approaches are restricted by the assumptions of the fitting functions. Furthermore, model based clustering requires serial clustering for all cluster numbers within a user defined interval. The final cluster number is then selected by various criteria. These supervised serial clustering methods are time consuming and frequently different criteria result in different optimal cluster numbers. Various unsupervised heuristic approaches that have been developed such as affinity propagation are too expensive to be applied to datasets on the order of 106 points that are often generated by high throughput experiments. Results To circumvent these limitations, we developed a new, unsupervised density contour clustering algorithm, called Misty Mountain, that is based on percolation theory and that efficiently analyzes large data sets. The approach can be envisioned as a progressive top-down removal of clouds covering a data histogram relief map to identify clusters by the appearance of statistically distinct peaks and ridges. This is a parallel clustering method that finds every cluster after analyzing only once the cross sections of the histogram. The overall run time for the composite steps of the algorithm increases linearly by the number of data points. The clustering of 106 data points in 2D data space takes place within about 15 seconds on a standard laptop PC. Comparison of the performance of this algorithm with other state of the art automated flow cytometry gating methods indicate that Misty Mountain provides substantial improvements in both run time and in the accuracy of cluster assignment. Conclusions Misty Mountain is fast, unbiased

  6. Identification of contact and respiratory sensitizers using flow cytometry

    SciTech Connect

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

    2005-06-15

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

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

    DOEpatents

    Mariella, Jr., Raymond P.

    2000-01-01

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

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

    PubMed

    Johansson, Ulrika; Macey, Marion

    2011-07-01

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

  9. Analysis of receptor tyrosine kinase internalization using flow cytometry.

    PubMed

    Li, Ning; Hill, Kristen S; Elferink, Lisa A

    2008-01-01

    The internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization.

  10. Automated nanoscale flow cytometry for assessing protein-protein interactions.

    PubMed

    von Kolontaj, Kerstin; Horvath, Gabor L; Latz, Eicke; Büscher, Martin

    2016-09-01

    Despite their importance for signalling events, protein-protein interactions cannot easily be analyzed on a single cell level. We developed a robust automated FRET measurement system implemented on a commercial flow cytometer allowing for rapid profiling of molecular associations in living cells. We used this method to measure the most proximal signaling events on human T lymphocyte activation, which preceded calcium influx, and could automatically detect T cell receptor/CD3 complex clustering defects in immunocompromised patients. © 2016 International Society for Advancement of Cytometry. PMID:27584593

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  12. Clinico-cytopathological spectrum of hepatocellular carcinoma, its correlation with serum alpha-fetoprotein level, and hepatitis B and C viral markers.

    PubMed

    Radhika, Nitin Shriniwas; Duseja, Ajay; Rajwanshi, Arwind; Gupta, Subhash Kumari; Sehgal, Shobha; Suri, Sudha; Chawla, Yogesh

    2004-01-01

    Fine-needle aspirationbiopsy (FNAB) is now widely accepted as a diagnostic modality for the treatment of hepatocellular carcinoma (HCC). The most common diagnostic problem in HCC is distinguishing it from a metastatic carcinoma. The literature from India on HCC is scanty. Hence, we studied the cytomorphological features of HCC and metastatic carcinoma. The study included 37 cases of space-occupying lesions (SOLs) of the liver as demonstrated by ultrasound or computed tomography (CT) scan. Cytomorphological features of these SOLs were analyzed in all subsequent to FNAB. Hepatitis B surface antigen (HBsAg), anti-hepatitis C virus antibody (anti-HCV) and alpha-fetoprotein (AFP) were determined in all the cases by enzyme-linked immunosorbent assay (ELISA). The cytopathological diagnosis was HCC in 22 and metastatic carcinoma of the liver in 15. The individual cytomorphological features and which helped to make a definite diagnosis of HCC were: a high nuclear cytoplasmic ratio (81.8%), predominantly trabecular pattern (63.6%) and atypical naked nuclei (100%). Other features were prominent multiple nucleoli (63.3%), hyperchromasia (100%) and moderate anisonucleosis (59%). AFP was elevated in 81.8% of the cases with a mean of 634.8+812.7 ng/ml. HBsAg by ELISA was found to be positive in 72.7% of cases while only 1 case (4.5%) was positive for anti-HCV. In 1 case (4.5%), there was dual infection due to hepatitis B virus (HBV) and HCV. No viral cause was found in 18.3% of cases. PMID:15682657

  13. Quantitative assessment of neutrophil phagocytosis using flow cytometry.

    PubMed

    Nordenfelt, Pontus

    2014-01-01

    Neutrophils have an incredible ability to find and eradicate intruders such as bacteria and fungi. They do this largely through the process of phagocytosis, where the target is internalized into a phagosome, and eventually destroyed by the hostile phagosomal environment. It is important to study phagocytosis in order to understand how neutrophils interact with various pathogens and how they respond to different stimuli. Here, I describe a method to study neutrophil phagocytosis of bacteria using flow cytometry. The bacteria are fluorescently labeled before being introduced to neutrophils. After phagocytosis, both any remaining extracellular bacteria and neutrophils are labeled using one-step staining before three-color analysis. To assess phagocytosis, first the average time it takes for the neutrophils to internalize all bound bacteria is determined. Experiments are then performed using that time point while varying the bacteria-to-neutrophil ratio for full control of the analysis. Due to the ease with which multiple samples can be analyzed, and the quantitative nature of flow cytometry, this approach is both reproducible and sensitive.

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

    PubMed Central

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

    2010-01-01

    We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells. PMID:20390041

  15. Measurement and Characterization of Apoptosis by Flow Cytometry.

    PubMed

    Telford, William; Tamul, Karen; Bradford, Jolene

    2016-01-01

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

  16. Expert systems for flow cytometry data analysis: A preliminary report

    SciTech Connect

    Salzman, G.C. ); Stewart, C.C. . Lab. of Flow Cytometry); Duque, R.E. )

    1990-01-01

    Flow Cytometry has become an accepted technique in the clinical laboratory for rapid immunophenotyping of patient blood samples. Multiple, fluorescent labeled monoclonal antibodies are used to tag the cells, which are then analyzed one at a time at rates of several thousand cells a second. Patient samples are processed through the flow cytometer at more than one a minute. Clinicians are being overwhelmed by the large amount of data that must be analyzed to provide the information needed to assist in disease diagnosis. An expert system is being developed to assist clinicians in analyzing this multivariate flow cytometry data. The data from each sample are processed by a clustering algorithm, which finds the means of the distinct cell subpopulations in a sample. These mean values of fluorescence are translated into words such as negative,'' dim'' and bright'' and the words are combined into patterns that are matched against the premises on the left hand side of the rules used to identify the disease categories. This is a report of work in progress. 13 refs., 4 figs.

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

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

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

  18. Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes

    PubMed Central

    Aanei, Carmen Mariana; Picot, Tiphanie; Tavernier, Emmanuelle; Guyotat, Denis; Campos Catafal, Lydia

    2016-01-01

    Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoiesis that exhibit heterogeneous clinical presentation and morphological findings, which complicates diagnosis, especially in early stages. Recently, refined definitions and standards in the diagnosis and treatment of MDS were proposed, but numerous questions remain. Multiparameter flow cytometry (MFC) is a helpful tool for the diagnostic workup of patients with suspected MDS, and various scores using MFC data have been developed. However, none of these methods have achieved the sensitivity that is required for a reassuring diagnosis in the absence of morphological abnormalities. One reason may be that each score evaluates one or two lineages without offering a broad view of the dysplastic process. The combination of two scores (e.g., Ogata and Red Score) improved the sensitivity from 50–60 to 88%, but the positive (PPV) and negative predictive values (NPV) must be improved. There are prominent differences between study groups when these scores are tested. Further research is needed to maximize the sensitivity of flow cytometric analysis in MDS. This review focuses on the application of flow cytometry for MDS diagnosis and discusses the advantages and limitations of different approaches. PMID:27446807

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

    SciTech Connect

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

    1998-07-01

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

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

    PubMed

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

    2015-01-01

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

  1. Recent applications of flow cytometry in aquatic microbial ecology.

    PubMed

    Troussellier, M; Courties, C; Vaquer, A

    1993-01-01

    Microorganisms (unicellular algae, bacteria) constitute fundamental compartments of aquatic ecosystems because of their high concentrations and activities. The evaluation and understanding of their behavior and role raise different problems for which traditional methodologies are often inadequate, whether they refer to global or classical microscopic analyses. Flow cytometry (FCM) has been recently used to study microorganisms in aquatic environments. Although this technology is still applied on a limited scale in our field, a large number of works has been done showing that FCM seems to be a promising tool for aquatic microbial ecology. This paper summarizes, from the literature produced during the last decade and with original data obtained in our laboratory, the main questions related to the cell identification, the evaluation of cell viability, biomasses and productions and the measurements of bacterial and phytoplanktonic activities. The representatives of sampling and observation scales is also discussed within the framework of the FCM measurements. PMID:8220221

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

    PubMed

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

    2016-01-01

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

  3. Applications of Flow Cytometry to Characterize Bacterial Physiological Responses

    PubMed Central

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

    2014-01-01

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

  4. An automated microfluidic sample preparation system for laser scanning cytometry.

    PubMed

    Wu, Eric; Menon, Vidya; Geddie, William; Sun, Yu

    2011-04-01

    Laser scanning cytometry (LSC) is emerging as a clinical tool. In one application a "Clatch" slide, named after the inventor, is used in conjunction with LSC for cell surface marker immunophenotyping of patient samples. The slide requires time consuming and laborious pipetting steps, making a test tedious and prone to handling errors. The Clatch slide also uses a significant number of cells, limiting the number of analyses on paucicellular samples. This paper presents an automated microfluidic system consisting of a control circuit, a microfluidic system, and an aluminum frame, capable of performing immunophenotyping procedures. This prototype system reduces 36 pipetting steps to 1, reduces the amount of cell sample from 180 μL to 56 μL, and shortens the time used by technicians.

  5. [Flow cytometry in immunology and hematology: some essential practical aspects].

    PubMed

    Doinel, C; Bourin, P

    1989-12-01

    Flow cytometry, supported by monoclonal antibodies, has widely contributed in the cellular identification, notably in the clinical and haematological fields. This technique has found several applications since the qualitative phenotyping and the quantitative analysis of the immune system's cell populations are helpful in the diagnosis and the therapy. Besides, these indications require an appropriate knowledge of several methodological aspects including factors related to the sample donor: age and sex; sampling: time and quantity; and the sample preparation conditions. References values, needed for the results interpretation, have a meaning only if they are defined within these validity limits. Previous trials have been done in order to define a biological value representative of the immunological status, such as the CD4/CD8 ratio. Unfortunately this ratio is not justified in the scope of new knowledge concerning the cellular interactions and the functional heterogeneity of cells involved in the immune system.

  6. Flow Cytometry in the Diagnosis of Myelodysplastic Syndromes

    PubMed Central

    Szánthó, Eszter; Kappelmayer, János; Hevessy, Zsuzsa

    2013-01-01

    Myelodysplastic syndromes are clonal hematopoietic stem cell disorders. Their exact etiology is unknown. Myelodysplastic syndromes cause progressive bone marrow failure resulting in pancytopenia and refractory, transfusion-dependent anemia. One can observe typical morphological alterations in the erythroid, myeloid and/or megakaryocytic cell lineage. Blast counts may also be increased. The pathologic cells are genetically unstable, and a myelodysplastic syndrome might transform into acute myeloid leukemia. The overall survival of these diseases range between few months to around ten years. Correct diagnosis and accurate prognostic classification is essential. In the past decades several scoring systems were established beginning with the French-American-British classification to the most recent Revised International Prognostic Scoring System. In all of these classifications bone marrow morphology is still the most important factor, though nowadays the genetic aberrations and flow cytometry findings are also included. The diagnosis and prognostic classification of myelodysplastic syndromes remain a great challenge for hematologists.

  7. Rapid Cell Population Identification in Flow Cytometry Data*

    PubMed Central

    Aghaeepour, Nima; Nikolic, Radina; Hoos, Holger H.; Brinkman, Ryan R.

    2011-01-01

    We have developed flowMeans, a time-efficient and accurate method for automated identification of cell populations in flow cytometry (FCM) data based on K-means clustering. Unlike traditional K-means, flowMeans can identify concave cell populations by modelling a single population with multiple clusters. flowMeans uses a change point detection algorithm to determine the number of sub-populations, enabling the method to be used in high throughput FCM data analysis pipelines. Our approach compares favourably to manual analysis by human experts and current state-of-the-art automated gating algorithms. flowMeans is freely available as an open source R package through Bioconductor. PMID:21182178

  8. A Survey of Flow Cytometry Data Analysis Methods

    PubMed Central

    Bashashati, Ali; Brinkman, Ryan R.

    2009-01-01

    Flow cytometry (FCM) is widely used in health research and in treatment for a variety of tasks, such as in the diagnosis and monitoring of leukemia and lymphoma patients, providing the counts of helper-T lymphocytes needed to monitor the course and treatment of HIV infection, the evaluation of peripheral blood hematopoietic stem cell grafts, and many other diseases. In practice, FCM data analysis is performed manually, a process that requires an inordinate amount of time and is error-prone, nonreproducible, nonstandardized, and not open for re-evaluation, making it the most limiting aspect of this technology. This paper reviews state-of-the-art FCM data analysis approaches using a framework introduced to report each of the components in a data analysis pipeline. Current challenges and possible future directions in developing fully automated FCM data analysis tools are also outlined. PMID:20049163

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

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

    PubMed Central

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

    2010-01-01

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

  12. AutoGate: automating analysis of flow cytometry data.

    PubMed

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

    2014-05-01

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

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

    PubMed Central

    Schmutz, Sandrine; Valente, Mariana

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2012-01-01

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

  17. Evaluation of slide based cytometry (SBC) for concentration measurements of fluorescent dyes in solution

    NASA Astrophysics Data System (ADS)

    Pierzchalski, Arkadiusz; Marecka, Monika; Müller, Hans-Willy; Bocsi, József; Tárnok, Attila

    2009-02-01

    Flow cytometers (FCM) are built for particle measurements. In principle, concentration measurement of a homogeneous solution is not possible with FCM due to the lack of a trigger signal. In contrast to FCM slide based cytometry systems could act as tools for the measurement of concentrations using volume defined cell counting chambers. These chambers enable to analyze a well defined volume. Sensovation AG (Stockach, Germany) introduced an automated imaging system that combines imaging with cytometric features analysis. Aim of this study was to apply this imaging system to quantify the fluorescent molecule concentrations. The Lumisens (Sensovation AG) slide-based technology based on fluorescence digital imaging microscopy was used. The instrument is equipped with an inverted microscope, blue and red LEDs, double band-pass filters and a high-resolution cooled 16-bit digital camera. The instrument was focussed on the bottom of 400μm deep 6 chamber slides (IBIDI GmbH, Martinsried, Germany) or flat bottom 96 well plates (Greiner Bio One GmbH, Frickenhausen, Germany). Fluorescent solutions were imaged under 90% pixel saturation in a broad concentration range (FITC: 0.0002-250 μg/ml, methylene blue (MethB): 0.0002-250 μg/ml). Exposition times were recorded. Images were analysed by the iCys (CompuCyte Corp., Cambridge, MA, USA) image analysis software with the phantom contour function. Relative fluorescence intensities were calculated from mean fluorescence intensities per phantom contours divided by the exposition time. Solution concentrations could be distinguished over a broad dynamic range of 3.5 to 5.5 decades log (range FITC: 0.0002-31.25μg/ml, MethB: 0.0076-31.25μg/ml) with a good linear relationship between dye concentration and relative fluorescence intensity. The minimal number of fluorescent molecules per pixel as determined by the mean fluorescence intensity and the molecular weight of the fluorochrome were about 800 molecules FITC and ~2.000 MethB. The

  18. Rapid titration of retroviral vectors encoding intracellular antigens by flow cytometry.

    PubMed

    Sladek, T L; Jacobberger, J W

    1990-06-01

    Flow cytometry was used to detect cells infected with retroviral vectors encoding both simian virus 40 large T antigen and G418 resistance after indirect immunofluorescence staining using a T-antigen-specific monoclonal antibody and a fluorescein-conjugated secondary antibody. Titers of viral stocks determined by flow cytometry were equivalent to those determined by quantitation of G418-resistant colonies.

  19. Flow cytometry reliability analysis and variations in sugarcane DNA content.

    PubMed

    Oliveira, A C L; Pasqual, M; Bruzi, A T; Pio, L A S; Mendonça, P M S; Soares, J D R

    2015-01-01

    The aim of this study was to evaluate the reliability of flow cytometry analysis and the use of this technique to differentiate species and varieties of sugarcane (Saccharum spp) according to their relative DNA content. We analyzed 16 varieties and three species belonging to this genus. To determine a reliable protocol, we evaluated three extraction buffers (LB01, Marie, and Tris·MgCl2), the presence and absence of RNase, six doses of propidium iodide (10, 15, 20, 25, and 30 μg), four periods of exposure to propidium iodide (0, 5, 10, and 20 min), and seven external reference standards (peas, beans, corn, radish, rye, soybean, and tomato) with reference to the coefficient of variation and the DNA content. For statistical analyses, we used the programs Sisvar(®) and Xlstat(®). We recommend using the Marie extraction buffer and at least 15 μg propidium iodide. The samples should not be analyzed immediately after the addition of propidium iodide. The use of RNase is optional, and tomato should be used as an external reference standard. The results show that sugarcane has a variable genome size (8.42 to 12.12 pg/2C) and the individuals analyzed could be separated into four groups according to their DNA content with relative equality in the genome sizes of the commercial varieties.

  20. Resonant-cavity apparatus for cytometry or particle analysis

    DOEpatents

    Gourley, P.L.

    1998-08-11

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

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

    PubMed

    Ogata, Kiyoyuki

    2008-12-01

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

  2. Flow cytometry for the diagnosis of autoimmune thrombocytopenia.

    PubMed

    Tomer, Aaron

    2006-03-01

    Autoimmune thrombocytopenia is a disorder characterized by antibody-mediated accelerated platelet destruction. Despite its clinical importance, the diagnosis of autoimmune thrombocytopenia is one of exclusion, thus inevitably associated with potential difficulties. Current clinically applicable methods used to determine antigen-specific antibodies, primarily directed to GPIIb/IIIa (CD41a) and GPIb (CD42b), include the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay and the radioactive immunobead assay. Neither of these assays is commonly used by clinical laboratories, however, because of methodologic and practical limitations. As a result, diagnoses are generally based on clinical impression despite patient presentations that are sometimes complex. To overcome some of these difficulties, flow cytometric techniques have been developed, employing standard methods and equipment suitable for testing a single sample or multiple samples, as may occur in cases of autoimmune thrombocytopenia. The availability of a feasible technique such as flow cytometry, with improved sensitivity and specificity, should facilitate the routine use of a diagnostic method in the evaluation of thrombo-cytopenic patients suspected of having an autoimmune disorder and permit follow-up to determine immune remission. PMID:16537048

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

    PubMed

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

    2016-01-01

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

  4. Cell deformation cytometry using diode-bar optical stretchers

    PubMed Central

    Sraj, Ihab; Eggleton, Charles D.; Jimenez, Ralph; Hoover, Erich; Squier, Jeff; Chichester, Justin; Marr, David W.M.

    2010-01-01

    The measurement of cell elastic parameters using optical forces has great potential as a reagent-free method for cell classification, identification of phenotype, and detection of disease; however, the low throughput associated with the sequential isolation and probing of individual cells has significantly limited its utility and application. We demonstrate a single-beam, high-throughput method where optical forces are applied anisotropically to stretch swollen erythrocytes in microfluidic flow. We also present numerical simulations of model spherical elastic cells subjected to optical forces and show that dual, opposing optical traps are not required and that even a single linear trap can induce cell stretching, greatly simplifying experimental implementation. Last, we demonstrate how the elastic modulus of the cell can be determined from experimental measurements of the equilibrium deformation. This new optical approach has the potential to be readily integrated with other cytometric technologies and, with the capability of measuring cell populations, enabling true mechanical-property-based cell cytometry. PMID:20799841

  5. Resonant-cavity apparatus for cytometry or particle analysis

    DOEpatents

    Gourley, Paul L.

    1998-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

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

    PubMed Central

    Tario, Joseph D.; Muirhead, Katharine A.; Pan, Dalin; Munson, Mark E.; Wallace, Paul K.

    2015-01-01

    In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8+ T cells. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in the T-cell pool that are able to bind tetramer, synthesize cytokines, undergo antigen-driven proliferation, and/or carry out various combinations of these functional responses. Analysis of antigen-specific proliferative responses represents just one of many functions that can be monitored using cell tracking dyes and flow cytometry. In this third edition, we address issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T-cell functions. We summarize key characteristics of and differences between general protein- and membrane-labeling dyes, discuss determination of optimal staining concentrations, and provide detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking are provided in the form of protocols for (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay and (b) simultaneous monitoring of proliferative responses in effector and regulatory T cells. PMID:21116982

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

    PubMed

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

    2014-12-01

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

  10. Reticulocyte count using thiazole orange. A flow cytometry method.

    PubMed

    Van Hove, L; Goossens, W; Van Duppen, V; Verwilghen, R L

    1990-01-01

    Recently flow cytometry techniques have been developed to replace the microscope reticulocyte count. We used thiazole orange, a RNA binding fluorochrome, to discriminate reticulocytes from mature erythrocytes. Thiazole orange and the Retic-COUNT software package were evaluated for performance of routine analysis on different flow instruments. The applied methodology analysed 10(4) cells semi-automatically in an easily performed manner. Consistent results were obtained with dipotassium EDTA anticoagulated blood (stable for 30 h after venesection), with incubation times in thiazole orange solution ranging from 2 to 7 h at 25 degrees C. This allowed flexibility in specimen collection and storage and assay performance with no change in results. Changes of incubation temperature up to 30 degrees C had no measurable effect. The values obtained showed good linearity, precision and accuracy for normal, low and high reticulocyte counts. However interferences were observed: RBC autofluorescence, nucleated RBC, Howell-Jolly bodies, high leucocyte count, high platelet count and giant platelets, all falsely increased the number of reticulocytes. These artifacts were eliminated by software gate corrections, thus leaving less than 5% of the specimen to be reanalysed by the microscopic method. The thiazole orange flow cytometric method was determined to be a fast, reliable method for the routine clinical quantitation of reticulocytes.

  11. Cell-based flow cytometry assay to measure cytotoxic activity.

    PubMed

    Noto, Alessandra; Ngauv, Pearline; Trautmann, Lydie

    2013-12-17

    Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10(6) cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

  12. The cytopathology of mycobacterial infection.

    PubMed

    Michelow, Pamela; Omar, Tanvier; Field, Andrew; Wright, Colleen

    2016-03-01

    Mycobacterial infection, tuberculosis (TB) in particular, remains one of the world's deadliest communicable diseases in adults and particularly in children, in low and middle income countries. The combination of human immunodeficiency virus (HIV) and TB is often lethal with TB accounting for 25% of deaths in the HIV population. One of the cornerstones for reducing the TB epidemic is early case detection using high quality diagnostic techniques. Cytology, especially fine needle aspiration biopsy (FNAB) is able to diagnose mycobacterial infection in a rapid and cost-effective manner without requiring surgery, thus allowing appropriate management to be quickly instituted. Confirmatory ancillary tests can effectively be performed on cytologic material. In this review, the pertinent cytomorphology of mycobacterial infection in various exfoliative and FNAB specimens is presented, in both immunocompetent and immunosuppressed patients. In the immunosuppressed, the typical cytomorphology of caseating granulomatous inflammation may not be seen but suppurative necrotic inflammation, mycobacterial spindle pseudotumour or a specimen comprised entirely of necrosis may be seen instead. This review includes discussion of currently available ancillary tests that can be performed on cytologic specimens. PMID:26800030

  13. Regression analysis of cytopathological data

    SciTech Connect

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

    1982-12-01

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

  14. Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.

    PubMed Central

    Ginaldi, L; Farahat, N; Matutes, E; De Martinis, M; Morilla, R; Catovsky, D

    1996-01-01

    AIMS: To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both. METHODS: Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC). RESULTS: CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells. CONCLUSIONS: Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts. Images PMID:8813949

  15. Aequorea green fluorescent protein analysis by flow cytometry

    SciTech Connect

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

    1995-12-01

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

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

    PubMed

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

    2007-10-01

    Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise < or = 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and < or =1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times. PMID:17712796

  17. Performance of calibration standards for antigen quantitation with flow cytometry.

    PubMed

    Lenkei, R; Gratama, J W; Rothe, G; Schmitz, G; D'hautcourt, J L; Arekrans, A; Mandy, F; Marti, G

    1998-10-01

    In the frame of the activities initiated by the Task Force for Antigen Quantitation of the European Working Group on Clinical Cell Analysis (EWGCCA), an experiment was conducted to evaluate microbead standards used for quantitative flow cytometry (QFCM). An unified window of analysis (UWA) was established on three different instruments (EPICS XL [Coulter Corporation, Miami, FL], FACScan and FACS Calibur [Becton Dickinson, San Jose, CA]) with QC3 microbeads (FCSC, PR). By using this defined fluorescence intensity scale, the performance of several monoclonal antibodies directed to CD3, CD4, and CD8 (conjugated and unconjugated), from three manufacturers (BDIS, Coulter [Immunotech], and DAKO) was tested. In addition, the QIFI system (DAKO) and QuantiBRITE (BDIS), and a method of relative fluorescence intensity (RFI, method of Giorgi), were compared. mAbs reacting with three more antigens, CD16, CD19, and CD38 were tested on the FACScan instrument. Quantitation was carried out using a single batch of cryopreserved peripheral blood leukocytes, and all tests were performed as single color analyses. Significant correlations were observed between the antibody-binding capacity (ABC) values of the same CD antigen measured with various calibrators and with antibodies differing in respect to vendor, labeling and possible epitope recognition. Despite the significant correlations, the ABC values of most monoclonal antibodies differed by 20-40% when determined by the different fluorochrome conjugates and different calibrators. The results of this study indicate that, at the present stage of QFCM consistent ABC values may be attained between laboratories provided that a specific calibration system is used including specific calibrators, reagents, and protocols.

  18. Detection of CFTR protein in human leukocytes by flow cytometry.

    PubMed

    Johansson, Jan; Vezzalini, Marzia; Verzè, Genny; Caldrer, Sara; Bolognin, Silvia; Buffelli, Mario; Bellisola, Giuseppe; Tridello, Gloria; Assael, Baroukh Maurice; Melotti, Paola; Sorio, Claudio

    2014-07-01

    Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 μL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

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

    PubMed

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

    2015-05-21

    In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of non-productive and staggered productive intermediates arise at different reprogramming time points. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells, prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that, during reprogramming, cells progressively lose donor cell identity and gradually acquire iPS cell properties. Here we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen, we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200, that are absent in both fibroblasts and iPS cells. Single-cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic, reprogramming phase. Expression profiling reveals early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1 (also known as Zfp42), Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus represent some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. PMID:25830878

  2. Impedance microflow cytometry for viability studies of microorganisms

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

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

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

    PubMed

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

    2007-01-01

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

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

    PubMed Central

    Chattopadhyay, Pratip K.; Roederer, Mario

    2010-01-01

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

  5. Image

    SciTech Connect

    Marsh, Amber; Harsch, Tim; Pitt, Julie; Firpo, Mike; Lekin, April; Pardes, Elizabeth

    2007-08-31

    The computer side of the IMAGE project consists of a collection of Perl scripts that perform a variety of tasks; scripts are available to insert, update and delete data from the underlying Oracle database, download data from NCBI's Genbank and other sources, and generate data files for download by interested parties. Web scripts make up the tracking interface, and various tools available on the project web-site (image.llnl.gov) that provide a search interface to the database.

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

    PubMed

    Spidlen, Josef; Breuer, Karin; Brinkman, Ryan

    2012-07-01

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

  7. Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.

    PubMed

    Chen, Tiffany J; Kotecha, Nikesh

    2014-01-01

    Cytometry is used extensively in clinical and laboratory settings to diagnose and track cell subsets in blood and tissue. High-throughput, single-cell approaches leveraging cytometry are developed and applied in the computational and systems biology communities by researchers, who seek to improve the diagnosis of human diseases, map the structures of cell signaling networks, and identify new cell types. Data analysis and management present a bottleneck in the flow of knowledge from bench to clinic. Multi-parameter flow and mass cytometry enable identification of signaling profiles of patient cell samples. Currently, this process is manual, requiring hours of work to summarize multi-dimensional data and translate these data for input into other analysis programs. In addition, the increase in the number and size of collaborative cytometry studies as well as the computational complexity of analytical tools require the ability to assemble sufficient and appropriately configured computing capacity on demand. There is a critical need for platforms that can be used by both clinical and basic researchers who routinely rely on cytometry. Recent advances provide a unique opportunity to facilitate collaboration and analysis and management of cytometry data. Specifically, advances in cloud computing and virtualization are enabling efficient use of large computing resources for analysis and backup. An example is Cytobank, a platform that allows researchers to annotate, analyze, and share results along with the underlying single-cell data.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-02-01

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

  10. Confocal 3D DNA Cytometry: Assessment of Required Coefficient of Variation by Computer Simulation

    PubMed Central

    Ploeger, Lennert S.; Beliën, Jeroen A.M.; Poulin, Neal M.; Grizzle, William; van Diest, Paul J.

    2004-01-01

    Background: Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. So far, sample size has been limited by the time consuming nature of the technology. Since the power of DNA histograms to resolve different stemlines depends on both the sample size and the coefficient of variation (CV) of histogram peaks, interpretation of 3D CLSM DNA histograms might be hampered by both a small sample size and a large CV. The aim of this study was to analyze the required CV for 3D CLSM DNA histograms given a realistic sample size. Methods: By computer simulation, virtual histograms were composed for sample sizes of 20000, 10000, 5000, 1000, and 273 cells and CVs of 30, 25, 20, 15, 10 and 5%. By visual inspection, the histogram quality with respect to resolution of G0/1 and G2/M peaks of a diploid stemline was assessed. Results: As expected, the interpretability of DNA histograms deteriorated with decreasing sample sizes and higher CVs. For CVs of 15% and lower, a clearly bimodal peak pattern with well distinguishable G0/1 and G2/M peaks were still seen at a sample size of 273 cells, which is our current average sample size with 3D CLSM DNA cytometry. Conclusions: For unambiguous interpretation of DNA histograms obtained using 3D CLSM, a CV of at most 15% is tolerable at currently achievable sample sizes. To resolve smaller near diploid stemlines, a CV of 10% or better should be aimed at. With currently available 3D imaging technology, this CV is achievable. PMID:15371645

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

    PubMed

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

    2015-07-01

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

  12. Cytofkit: A Bioconductor Package for an Integrated Mass Cytometry Data Analysis Pipeline

    PubMed Central

    Wong, Michael Thomas

    2016-01-01

    Single-cell mass cytometry significantly increases the dimensionality of cytometry analysis as compared to fluorescence flow cytometry, providing unprecedented resolution of cellular diversity in tissues. However, analysis and interpretation of these high-dimensional data poses a significant technical challenge. Here, we present cytofkit, a new Bioconductor package, which integrates both state-of-the-art bioinformatics methods and in-house novel algorithms to offer a comprehensive toolset for mass cytometry data analysis. Cytofkit provides functions for data pre-processing, data visualization through linear or non-linear dimensionality reduction, automatic identification of cell subsets, and inference of the relatedness between cell subsets. This pipeline also provides a graphical user interface (GUI) for ease of use, as well as a shiny application (APP) for interactive visualization of cell subpopulations and progression profiles of key markers. Applied to a CD14−CD19− PBMCs dataset, cytofkit accurately identified different subsets of lymphocytes; applied to a human CD4+ T cell dataset, cytofkit uncovered multiple subtypes of TFH cells spanning blood and tonsils. Cytofkit is implemented in R, licensed under the Artistic license 2.0, and freely available from the Bioconductor website, https://bioconductor.org/packages/cytofkit/. Cytofkit is also applicable for flow cytometry data analysis. PMID:27662185

  13. Enumeration and Biomass Estimation of Bacteria in Aquifer Microcosm Studies by Flow Cytometry

    PubMed Central

    DeLeo, P. C.; Baveye, P.

    1996-01-01

    Flow cytometry was used to enumerate and characterize bacteria from a sand column microcosm simulating aquifer conditions. Pure cultures of a species of Bacillus isolated from subsurface sediments or Bacillus megaterium were first evaluated to identify these organisms' characteristic histograms. Counting was then carried out with samples from the aquifer microcosms. Enumeration by flow cytometry was compared with more-traditional acridine orange direct counting. These two techniques gave statistically similar results. However, counting by flow cytometry, in this case, surveyed a sample size 700 times greater than did acridine orange direct counting (25 (mu)l versus 0.034 (mu)l) and required 1/10 the time (2 h versus 20 h). Flow cytometry was able to distinguish the same species of bacteria grown under different nutrient conditions, and it could distinguish changes in cell growth patterns, specifically single cell growth versus chained cell growth in different regions of an aquifer microcosm. A biomass estimate was calculated by calibrating the total fluorescence of a sample from a pure culture with the dry weight of a freeze-dried volume from the original pure culture. Growth conditions significantly affected histograms and biomass estimates, so the calibration was carried out with cells grown under conditions similar to those in the aquifer microcosm. Costs associated with using flow cytometry were minimal compared with the amount of time saved in counting cells and estimating biomass. PMID:16535470

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

    NASA Astrophysics Data System (ADS)

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

    2012-08-01

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

  15. Cytometry in malaria--a practical replacement for microscopy?

    PubMed

    Shapiro, Howard M; Apte, Simon H; Chojnowski, Grace M; Hänscheid, Thomas; Rebelo, Maria; Grimberg, Brian T

    2013-07-01

    Malaria, caused by protozoan Plasmodium parasites, kills ~800,000 people each year. Exact figures are uncertain because presumptive diagnoses are often made without identifying parasites in patients' blood either by microscopy, using Giemsa's century-old stain, or by simpler tests that are ultimately dependent on microscopy for quality control. Microscopy itself relies on trained observers' ability to detect subtle morphological features of parasitized red blood cells, only a few of which may be present on a slide. Quantitative and objective flow cytometric measurements of cellular constituents such as DNA, RNA, and the malaria pigment hemozoin are now useful in research in malaria biology and pharmacology, and can provide more reliable identification of parasite species and developmental stages and better detection of low-density parasitemia than could microscopy. The same measurements can now be implemented in much smaller, simpler, cheaper imaging cytometers, potentially providing a more accurate and precise diagnostic modality.

  16. First proposed panels on acute leukemia for four-color immunophenotyping by flow cytometry from the Brazilian group of flow cytometry-GBCFLUX.

    PubMed

    Ikoma, Maura R V; Sandes, Alex F; Thiago, Leandro S; Cavalcanti Júnior, Geraldo B; Lorand-Metze, Irene G H; Costa, Elaine S; Pimenta, Glicinia; Santos-Silva, Maria C; Bacal, Nydia S; Yamamoto, Mihoko; Souto, Elizabeth X

    2015-01-01

    Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2).

  17. A pilot study for the integration of cytometry reports in digital cytology telemedicine applications.

    PubMed

    Giansanti, Daniele; Cerroni, Fabio; Amodeo, Rachele; Filoni, Marco; Giovagnoli, Maria Rosaria

    2010-01-01

    Up to date, tele-pathology in the three different forms of application, "dynamic", "static" and "virtual microscopy" has been mainly based on tele-hystology remote consulting. Today the diffusion of specialized WAN connections is guiding the research of new applications of tele-pathology. A specific analysis has been conducted, focused on digital cytology, in the biomedical laboratory of Sant'Andrea Hospital to investigate the technologies potentially useful to integrate in the LAN/WAN for telemedicine applications. Among the possible tools useful to be integrated in the LAN/WAN for telemedicine applications, the cytometry equipment available in the technical unity of cytometry has been considered important. The study finally provides a proposal for a tele-consulting architecture for the integration of cytometry reports both in the hospital LAN and the WAN for possible cooperative diagnosis and second opinion support.

  18. Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development

    PubMed Central

    Schwickart, Martin; Schneider, Amy K.; Vainshtein, Inna; Del Nagro, Christopher; Standifer, Nathan; Roskos, Lorin K.

    2015-01-01

    Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on the cell surface and are frequently used to generate pharmacodynamic (PD) biomarker data in nonclinical and clinical studies of biopharmaceuticals. When combined with the pharmacokinetic (PK) profile, RO data can establish PKPD relationships, which are crucial for informing dose decisions. RO is commonly measured by flow cytometry on fresh blood specimens and is subject to numerous technical and logistical challenges. To ensure that reliable and high quality results are generated from RO assays, careful assay design, key reagent characterization, data normalization/reporting, and thorough planning for implementation are of critical importance during development. In this article, the authors share their experiences and perspectives in these areas and discuss challenges and potential solutions when developing and implementing a flow cytometry‐based RO method in support of biopharmaceutical drug development. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc. PMID:26054054

  19. Single Cell Mass Cytometry for Analysis of Immune System Functional States

    PubMed Central

    Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

    2013-01-01

    Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316

  20. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

    PubMed Central

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

    2011-01-01

    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  1. Beyond conventional cell analysis: the latest science and technology in flow cytometry.

    PubMed

    Wright, Sharlene

    2016-01-01

    Combining powerful performance and innovative design and technology, it is possible to deliver a compact, easy-to-use flow cytometry system. Pushing the 'norms' of conventional flow cytometry, today's--and tomorrow's--systems enable complex research into high-content applications in cell biology, as well as a deeper understanding of the advantages gained from the emerging nanoparticle frontier. Flow cytometry is a powerful tool for interrogating complex biological questions at the forefront of biomedical and life science research and increasingly for clinical laboratory applications. Today's investigators want to harness that power and are demanding smaller and more powerful instruments that are more affordable and easier to use. Using innovation, engineers are able to deliver solutions to meet the challenge.

  2. Methodology and Application of Flow Cytometry for Investigation of Human Malaria Parasites

    PubMed Central

    Grimberg, Brian T.

    2011-01-01

    Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries. PMID:21296083

  3. Methodology and application of flow cytometry for investigation of human malaria parasites.

    PubMed

    Grimberg, Brian T

    2011-03-31

    Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries.

  4. Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies

    PubMed Central

    Aubert, Geraldine

    2016-01-01

    Genetic defects of the immune system are referred to as primary immunodeficiencies (PIDs). These immunodeficiencies are clinically and immunologically heterogeneous and, therefore, pose a challenge not only for the clinician but also for the diagnostic immunologist. There are several methodological tools available for evaluation and monitoring of patients with PIDs, and of these tools, flow cytometry has gained prominence, both for phenotyping and functional assays. Flow cytometry allows real-time analysis of cellular composition, cell signaling, and other relevant immunological pathways, providing an accessible tool for rapid diagnostic and prognostic assessment. This minireview provides an overview of the use of flow cytometry in disease-specific diagnosis of PIDs, in addition to other broader applications, which include immune phenotyping and cellular functional measurements. PMID:26912782

  5. High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

    2016-02-01

    Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

  6. Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

    PubMed

    Käser, T; Pasternak, J A; Hamonic, G; Rieder, M; Lai, K; Delgado-Ortega, M; Gerdts, V; Meurens, F

    2016-05-01

    Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry. PMID:26849001

  7. Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

    PubMed

    Käser, T; Pasternak, J A; Hamonic, G; Rieder, M; Lai, K; Delgado-Ortega, M; Gerdts, V; Meurens, F

    2016-05-01

    Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry.

  8. The Bethesda system for reporting thyroid cytopathology: An experience of 1,382 cases in a community practice setting with the implication for risk of neoplasm and risk of malignancy.

    PubMed

    Wu, Howard Her-Juing; Rose, Crystal; Elsheikh, Tarik M

    2012-05-01

    The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) has provided a set of uniform diagnostic terminology including benign (B), atypia of undetermined significance (AUS), follicular neoplasm (FN), suspicious for malignancy (SM), malignancy (M), and nondiagnostic (ND) for the interpretation of thyroid fine-needle aspiration (FNA). We applied this terminology on our 1,382 thyroid aspirates in a community practice setting, which included 539 cases of B (39%), 376 cases of AUS (27.2%), 116 cases of FN (8.4%), 37 cases of malignant (2.7%), 36 cases of SM (2.6%), and 278 cases of ND (20.1%). Two hundred twenty-one cases (16%) of thyroid FNA had corresponding follow-up thyroidectomies. Each diagnostic category represented a unique association with risk of malignancy and risk of neoplasm. Based on histologic follow-up, the risk of neoplasm (including benign and malignant neoplasm) was B 14%, AUS 44%, FN 67%, SM 77%, and M 100% and the risk of malignancy was B 3%, AUS 6%, FN 22%, SM 56%, and M 100%. The classification and follow-up recommendation of TBSRTC are appropriate for each category. Both B and AUS are low-risk lesions with low probability of malignancy. FN predicts a higher rate for neoplasm but an intermediate rate for malignancy while SM carries a high risk for malignancy.

  9. A Different Perspective on Evaluating the Malignancy Rate of the Non-Diagnostic Category of the Bethesda System for Reporting Thyroid Cytopathology: A Single Institute Experience and Review of the Literature

    PubMed Central

    Onenerk, Mine; Erkan, Murat; Gursan, Nilufer; Kilinc, Emine; Kilicoglu, Gamze Zeynep

    2016-01-01

    Objective To determine the malignancy rate in the non-diagnostic (ND) category of the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) based on a different approach in relation to histopathology diagnoses. Study Design All ND fine needle aspirations (FNAs) that were performed under ultrasound guidance by an interventional radiologist with rapid on-site evaluation were included in the study. Slides were reevaluated to identify the cause of inadequacy as “qualitative” or “quantitative.” The malignancy rate of the ND category was assessed. Nodule/patient characteristics were compared between benign and malignant cases within the study cohort. Results The study cohort consisted of 192 ND aspirations. Overall there were 156 (81.3%) women and 36 (18.7%) men with a mean age of 50.6 years (range 24–82 years). The malignancy rate was 4.7%. None of the nodules (size, consistency, and number) or patient characteristics (gender and age) were found to be predictive of malignancy. Conclusion The malignancy rate of the ND category was high when compared to BSRTC predictions, but at the low end of the reported malignancy rates in the literature. Our results revealed that cyto-histopathologic correlation and method of malignancy rate estimation could have an effect on a wide range of reported malignancy rates. Furthermore, patient/nodule dependent factors were not statistically found to be predictive of malignancy. PMID:27627674

  10. Role of Flow Cytometry in the Diagnosis and Prognosis of Plasma Cell Myeloma.

    PubMed

    Olteanu, Horatiu

    2016-03-01

    This article provides an overview of the role of flow cytometry in the diagnosis and follow-up of plasma cell myeloma. A brief introduction to the general immunophenotypic features of normal and myeloma plasma cells is provided, followed by a discussion of technical issues as they relate to the application of flow cytometry in this entity. The prognostic and therapeutic utility of flow cytometric immunophenotyping in myeloma is also analyzed, with an emphasis on the growing role of minimal residual analysis as potential biomarker for evaluating treatment efficacy and for tailoring risk-adapted treatment, in prospective clinical trials. PMID:26940271

  11. Differentiation of normal and leukemic cells by 2D light scattering label-free static cytometry.

    PubMed

    Xie, Linyan; Liu, Qiao; Shao, Changshun; Su, Xuantao

    2016-09-19

    Two-dimensional (2D) light scattering patterns of single microspheres, normal granulocytes and leukemic cells are obtained by label-free static cytometry. Statistical results of experimental 2D light scattering patterns obtained from standard microspheres with a mean diameter of 4.19 μm agree well with theoretical simulations. High accuracy rates (greater than 92%) for label-free differentiation of normal granulocytes and leukemic cells, both the acute and chronic leukemic cells, are achieved by analyzing the 2D light scattering patterns. Our label-free static cytometry is promising for leukemia screening in clinics. PMID:27661908

  12. Circulation times of cancer cells by in vivo flow cytometry

    NASA Astrophysics Data System (ADS)

    Zhang, Li; Li, Yan; Gu, Zhengqin; Chen, Tong; Wang, Cheng; Wei, Xunbin

    2012-03-01

    Liver cancer is one of the most common malignancies in the world, with approximately 1,000,000 cases reported every year. Hepatocellular carcinoma may metastasize to lung, bones, kidney, and many other organs. Surgical resection, liver transplantation, chemotherapy and radiation therapy are the foundation of current HCC therapies. However the outcomes are poor: the survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. A recently developed "in vivo flow cytometer" combined with real-time confocal fluorescence imaging are used to assess spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labeled cells in vivo in real time without extracting blood sample. We have measured the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which were from the same origin and obtained by repetitive screenings in mice. >60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

  13. Computational prediction of manually gated rare cells in flow cytometry data.

    PubMed

    Qiu, Peng

    2015-07-01

    Rare cell identification is an interesting and challenging question in flow cytometry data analysis. In the literature, manual gating is a popular approach to distill flow cytometry data and drill down to the rare cells of interest, based on prior knowledge of measured protein markers and visual inspection of the data. Several computational algorithms have been proposed for rare cell identification. To compare existing algorithms and promote new developments, FlowCAP-III put forward one computational challenge that focused on this question. The challenge provided flow cytometry data for 202 training samples and two manually gated rare cell types for each training sample, roughly 0.02 and 0.04% of the cells, respectively. In addition, flow cytometry data for 203 testing samples were provided, and participants were invited to computationally identify the rare cells in the testing samples. Accuracy of the identification results was evaluated by comparing to manual gating of the testing samples. We participated in the challenge, and developed a method that combined the Hellinger divergence, a downsampling trick and the ensemble SVM. Our method achieved the highest accuracy in the challenge.

  14. Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.

    PubMed

    Leelatian, Nalin; Diggins, Kirsten E; Irish, Jonathan M

    2015-01-01

    Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass cytometry experiments routinely measure 25-35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two mass cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a mass cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank ( http://www.cytobank.org/irishlab/ ). PMID:26542718

  15. Application of flow cytometry to detection and characterization of Legionella spp

    SciTech Connect

    Tyndall, R.L.; Hand, R.E. Jr.; Mann, R.C.; Evans, C.; Jernigan, R.

    1985-04-01

    Flow cytometry, using fluorescein-bound specific antibodies and propidium iodide, was shown to be effective in detecting Legionella spp. in cooling tower waters. The procedure was quicker and less labor intensive than fluorescent microscopy. The use of these procedures also identified qualitative differences, perhaps related to infectivity, in Legionella populations.

  16. Improved Method for Bacterial Cell Capture after Flow Cytometry Cell Sorting ▿

    PubMed Central

    Guillebault, D.; Laghdass, M.; Catala, P.; Obernosterer, I.; Lebaron, P.

    2010-01-01

    Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification. PMID:20817799

  17. Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.

    PubMed

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

    2015-02-15

    Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 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 it reduces wet work and Ab 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 it should be applicable to fluorescence flow cytometry as well.

  18. In vivo plant flow cytometry: A first proof-of-concept

    PubMed Central

    Nedosekin, Dmitry A.; Khodakovskaya, Mariya V.; Biris, Alexandru S.; Wang, Daoyuan; Xu, Yang; Villagarcia, Hector; Galanzha, Ekaterina I.; Zharov, Vladimir P.

    2011-01-01

    In vivo flow cytometry has facilitated advances in the ultrasensitive detection of tumor cells, bacteria, nanoparticles, dyes, and other normal and abnormal objects directly in blood and lymph circulatory systems. Here, we propose in vivo plant flow cytometry for the real-time noninvasive study of nanomaterial transport in xylem and phloem plant vascular systems. As a proof of this concept, we demonstrate in vivo real-time photoacoustic monitoring of quantum dot-carbon nanotube conjugate uptake and uptake by roots and spreading through stem to leaves in a tomato plant. In addition, in vivo scanning cytometry using multimodal photoacoustic, photothermal, and fluorescent detection schematics provided multiplex detection and identification of nanoparticles accumulated in plant leaves in the presence of intensive absorption, scattering, and autofluorescent backgrounds. The use of a portable fiber-based photoacoustic flow cytometer for studies of plant vasculature was demonstrated. These integrated cytometry modalities using both endogenous and exogenous contrast agents have a potential to open new avenues of in vivo study of the nutrients, products of photosynthesis and metabolism, nanoparticles, infectious agents, and other objects transported through plant vasculature. PMID:21905208

  19. Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

    PubMed Central

    Hossain, Md. Sharoare; Johannisson, Anders; Wallgren, Margareta; Nagy, Szabolcs; Siqueira, Amanda Pimenta; Rodriguez-Martinez, Heriberto

    2011-01-01

    Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of ‘bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa. PMID:21478895

  20. Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways**

    EPA Science Inventory

    Rationale: The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. ...

  1. Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.

    PubMed

    Leelatian, Nalin; Diggins, Kirsten E; Irish, Jonathan M

    2015-01-01

    Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass cytometry experiments routinely measure 25-35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two mass cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a mass cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank ( http://www.cytobank.org/irishlab/ ).

  2. Teaching the Microbial Growth Curve Concept Using Microalgal Cultures and Flow Cytometry

    ERIC Educational Resources Information Center

    Forget, Nathalie; Belzile, Claude; Rioux, Pierre; Nozais, Christian

    2010-01-01

    The microbial growth curve is widely studied within microbiology classes and bacteria are usually the microbial model used. Here, we describe a novel laboratory protocol involving flow cytometry to assess the growth dynamics of the unicellular microalgae "Isochrysis galbana." The algal model represents an appropriate alternative to bacteria…

  3. A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots

    SciTech Connect

    Kovtun, Oleg; Ross, Emily; Tomlinson, Ian; Rosenthal, Sandra

    2012-01-01

    Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs).We anticipate that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.

  4. Data File Standard for Flow Cytometry, Version FCS 3.1

    SciTech Connect

    Spidlen, Josef; Moore, Wayne; Parks, David; Goldberg, Michael; Bray, Chris; Gorombey, Peter; Hyun, Bill; Hubbard, Mark; Lange, Simon; Lefebvre, Ray; Leif, Robert; Novo, David; Ostruszka, Leo; Treister, Adam; Wood, James; Murphy, Robert F.; Roederer, Mario; Sudar, Damir; Zigon, Robert; Brinkman, Ryan R.; Brierre, Pierre

    2009-11-10

    The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type. The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.

  5. Computational prediction of manually gated rare cells in flow cytometry data1

    PubMed Central

    Qiu, Peng

    2015-01-01

    Rare cell identification is an interesting and challenging question in flow cytometry data analysis. In the literature, manual gating is a popular approach to distill flow cytometry data and drill down to the rare cells of interest, based on prior knowledge of measured protein markers and visual inspection of the data. Several computational algorithms have been proposed for rare cell identification. To compare existing algorithms and promote new developments, FlowCAP-III put forward one computational challenge that focused on this question. The challenge provided flow cytometry data for 202 training samples and two manually gated rare cell types for each training sample, roughly 0.02% and 0.04% of the cells, respectively. In addition, flow cytometry data for 203 testing samples were provided, and participants were invited to computationally identify the rare cells in the testing samples. Accuracy of the identification results was evaluated by comparing to manual gating of the testing samples. We participated in the challenge, and developed a method that combined the Hellinger divergence, a downsampling trick and the ensemble SVM. Our method achieved the highest accuracy in the challenge. PMID:25755118

  6. Minimal residual disease analysis by eight-color flow cytometry in relapsed childhood acute lymphoblastic leukemia.

    PubMed

    Karawajew, Leonid; Dworzak, Michael; Ratei, Richard; Rhein, Peter; Gaipa, Giuseppe; Buldini, Barbara; Basso, Giuseppe; Hrusak, Ondrej; Ludwig, Wolf-Dieter; Henze, Günter; Seeger, Karl; von Stackelberg, Arend; Mejstrikova, Ester; Eckert, Cornelia

    2015-07-01

    Multiparametric flow cytometry is an alternative approach to the polymerase chain reaction method for evaluating minimal residual disease in treatment protocols for primary acute lymphoblastic leukemia. Given considerable differences between primary and relapsed acute lymphoblastic leukemia treatment regimens, flow cytometric assessment of minimal residual disease in relapsed leukemia requires an independent comprehensive investigation. In the present study we addressed evaluation of minimal residual disease by flow cytometry in the clinical trial for childhood relapsed acute lymphoblastic leukemia using eight-color flow cytometry. The major challenge of the study was to reliably identify low amounts of residual leukemic cells against the complex background of regeneration, characteristic of follow-up samples during relapse treatment. In a prospective study of 263 follow-up bone marrow samples from 122 patients with B-cell precursor acute lymphoblastic leukemia, we tested various B-cell markers, adapted the antibody panel to the treatment protocol, and evaluated its performance by a blinded parallel comparison with the polymerase chain reaction data. The resulting eight-color single-tube panel showed a consistently high overall concordance (P<0.001) and, under optimal conditions, sensitivity similar to that of the reference polymerase chain reaction method. Overall, evaluation of minimal residual disease by flow cytometry can be successfully integrated into the clinical management of relapsed childhood acute lymphoblastic leukemia either as complementary to the polymerase chain reaction or as an independent risk stratification tool. ALL-REZ BFM 2002 clinical trial information: NCT00114348.

  7. Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species.

    PubMed

    Catena, Raúl; Özcan, Alaz; Zivanovic, Nevena; Bodenmiller, Bernd

    2016-05-01

    Mass cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in mass cytometry, called mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO4 and RuO4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO4 and RuO4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO4 and RuO4 were used in a 20-sample barcoding protocol together with palladium isotopes. As mass channels occupied by osmium and ruthenium are not used for antibody detection the number of masses effectively utilized in a single experiment is expanded. OsO4 and RuO4 can therefore be used as MCB reagents for a wide range of mass cytometry workflows. © 2016 International Society for Advancement of Cytometry. PMID:27018769

  8. American Society of Cytopathology workload recommendations for automated Pap test screening: developed by the productivity and quality assurance in the era of automated screening task force.

    PubMed

    Elsheikh, Tarik M; Austin, R Marshall; Chhieng, David F; Miller, Fern S; Moriarty, Ann T; Renshaw, Andrew A

    2013-02-01

    Based on current literature and the best available research to date, the current FDA workload limits for automated image-assisted screening, including the ThinPrep Imaging System and the FocalPoint GS, of 100 slides/day (imaged only slides counted as 0.5) are extremely high and may be associated with significant reduction in sensitivity. This task force has proposed six recommendations relating to cytotechnologist (CT) workload in automated image-guided Pap test screening, which have already been endorsed by major pathology professional societies. These evidence-based recommendations, however, pertain only to gynecologic specimens with image-assisted screening, as there is no current available data to justify modifying screening practices regarding non-gynecologic specimens. The proposed recommendations are as follow: 1) CT workday should not include more than 7 hours of Pap test screening in a 24-hr period, and an 8-hr shift day must include at least 2 paid mini-breaks of 15 minutes each and a 30-minute lunch break. 2) Future Studies examining CT workload should use actual hours of screening rather than lesser number of hours extrapolated to 8-hour days. 3) Average laboratory CT workload should NOT exceed 70 slides/day (slides counted per 2010 FDA bulletin). 4) Proportion of imaged slides that undergo full manual review should be at least either 15%, or twice (2×) the epithelial cell abnormality (ECA) rate, whichever is greater. 5) ECA-adjusted workload measure is a promising method for calculating and monitoring CT workload, but further studies of this method are necessary before full endorsement. 6) CT productivity and workload limits are just one aspect of a good quality assurance program in a cytology laboratory, so other quality indicators to assess CT performance are essential. PMID:22351120

  9. Gel microdrop flow cytometry assay for low-dose studies of chemical and radiation cytotoxicity.

    PubMed

    Bogen, K T; Enns, L; Hall, L C; Keating, G A; Weinfeld, M; Murphy, G; Wu, R W; Panteleakos, F N

    2001-03-01

    Low-level cytotoxicity may affect low-dose dose-response relations for cancer and other endpoints. Conventional colony-forming assays are rarely sensitive enough to examine small changes in cell survival and growth. Automated image-analysis techniques are limited to ca. 10(4) cells/plate. An alternative method involves encapsulation of single proliferating cells into ca. 35-75-microm-diameter agarose gel microdrops (GMDs) that are randomly grouped, differential exposure of these groups, culture at 37 degrees C for 3-5 days, and finally GMD analysis by flow cytometry (FC) to determine the ratio of GMDs containing multiple versus single cells as a measure of clonogenic survival. This GMD/FC assay was used to examine low-dose cell killing induced by a cooked-meat mutagen/rodent-carcinogen (MeIQx) in DNA-repair-deficient/metabolically-sensitive CHO cells. Results of conventional colony-forming assays using up to 30 replicate plates indicate a shouldered, threshold-like dose-response; in contrast, those obtained using the GMD/FC assay suggest "hypersensitivity"-like nonlinearity in dose-response. The GMD/FC assay was also applied to human A549 lung cells after GMD-encapsulation and gamma radiation followed by culture for a total of 4 days, to examine survival after exposure to > or =100 cGy delivered at a relatively low dose rate (0.18 cGy/min). Dose-response for clonogenic growth was again observed to be reduced with apparent nonlinear suggesting hypersensitivity between 0 and 50 cGy, insofar as doses of 5 and 10 cGy appear to be ca. fivefold more effective per unit dose than the 50- or 100-cGy doses used. The GMD/FC assay may thus reveal low-dose dose-response relations for chemical and radiation effects on cell proliferation/killing with implications for low-dose risk assessment.

  10. Generalized Unmixing Model for Multispectral Flow Cytometry Utilizing Nonsquare Compensation Matrices

    PubMed Central

    Novo, David; Grégori, Gérald; Rajwa, Bartek

    2014-01-01

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

  11. Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry.

    PubMed

    Peng, Yan; Lee-Pullen, Tracey F; Heel, Kathy; Millar, A Harvey; Baer, Boris

    2014-05-01

    Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n = 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods.

  12. Flow cytometry detection of vitamin D receptor changes during vitamin D treatment in Crohn's disease.

    PubMed

    Bendix, M; Dige, A; Deleuran, B; Dahlerup, J F; Jørgensen, S P; Bartels, L E; Husted, L B; Harsløf, T; Langdahl, B; Agnholt, J

    2015-07-01

    Crohn's disease (CD) is a chronic inflammatory disease associated with a dysregulated T cell response towards intestinal microflora. Vitamin D has immune modulatory effects on T cells through the nuclear vitamin D receptor (VDR) in vitro. It is unclear how oral vitamin D treatment affects VDR expression. The aim of this study was to establish a flow cytometry protocol, including nuclear and cytoplasmic VDR expression, and to investigate the effects of vitamin D treatment on T cell VDR expression in CD patients. The flow cytometry protocol for VDR staining was developed using the human acute monocytic leukaemia cell line (THP-1). The protocol was evaluated in anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) from vitamin D3- (n = 9) and placebo-treated (n = 9) CD patients. Anti-VDR-stained PBMCs were examined by flow cytometry, and their cytokine production was determined by cytokine bead array. VDR, CYP27B1 and RXRα mRNA expression levels in CD4(+) T cells were measured by quantitative reverse transcriptase polymerase chain reaction. The flow cytometry protocol enabled detection of cytoplasmic and nuclear VDR expression. The results were confirmed by confocal microscopy and supported by correlation with VDR mRNA expression. VDR expression in CD4(+) T cells increased following stimulation. This VDR up-regulation was inhibited with 30% by vitamin D treatment compared to placebo in CD patients (P = 0027). VDR expression was correlated with in-vitro interferon-γ production in stimulated PBMCs (P = 0.01). Flow cytometry is a useful method with which to measure intracellular VDR expression. Vitamin D treatment in CD patients reduces T cell receptor-mediated VDR up-regulation.

  13. National flow cytometry and sorting research resource. Annual progress report, July, 1, 1994--June 30, 1995, Year 12

    SciTech Connect

    Jett, J.H.

    1995-04-27

    Research progress utilizing flow cytometry is described. Topics include: rapid kinetics flow cytometry; characterization of size determinations for small DNA fragments; statistical analysis; energy transfer measurements of molecular confirmation in micelles; and enrichment of Mus spretus chromosomes by dual parameter flow sorting and identification of sorted fractions by fluorescence in-situ hybridization onto G-banded mouse metaphase spreads.

  14. Electromagnetic field interactions with micro channels, particles and cells: Application to advanced cytometry

    NASA Astrophysics Data System (ADS)

    Venkatapathi, Murugesan

    This thesis involves a study of the interaction of laser beams with micro channels and micro particles/cells using the electromagnetic field approach. This problem is relevant to the next generation cytometry, in particular to model based design of flow cytometers. The field approach is applied to study light scatter from particles/cells and also internal and scattered fields of cylindrical micro channels that are important for optical interrogation of particles and cells flowing through. Though current flow cytometers use qualitative fluorescence measurements for biological analysis, other viable optical interrogation techniques like light scatter, quantitative fluorescence and Coherent anti-stokes Raman scatter (CARS) are being studied for application to flow cytometry. The light scatter from particles and cells in a flow cytometer has been studied with the objective of extracting useful information about the particles using scatter measurements. First, the correlation between the size of particles and the current forward scatter measurements was both analytically modeled and experimentally determined. These results indicated that integrated scatter measurements currently used in flow cytometry (forward and side scatter) cannot be used to unambiguously estimate size, shape or refractive index of particles for classification. It is shown that multi-angle scatter measurements can be used to classify micro spheres of different sizes/refractive indices and different bacteria species, provided the scatter measurements are designed based on numerical scatter models. The numerical scatter models were then also used to do a preliminary study of correlation of scatter with internal structure of simple cells like stem cells. A few multivariate statistical methods have been applied for the classification of such particles in flow cytometry using scatter and multi-spectral fluorescence measurements. Typically the micro channels used in flow cytometry have square or circular

  15. Sources of error in screening by cytometry for the effects of environmental mutagens

    SciTech Connect

    Tiersch, T.R. ); Wachtel, S.S. )

    1993-01-01

    Flow cytometry is often used to detect DNA aneuploidy and mosaicism associated with malignancy or genetic damage. Yet DNA aneuploidy and mosaicism detected by flow cytometry may be more apparent than real. In contrast to the DNA mass observed for blood, the authors consistently found markedly different values and higher variability for DNA mass among other tissues collected from the same animal. Prepared mixtures of blood and other cells generated multiple fluorescence peaks identical to those that might be expected for aneuploid mosaicism. Moreover, analysis of tissues such as feather pulp, which contains a combination of cell types, yielded multiple fluorescence peaks that were not observed when blood alone was analyzed. Thus care should be exercised in classifying DNA values from different tissues as normal or abnormal, because the appearance of supernumerary fluorescence peaks might not always indicate the presence of abnormal cell populations.

  16. MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions

    NASA Astrophysics Data System (ADS)

    Shipunova, V. O.; Nikitin, M. P.; Nikitin, P. I.; Deyev, S. M.

    2016-06-01

    Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method

  17. Metabolic activity in filamentous fungi can be analysed by flow cytometry.

    PubMed

    Bradner, J R; Nevalainen, K M H

    2003-08-01

    The use of flow cytometry in combination with fluorescent dyes as a technique to rapidly differentiate and enumerate bacterial and yeast cells is well established. We have shown that through the judicial choice of stains, the nondestructive screening and sorting of fungal material is possible. The early stages of growth, from germination through hyphal development of three filamentous fungal species, Penicillium, Phoma and Trichoderma, have been followed using forward- and side-angle scatter on a Becton Dickinson FACSCalibur flow cytometer. By staining isolates with the permeant fluorogenic substrates, dihydroethidium and hexidium iodide metabolic activity in the developing hyphae has been measured. We have been able to demonstrate that there is a 12-13 h window of opportunity during which germination and the early stages of hyphal development of filamentous fungi can be analysed by flow cytometry.

  18. Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment.

    PubMed

    Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

    2015-11-17

    Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible.

  19. FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data.

    PubMed

    Van Gassen, Sofie; Callebaut, Britt; Van Helden, Mary J; Lambrecht, Bart N; Demeester, Piet; Dhaene, Tom; Saeys, Yvan

    2015-07-01

    The number of markers measured in both flow and mass cytometry keeps increasing steadily. Although this provides a wealth of information, it becomes infeasible to analyze these datasets manually. When using 2D scatter plots, the number of possible plots increases exponentially with the number of markers and therefore, relevant information that is present in the data might be missed. In this article, we introduce a new visualization technique, called FlowSOM, which analyzes Flow or mass cytometry data using a Self-Organizing Map. Using a two-level clustering and star charts, our algorithm helps to obtain a clear overview of how all markers are behaving on all cells, and to detect subsets that might be missed otherwise. R code is available at https://github.com/SofieVG/FlowSOM and will be made available at Bioconductor. PMID:25573116

  20. In Vivo Monitoring of Multiple Circulating Cell Populations Using Two-photon Flow Cytometry

    PubMed Central

    Tkaczyk, Eric R.; Zhong, Cheng Frank; Ye, Jing Yong; Myc, Andrzej; Thomas, Thommey; Cao, Zhengyi; Duran-Struuck, Raimon; Luker, Kathryn E.; Luker, Gary D.; Norris, Theodore B.; Baker, James R.

    2008-01-01

    To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes. PMID:19221581

  1. In Vivo Monitoring of Multiple Circulating Cell Populations Using Two-photon Flow Cytometry.

    PubMed

    Tkaczyk, Eric R; Zhong, Cheng Frank; Ye, Jing Yong; Myc, Andrzej; Thomas, Thommey; Cao, Zhengyi; Duran-Struuck, Raimon; Luker, Kathryn E; Luker, Gary D; Norris, Theodore B; Baker, James R

    2008-02-15

    To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes.

  2. Validation of algal viability treated with total residual oxidant and organic matter by flow cytometry.

    PubMed

    Lee, Junghyun; Choi, Eun Joo; Rhie, Kitae

    2015-08-15

    Algal cell growth after starch and oxidant treatments in seawater species (Isochrysis galbana and Phaeodactylum tricornutum) and freshwater species (Selenastrum capricornutum and Scenedesmus obliquus) were evaluated by flow cytometry with fluorescein diacetate (FDA) staining to determine algal viability. Growth of algal cell was found to be significantly different among groups treated with NaOCl, starch and/or sodium thiosulfate, which are active substance (Total Residual Oxidant; TRO as Cl2), organic compound to meet efficacy testing standard and neutralizer of TRO by Ballast Water Management Convention of International Maritime Organization, respectively. The viability of algal cell treated with TRO in starch-add culture of 5days after treatment and neutralization was decreased significantly. ATP contents of the treated algal cells corresponded to the FL1 fluorescent signal of flow cytometry with FDA staining. I. galbana was the most sensitive to TRO-neutralized cultures during viability analysis.

  3. Use of Flow Cytometry to Measure Biogeochemical Rates and Processes in the Ocean

    NASA Astrophysics Data System (ADS)

    Lomas, Michael W.; Bronk, Deborah A.; van den Engh, Ger

    2011-01-01

    An important goal of marine biogeochemists is to quantify the rates at which elements cycle through the ocean's diverse microbial assemblage, as well as to determine how these rates vary in time and space. The traditional view that phytoplankton are producers and bacteria are consumers has been found to be overly simplistic, and environmental metagenomics is discovering new and important microbial metabolisms at an accelerating rate. Many nutritional strategies previously attributed to one microorganism or functional group are also or instead carried out by other groups. To tease apart which organism is doing what will require new analytical approaches. Flow cytometry, when combined with other techniques, has great potential for expanding our understanding of microbial interactions because groups can be distinguished optically, sorted, and then collected for subsequent analyses. Herein, we review the advances in our understanding of marine biogeochemistry that have arisen from the use of flow cytometry.

  4. MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions.

    PubMed

    Shipunova, V O; Nikitin, M P; Nikitin, P I; Deyev, S M

    2016-07-01

    Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.

  5. Addressing the malaria drug resistance challenge using flow cytometry to discover new antimalarials.

    PubMed

    Grimberg, Brian T; Jaworska, Maria M; Hough, Lindsay B; Zimmerman, Peter A; Phillips, James G

    2009-09-15

    A new flow cytometry method that uses an optimized DNA and RNA staining strategy to monitor the growth and development of the Plasmodium falciparum strain W2mef has been used in a pilot study and has identified Bay 43-9006 1, SU 11274 2, and TMC 125 5 as compounds that exhibit potent (<1 microM) overall and ring stage in vitro antimalarial activity.

  6. Detection of antibodies to Staphylococcus aureus in water buffalo milk by flow cytometry.

    PubMed

    D'Apice, L; Fenizia, D; Capparelli, R; Scala, F; Iannelli, D

    1996-03-01

    An assay has been developed to detect antibodies to Staphylococcus aureus in water buffalo milk by flow cytometry. The method was the protein A-deficient strain Wood 46 of S aureus incubated with milk samples and fluorescein-labelled rabbit anti-water buffalo antiserum. The assay can detect antibodies when the pathogen is not detectable by bacterial tests and can determine the antibody titre directly on undiluted samples.

  7. Rapid parallel flow cytometry assays of active GTPases using effector beads

    PubMed Central

    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela

    2013-01-01

    We describe a rapid assay for measuring the cellular activity of small GTPases in response to a specific stimulus. Effector functionalized beads are used to quantify in parallel multiple, GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus. PMID:23928044

  8. In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues

    PubMed Central

    Biris, Alexandru S.; Galanzha, Ekaterina I.; Li, Zhongrui; Mahmood, Meena; Xu, Yang; Zharov, Vladimir P.

    2016-01-01

    Nanoparticles are intensively being explored as contrast agents for medical diagnostics and therapies using various optical methods. We present the first demonstration of the use of time-resolved Raman spectroscopy for in vivo real-time detection of circulating carbon nanotubes (CNTs) or cancer cells labeled with CNTs in the lymph, blood, and tissues of live animals with fast spectral acquisition times of down to few milliseconds. After intravenously administering CNTs in the tail vein of the rat, this technique provides the ability to detect the circulation of CNTs in the blood microvessels of the intact rat ear. The capability of Raman spectroscopy is also demonstrated to monitor, identify, and image the CNTs during their transportation by lymphatics in the rat ear and mesentery. The strong and specific Raman scattering properties of CNTs make it possible to detect in vitro and in vivo single cancer cells (HeLa) tagged with CNTs. In vivo Raman flow cytometry opens a new avenue for multiparameter analysis of circulating nanoparticles with strong Raman scattering properties and their pharmokinetics in blood and lymph systems. Moreover, this technology has the potential for molecular detection and identification of circulating tumor cells, and infections labeled with CNTs. PMID:19405719

  9. Characterisation of the green turtle's leukocyte subpopulations by flow cytometry and evaluation of their phagocytic activity.

    PubMed

    Muñoz, F A; Franco-Noguez, S Y; Gonzalez-Ballesteros, E; Negrete-Philippe, A C; Flores-Romo, L

    2014-06-01

    Phagocytosis is a fundamental aspect of innate immunity that is conserved across many species making it a potentially useful health-assessment tool for wildlife. In non-mammalian vertebrates, heterophils, monocytes, macrophages, melanomacrophages, and thrombocytes all have phagocytic properties. Recently, B lymphocytes from fish, amphibians, and aquatic turtles have also showed phagocytic capacity. Phagocytes can be studied by flow cytometry; however, the use of this tool is complicated in reptiles partly because nucleated erythrocytes complicate the procedure. We separated green turtle leukocytes by density gradient centrifugation and identified subpopulations by flow cytometry and confocal microscopy. Additionally, we assessed their ability to phagocytize Fluorspheres and Ovoalbumin-Alexa. We found that heterophils and lymphocytes but not monocytes could be easily identified by flow cytometry. While heterophils from adults and juvenile turtles were equally able to phagocytize fluorspheres, adults had significantly more phagocytic ability for OVA-Alexa. Lymphocytes had a mild phagocytic activity with fluorospheres (27-38 %; 39-45 %) and OVA-Alexa (35-46 %; 14-22 %) in juvenile and adult green turtles, respectively. Confocal microscopy confirmed phagocytosis of fluorospheres in both heterophils and lymphocytes. This provides the first evidence that green turtle lymphocytes have phagocytic activity and that this assay could potentially be useful to measure one aspect of innate immunity in this species. PMID:24570347

  10. Micro flow cytometry utilizing a magnetic bead-based immunoassay for rapid virus detection.

    PubMed

    Yang, Sung-Yi; Lien, Kang-Yi; Huang, Kao-Jean; Lei, Huan-Yao; Lee, Gwo-Bin

    2008-12-01

    The current study presents a new miniature microfluidic flow cytometer integrated with several functional micro-devices capable of viral sample purification and detection by utilizing a magnetic bead-based immunoassay. The magnetic beads were conjugated with specific antibodies, which can recognize and capture target viruses. Another dye-labeled anti-virus antibody was then used to mark the bead-bound virus for the subsequent optical detection. Several essential components were integrated onto a single chip including a sample incubation module, a micro flow cytometry module and an optical detection module. The sample incubation module consisting of pneumatic micropumps and a membrane-type, active micromixer was used for purifying and enriching the target virus-bound magnetic beads with the aid of a permanent magnet. The micro flow cytometry module and the optical detection module were used to perform the functions of virus counting and collection. Experimental results showed that virus samples with a concentration of 10(3)PFU/ml can be automatically detected successfully by the developed system. In addition, the entire diagnosis procedure including sample incubation and virus detection took only about 40min. Consequently, the proposed micro flow cytometry may provide a powerful platform for rapid diagnosis and future biological applications.

  11. A novel procedure of quantitation of virus based on microflow cytometry analysis.

    PubMed

    Vazquez, Diego; López-Vázquez, Carmen; Cutrín, Juan Manuel; Dopazo, Carlos P

    2016-03-01

    The accurate and fast titration of viruses is a critical step in research laboratories and biotechnology industries. Different approaches are commonly applied which either are time consuming (like the plaque and endpoint dilution assays) or do not ensure quantification of only infective particles (like quantitative real-time PCR). In the last decade, a methodology based on the analysis of infected cells by flow cytometry and fluorescence-activated cell sorting (FACS) has been reported as a fast and reliable test for the titration of some viruses. However, this technology needs expensive equipment and expert technicians to operate it. Recently, the "lab on a chip" integrated devices have brought about the miniaturization of this equipment, turning this technology into an affordable and easy-to-use alternative to traditional flow cytometry. In the present study, we have designed a microflow cytometry (μFC) procedure for the quantitation of viruses, using the infectious pancreatic necrosis virus (IPNV) as a model. The optimization of conditions and validation of the method are reported here.

  12. Flow cytometry community fingerprinting and amplicon sequencing for the assessment of landfill leachate cellulolytic bioaugmentation.

    PubMed

    Kinet, R; Dzaomuho, P; Baert, J; Taminiau, B; Daube, G; Nezer, C; Brostaux, Y; Nguyen, F; Dumont, G; Thonart, P; Delvigne, F

    2016-08-01

    Flow cytometry (FCM) is a high throughput single cell technology that is actually becoming widely used for studying phenotypic and genotypic diversity among microbial communities. This technology is considered in this work for the assessment of a bioaugmentation treatment in order to enhance cellulolytic potential of landfill leachate. The experimental results reveal the relevant increase of leachate cellulolytic potential due to bioaugmentation. Cytometric monitoring of microbial dynamics along these assays is then realized. The flow FP package is used to establish microbial samples fingerprint from initial 2D cytometry histograms. This procedure allows highlighting microbial communities' variation along the assays. Cytometric and 16S rRNA gene sequencing fingerprinting methods are then compared. The two approaches give same evidence about microbial dynamics throughout digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at genus or species level. Same phenotypical profiles of microbiota during assays matched to several 16S rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses. PMID:27160955

  13. Simultaneous measurement of human hematopoietic stem and progenitor cells in blood using multicolor flow cytometry.

    PubMed

    Cimato, Thomas R; Furlage, Rosemary L; Conway, Alexis; Wallace, Paul K

    2016-09-01

    Hematopoietic stem cells are the source of all inflammatory cell types. Discovery of specific cell surface markers unique to human hematopoietic stem (HSC) and progenitor (HSPC) cell populations has facilitated studies of their development from stem cells to mature cells. The specific marker profiles of HSCs and HSPCs can be used to understand their role in human inflammatory diseases. The goal of this study is to simultaneously measure HSCs and HSPCs in normal human venous blood using multicolor flow cytometry. Our secondary aim is to determine how G-CSF mobilization alters the quantity of each HSC and HSPC population. Here we show that cells within the CD34+ fraction of human venous blood contains cells with the same cell surface markers found in human bone marrow samples. Mobilization with G-CSF significantly increases the quantity of total CD34+ cells, blood borne HSCs, multipotent progenitors, common myeloid progenitors, and megakaryocyte erythroid progenitors as a percentage of total MNCs analyzed. The increase in blood borne common lymphoid and granulocyte macrophage progenitors with G-CSF treatment did not reach significance. G-CSF treatment predominantly increased the numbers of HSCs and multipotent progenitors in the total CD34+ cell population; common myeloid progenitors and megakaryocyte erythroid progenitors were enriched relative to total MNCs analyzed, but not relative to total CD34+ cells. Our findings illustrate the utility of multicolor flow cytometry to quantify circulating HSCs and HSPCs in venous blood samples from human subjects. © 2016 International Clinical Cytometry Society. PMID:26663713

  14. Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts

    PubMed Central

    Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I.; Sawant, Rupa; Torchilin, Vladimir P.; Verkhusha, Vladislav V.; Ma, Jie; Frank, Markus H.; Biris, Alexandru S.; Zharov, Vladimir P.

    2012-01-01

    In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real-time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa-660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co-expressing melanin and green fluorescent protein (GFP), C8161-GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near-infrared iRFP protein, and quantum dot-carbon nanotube conjugates. Negative contrast flow cytometry provided label-free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time-resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique. The principles of integrated photoacoustic and fluorescence flow cytometry using positive contrast for detection of strongly absorbing and fluorescent cells and negative contrast for detection of weakly absorbing and fluorescent cells in blood absorption and autofluorescence background, respectively. PMID:22903924

  15. Methods for discovery and characterization of cell subsets in high dimensional mass cytometry data.

    PubMed

    Diggins, Kirsten E; Ferrell, P Brent; Irish, Jonathan M

    2015-07-01

    The flood of high-dimensional data resulting from mass cytometry experiments that measure more than 40 features of individual cells has stimulated creation of new single cell computational biology tools. These tools draw on advances in the field of machine learning to capture multi-parametric relationships and reveal cells that are easily overlooked in traditional analysis. Here, we introduce a workflow for high dimensional mass cytometry data that emphasizes unsupervised approaches and visualizes data in both single cell and population level views. This workflow includes three central components that are common across mass cytometry analysis approaches: (1) distinguishing initial populations, (2) revealing cell subsets, and (3) characterizing subset features. In the implementation described here, viSNE, SPADE, and heatmaps were used sequentially to comprehensively characterize and compare healthy and malignant human tissue samples. The use of multiple methods helps provide a comprehensive view of results, and the largely unsupervised workflow facilitates automation and helps researchers avoid missing cell populations with unusual or unexpected phenotypes. Together, these methods develop a framework for future machine learning of cell identity.

  16. Unfold High-Dimensional Clouds for Exhaustive Gating of Flow Cytometry Data.

    PubMed

    Qiu, Peng

    2014-01-01

    Flow cytometry is able to measure the expressions of multiple proteins simultaneously at the single-cell level. A flow cytometry experiment on one biological sample provides measurements of several protein markers on or inside a large number of individual cells in that sample. Analysis of such data often aims to identify subpopulations of cells with distinct phenotypes. Currently, the most widely used analytical approach in the flow cytometry community is manual gating on a sequence of nested biaxial plots, which is highly subjective, labor intensive, and not exhaustive. To address those issues, a number of methods have been developed to automate the gating analysis by clustering algorithms. However, completely removing the subjectivity can be quite challenging. This paper describes an alternative approach. Instead of automating the analysis, we develop novel visualizations to facilitate manual gating. The proposed method views single-cell data of one biological sample as a high-dimensional point cloud of cells, derives the skeleton of the cloud, and unfolds the skeleton to generate 2D visualizations. We demonstrate the utility of the proposed visualization using real data, and provide quantitative comparison to visualizations generated from principal component analysis and multidimensional scaling.

  17. Flow cytometry as an auxiliary tool for the selection of probiotic bacteria.

    PubMed

    Mudroňová, D

    2015-01-01

    Selection of appropriate bacterial strains is crucial for development of new probiotic preparations. The fundamental prerequisite for potential efficacy of a probiotic preparation for oral application is the selection of appropriate bacterial strains with good gastrointestinal colonisation abilities, antimicrobial activity, and tolerance of conditions in the gastrointestinal tract, resistance to different antimicrobial agents, survival during processing and storage. The strain should be genetically stable, it should have good growth properties, to maintain its high viability at processing and when in storage. Mostly, the properties of promising strains are tested in the first phase in vitro, and only the best ones undergo subsequent in vivo testing. in vitro tests are often performed by classical microbiological cultivation methods which are material and time consuming, and they are not able to distinguish between 'viable but nonculturable' and dead bacteria. Flow cytometry is usually used for counting, phenotyping or functional characterisation of immune cells. Nowadays, flow cytometry is increasingly used in microbiology for counting bacteria, determining their viability and metabolic activity, detecting specific strains or testing their adherence abilities. The utilisation of flow cytometry in combination with an appropriate fluorescent labelling represents an effective and rapid method for the selection of probiotic bacteria.

  18. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    PubMed Central

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-01-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis. PMID:27596736

  19. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood.

    PubMed

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-01-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis. PMID:27596736

  20. Discovering cell types in flow cytometry data with random matrix theory

    NASA Astrophysics Data System (ADS)

    Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

    Flow cytometry is a widely used experimental technique in immunology research. During the experiments, peripheral blood mononuclear cells (PBMC) from a single patient, labeled with multiple fluorescent stains that bind to different proteins, are illuminated by a laser. The intensity of each stain on a single cell is recorded and reflects the amount of protein expressed by that cell. The data analysis focuses on identifying specific cell types related to a disease. Different cell types can be identified by the type and amount of protein they express. To date, this has most often been done manually by labelling a protein as expressed or not while ignoring the amount of expression. Using a cross correlation matrix of stain intensities, which contains both information on the proteins expressed and their amount, has been largely ignored by researchers as it suffers from measurement noise. Here we present an algorithm to identify cell types in flow cytometry data which uses random matrix theory (RMT) to reduce noise in a cross correlation matrix. We demonstrate our method using a published flow cytometry data set. Compared with previous analysis techniques, we were able to rediscover relevant cell types in an automatic way. Department of Physics, University of Maryland, College Park, MD 20742.

  1. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    NASA Astrophysics Data System (ADS)

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-09-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

  2. International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards

    PubMed Central

    Holmes, Kevin L.; Fontes, Benjamin; Hogarth, Philip; Konz, Richard; Monard, Simon; Pletcher, Charles H.; Wadley, Robert B.; Schmid, Ingrid; Perfetto, Stephen P.

    2014-01-01

    Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. PMID:24634405

  3. Methods for discovery and characterization of cell subsets in high dimensional mass cytometry data

    PubMed Central

    Diggins, Kirsten E.; Ferrell, P. Brent; Irish, Jonathan M.

    2015-01-01

    The flood of high-dimensional data resulting from mass cytometry experiments that measure more than 40 features of individual cells has stimulated creation of new single cell computational biology tools. These tools draw on advances in the field of machine learning to capture multi-parametric relationships and reveal cells that are easily overlooked in traditional analysis. Here, we introduce a workflow for high dimensional mass cytometry data that emphasizes unsupervised approaches and visualizes data in both single cell and population level views. This workflow includes three central components that are common across mass cytometry analysis approaches: 1) distinguishing initial populations, 2) revealing cell subsets, and 3) characterizing subset features. In the implementation described here, viSNE, SPADE, and heatmaps were used sequentially to comprehensively characterize and compare healthy and malignant human tissue samples. The use of multiple methods helps provide a comprehensive view of results, and the largely unsupervised workflow facilitates automation and helps researchers avoid missing cell populations with unusual or unexpected phenotypes. Together, these methods develop a framework for future machine learning of cell identity. PMID:25979346

  4. Flow cytometry community fingerprinting and amplicon sequencing for the assessment of landfill leachate cellulolytic bioaugmentation.

    PubMed

    Kinet, R; Dzaomuho, P; Baert, J; Taminiau, B; Daube, G; Nezer, C; Brostaux, Y; Nguyen, F; Dumont, G; Thonart, P; Delvigne, F

    2016-08-01

    Flow cytometry (FCM) is a high throughput single cell technology that is actually becoming widely used for studying phenotypic and genotypic diversity among microbial communities. This technology is considered in this work for the assessment of a bioaugmentation treatment in order to enhance cellulolytic potential of landfill leachate. The experimental results reveal the relevant increase of leachate cellulolytic potential due to bioaugmentation. Cytometric monitoring of microbial dynamics along these assays is then realized. The flow FP package is used to establish microbial samples fingerprint from initial 2D cytometry histograms. This procedure allows highlighting microbial communities' variation along the assays. Cytometric and 16S rRNA gene sequencing fingerprinting methods are then compared. The two approaches give same evidence about microbial dynamics throughout digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at genus or species level. Same phenotypical profiles of microbiota during assays matched to several 16S rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses.

  5. Automated gating of flow cytometry data via robust model-based clustering.

    PubMed

    Lo, Kenneth; Brinkman, Ryan Remy; Gottardo, Raphael

    2008-04-01

    The capability of flow cytometry to offer rapid quantification of multidimensional characteristics for millions of cells has made this technology indispensable for health research, medical diagnosis, and treatment. However, the lack of statistical and bioinformatics tools to parallel recent high-throughput technological advancements has hindered this technology from reaching its full potential. We propose a flexible statistical model-based clustering approach for identifying cell populations in flow cytometry data based on t-mixture models with a Box-Cox transformation. This approach generalizes the popular Gaussian mixture models to account for outliers and allow for nonelliptical clusters. We describe an Expectation-Maximization (EM) algorithm to simultaneously handle parameter estimation and transformation selection. Using two publicly available datasets, we demonstrate that our proposed methodology provides enough flexibility and robustness to mimic manual gating results performed by an expert researcher. In addition, we present results from a simulation study, which show that this new clustering framework gives better results in terms of robustness to model misspecification and estimation of the number of clusters, compared to the popular mixture models. The proposed clustering methodology is well adapted to automated analysis of flow cytometry data. It tends to give more reproducible results, and helps reduce the significant subjectivity and human time cost encountered in manual gating analysis.

  6. Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry.

    PubMed

    Freire, João Miguel; Gaspar, Diana; de la Torre, Beatriz Garcia; Veiga, Ana Salomé; Andreu, David; Castanho, Miguel A R B

    2015-02-01

    Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live-dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria. PMID:25445678

  7. Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity

    PubMed Central

    Ayers, Lisa; Harrison, Paul; Kohler, Malcolm; Ferry, Berne

    2014-01-01

    Background Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results. Methods One hundred samples from healthy individuals and patients with obstructive sleep apnoea were analysed by conventional flow cytometry (FACSCalibur) and by three functional MV assays: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA® Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation. Results Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001); correlated with ETP (r=0.7444, p<0.0001); negatively correlated with STA® Phospholipid Procoag Assay clotting time (−0.7872, p<0.0001), reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results. Conclusions Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive

  8. Discrepancy of target sites between clinician and cytopathological reports in head neck fine needle aspiration: Did I miss the target or did the clinician mistake the organ site?

    PubMed Central

    Khanlari, Mahsa; Daneshbod, Yahya; Shaterzadeh Yazdi, Hanieh; Shirian, Sadegh; Negahban, Shahrzad; Aledavood, Azita; Oryan, Ahmad; Khademi, Bijan; Daneshbod, Khosrow; Field, Andrew

    2015-01-01

    The diagnostic accuracy of fine needle aspiration cytology (FNAC) of head and neck lesions is relatively high, but cytologic interpretation might be confusing if the sample is lacking typical cytologic features according to labeled site by physician. These errors may have an impact on pathology search engines, healthcare costs or even adverse outcomes. The cytology archive database of multiple institutions in southern Iran and Australia covering the period 2001–2011, were searched using keywords: salivary gland, head, neck, FNAC, and cytology. All the extracted reports were reviewed. The reports which showed discordance between the clinician's impression of the organ involved and subsequent fine needle biopsy request, and the eventual cytological diagnosis were selected. The cytological diagnosis was confirmed by histology or cell block, with assistance from imaging, clinical outcome, physical examination, molecular studies, or microbiological culture. The total number of 10,200 head and neck superficial FNAC were included in the study, from which 48 cases showed discordance between the clinicians request and the actual site of pathology. Apart from the histopathology, the imaging, clinical history, physical examination, immunohistochemical study, microbiologic culture and molecular testing helped to finalize the target organ of pathology in 23, 6, 7, 8, 2, and 1 cases respectively. The commonest discrepancies were for FNAC of “salivary gland” [total: 20 with actual final pathology in: bone (7), soft tissue (5), lymph node (3), odontogenic (3) and skin (2)], “lymph node” [total: 12 with final pathology in: soft tissue (3), skin (3), bone (1) and brain (1)], “soft tissue” [total: 11 with final pathology in: bone (5), skin (2), salivary gland (1), and ocular region (1)] and “skin” [total: 5 with final pathology in: lymph node (2), bone (1), soft tissue (1) and salivary gland (1)]. The primary physician requesting FNAC of head and neck lesions are

  9. Endoscopic ultrasound-guided fine-needle aspiration for the diagnosis of pancreatic cysts by combined cytopathology and cystic content analysis

    PubMed Central

    Martin, Amanda K; Zhou, Zhongren

    2015-01-01

    Recent advances in imaging technology have resulted in an increase in incidental discoveries of pancreatic cystic lesions. Pancreatic cysts comprise a wide variety of lesions and include non-neoplastic cysts and neoplastic cysts. Because some pancreatic cysts have more of a malignant potential than others, it is absolutely essential that an accurate diagnosis is rendered so that effective care can be given to each patient. In many centers, endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) has emerged as the modality of choice that enables one to distinguish between mucinous and non-mucinous lesion, diagnose malignancy and collect cyst fluid for further diagnostic studies, such as pancreatic enzyme levels, molecular analysis and other tumor biomarkers. The current review will focus on EUS-guided FNA and the cytological diagnosis for pancreatic cysts. PMID:26504505

  10. Flow Cytometry with Gold Nanoparticles and their Clusters as scattering Contrast Agents: FDTD Simulation of Light-Cell Interaction

    PubMed Central

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

    2010-01-01

    The formulation of the Finite-Difference Time-Domain (FDTD) approach is presented in the framework of its potential applications to in vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. PMID:19670359

  11. High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA.

    PubMed

    Torres, Alex José Leite; Brígido, Luis Fernando de Macedo; Abrahão, Marcos Herculano Nunes; Angelo, Ana Luiza Dias; de Jesus Ferreira, Gilcivaldo; Coelho, Luana Portes; Ferreira, João Leandro; Jorge, Célia Regina Mayoral Pedroso; Netto, Eduardo Martins; Brites, Carlos

    2015-01-01

    Use of CCR5 antagonists requires previous viral tropism determination. The available methods have high cost, are time-consuming, or require highly trained personnel, and sophisticated equipment. We compared a flow cytometry-based tropism assay with geno2pheno method to determine HIV-1 tropism in AIDS patients, in Bahia, Brazil. We tested peripheral blood mononuclear cells of 102 AIDS patients under antiretroviral therapy by using a cytometry-based tropism assay and geno2pheno assay. Cellular membrane receptors were identified by using CXCR4, CCR5 and CD4 monoclonal antibodies, while detection of cytoplasmic mRNAs for gag and pol HIV regions was achieved by using a labeled probe. Genotypic identification of X4 and R5 tropic viruses was attempted by geno2pheno algorithm. There was a high degree of concordance between cytometry-based tropism assay and geno2pheno algorithm in determination of HIV-1 tropism. Cytometry-based tropism assay demonstrated higher sensitivity and specificity in comparison to geno2pheno, which was used as a gold-standard. One sample could not be amplified by geno2pheno method, but was classified as duotropic by cytometry-based tropism assay. We did not find any association between CD4+ count or plasma HIV-1 RNA viral load and tropism results. The overall performances of cytometry-based tropism assay and geno2pheno assay were almost identical in determination of HIV-1 tropism.

  12. A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action

    PubMed Central

    Hendon-Dunn, Charlotte Louise; Doris, Kathryn Sarah; Thomas, Stephen Richard; Allnutt, Jonathan Charles; Marriott, Alice Ann Neville; Hatch, Kim Alexandra; Watson, Robert James; Bottley, Graham; Marsh, Philip David; Taylor, Stephen Charles

    2016-01-01

    Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic. PMID:26902767

  13. A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action.

    PubMed

    Hendon-Dunn, Charlotte Louise; Doris, Kathryn Sarah; Thomas, Stephen Richard; Allnutt, Jonathan Charles; Marriott, Alice Ann Neville; Hatch, Kim Alexandra; Watson, Robert James; Bottley, Graham; Marsh, Philip David; Taylor, Stephen Charles; Bacon, Joanna

    2016-07-01

    Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

  14. Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

    PubMed Central

    Friedrich, Ralf P; Janko, Christina; Poettler, Marina; Tripal, Philipp; Zaloga, Jan; Cicha, Iwona; Dürr, Stephan; Nowak, Johannes; Odenbach, Stefan; Slabu, Ioana; Liebl, Maik; Trahms, Lutz; Stapf, Marcus; Hilger, Ingrid; Lyer, Stefan; Alexiou, Christoph

    2015-01-01

    Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. PMID:26170658

  15. Determination of cluster composition in heteroaggregation of binary particle systems by flow cytometry.

    PubMed

    Rollié, Sascha; Sundmacher, Kai

    2008-12-01

    Cluster composition in aggregation processes of multiple particle species can be dynamically determined by flow cytometry if particle populations are fluorescently labeled. By flow cytometric single particle analysis, aggregates can be characterized according to the exact amount of constituent particles, allowing the detailed and separate quantification of homo- and heteroaggregation. This contribution demonstrates the application of flow cytometry for the experimental detection of heteroaggregation in a binary particle mixture of oppositely charged polystyrene (PS) particles and Rhodamine-B labeled melamine-formaldehyde (MF-RhB) particles. Experiments with different particle concentration, temperature, mixing mode, ionic strength and particle mixing ratio are presented. Aggregation kinetics are enhanced with increasing particle concentration and temperature as well as by increased shear of mixing. These results represent well-known behavior published in previous investigations and validate the performance of flow cytometry for probing heteroaggregation processes. Physical insight with a novel level of detail is gained by the quantification of de- and restabilization phenomena. At low ionic strength, "raspberry"-type aggregates with PS cores are formed by primary heteroaggregation. At moderate particle number ratios, these aggregates are electrostatically destabilized and form more complex aggregates in a secondary heteroaggregation process. At high particle number ratios (> or =50:1), the raspberry-type aggregates are electrostatically restabilized and secondary heteroaggregation is prevented. The dynamic change of aggregate charge was verified by zeta-potential measurements. The elevation of salt concentration over several orders of magnitude retards aggregation dynamics, since attractive interparticle forces are diminished by an electrostatic double layer. This indicates that heteroaggregation induced by attractive interparticle forces is faster than aggregation

  16. Fluorogen Activating Proteins in Flow Cytometry for the Study of Surface Molecules and Receptors

    PubMed Central

    Saunders, Matthew J.; Szent-Gyorgyi, Christopher; Fisher, Gregory W.; Jarvik, Jonathan W.; Bruchez, Marcel P.; Waggoner, Alan S.

    2012-01-01

    The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g. EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extra cellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes (‘activate the fluorogen’). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000 fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (β2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well. PMID:22366230

  17. An Improved Flow Cytometry Method For Precise Quantitation Of Natural-Killer Cell Activity

    NASA Technical Reports Server (NTRS)

    Crucian, Brian; Nehlsen-Cannarella, Sandra; Sams, Clarence

    2006-01-01

    The ability to assess NK cell cytotoxicity using flow cytometry has been previously described and can serve as a powerful tool to evaluate effector immune function in the clinical setting. Previous methods used membrane permeable dyes to identify target cells. The use of these dyes requires great care to achieve optimal staining and results in a broad spectral emission that can make multicolor cytometry difficult. Previous methods have also used negative staining (the elimination of target cells) to identify effector cells. This makes a precise quantitation of effector NK cells impossible due to the interfering presence of T and B lymphocytes, and the data highly subjective to the variable levels of NK cells normally found in human peripheral blood. In this study an improved version of the standard flow cytometry assay for NK activity is described that has several advantages of previous methods. Fluorescent antibody staining (CD45FITC) is used to positively identify target cells in place of membranepermeable dyes. Fluorescent antibody staining of target cells is less labor intensive and more easily reproducible than membrane dyes. NK cells (true effector lymphocytes) are also positively identified by fluorescent antibody staining (CD56PE) allowing a simultaneous absolute count assessment of both NK cells and target cells. Dead cells are identified by membrane disruption using the DNA intercalating dye PI. Using this method, an exact NK:target ratio may be determined for each assessment, including quantitation of NK target complexes. Backimmunoscatter gating may be used to track live vs. dead Target cells via scatter properties. If desired, NK activity may then be normalized to standardized ratios for clinical comparisons between patients, making the determination of PBMC counts or NK cell percentages prior to testing unnecessary. This method provides an exact cytometric determination of NK activity that highly reproducible and may be suitable for routine use in the

  18. Multispectral cytometry of single bio-particles using a 32-channel detector

    NASA Astrophysics Data System (ADS)

    Robinson, J. Paul; Rajwa, Bartek; Gregori, Gerald; Jones, James; Patsekin, Valeri

    2005-04-01

    Detecting biological particles and subsequently identifying them in a very short period of time is highly desirable, but a very difficult task. There are several pathways for developing rapid detection systems. For example, one can reduce sample size to a very small volume, and amplify cellular components by PCR technology with a view to identifying antigen-specific molecules. Alternatively, antibody-based assays allow for detection and identification of a variety of well-characterized pathogens. The system we propose utilizes flow cytometry technology to rapidly detect spectral fingerprints or organisms. However, the current limit for simultaneously detectable fluorescence signals in flow cytometry is around 12-15. Making these measurements is very complex and the necessity for advanced spectral overlap calculations creates a number of difficult problems to solve in a short period of time. Next-generation instruments can either increase the number of detectors or modify the principles of collection. If the detector system were simplified, the overall cost and complexity of single-cell analytical systems might be reduced. This requires changes in both hardware and software that allow for the analysis of 30 or more spectral signals. Further, analysis of complex data sets requires some completely new approaches, particularly in the area of multispectral analysis. This presentation describes the key components and principles involved in building a next-generation instrument which can collect simultaneously 32 bands of fluorescence from a particle in less than 5 microseconds. This would allow the analysis of several thousand bioparticles per second. The flow cytometry system based on our new detector would be designed to be portable and low cost.

  19. Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species

    PubMed Central

    Loureiro, João; Rodriguez, Eleazar; Doležel, Jaroslav; Santos, Conceição

    2007-01-01

    Background and Aims After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry. Methods GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species. Key Results In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed. Conclusions WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high

  20. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    PubMed Central

    Pospichalova, Vendula; Svoboda, Jan; Dave, Zankruti; Kotrbova, Anna; Kaiser, Karol; Klemova, Dobromila; Ilkovics, Ladislav; Hampl, Ales; Crha, Igor; Jandakova, Eva; Minar, Lubos; Weinberger, Vit; Bryja, Vitezslav

    2015-01-01

    Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for

  1. Rapid Analysis of the Internal Configurations of Droplets of Liquid Crystal using Flow Cytometry

    PubMed Central

    Miller, Daniel S.; Wang, Xiaoguang; Buchen, James; Lavrentovich, Oleg D.; Abbott, Nicholas L.

    2014-01-01

    We report the use of flow cytometry to identify the internal ordering (director configurations) of micrometer-sized droplets of thermotropic liquid crystals (LCs) dispersed in aqueous solutions of adsorbates (surfactants and phospholipids). We reveal that changes in the configurations of the LC droplets induced by the adsorbates generate distinct changes in light scattering plots (side versus forward scattering). Specifically, when compared to bipolar droplets, radial droplets generate a narrower distribution of side scattering intensities (SSC, large angle light scattering) for a given intensity of forward scattering (FSC, small angle light scattering). This difference is shown to arise from the rotational symmetry of a radial LC droplet which is absent for the bipolar configuration of the LC droplet. In addition, the scatter plots for radial droplets possess a characteristic “S-shape”, with two or more SSC intensities observed for each intensity of FSC. The origin of the experimentally observed S-shape is investigated via calculation of form factors and established to be due to size-dependent interference effects that differ for the forward and side scattered light. Finally, by analyzing emulsions comprised of mixtures of bipolar and radial droplets at rates of up to 10,000 droplets per second, we demonstrate that flow cytometry permits precise determination of the percentage of radial droplets within the mixture with a coefficient of determination of 0.98 (as validated by optical microscopy). Overall, the results presented in this paper demonstrate that flow cytometry provides a promising approach for high throughput quantification of the internal configurations of LC emulsion microdroplets. Because large numbers of droplets can be characterized, it enables statistically robust analyses of LC droplets. The methodology also appears promising for quantification of chemical and biological assays based on adsorbate-induced ordering transitions within LC droplets

  2. Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

    PubMed

    Friedrich, Ralf P; Janko, Christina; Poettler, Marina; Tripal, Philipp; Zaloga, Jan; Cicha, Iwona; Dürr, Stephan; Nowak, Johannes; Odenbach, Stefan; Slabu, Ioana; Liebl, Maik; Trahms, Lutz; Stapf, Marcus; Hilger, Ingrid; Lyer, Stefan; Alexiou, Christoph

    2015-01-01

    Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEON(LA)) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEON(LA) with an additional protein corona formed by bovine serum albumin (SEON(LA-BSA)) and commercially available Rienso(®) particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. PMID:26170658

  3. Using Lanthanide Nanoparticles as Isotopic Tags for Biomarker Detection by Mass Cytometry

    NASA Astrophysics Data System (ADS)

    Cao, Pengpeng

    The development of robust, versatile, and high-throughput biosensing techniques has widespread implications for early disease detection and accurate diagnosis. An innovative technology, mass cytometry, has been developed to use isotopically-labelled antibodies to simultaneously study multiple parameters of single cells. The current detection sensitivity of mass cytometry is limited by the number of copies of a given isotope that can be attached to a given antibody. This thesis describes research on the synthesis, characterization, and bioconjugation of a new class of nanoparticle-based labelling agents to be employed for the detection of low-abundance biomarkers by mass cytometry. Hydrophobic lanthanide nanoparticles (Ln NPs) have been prepared by the Winnik group. To render the NPs water-soluble for biological applications, we coated the NP surface with a first generation of multidentate poly(ethylene glycol) (PEG)-based ligands via ligand exchange. We measured the size, morphology, and polydispersity of these hydrophilic NPs by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The colloidal stability of the NPs was determined at various pH and in phosphate buffered saline (PBS) solutions. Tetradentate-PEG-coated NPs (Tetra-NPs) exhibited the best stability at pH 3 to 9, and in PBS. However, when cells were treated with Tetra-NPs in preliminary in vitro studies, significant undesirable non-specific binding (NSB) was observed. In order to tackle the NSB issue presented in the Tetra-NPs, we prepared a second generation of polymer-based ligands using ring-opening metathesis polymerization (ROMP). A small library of ROMP polymers was synthesized, characterized, and used to stabilize NPs in aqueous solutions. The ROMP-NPs were found to have significantly reduced NSB to cells by inductively coupled plasma-mass spectrometry (ICP-MS). To further modify the NPs, amine groups were introduced as functional handles to both the tetradentate-PEG and

  4. Applications and perspectives of multi-parameter flow cytometry to microbial biofuels production processes.

    PubMed

    da Silva, Teresa Lopes; Roseiro, José Carlos; Reis, Alberto

    2012-04-01

    Conventional microbiology methods used to monitor microbial biofuels production are based on off-line analyses. The analyses are, unfortunately, insufficient for bioprocess optimization. Real time process control strategies, such as flow cytometry (FC), can be used to monitor bioprocess development (at-line) by providing single cell information that improves process model formulation and validation. This paper reviews the current uses and potential applications of FC in biodiesel, bioethanol, biomethane, biohydrogen and fuel cell processes. By highlighting the inherent accuracy and robustness of the technique for a range of biofuel processing parameters, more robust monitoring and control may be implemented to enhance process efficiency.

  5. Determination of internalization of chromium oxide nano-particles in Escherichia coli by flow cytometry.

    PubMed

    Khatoon, Imrana; Vajpayee, Poornima; Singh, Gulshan; Pandey, Alok K; Dhawan, Alok; Gupta, K C; Shanker, Rishi

    2011-02-01

    In this study, Escherichia coil DH5alpha (ATCC 35218) were exposed to 0-100 microg/mL chromium oxide nanoparticles (Cr2O3, Nps) for 15-120 min to study the internalization of Nps by flowcytometry. A concentration-duration dependent increased side scatter (SSC) confirmed the internalization of Cr2O3 NPs by the E. coli. This study suggests that the uptake of Nps by bacterial cells can be rapidly monitored with flow cytometry for toxicity and risk assessment. PMID:21485855

  6. Cutaneous T cell lymphoma mimicking cutaneous histiocytosis: differentiation by flow cytometry.

    PubMed

    Baines, S J; McCormick, D; McInnes, E; Dunn, J K; Dobson, J M; McConnell, I

    2000-07-01

    A two-year-old, neutered female cross-bred labrador had multiple cutaneous nodules, biopsies of which revealed pathological changes consistent with cutaneous histiocytosis. During a period of one month the dog developed multicentric lymphadenopathy, a retrobulbar mass and masses within the quadriceps and cervical muscles. Fine needle aspiration cytology of the cutaneous nodules and lymph nodes and histological examination of the cutaneous nodules and muscle masses suggested the presence of lymphoblastic lymphoma. A definitive diagnosis of CD8+ T cell lymphoma was achieved by immunophenotyping the tumour cells by flow cytometry.

  7. A structured population modeling framework for quantifying and predicting gene expression noise in flow cytometry data.

    PubMed

    Flores, Kevin B

    2013-07-01

    We formulated a structured population model with distributed parameters to identify mechanisms that contribute to gene expression noise in time-dependent flow cytometry data. The model was validated using cell population-level gene expression data from two experiments with synthetically engineered eukaryotic cells. Our model captures the qualitative noise features of both experiments and accurately fit the data from the first experiment. Our results suggest that cellular switching between high and low expression states and transcriptional re-initiation are important factors needed to accurately describe gene expression noise with a structured population model.

  8. [Nuclear morphometry and DNA cytometry in the grading of malignant tumors of the salivary gland].

    PubMed

    Zhang, W Z

    1992-05-01

    Nuclear morphometry and DNA cytometry were performed in 6 normal salivary glands and 37 malignant tumors of the salivary gland. Multivariate discrimination analysis was used to grade the malignant salivary gland tumors. The discrimination rate was 100% for normal salivary gland, benign tumor, high malignant carcinoma and low malignant carcinoma. It was 66.7% for borderline malignancies. These results indicate that quantitative cytological analysis is effective and reproducible in the grading of salivary gland tumors. Stepwise multivariate regression analysis showed that there was a very complicated correlation between DNA content and nuclear morphometric parameters of salivary gland tumors.

  9. Lensless Imaging and Sensing.

    PubMed

    Ozcan, Aydogan; McLeod, Euan

    2016-07-11

    High-resolution optical microscopy has traditionally relied on high-magnification and high-numerical aperture objective lenses. In contrast, lensless microscopy can provide high-resolution images without the use of any focusing lenses, offering the advantages of a large field of view, high resolution, cost-effectiveness, portability, and depth-resolved three-dimensional (3D) imaging. Here we review various approaches to lensless imaging, as well as its applications in biosensing, diagnostics, and cytometry. These approaches include shadow imaging, fluorescence, holography, superresolution 3D imaging, iterative phase recovery, and color imaging. These approaches share a reliance on computational techniques, which are typically necessary to reconstruct meaningful images from the raw data captured by digital image sensors. When these approaches are combined with physical innovations in sample preparation and fabrication, lensless imaging can be used to image and sense cells, viruses, nanoparticles, and biomolecules. We conclude by discussing several ways in which lensless imaging and sensing might develop in the near future. PMID:27420569

  10. Lensless Imaging and Sensing.

    PubMed

    Ozcan, Aydogan; McLeod, Euan

    2016-07-11

    High-resolution optical microscopy has traditionally relied on high-magnification and high-numerical aperture objective lenses. In contrast, lensless microscopy can provide high-resolution images without the use of any focusing lenses, offering the advantages of a large field of view, high resolution, cost-effectiveness, portability, and depth-resolved three-dimensional (3D) imaging. Here we review various approaches to lensless imaging, as well as its applications in biosensing, diagnostics, and cytometry. These approaches include shadow imaging, fluorescence, holography, superresolution 3D imaging, iterative phase recovery, and color imaging. These approaches share a reliance on computational techniques, which are typically necessary to reconstruct meaningful images from the raw data captured by digital image sensors. When these approaches are combined with physical innovations in sample preparation and fabrication, lensless imaging can be used to image and sense cells, viruses, nanoparticles, and biomolecules. We conclude by discussing several ways in which lensless imaging and sensing might develop in the near future.

  11. Minimal residual disease monitoring by 8-color flow cytometry in mantle cell lymphoma: an EU-MCL and LYSA study.

    PubMed

    Cheminant, Morgane; Derrieux, Coralie; Touzart, Aurore; Schmit, Stéphanie; Grenier, Adrien; Trinquand, Amélie; Delfau-Larue, Marie-Hélène; Lhermitte, Ludovic; Thieblemont, Catherine; Ribrag, Vincent; Cheze, Stéphane; Sanhes, Laurence; Jardin, Fabrice; Lefrère, François; Delarue, Richard; Hoster, Eva; Dreyling, Martin; Asnafi, Vahid; Hermine, Olivier; Macintyre, Elizabeth

    2016-03-01

    Quantification of minimal residual disease may guide therapeutic strategies in mantle cell lymphoma. While multiparameter flow cytometry is used for diagnosis, the gold standard method for minimal residual disease analysis is real-time quantitative polymerase chain reaction (RQ-PCR). In this European Mantle Cell Lymphoma network (EU-MCL) pilot study, we compared flow cytometry with RQ-PCR for minimal residual disease detection. Of 113 patients with at least one minimal residual disease sample, RQ-PCR was applicable in 97 (86%). A total of 284 minimal residual disease samples from 61 patients were analyzed in parallel by flow cytometry and RQ-PCR. A single, 8-color, 10-antibody flow cytometry tube allowed specific minimal residual disease assessment in all patients, with a robust sensitivity of 0.01%. Using this cut-off level, the true-positive-rate of flow cytometry with respect to RQ-PCR was 80%, whereas the true-negative-rate was 92%. As expected, RQ-PCR frequently detected positivity below this 0.01% threshold, which is insufficiently sensitive for prognostic evaluation and would ideally be replaced with robust quantification down to a 0.001% (10-5) threshold. In 10 relapsing patients, the transition from negative to positive by RQ-PCR (median 22.5 months before relapse) nearly always preceded transition by flow cytometry (4.5 months), but transition to RQ-PCR positivity above 0.01% (5 months) was simultaneous. Pre-emptive rituximab treatment of 2 patients at minimal residual disease relapse allowed re-establishment of molecular and phenotypic complete remission. Flow cytometry minimal residual disease is a complementary approach to RQ-PCR and a promising tool in individual mantle cell lymphoma therapeutic management. (clinicaltrials identifiers: 00209209 and 00209222).

  12. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    PubMed

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J; Vaulot, Daniel

    2011-04-28

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  13. Plastid 16S rRNA Gene Diversity among Eukaryotic Picophytoplankton Sorted by Flow Cytometry from the South Pacific Ocean

    PubMed Central

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J.; Vaulot, Daniel

    2011-01-01

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image. PMID:21552558

  14. Novel fluorescent antagonist as a molecular probe in A(3) adenosine receptor binding assays using flow cytometry.

    PubMed

    Kozma, Eszter; Kumar, T Santhosh; Federico, Stephanie; Phan, Khai; Balasubramanian, Ramachandran; Gao, Zhan-Guo; Paoletta, Silvia; Moro, Stefano; Spalluto, Giampiero; Jacobson, Kenneth A

    2012-06-01

    The physiological role of the A(3) adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A(3)AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding K(i) value of 6.4±2.5nM in hA(3)AR-expressing CHO cell membranes. MRS5449 antagonized hA(3)AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (K(B)=4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA(3)ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the K(d) value of 6.65nM calculated from kinetic experiments. K(i) values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5-20 fold weaker than obtained with agonist radioligand [(125)I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A(3)AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA(3)AR characterization.

  15. Novel Fluorescent Antagonist as a Molecular Probe in A3 Adenosine Receptor Binding Assays Using Flow Cytometry

    PubMed Central

    Kozma, Eszter; Kumar, T. Santhosh; Federico, Stephanie; Phan, Khai; Balasubramanian, Ramachandran; Gao, Zhan-Guo; Paoletta, Silvia; Moro, Stefano; Spalluto, Giampiero; Jacobson, Kenneth A.

    2012-01-01

    The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding Ki value of 6.4 ± 2.5 nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (KB 4.8 nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15 nM, comparable to the Kd value of 6.65 nM calculated from kinetic experiments. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5–20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization. PMID:22402302

  16. Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy.

    PubMed

    Fleck, Roland A; Pickup, Roger W; Day, John G; Benson, Erica E

    2006-04-01

    Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C). PMID:16455069

  17. Tree-Based Methods for Discovery of Association between Flow Cytometry Data and Clinical Endpoints.

    PubMed

    Eliot, M; Azzoni, L; Firnhaber, C; Stevens, W; Glencross, D K; Sanne, I; Montaner, L J; Foulkes, A S

    2009-01-01

    We demonstrate the application and comparative interpretations of three tree-based algorithms for the analysis of data arising from flow cytometry: classification and regression trees (CARTs), random forests (RFs), and logic regression (LR). Specifically, we consider the question of what best predicts CD4 T-cell recovery in HIV-1 infected persons starting antiretroviral therapy with CD4 count between 200 and 350 cell/muL. A comparison to a more standard contingency table analysis is provided. While contingency table analysis and RFs provide information on the importance of each potential predictor variable, CART and LR offer additional insight into the combinations of variables that together are predictive of the outcome. In all cases considered, baseline CD3-DR-CD56+CD16+ emerges as an important predictor variable, while the tree-based approaches identify additional variables as potentially informative. Application of tree-based methods to our data suggests that a combination of baseline immune activation states, with emphasis on CD8 T-cell activation, may be a better predictor than any single T-cell/innate cell subset analyzed. Taken together, we show that tree-based methods can be successfully applied to flow cytometry data to better inform and discover associations that may not emerge in the context of a univariate analysis.

  18. The role of multiparametric flow cytometry in the detection of minimal residual disease in acute leukaemia.

    PubMed

    Lee, Denise; Grigoriadis, George; Westerman, David

    2015-12-01

    Flow cytometry is the most accessible method for minimal residual disease (MRD) detection due to its availability in most haematological centres. Using a precise combination of different antibodies, immunophenotypic detection of MRD in acute leukaemia can be performed by identifying abnormal combinations or expressions of antigens on malignant cells at diagnosis, during and post treatment. These abnormal phenotypes, referred to as leukaemia-associated immunophenotypes (LAIPs) are either absent or expressed at low frequency in normal bone marrow (BM) cells and are used to monitor the behaviour and quantitate the amount of residual disease following treatment. In paediatric acute lymphoblastic leukaemia (ALL), the level of MRD by multiparametric flow cytometry (MPFC) during therapy is recognised as an important predictor of outcome. Although less extensively studied, adult ALL and adult and paediatric acute myeloid leukaemia (AML) have also demonstrated similar findings. The challenge now is incorporating this information for risk-stratification so that therapy can be tailored individually and ultimately improve outcome while also limiting treatment-related toxicity. In this review we will elaborate on the current and future role of MPFC in MRD in acute leukaemia while also addressing its limitations.

  19. Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry

    PubMed Central

    Gump, Jacob M; Thorburn, Andrew

    2014-01-01

    We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers. PMID:24915460

  20. The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy.

    PubMed

    Wu, Yang; Campos, Samuel K; Lopez, Gabriel P; Ozbun, Michelle A; Sklar, Larry A; Buranda, Tione

    2007-05-15

    The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.

  1. Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    NASA Astrophysics Data System (ADS)

    Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

    2015-03-01

    Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

  2. Enumerating Microorganism Surrogates for Groundwater Transport Studies Using Solid-Phase Cytometry.

    PubMed

    Stevenson, Margaret E; Blaschke, A Paul; Schauer, Sonja; Zessner, Matthias; Sommer, Regina; Farnleitner, Andreas H; Kirschner, Alexander K T

    2014-01-01

    Investigations on the pollution of groundwater with pathogenic microorganisms, e.g. tracer studies for groundwater transport, are constrained by their potential health risk. Thus, microspheres are often used in groundwater transport studies as non-hazardous surrogates for pathogenic microorganisms. Even though pathogenic microorganisms occur at low concentrations in groundwater, current detection methods of microspheres (spectrofluorimetry, flow cytometry and epifluorescence microscopy) have rather high detection limits and are unable to detect rare events. Solid-phase cytometry (SPC) offers the unique capability of reliably quantifying extremely low concentrations of fluorescently labelled microorganisms or microspheres in natural waters, including groundwater. Until now, microspheres have been used in combination with SPC only for instrument calibration purposes and not for environmental applications. In this study, we explored the limits of the SPC methodology for its applicability to groundwater transport studies. The SPC approach proved to be a highly sensitive and reliable enumeration system for microorganism surrogates down to a minimum size of 0.5 μm, in up to 500 ml of groundwater, and 0.75 μm, in up to 1 ml of turbid surface water. Hence, SPC is proposed to be a useful method for enumerating microspheres for groundwater transport studies in the laboratory, as well as in the field when non-toxic, natural products are used. PMID:24578583

  3. Alternative flow cytometry strategies to analyze stem cells and cell death in planarians.

    PubMed

    Peiris, Tanuja Harshani; García-Ojeda, Marcos E; Oviedo, Néstor J

    2016-04-01

    Planarians possess remarkable stem cell populations that continuously support cellular turnover and are instrumental in the regeneration of tissues upon injury. Cellular turnover and tissue regeneration in planarians rely on the proper integration of local and systemic signals that regulate cell proliferation and cell death. Thus, understanding the signals controlling cellular proliferation and cell death in planarians could provide valuable insights for maintenance of adult body homeostasis and the biology of regeneration. Flow cytometry techniques have been utilized widely to identify, isolate, and characterize planarian stem cell populations. We developed alternative flow cytometry strategies that reduce the number of reagents and the time of sample preparation to analyze stem cells and cell death in planarians. The sensitivity of these methods is validated with functional studies using RNA interference and treatment with  γ irradiation or stressful conditions that are known to trigger cell death. Altogether, we provide a community resource intended to minimize adverse effects during ex vivo studies of stem cells and cell death in planarians.

  4. Effect of acetic acid on Saccharomyces carlsbergensis ATCC 6269 batch ethanol production monitored by flow cytometry.

    PubMed

    Freitas, Cláudia; Neves, Elisabete; Reis, Alberto; Passarinho, Paula C; da Silva, Teresa Lopes

    2012-11-01

    Bioethanol produced from lignocellulosic materials has been considered a sustainable alternative fuel. Such type of raw materials have a huge potential, but their hydrolysis into mono-sugars releases toxic compounds such as weak acids, which affect the microorganisms' physiology, inhibiting the growth and ethanol production. Acetic acid (HAc) is the most abundant weak acid in the lignocellulosic materials hydrolysates. In order to understand the physiological changes of Saccharomyces carlsbergensis when fermenting in the presence of different acetic acid (HAc) concentrations, the yeast growth was monitored by multi-parameter flow cytometry at same time that the ethanol production was assessed. The membrane potential stain DiOC(6)(3) fluorescence intensity decreased as the HAc concentration increased, which was attributed to the plasmic membrane potential reduction as a result of the toxic effect of the HAc undissociated form. Nevertheless, the proportion of cells with permeabilized membrane did not increase with the HAc concentration increase. Fermentations ending at lower external pH and higher ethanol concentrations depicted the highest proportions of permeabilized cells and cells with increased reactive oxygen species levels. Flow cytometry allowed monitoring, near real time (at-line), the physiological states of the yeast during the fermentations. The information obtained can be used to optimize culture conditions to improve bioethanol production. PMID:22971830

  5. Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

    PubMed Central

    Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

    2013-01-01

    The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

  6. Effect of acetic acid on Saccharomyces carlsbergensis ATCC 6269 batch ethanol production monitored by flow cytometry.

    PubMed

    Freitas, Cláudia; Neves, Elisabete; Reis, Alberto; Passarinho, Paula C; da Silva, Teresa Lopes

    2012-11-01

    Bioethanol produced from lignocellulosic materials has been considered a sustainable alternative fuel. Such type of raw materials have a huge potential, but their hydrolysis into mono-sugars releases toxic compounds such as weak acids, which affect the microorganisms' physiology, inhibiting the growth and ethanol production. Acetic acid (HAc) is the most abundant weak acid in the lignocellulosic materials hydrolysates. In order to understand the physiological changes of Saccharomyces carlsbergensis when fermenting in the presence of different acetic acid (HAc) concentrations, the yeast growth was monitored by multi-parameter flow cytometry at same time that the ethanol production was assessed. The membrane potential stain DiOC(6)(3) fluorescence intensity decreased as the HAc concentration increased, which was attributed to the plasmic membrane potential reduction as a result of the toxic effect of the HAc undissociated form. Nevertheless, the proportion of cells with permeabilized membrane did not increase with the HAc concentration increase. Fermentations ending at lower external pH and higher ethanol concentrations depicted the highest proportions of permeabilized cells and cells with increased reactive oxygen species levels. Flow cytometry allowed monitoring, near real time (at-line), the physiological states of the yeast during the fermentations. The information obtained can be used to optimize culture conditions to improve bioethanol production.

  7. Monitoring Rhodosporidium toruloides NCYC 921 batch fermentations growing under carbon and nitrogen limitation by flow cytometry.

    PubMed

    Andrade, Raoni; Leal, Rodrigo; Roseiro, José; Reis, Alberto; da Silva, Teresa Lopes

    2012-03-01

    The yeast Rhodosporidium toruloides NCYC 921 was grown on carbon or nitrogen limited batch cultures. The fermentations were monitored using traditional techniques and multi-parameter flow cytometry. The lipid content was assessed by flow cytometry in association with the fluorocrome Nile Red which emits yellow gold fluorescence when dissolved in neutral lipids and red fluorescence when dissolved in polar lipids. In this way, it was possible to at-line monitor the yeast lipid composition in terms of polarity classes throughout the batch growths. It was found that the neutral lipids decreased during the carbon-limited stationary phase, and increased during the nitrogen-limited batch growth. The maximum lipid content was obtained for the nitrogen-limited yeast culture (24% w/w lipids). The yeast cells with permeabilised membranes profile remained almost unchanged during the time course of both fermentations. The scatter light measurements (forward and side scatter signals) provided information on the yeast growth phase. The multi-parameter flow cytometric approach here reported represents a better control system based on measurements made at the single cell level for optimization of the yeast lipid production bioprocess performance.

  8. Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts.

    PubMed

    Nedosekin, Dmitry A; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I; Sawant, Rupa; Torchilin, Vladimir P; Verkhusha, Vladislav V; Ma, Jie; Frank, Markus H; Biris, Alexandru S; Zharov, Vladimir P

    2013-05-01

    In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real-time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa-660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co-expressing melanin and green fluorescent protein (GFP), C8161-GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near-infrared iRFP protein, and quantum dot-carbon nanotube conjugates. Negative contrast flow cytometry provided label-free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time-resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique. PMID:22903924

  9. Bayesian clustering of flow cytometry data for the diagnosis of B-chronic lymphocytic leukemia.

    PubMed

    Lakoumentas, John; Drakos, John; Karakantza, Marina; Nikiforidis, George C; Sakellaropoulos, George C

    2009-04-01

    In the rapidly advancing field of flow cytometry, methodologies facilitating automated clinical decision support are increasingly needed. In the case of B-chronic lymphocytic leukemia (B-CLL), discrimination of the various subpopulations of blood cells is an important task. In this work, our objective is to provide a useful paradigm of computer-based assistance in the domain of flow-cytometric data analysis by proposing a Bayesian methodology for flow cytometry clustering. Using Bayesian clustering, we replicate a series of (unsupervised) data clustering tasks, usually performed manually by the expert. The proposed methodology is able to incorporate the expert's knowledge, as prior information to data-driven statistical learning methods, in a simple and efficient way. We observe almost optimal clustering results, with respect to the expert's gold standard. The model is flexible enough to identify correctly non canonical clustering structures, despite the presence of various abnormalities and heterogeneities in data; it offers an advantage over other types of approaches that apply hierarchical or distance-based concepts.

  10. Discriminative variable subsets in Bayesian classification with mixture models, with application in flow cytometry studies.

    PubMed

    Lin, Lin; Chan, Cliburn; West, Mike

    2016-01-01

    We discuss the evaluation of subsets of variables for the discriminative evidence they provide in multivariate mixture modeling for classification. The novel development of Bayesian classification analysis presented is partly motivated by problems of design and selection of variables in biomolecular studies, particularly involving widely used assays of large-scale single-cell data generated using flow cytometry technology. For such studies and for mixture modeling generally, we define discriminative analysis that overlays fitted mixture models using a natural measure of concordance between mixture component densities, and define an effective and computationally feasible method for assessing and prioritizing subsets of variables according to their roles in discrimination of one or more mixture components. We relate the new discriminative information measures to Bayesian classification probabilities and error rates, and exemplify their use in Bayesian analysis of Dirichlet process mixture models fitted via Markov chain Monte Carlo methods as well as using a novel Bayesian expectation-maximization algorithm. We present a series of theoretical and simulated data examples to fix concepts and exhibit the utility of the approach, and compare with prior approaches. We demonstrate application in the context of automatic classification and discriminative variable selection in high-throughput systems biology using large flow cytometry datasets.

  11. In vitro flow cytometry-based screening platform for cellulase engineering

    PubMed Central

    Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

  12. Quantitative studies of chicken somatotrophs during growth and development by morphometry, immunocytochemistry, and flow cytometry.

    PubMed

    Malamed, S; Deaver, D; Perez, F; Radecki, S; Gibney, J; Scanes, C G

    1997-10-01

    Changes in the male chicken somatotroph during growth and maturation have been examined by morphometric and immunocytochemical (ICC) analysis of serial sections of the anterior pituitary gland and by flow cytometry of dispersed anterior pituitary cells. ICC showed that somatotrophs are confined to the middle and caudal thirds of the anterior pituitary gland at all ages from 5 to 26 weeks. At a given age somatotrophs are of equal size at all positions along the cephalocaudal axis of the anterior pituitary gland. However, there are age-related changes: from 5 to 11 weeks rises occur in both the mean total somatotroph volume per gland (64%) and the mean number of somatotrophs (78%), while the mean volume of the single somatotroph is unchanged. From 11 to 18 weeks the mean volume of the single somatotroph decreases 41%. From 18 to 26 weeks the mean volume of the somatotroph, the mean total somatotroph volume, and the mean number per gland do not change. Flow cytometry studies suggested that somatotrophs from adults have less growth hormone (GH) than somatotrophs from young birds. The increases in total somatotroph volume and number from 5 to 11 weeks are consistent with the rise in anterior pituitary GH reported previously. Basic quantitative morphological information about age-related changes in somatotrophs is reported here. When combined with additional facts from future work, they may explain the well-documented sharp decline in circulating GH from 5 to 11 weeks.

  13. Discriminative variable subsets in Bayesian classification with mixture models, with application in flow cytometry studies.

    PubMed

    Lin, Lin; Chan, Cliburn; West, Mike

    2016-01-01

    We discuss the evaluation of subsets of variables for the discriminative evidence they provide in multivariate mixture modeling for classification. The novel development of Bayesian classification analysis presented is partly motivated by problems of design and selection of variables in biomolecular studies, particularly involving widely used assays of large-scale single-cell data generated using flow cytometry technology. For such studies and for mixture modeling generally, we define discriminative analysis that overlays fitted mixture models using a natural measure of concordance between mixture component densities, and define an effective and computationally feasible method for assessing and prioritizing subsets of variables according to their roles in discrimination of one or more mixture components. We relate the new discriminative information measures to Bayesian classification probabilities and error rates, and exemplify their use in Bayesian analysis of Dirichlet process mixture models fitted via Markov chain Monte Carlo methods as well as using a novel Bayesian expectation-maximization algorithm. We present a series of theoretical and simulated data examples to fix concepts and exhibit the utility of the approach, and compare with prior approaches. We demonstrate application in the context of automatic classification and discriminative variable selection in high-throughput systems biology using large flow cytometry datasets. PMID:26040910

  14. Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

    PubMed Central

    Pickles, Sarah; Arbour, Nathalie; Vande Velde, Christine

    2014-01-01

    Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver. PMID:25285411

  15. Evaluating the morphology of erythrocyte population: An approach based on atomic force microscopy and flow cytometry.

    PubMed

    Ghosh, Sayari; Chakraborty, Ishita; Chakraborty, Monojit; Mukhopadhyay, Ashis; Mishra, Raghwendra; Sarkar, Debasish

    2016-04-01

    Erythrocyte morphology is gaining importance as a powerful pathological index in identifying the severity of any blood related disease. However, the existing technique of quantitative microscopy is highly time consuming and prone to personalized bias. On the other hand, relatively unexplored, complementary technique based on flow cytometry has not been standardized till date, particularly due to the lack of a proper morphological scoring scale. In this article, we have presented a new approach to formulate a non-empirical scoring scale based on membrane roughness (R(rms)) data obtained from atomic force microscopy. Subsequently, the respective morphological quantifier of the whole erythrocyte population, commonly known as morphological index, was expressed as a function of highest correlated statistical parameters of scattered signal profiles generated by flow cytometry. Feed forward artificial neural network model with multilayer perceptron architecture was used to develop the intended functional form. High correlation coefficient (R(2) = 0.95), even for model-formulation exclusive samples, clearly indicates the universal validity of the proposed model. Moreover, a direct pathological application of the proposed model has been illustrated in relation to patients, diagnosed to be suffering from a wide variety of cancer.

  16. Antimicrobial Activity of Rhoeo discolor Phenolic Rich Extracts Determined by Flow Cytometry.

    PubMed

    García-Varela, Rebeca; García-García, Rebeca M; Barba-Dávila, Bertha A; Fajardo-Ramírez, Oscar R; Serna-Saldívar, Sergio O; Cardineau, Guy A

    2015-01-01

    Traditional medicine has led to the discovery of important active substances used in several health-related areas. Phytochemicals in Rhoeo discolor extracts have proven to have important antimicrobial activity. In the present study, our group determined the antimicrobial effects of extracts of Rhoeo discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the in vitro activity of phenolic rich extracts against specifically chosen microorganisms of human health importance by measuring their susceptibility via agar-disc diffusion assay and flow cytometry: Gram-positive Listeria innocua and Streptococcus mutans, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and lastly a fungal pathogen Candida albicans. Ten different extracts were tested in eight different doses on all the microorganisms. Analytical data revealed a high content of phenolic compounds. Both agar-disc diffusion assay and flow cytometry results demonstrated that Pseudomonas aeruginosa was the least affected by extract exposure. However, low doses of these extracts (predominantly polar), in a range from 1 to 4 μg/mL, did produce a statistically significant bacteriostatic and bactericidal effect on the rest of the microorganisms. These results suggest the addition of certain natural extracts from Rhoeo discolor could act as antibacterial and antimycotic drugs or additives for foods and cosmetics.

  17. Recent Advances on Multi-Parameter Flow Cytometry to Characterize Antimicrobial Treatments

    PubMed Central

    Léonard, Lucie; Bouarab Chibane, Lynda; Ouled Bouhedda, Balkis; Degraeve, Pascal; Oulahal, Nadia

    2016-01-01

    The investigation on antimicrobial mechanisms is a challenging and crucial issue in the fields of food or clinical microbiology, as it constitutes a prerequisite to the development of new antimicrobial processes or compounds, as well as to anticipate phenomenon of microbial resistance. Nowadays it is accepted that a cells population exposed to a stress can cause the appearance of different cell populations and in particular sub-lethally compromised cells which could be defined as viable but non-culturable (VBNC). Recent advances on flow cytometry (FCM) and especially on multi-parameter flow cytometry (MP-FCM) provide the opportunity to obtain high-speed information at real time on damage at single-cell level. This review gathers MP-FCM methodologies based on individual and simultaneous staining of microbial cells employed to investigate their physiological state following different physical and chemical antimicrobial treatments. Special attention will be paid to recent studies exploiting the possibility to corroborate MP-FCM results with additional techniques (plate counting, microscopy, spectroscopy, molecular biology techniques, membrane modeling) in order to elucidate the antimicrobial mechanism of action of a given antimicrobial treatment or compound. The combination of MP-FCM methodologies with these additional methods is namely a promising and increasingly used approach to give further insight in differences in microbial sub-population evolutions in response to antimicrobial treatments. PMID:27551279

  18. Bead-Based Assays for Biodetection: From Flow-Cytometry to Microfluidics

    SciTech Connect

    Ozanich, Richard M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby J.

    2009-05-04

    ABSTRACT The potential for the use of biological agents by terrorists is a real threat. Two approaches for detection of biological species will be described: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. The methods and automated fluidic systems used for trapping functionalized microbeads will be described. This approach allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive assays. The automated fluidic approach resulted in up to five-fold improvements in assay sensitivity/speed as compared to identical assays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based assays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (> 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100’s of picomolar range (10’s of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach. Video taping magnetic nanoparticle capture and release was used to improve understanding of the process and revealed interesting behavior.

  19. Integrated optical waveguides and inertial focussing microfluidics in silica for microflow cytometry applications

    NASA Astrophysics Data System (ADS)

    Butement, Jonathan T.; Hunt, Hamish C.; Rowe, David J.; Sessions, Neil P.; Clark, Owain; Hua, Ping; Senthil Murugan, G.; Chad, John E.; Wilkinson, James S.

    2016-10-01

    A key challenge in the development of a microflow cytometry platform is the integration of the optical components with the fluidics as this requires compatible micro-optical and microfluidic technologies. In this work a microflow cytometry platform is presented comprising monolithically integrated waveguides and deep microfluidics in a rugged silica chip. Integrated waveguides are used to deliver excitation light to an etched microfluidic channel and also collect transmitted light. The fluidics are designed to employ inertial focussing, a particle positioning technique, to reduce signal variation by bringing the flowing particles onto the same plane as the excitation light beam. A fabrication process is described which exploits microelectronics mass production techniques including: sputtering, ICP etching and PECVD. Example devices were fabricated and the effectiveness of inertial focussing of 5.6 µm fluorescent beads was studied showing lateral and vertical confinement of flowing beads within the microfluidic channel. The fluorescence signals from flowing calibration beads were quantified demonstrating a CV of 26%. Finally the potential of this type of device for measuring the variation in optical transmission from input to output waveguide as beads flowed through the beam was evaluated.

  20. Determination of aneuploids in hop (Humulus lupulus L.) using flow cytometry.

    PubMed

    Sesek, P; Sustar-Vozlic, J; Bohanec, B

    2000-01-01

    In order to study the possibility that high-resolution flow cytometry can be used for determination of aneuploids, different genotypes of Humulus lupulus were analyzed. Triploid cultivars are bred by hybridization between diploid and tetraploid lines, and as the result of this process, some aneuploids are occasionally also formed. We analyzed eight triploid cultivars and seven putative aneuploids. Triploid cultivars Cerera, Cicero, Celeia, Cekin, Blisk, Mt. Hood, Huller Bit. and Willamette (3x = 30) were measured for nuclear DNA content using Trifolium repens as reference. No significant differences among peak positions of triploid cultivars (having an average CV value per peak of 1.94%) were found. Measurement of nuclear DNA content was also performed for seven lines: 175/75, 89/113, 89/154, 91/215, 175/17, 89/87 and 91/74 previously determined by chromosome counting to be aneuploids (CV per peak was 1.41%). A statistically lower DNA content was found for line 175/75 and higher values were measured for lines 89/154, 89/113 and 91/215. Repeated chromosome counting revealed that the number of chromosomes in line 175/75 was 29, while lines 89/154, 89/113 and 91/215 possessed 31 chromosomes. The other lines were identified as triploids. We conclude that flow cytometry can be efficiently used for determination of aneuploidy in Humulus lupulus. PMID:10653127

  1. Alternative flow cytometry strategies to analyze stem cells and cell death in planarians

    PubMed Central

    Peiris, Tanuja Harshani; García‐Ojeda, Marcos E.

    2016-01-01

    Abstract Planarians possess remarkable stem cell populations that continuously support cellular turnover and are instrumental in the regeneration of tissues upon injury. Cellular turnover and tissue regeneration in planarians rely on the proper integration of local and systemic signals that regulate cell proliferation and cell death. Thus, understanding the signals controlling cellular proliferation and cell death in planarians could provide valuable insights for maintenance of adult body homeostasis and the biology of regeneration. Flow cytometry techniques have been utilized widely to identify, isolate, and characterize planarian stem cell populations. We developed alternative flow cytometry strategies that reduce the number of reagents and the time of sample preparation to analyze stem cells and cell death in planarians. The sensitivity of these methods is validated with functional studies using RNA interference and treatment with  γ irradiation or stressful conditions that are known to trigger cell death. Altogether, we provide a community resource intended to minimize adverse effects during ex vivo studies of stem cells and cell death in planarians. PMID:27307993

  2. Bead-based assays for biodetection: from flow-cytometry to microfluidics

    NASA Astrophysics Data System (ADS)

    Ozanich, Richard M., Jr.; Antolick, Kathryn; Bruckner-Lea, Cynthia J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby; Warner, Cynthia L.; Warner, Marvin G.

    2009-05-01

    The potential for the use of biological agents by terrorists is a real threat. Two approaches for antibody-based detection of biological species are described in this paper: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. These approaches both involve the use of automated fluidic systems for trapping antibody-functionalized microbeads, which allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive immunoassays. The automated fluidic approach resulted in up to five-fold improvements in immunoassay sensitivity/speed as compared to identical immunoassays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based immunoassays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (>= 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100's of picomolar range (10's of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach.

  3. Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction

    PubMed Central

    Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela

    2016-01-01

    Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascular manifestations. A number of studies highlighted the role of these cells in age-related diseases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentiation markers in healthy subjects and in patients affected by peripheral vascular manifestations associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accurately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vascular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context. PMID:27560136

  4. Enumerating Microorganism Surrogates for Groundwater Transport Studies Using Solid-Phase Cytometry.

    PubMed

    Stevenson, Margaret E; Blaschke, A Paul; Schauer, Sonja; Zessner, Matthias; Sommer, Regina; Farnleitner, Andreas H; Kirschner, Alexander K T

    2014-01-01

    Investigations on the pollution of groundwater with pathogenic microorganisms, e.g. tracer studies for groundwater transport, are constrained by their potential health risk. Thus, microspheres are often used in groundwater transport studies as non-hazardous surrogates for pathogenic microorganisms. Even though pathogenic microorganisms occur at low concentrations in groundwater, current detection methods of microspheres (spectrofluorimetry, flow cytometry and epifluorescence microscopy) have rather high detection limits and are unable to detect rare events. Solid-phase cytometry (SPC) offers the unique capability of reliably quantifying extremely low concentrations of fluorescently labelled microorganisms or microspheres in natural waters, including groundwater. Until now, microspheres have been used in combination with SPC only for instrument calibration purposes and not for environmental applications. In this study, we explored the limits of the SPC methodology for its applicability to groundwater transport studies. The SPC approach proved to be a highly sensitive and reliable enumeration system for microorganism surrogates down to a minimum size of 0.5 μm, in up to 500 ml of groundwater, and 0.75 μm, in up to 1 ml of turbid surface water. Hence, SPC is proposed to be a useful method for enumerating microspheres for groundwater transport studies in the laboratory, as well as in the field when non-toxic, natural products are used.

  5. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    PubMed Central

    Rowan, Beth A; Oldenburg, Delene J; Bendich, Arnold J

    2007-01-01

    Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI), SYBR Green I (SG), SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR) were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA) content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content. PMID:17381841

  6. Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

    PubMed

    Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

    2014-01-01

    The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis.

  7. In vitro flow cytometry-based screening platform for cellulase engineering.

    PubMed

    Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 10(7) events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

  8. Using dual laser flow cytometry for monitoring phytoplankton composition and integrity

    SciTech Connect

    Schaefer, H.; Beisker, W.; Steinberg, C.

    1995-12-31

    Dual laser flow cytometry can be used for determining phytoplankton populations in lakes and lowland rivers and streams. Apart from answering basic limnological questions such as the time course of algal blooms or the annual succession of phytoplankton composition further investigations can be made for estimating the integrity of phytoplankton community using biomass distribution spectra. Thus anthropogenic influence such as eutrophication, acidification or effects of xenobiotica can be monitored. Dual laser flow cytometry with excitation wavelengths of 458 and 528 nm was used to measure photosynthesis pigment fluorescence (chlorophyll a (CHLa), Em>665 nm) and phycoerythrin (PE, Em 575 nm) and cell density of phytoplankton organisms in water samples. CHLa is excited directly by 458 nm and by energy transfer from carotenoids (Ex 528 nm). The ratio of the two fluorescence parameters (CFR) allows to identify pigment groups in the phytoplankton population (chlorophytes and euglenophytes from chrysophytes, diatoms and dinophytes). PE-containing cyanophytes and cryptophytes can be detected by their PE fluorescence (Ex 528 nm). As a result of preliminary studies for preparing biomass spectra of phytoplankton communities measurements of protein content by staining with fluorescein isothiocyanate (FITC, ex 488 nm, Em 530 nm) are also shown.

  9. Recent Advances on Multi-Parameter Flow Cytometry to Characterize Antimicrobial Treatments.

    PubMed

    Léonard, Lucie; Bouarab Chibane, Lynda; Ouled Bouhedda, Balkis; Degraeve, Pascal; Oulahal, Nadia

    2016-01-01

    The investigation on antimicrobial mechanisms is a challenging and crucial issue in the fields of food or clinical microbiology, as it constitutes a prerequisite to the development of new antimicrobial processes or compounds, as well as to anticipate phenomenon of microbial resistance. Nowadays it is accepted that a cells population exposed to a stress can cause the appearance of different cell populations and in particular sub-lethally compromised cells which could be defined as viable but non-culturable (VBNC). Recent advances on flow cytometry (FCM) and especially on multi-parameter flow cytometry (MP-FCM) provide the opportunity to obtain high-speed information at real time on damage at single-cell level. This review gathers MP-FCM methodologies based on individual and simultaneous staining of microbial cells employed to investigate their physiological state following different physical and chemical antimicrobial treatments. Special attention will be paid to recent studies exploiting the possibility to corroborate MP-FCM results with additional techniques (plate counting, microscopy, spectroscopy, molecular biology techniques, membrane modeling) in order to elucidate the antimicrobial mechanism of action of a given antimicrobial treatment or compound. The combination of MP-FCM methodologies with these additional methods is namely a promising and increasingly used approach to give further insight in differences in microbial sub-population evolutions in response to antimicrobial treatments. PMID:27551279

  10. Mathematical analysis of mis-estimation of cell subsets in flow cytometry: viability staining revisited.

    PubMed

    Petrunkina, A M; Harrison, R A P

    2011-05-31

    Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labeled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. We demonstrate that in all cases in which dead cells are present within the population, percentages of live sub-populations in different subsets are mis-estimated. In cases where the pattern of marker expression differs greatly between live and dead cells, or where the proportion of dead cells is high, this mis-estimation will be aggravated; the subsets pattern will therefore be biased in a population selected only on the basis of light-scatter behavior. The importance of accurately detecting and gating out dead cells is illustrated by an experimental example accompanying the mathematical analysis. To conclude, identification of dead cells by means of viability stains should be an absolute routine in practical flow cytometry, so as to avoid mis-estimation in sorting or analysis.

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

    PubMed

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

    2015-01-01

    In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols. PMID:26267664

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

    PubMed Central

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

    2015-01-01

    In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols. PMID:26267664

  13. Amiodarone pulmonary toxicity: cytopathology, ultrastructure, and immunocytochemistry.

    PubMed

    Bedrossian, C W; Warren, C J; Ohar, J; Bhan, R

    1997-10-01

    One hundred ninety cardiac patients were prospectively enrolled in an amiodarone protocol. Over a 10-year period, 16 patients developed new or progressive respiratory symptoms while taking amiodarone. These symptoms included dyspnea associated with abnormal chest radiographs or new or worsening abnormalities on pulmonary function testing. Specimens for microscopic examination were obtained by fiberoptic bronchoscopy with transbronchial lung biopsy (TBB), bronchoalveolar lavage (BAL), open lung biopsy (OLB), or autopsy. Large foamy macrophages with characteristic lamellated cytoplasmic inclusions were noted in all specimens, regardless of other evidence of pulmonary toxicity, suggesting that foamy macrophages represent a routine drug effect. Foamy macrophages were not present in BAL specimens from 53 normal controls and were rarely seen in specimens from 27 patients who had other interstitial lung diseases. When present, the foamy macrophages were less prominent than those seen in specimens from patients receiving amiodarone. Fibrosis was noted in 11 of 16 histological specimens, whereas type II-cell-hyperplasia was observed in 7 of the 16 specimens. Four of the 16 patients with respiratory symptoms died, and their autopsy revealed a combination of foamy macrophages with fibrosis and type II cell hyperplasia reflective of amiodarone pulmonary toxicity. Hyperplastic type II cells were not found in the absence of fibrosis. Immunocytochemistry allowed differentiation between foamy macrophages and type II cells and represents a useful tool for future investigations of the pathogenesis of amiodarone-induced pulmonary disease.

  14. Error reduction and risk management in cytopathology.

    PubMed

    Frable, William J

    2007-05-01

    Currently, tort reform is not a major priority in either the Congress of the United States or in state legislatures. Thus, it is fortunate that medical negligence claims against pathologists are relatively infrequent, at 8.3% per year per 100 insured pathologists (data from the Doctors' Company, 2000-2003). However, claims for "missed" cervical cytology specimens rank third, behind those for alleged misinterpretation of breast biopsies and pigmented skin lesions. The severity of cervical cytology errors is high, at almost $700,000 per claim, surpassed only by those concerning melanoma. There are common threads that appear consistently in the analysis of slides from allegedly misdiagnosed cervical cytology cases, including small-cell variants of high-grade squamous intraepithelial neoplasia (HGSIL), present in small numbers; hyperchromatic crowded cell groups; atypical squamous cells of undetermined significance (ASCUS); smears taken during menses; other bloody smears, particularly with degenerative features or excessive inflammation; others showing atypical repair; and unsatisfactory samples. It is important for pathologists to spend time with cytotechnologists to emphasize the patterns of abnormal smears at low microscopic magnification and those backgrounds featuring blood and inflammation which require particular attention. Managing the "look-back" requirement of the Clinical Laboratory Amendments of 1988 (CLIA88) is also crucial; the need to issue amended reports as a consequence of that provision is quite rare. Procedures for administrating and reporting retrospective reviews under the CLIA88 should be clearly outlined in a peer-reviewed procedure document in each laboratory. They should be reviewed and approved by risk managers or insurance carriers, and documented in such a manner that one obtains maximal protection from legal discovery. Consumer education is particularly important in maintaining laboratory performance and reducing risk from error in cytology. Periodic feedback to clinicians on the quality of their smear preparations, the use of ancillary techniques (eg, human papillomavirus testing), and discussion of reporting terminology are important. Moreover, one should stress the need for pertinent clinical history that is often required to initiate quality control measures for evaluation and reporting of cervical cytology specimens. The incidence of cervical cancer in the United States, at only 9700 new cases per year, is low, emphasizing the need for clinical vigilance, attention to unexplained symptoms and signs, and biopsies of any cervical abnormality. These and other efforts may assist in reducing the risk of litigation attached to allegedly false-negative gynecologic and nongynecologic cytology samples.

  15. Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry.

    PubMed

    Garner, D L; Gledhill, B L; Pinkel, D; Lake, S; Stephenson, D; Van Dilla, M A; Johnson, L A

    1983-03-01

    The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than

  16. Femtosecond laser pulse optimization for multiphoton cytometry and control of fluorescence

    NASA Astrophysics Data System (ADS)

    Tkaczyk, Eric Robert

    This body of work encompasses optimization of near infrared femtosecond laser pulses both for enhancement of flow cytometry as well as adaptive pulse shaping to control fluorescence. A two-photon system for in vivo flow cytometry is demonstrated, which allows noninvasive quantification of circulating cell populations in a single live mouse. We monitor fluorescently-labeled red blood cells for more than two weeks, and are also able to noninvasively measure circulation times of two distinct populations of breast cancer cells simultaneously in a single mouse. We build a custom laser excitation source in the form of an extended cavity mode-locked oscillator, which enables superior detection in whole blood or saline of cell lines expressing fluorescent proteins including the green fluorescent protein (GFP), tdTomato and mPlum. A mathematical model explains unique features of the signals. The ability to distinguish different fluorescent species is central to simultaneous measurement of multiple molecular targets in high throughput applications including the multiphoton flow cytometer. We demonstrate that two dyes which are not distinguishable to one-photon measurements can be differentiated and in fact quantified in mixture via phase-shaped two-photon excitation pulses found by a genetic algorithm. We also selectively enhance or suppress two-photon fluorescence of numerous common dyes with tailored pulse shapes. Using a multiplicative (rather than ratiometric) fitness parameter, we are able to control the fluorescence while maintaining a strong signal. With this method, we control the two-photon fluorescence of the blue fluorescent protein (BFP), which is of particular interest in investigations of protein-protein interactions, and has frustrated previous attempts of control. Implementing an acousto-optic interferometer, we use the same experimental setup to measure two-photon excitation cross-sections of dyes and prove that photon-photon interferences are the

  17. Toxinological studies of the venom from Cassiopea xamachana nematocysts isolated by flow cytometry.

    PubMed

    Radwan, F F; Burnett, J W

    2001-01-01

    The tentacle epithelial tissue of Cassiopea xamachana contains nematocysts and symbiotic algal particles. These two structures were dissociated, analyzed and sorted by flow cytometry. A simple separating method was developed utilizing the algal chlorophyll autofluorescence and the nematocysts' fluorescence after the uptake of fluorescent stains. A five-fold increase in mouse lethality; significantly more potent hemolytic and cytosensing activities; as well as a cleanup in the capillary electropherogram and SDS gel profiles for the crude nematocyst venom preparations prepared by fluorescence activated cell sorting (FACS), was observed relative to alternative methods. Because the hemolytic potency of pre-sorting nematocyst venom was minimal and the post-sorting counterpart was significantly positive, the possibility that algae inhibited the venom's toxinological activity was considered. PMID:11166675

  18. Characterization of aggregate load and pattern in living yeast cells by flow cytometry.

    PubMed

    Hidalgo, Itahisa Hernández; Fleming, Thomas; Eckstein, Volker; Herzig, Stephan; Nawroth, Peter P; Tyedmers, Jens

    2016-01-01

    Protein aggregation is both a hallmark of and a driving force for a number of diseases. It is therefore important to identify the nature of these aggregates and the mechanism(s) by which the cell counteracts their detrimental properties. Currently, the study of aggregation in vivo is performed primarily using fluorescently tagged versions of proteins and analyzing the aggregates by fluorescence microscopy. While this strategy is considered the gold standard, it has several limitations, particularly with respect to its suitability for high-throughput screening (HTS). Here, using a GFP fusion of the well-characterized yeast prion amyloid protein [PSI+], we demonstrate that flow cytometry, which utilizes the same physical principles as fluorescence microscopy, can be used to determine the aggregate load and pattern in live and fixed yeast cells. Furthermore, our approach can easily be applied to high-throughput analyses such as screenings with a yeast deletion library. PMID:27625208

  19. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons

    USGS Publications Warehouse

    Custer, T.W.; Bickham, J.W.; Lyne, T.B.; Lewis, T.; Ruedas, L.A.; Custer, Christine M.; Melancon, M.J.

    1994-01-01

    The flow cytometry method (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a site in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. The CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

  20. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons

    USGS Publications Warehouse

    Custer, T.W.; Bickham, J.W.; Lyne, T.B.; Lewis, T.; Ruedas, L.A.; Custer, Christine M.; Melancon, M.J.

    1994-01-01

    The flow cytometry method (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a site in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. the CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

  1. A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity

    PubMed Central

    O’Brien-Simpson, Neil M.; Pantarat, Namfon; Attard, Troy J.; Walsh, Katrina A.; Reynolds, Eric C.

    2016-01-01

    We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy. PMID:26986223

  2. Monitoring of dynamic microbiological processes using real-time flow cytometry.

    PubMed

    Arnoldini, Markus; Heck, Tobias; Blanco-Fernández, Alfonso; Hammes, Frederik

    2013-01-01

    We describe a straightforward approach to continuously monitor a variety of highly dynamic microbiological processes in millisecond resolution with flow cytometry, using standard bench-top instrumentation. Four main experimental examples are provided, namely: (1) green fluorescent protein expression by antibiotic-stressed Escherichia coli, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary E. coli cells inoculated into fresh growth media, and (4) oxidative disinfection of a mixed culture of auto-fluorescent microorganisms. These examples demonstrate the broad applicability of the method to diverse biological experiments, showing that it allows the collection of detailed, time-resolved information on complex processes. PMID:24244624

  3. An EPROM-based programmable contour generator for use in flow cytometry.

    PubMed

    Wheeless, D M; Cambier, J L; Wheeless, L L

    1988-09-01

    An erasable programmable read-only memory (EPROM) contour generator has been fabricated to produce contours for use in flow cytometry. Contours are analog waveforms representing the fluorescence or light-scatter intensity distribution along a cell or object. The generator has particular utility in the development and testing of slit-scan instrumentation and analysis algorithms. Contours are generated without the requirement of specimens or full operation of the flow instrumentation. The generator provides control of contour height, width, offset, and rate. The EPROM may be custom programmed to produce contours for specific test applications or for reproducing "real" contour events. The generator is useful in situations where constant repetitive contours of predetermined characteristics are required.

  4. Flow Cytometry Analysis of NK Cell Phenotype and Function in Aging.

    PubMed

    Tarazona, Raquel; Campos, Carmen; Pera, Alejandra; Sanchez-Correa, Beatriz; Solana, Rafael

    2015-01-01

    Natural killer (NK) cells represent a subpopulation of lymphocytes involved in innate immunity, defined recently as group 1 of innate lymphoid cells (ILCs). NK cells are cytotoxic lymphocytes with a relevant role in the destruction of transformed cells as virus-infected or tumor cells, as well as the regulation of the immune response through cytokine and chemokine production that activates other cellular components of innate and adaptive immunity. In humans, NK cell subsets have been defined according to the level of expression of CD56. Aging differentially affects NK cell subsets and NK cell function. Here, we describe protocols for the delineation of NK cell subsets and the analysis of their functional capacity using multiparametric flow cytometry.

  5. [Multi-parametric Flow Cytometry for Neuroblastoma, a new and possible diagnostic tool: case report].

    PubMed

    Manrique, Belén; López Marti, Jessica; Cacciavillano, Walter; Rossi, Jorge

    2016-04-01

    Neuroblastoma is the most frequent extracranial solid tumor in childhood, representing 5.6% according to the "Registro Oncopediatrico Hospitalario Argentino". For its diagnosis, several complementary methods (radiological, biological and biochemical) are required, and Multi-parametric Flow Cytometry (MFC) arises as a potential diagnostic method, despite not having been so far extensively explored. MFC is a method that allows to obtain several information about size, internal complexity and antigenic expression by the use of a laser and fluorescent monoclonal antibodies. There are an increasing number of reports in the literature, which reveal the importance of using MFC for diagnosis and monitoring of solid tumors. The aim in this presentation is to highlight the fundamental role that MFC had in the case of a patient affected by neuroblastoma, in which an early diagnosis using this methodology allowed prompt administration of adequate treatment.

  6. In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents

    NASA Astrophysics Data System (ADS)

    Zharov, Vladimir P.; Galanzha, Ekaterina I.; Shashkov, Evgeny V.; Khlebtsov, Nicolai G.; Tuchin, Valery V.

    2006-12-01

    A new photoacoustic flow cytometry was developed for real-time detection of circulating cells, nanoparticles, and contrast agents in vivo. Its capability, integrated with photothermal and optical clearing methods, was demonstrated using a near-infrared tunable laser to characterize the in vivo kinetics of Indocyanine Green alone and single cancer cells labeled with gold nanorods and Indocyanine Green in the vasculature of the mouse ear. In vivo applications are discussed, including selective nanophotothermolysis of metastatic squamous cells, label-free detection of melanoma cells, study of pharmokinetics, and immune response to apoptotic and necrotic cells, with potential translation to humans. The threshold sensitivity is estimated as one cancer cell in the background of 107 normal blood cells.

  7. Comparative analysis of hemocyte phagocytosis between six species of arthropods as measured by flow cytometry.

    PubMed

    Oliver, Jonathan D; Dusty Loy, J; Parikh, Grishma; Bartholomay, Lyric

    2011-10-01

    Phagocytosis of pathogens by hemocytes is a rapid-acting immune response and represents a primary means of limiting microbial infection in some species of arthropods. To survey the relative capacity of hemocyte phagocytosis as a function of the arthropod immune response, we examined the extent of phagocytosis among a wide taxonomic range of arthropod species including a decapod crustacean (Litopenaeus vannamei), three ixodid tick species (Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis), a mosquito species (Aedes aegypti), and a larval moth (Manduca sexta). Injected fluorescent beads were used as a model to elicit phagocytosis and were measured by flow cytometry, a technique provided in detail that may be adapted for use with any species of arthropod. The data indicated that smaller arthropods generally had a higher proportion of phagocytic cells than larger arthropods.

  8. Radiocarbon dating of sub-fossil pollen grans extracted from terrestrial sediments using flow Cytometry.

    NASA Astrophysics Data System (ADS)

    Jones, Richard; Tennant, Richard; Love, John

    2015-04-01

    Producing robust high-resolution radiocarbon chronologies for sediment archives is often hampered by a lack of suitable terrestrial plant macrofossils. Pollen is a viable alternative, readily identifiable as terrestrial in origin and often present in sufficient quantitates for AMS 14C dating. Producing reliable samples is challenging because of time-consuming methods of extraction and purification and possible contamination from other organic material. Here we report a new, rapid method using flow cytometry (FCM) to distinguish, sort and collect sufficient quantities of fossil pollen with minimal contamination from lake sediments. Indeed it is now possible to produce datable samples using a single species if that species is sufficiently abundant in a sample. FCM dating of microfossils shows considerable promise in generating robust geochronological frameworks for terrestrial sequences including those that have previously proved problematic.

  9. Flow cytometry for monitoring contaminant exposure in black-crowned night-herons.

    PubMed

    Custer, T W; Bickham, J W; Lyne, T B; Lewis, T; Ruedas, L A; Custer, C M; Melancon, M J

    1994-08-01

    The flow cytometry methods (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a side in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. The CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

  10. Improving survival of disassociated human embryonic stem cells by mechanical stimulation using acoustic tweezing cytometry.

    PubMed

    Chen, Di; Sun, Yubing; Deng, Cheri X; Fu, Jianping

    2015-03-24

    Dissociation-induced apoptosis of human embryonic stem cells (hESCs) hampers their large-scale culture. Herein we leveraged the mechanosensitivity of hESCs and employed, to our knowledge, a novel technique, acoustic tweezing cytometry (ATC), for subcellular mechanical stimulation of disassociated single hESCs to improve their survival. By acoustically actuating integrin-bound microbubbles (MBs) to live cells, ATC increased the survival rate and cloning efficiency of hESCs by threefold. A positive correlation was observed between the increased hESC survival rate and total accumulative displacement of integrin-anchored MBs during ATC stimulation. ATC may serve as a promising biocompatible tool to improve hESC culture.

  11. A java-based application for differential diagnosis of hematopoietic neoplasms using immunophenotyping by flow cytometry.

    PubMed

    Nguyen, A N; Milam, J D; Johnson, K A; Banez, E I

    2000-07-01

    We describe the implementation of a Java-based application for differential diagnosis of hematopoietic neoplasms using immunophenotyping by flow cytometry. The current version of this Java applet includes the knowledge-base for 33 hematopoietic neoplasms and 43 diagnostic immunophenotyping markers. Java, a new object-oriented computing language, helps facilitate development of this applet, a platform-independent module that can be implemented on the World Wide Web. As the Web rapidly becomes more accessible to users around the world, Web-based software may eventually form the core of decision-support systems in clinical settings. Java-based applications, such as the one described in this paper, are expected to contribute significantly in this area.

  12. Rapid flow cytometry analysis of antimicrobial properties of nettle powder and cranberry powder

    NASA Astrophysics Data System (ADS)

    Hattuniemi, Maarit; Korhonen, Johanna; Jaakkola, Mari; Räty, Jarkko; Virtanen, Vesa

    2010-11-01

    Both nettle (Urtica dioica) and cranberry (Vaccinium oxycoccus) are widely known to have good influence on health. The aim of this study was to investigate antimicrobial properties of nettle powder and cranberry powder against Escherichia coli (E. coli) and monitor the growth of the bacteria by a rapid flow cytometry (FCM) method. For FCM measurements samples were stained with fluorescent dyes. The inhibitory effects of plant material on growth of E. coli were estimated by comparing the results of control sample (E. coli) to E. coli samples with plant material. FCM offers both a brilliant tool to investigate the kinetics of the growth of bacterium, since subsamples can be taken from the same liquid medium during the growing period and with fluorescent dyes a rapid method to investigate viability of the bacterium.

  13. Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry.

    PubMed

    Xu, Li-Ming; Liu, Miao; Zhao, Jing-Zhuang; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

    2014-10-01

    The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments, except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.

  14. Quality control in the application of flow cytometry to studies of environmentally-induced genetic damage

    SciTech Connect

    McCreedy, C.D.; Robinson, J.P.; Dallas, C.E.; Jagoe, C.H.

    1999-07-01

    Flow cytometry (FCM) has been used to demonstrate altered DNA content in fish, reptiles, birds and mammals exposed to radionuclides, PAHs and other contaminants. However, artifacts resulting from sample preparation, handling, variations in instrument parameters or other factors may confound such measurements. Some artifacts resemble genotoxic responses and so could lead to erroneous positive conclusions. As part of ongoing studies of effects of various pollutants on DNA content in fishes, the authors tested sample handling and preparation methods for the induction of artifacts. The authors describe QA/QC methods, including control of staining, conditions, doublet discrimination by comparison of peak versus integral fluorescence, internal DNA standards, and the use of time versus fluorescence plots. Consistent application of these practices is essential to obtain valid measurements of DNA content in environmental samples, and neglect of these can result in poor quality data and the acceptance of incorrect hypotheses.

  15. Measuring antibody neutralization of dengue virus (DENV) using a flow cytometry-based technique.

    PubMed

    de Alwis, Ruklanthi; de Silva, Aravinda M

    2014-01-01

    Dengue virus (DENV) is an emerging virus that threatens over two-third of the world's population. The specific diagnosis of dengue infection by serology is based on assays that detect DENV-specific antibodies including neutralizing antibodies (Abs). Neutralizing Abs are an important, if not the main, mechanism of protection from natural dengue virus (DENV) infection as well. The current gold-standard assay for measuring neutralizing Ab responses against DENV is the plaque reduction neutralization assay (PRNT). However, this assay is slow and laborious and utilizes physiologically irrelevant cell lines. Here, we describe a relatively high-throughput, flow cytometry-based neutralization assay for DENV that has been optimized for use with a human monocytic suspension cell line, U937 + DC-SIGN, or the more commonly used adherent monkey kidney cells, Vero-81. PMID:24696329

  16. Impact of recent innovations in the use of mass cytometry in support of drug development

    PubMed Central

    Ogura, Hideki; Wisnewski, Adam V.

    2015-01-01

    Cytometry by time-of-flight (CyTOF) is a novel technology for the real-time analysis of single cells. CyTOF is a significant advance in fields including immunology, hematology, and oncology. It resolves multiple metal-conjugated probes per cell with minimal signal overlap, which maximizes the information obtained from each individual sample. CyTOF provides the ability to phenotypically and functionally profile cells from normal and diseased states. Single cell technologies enable researchers to measure the effects of a drug at the single cell level and better understand its mechanism of action. Here, we discuss novel instruments for the analysis of individual biological cells, the impact of recent innovations in support of drug development, and the important roles of CyTOF in drug profiling. PMID:26092491

  17. Monitoring of Dynamic Microbiological Processes Using Real-Time Flow Cytometry

    PubMed Central

    Arnoldini, Markus; Heck, Tobias; Blanco-Fernández, Alfonso; Hammes, Frederik

    2013-01-01

    We describe a straightforward approach to continuously monitor a variety of highly dynamic microbiological processes in millisecond resolution with flow cytometry, using standard bench-top instrumentation. Four main experimental examples are provided, namely: (1) green fluorescent protein expression by antibiotic-stressed Escherichia coli, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary E. coli cells inoculated into fresh growth media, and (4) oxidative disinfection of a mixed culture of auto-fluorescent microorganisms. These examples demonstrate the broad applicability of the method to diverse biological experiments, showing that it allows the collection of detailed, time-resolved information on complex processes. PMID:24244624

  18. Principles of minimal residual disease detection for hematopoietic neoplasms by flow cytometry.

    PubMed

    Wood, Brent L

    2016-01-01

    Flow cytometry has become an indispensible tool for the diagnosis and classification of hematopoietic neoplasms. The ability to rapidly distinguish cellular subpopulations via multiparametric assessment of quantitative differences in antigen expression on single cells and enumerate the relative sizes of the resulting subpopulations is a key feature of the technology. More recently, these capabilities have been expanded to include the identification and enumeration of rare subpopulations within complex cellular mixtures, for example, blood or bone marrow, leading to the application for post-therapeutic monitoring or minimal residual disease detection. This review will briefly present the principles to be considered in the construction and use of flow cytometric assays for minimal residual disease detection including the use of informative antibody combinations, the impact of immunophenotypic instability, enumeration, assay sensitivity, and reproducibility.

  19. Assessment of immune parameters of manila clam Ruditapes philippinarum in different physiological conditions using flow cytometry

    NASA Astrophysics Data System (ADS)

    Park, Kyung-Il; Donaghy, Ludovic; Kang, Hyun-Sil; Hong, Hyun-Ki; Kim, Young-Ok; Choi, Kwang-Sik

    2012-03-01

    Cellular and humoral immune parameters are often used as biomarkers to trace environmental and physiological stresses in marine bivalves. In this study, we compared various immune parameters of Manila clams ( Ruditapes philippinarum) under normal conditions and under a high level of desiccation, using flow cytometry. The immune parameters analyzed included, total hemocyte count, hemocyte mortality, hemocyte DNA damage, reactive oxygen species (ROS) production, and phagocytosis activity. Total hemocyte count, hemocyte DNA damage, and hemocyte mortality were significantly elevated among clams under high desiccation stress, while phagocytosis activity and spontaneous ROS production were significantly lower compared to those parameters of the control clams ( p<0.05). These data suggest that the immune parameters analyzed in this study well reflect the physiological status of clams.

  20. Flow cytometry evidence of human granulocytes interaction with polyhedral oligomeric silsesquioxanes: effect of nanoparticle charge

    NASA Astrophysics Data System (ADS)

    Renò, Filippo; Carniato, Fabio; Rizzi, Manuela; Olivero, Francesco; Pittarella, Pamela; Marchese, Leonardo

    2013-05-01

    Nanoparticles (NPs) entering the human body are immediately confronted with the innate part of human immune system. In particular, monocyte and neutrophil granulocytes readily clear particles by phagocytosis, even if in the case of NPs the uptake mechanism may be classified as macropinocytosis. Among engineered nanoparticles, in the last years, siliceous materials have emerged as promising materials for several applications ranging from catalysis to biomedical. The polyhedral oligomeric silsesquioxanes (POSS) are nanodimensional, easily synthesizable molecular compounds and POSS-based systems are promising carriers for biological molecules. In this work, the ability of human granulocytes to uptake positively and negatively charged POSS was measured using a simple flow cytometry analysis based on cell size modifications. The data obtained showed that after a 30 min exposure only positive NPs were uptaken by human granulocyte using both macropinocytosis and clathrin-mediated mechanisms as demonstrated by uptake inhibition mediated by amiloride and chlorpromazine.

  1. The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry

    PubMed Central

    Omiatek, Donna M.; Bressler, Amanda J.; Cans, Ann-Sofie; Andrews, Anne M.; Heien, Michael L.; Ewing, Andrew G.

    2013-01-01

    Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the “weak base hypothesis” that amphetamine reduces synaptic vesicle transmitter and quantal size. PMID:23486177

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

    PubMed Central

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

    2011-01-01

    Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing. PMID:22355635

  3. BeadCons: detection of nucleic acid sequences by flow cytometry.

    PubMed

    Horejsh, Douglas; Martini, Federico; Capobianchi, Maria Rosaria

    2005-11-01

    Molecular beacons are single-stranded nucleic acid structures with a terminal fluorophore and a distal, terminal quencher. These molecules are typically used in real-time PCR assays, but have also been conjugated with solid matrices. This unit describes protocols related to molecular beacon-conjugated beads (BeadCons), whose specific hybridization with complementary target sequences can be resolved by cytometry. Assay sensitivity is achieved through the concentration of fluorescence signal on discrete particles. By using molecular beacons with different fluorophores and microspheres of different sizes, it is possible to construct a fluid array system with each bead corresponding to a specific target nucleic acid. Methods are presented for the design, construction, and use of BeadCons for the specific, multiplexed detection of unlabeled nucleic acids in solution. The use of bead-based detection methods will likely lead to the design of new multiplex molecular diagnostic tools.

  4. A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis.

    PubMed

    Patra, Bishnubrata; Chen, Ying-Hua; Peng, Chien-Chung; Lin, Shiang-Chi; Lee, Chau-Hwang; Tung, Yi-Chung

    2013-01-01

    Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. PMID:24396525

  5. A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

    PubMed

    O'Brien-Simpson, Neil M; Pantarat, Namfon; Attard, Troy J; Walsh, Katrina A; Reynolds, Eric C

    2016-01-01

    We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy. PMID:26986223

  6. Spatial characterization of glutathione depletion in the KHT sarcoma using flow cytometry.

    PubMed

    Minchinton, A I; Chaplin, D J

    1991-06-01

    Intravenous administration of the fluorescent DNA stain Hoechst 33342 to tumour-bearing mice was used to label cells proportionally to their proximity from the vasculature. Flow cytometry was used to sort cells from the tumour into populations based on their Hoechst 33342-derived fluorescence. The cell populations were then assayed for their glutathione (GSH) content and their radiosensitivity. Tumours from mice pretreated with buthionine sulphoximine (BSO) were compared with untreated animals. The major findings of this study suggest that the cellular GSH concentration within tumours decreases with distance from the vasculature, and that the GSH concentration within cells from all locations in the tumour can be depleted by enzymatically inhibiting its synthesis using BSO. This depletion of GSH resulted in a small degree of hypoxic radiosensitization of cells both distal and proximal to the vasculature.

  7. Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes.

    PubMed

    Tracy, Bryan P; Gaida, Stefan M; Papoutsakis, Eleftherios T

    2010-02-01

    Flow cytometry (FC) and FC-based cell sorting have been established as critical tools in modern cell and developmental biology. Yet, their applications in bacteria, especially in the multiparametric mode, remain limited. We argue that FC technologies have the potential to greatly accelerate the analysis and development of microbial complex phenotypes through applications of metabolic engineering, synthetic biology, and evolutionary engineering. We demonstrate the importance of FC for elucidating population heterogeneity because of developmental processes or epigenetic regulation. FC can be engaged for both synthetic and analytical applications of complex phenotypes within a single species, multispecies, and microbial-library populations. Examples include methods to identify developmental microbial stages associated with productive metabolic phenotypes, select desirable promoters from a single species or metagenomic libraries, and to screen designer riboswitches for synthetic-biology applications. PMID:20206495

  8. Fluorescence Assisted Selection of Transformants (FAST): Using flow cytometry to select fungal transformants.

    PubMed

    Vlaardingerbroek, Ido; Beerens, Bas; Shahi, Shermineh; Rep, Martijn

    2015-03-01

    The availability of drug resistance markers for fungal transformation is often a limiting factor in both fungal genetics research and industrial applications. We describe a new technique using flow cytometry to select fungal transformants using well-known fluorescent proteins as markers for transformation. This new technique, Fluorescence-Assisted Selection of Transformants (FAST), was used for a transformation of Fusarium oxysporum with GFP as a marker targeted at a specific site on chromosome 14. The resulting strain was then transformed again with a gene replacement construct containing both RFP and a gene for drug resistance as markers. By directly comparing FAST with drug resistance selection we show that both methods yield comparable numbers of gene deletion mutants.

  9. Overview of clinical flow cytometry data analysis: recent advances and future challenges.

    PubMed

    Pedreira, Carlos E; Costa, Elaine S; Lecrevisse, Quentin; van Dongen, Jacques J M; Orfao, Alberto

    2013-07-01

    Major technological advances in flow cytometry (FC), both for instrumentation and reagents, have emerged over the past few decades. These advances facilitate simultaneous evaluation of more parameters in single cells analyzed at higher speed. Consequently, larger and more complex data files that contain information about tens of parameters for millions of cells are generated. This increasing complexity has challenged pre-existing data analysis tools and promoted the development of new algorithms and tools for data analysis and visualization. Here, we review the currently available (conventional and newly developed) data analysis and visualization strategies that aim for easier, more objective, and robust interpretation of FC data both in biomedical research and clinical diagnostic laboratories.

  10. Screening of Urine Samples by Flow Cytometry Reduces the Need for Culture▿

    PubMed Central

    Jolkkonen, Santra; Paattiniemi, Eeva-Liisa; Kärpänoja, Pauliina; Sarkkinen, Hannu

    2010-01-01

    Urine samples constitute a large proportion of samples tested in clinical microbiology laboratories. Culturing of the samples is fairly time- and labor-consuming, and most of the samples will yield no growth or insignificant growth. We analyzed the feasibility of the flow cytometry-based UF-500i instrument (Sysmex, Japan) to screen out urine samples with no growth or insignificant growth and reduce the number of samples to be cultured. A total of 1,094 urine specimens sent to our laboratory for culture during 4 months in the spring of 2009 in Lahti, Finland, were included in the study. After culture, all samples were analyzed with the Sysmex UF-500i for bacterial and leukocyte (white blood cell [WBC]) counts. Youden index and closest (0,1) methods were used to determine the cutoff values for bacterial and WBC counts in culture-positive and -negative groups. By flow cytometry, samples considered positive for UTI in culture had bacterial and WBC values that were significantly higher than those for samples considered negative. The flow cytometric screening worked best when both bacterial counts and WBC counts were used with age- and gender-specific cutoff values for all patient groups, excluding patients with urological disease or anomaly. By use of these cutoff values, 5/167 (3.0%) of culture-positive samples were missed by UF-500i and the percentage of samples that did not need to be cultured was 64.5%. Use of the UF-500i instrument is a reliable method for screening out a major part of the UTI-negative samples, significantly diminishing the amount of work required in the microbiology laboratory. PMID:20592157

  11. Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

    PubMed Central

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-01-01

    We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group of homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are

  12. Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

    PubMed Central

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-01-01

    We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group of homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are

  13. Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples.

    PubMed

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-01-01

    We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations' characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group of homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are

  14. Hierarchical modeling for rare event detection and cell subset alignment across flow cytometry samples.

    PubMed

    Cron, Andrew; Gouttefangeas, Cécile; Frelinger, Jacob; Lin, Lin; Singh, Satwinder K; Britten, Cedrik M; Welters, Marij J P; van der Burg, Sjoerd H; West, Mike; Chan, Cliburn

    2013-01-01

    Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less). Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to identify cell subsets of interest in an automated fashion. Two specific challenges for automated analysis are to detect extremely low frequency event subsets without biasing the estimate by pre-processing enrichment, and the ability to align cell subsets across multiple data samples for comparative analysis. In this manuscript, we develop hierarchical modeling extensions to the Dirichlet Process Gaussian Mixture Model (DPGMM) approach we have previously described for cell subset identification, and show that the hierarchical DPGMM (HDPGMM) naturally generates an aligned data model that captures both commonalities and variations across multiple samples. HDPGMM also increases the sensitivity to extremely low frequency events by sharing information across multiple samples analyzed simultaneously. We validate the accuracy and reproducibility of HDPGMM estimates of antigen-specific T cells on clinically relevant reference peripheral blood mononuclear cell (PBMC) samples with known frequencies of antigen-specific T cells. These cell samples take advantage of retrovirally TCR-transduced T cells spiked into autologous PBMC samples to give a defined number of antigen-specific T cells detectable by HLA-peptide multimer binding. We provide open source software that can take advantage of both multiple processors and GPU-acceleration to perform the numerically-demanding computations. We show that hierarchical modeling is a useful probabilistic approach that can provide a consistent labeling

  15. Quantification and Characterization of Phagocytosis in the Soil Amoeba Acanthamoeba castellanii by Flow Cytometry

    PubMed Central

    Avery, S. V.; Harwood, J. L.; Lloyd, D.

    1995-01-01

    Phagocytosis in the common grazing soil amoeba Acanthamoeba castellanii was characterized by flow cytometry. Uptake of fluorescently labelled latex microbeads by cells was quantified by appropriate setting of thresholds on light scatter channels and, subsequently, on fluorescence histograms. Confocal laser scanning microscopy was used to verify the effectiveness of sodium azide as a control for distinguishing between cell surface binding and internalization of beads. It was found that binding of beads at the cell surface was complete within 5 min and 80% of cells had beads associated with them after 10 min. However, the total number of phagocytosed beads continued to rise up to 2 h. The prolonged increase in numbers of beads phagocytosed was due to cell populations containing increasing numbers of beads peaking at increasing time intervals from the onset of phagocytosis. Fine adjustment of thresholds on light scatter channels was used to fractionate cells according to cell volume (cell cycle stage). Phagocytotic activity was approximately threefold higher in the largest (oldest) than in the smallest (newly divided) cells of A. castellanii and showed some evidence of periodicity. At no stage in the cell cycle did phagocytosis cease. Binding and phagocytosis of beads were also markedly influenced by culture age and rate of rotary agitation of cell suspensions. Saturation of phagocytosis (per cell) at increasing bead or decreasing cell concentrations occurred at bead/cell ratios exceeding 10:1. This was probably a result of a limitation of the vacuolar uptake system of A. castellanii, as no saturation of bead binding was evident. The advantages of flow cytometry for characterization of phagocytosis at the single-cell level in heterogeneous protozoal populations and the significance of the present results are discussed. PMID:16534962

  16. Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

    PubMed

    Steudler, Susanne; Böhmer, Ulrike; Weber, Jost; Bley, Thomas

    2015-02-01

    Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.

  17. A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

    PubMed

    Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

    2013-06-01

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting.

  18. Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry

    PubMed Central

    Tigges, John; Toxavidis, Vasilis; Camacho, Virginia; Felton, Edward J.; Khoory, Joseph; Kreimer, Simion; Ivanov, Alexander R.; Mantel, Pierre-Yves; Jones, Jennifer; Akuthota, Praveen; Das, Saumya; Ghiran, Ionita

    2016-01-01

    The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform

  19. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

    PubMed Central

    Davey, H M; Kell, D B

    1996-01-01

    The most fundamental questions such as whether a cell is alive, in the sense of being able to divide or to form a colony, may sometimes be very hard to answer, since even axenic microbial cultures are extremely heterogeneous. Analyses that seek to correlate such things as viability, which is a property of an individual cell, with macroscopic measurements of culture variables such as ATP content, respiratory activity, and so on, must inevitably fail. It is therefore necessary to make physiological measurements on individual cells. Flow cytometry is such a technique, which allows one to analyze cells rapidly and individually and permits the quantitative analysis of microbial heterogeneity. It therefore offers many advantages over conventional measurements for both routine and more exploratory analyses of microbial properties. While the technique has been widely applied to the study of mammalian cells, is use in microbiology has until recently been much more limited, largely because of the smaller size of microbes and the consequently smaller optical signals obtainable from them. Since these technical barriers no longer hold, flow cytometry with appropriate stains has been used for the rapid discrimination and identification of microbial cells, for the rapid assessment of viability and of the heterogeneous distributions of a wealth of other more detailed physiological properties, for the analysis of antimicrobial drug-cell interactions, and for the isolation of high-yielding strains of biotechnological interest. Flow cytometric analyses provide an abundance of multivariate data, and special methods have been devised to exploit these. Ongoing advances mean that modern flow cytometers may now be used by nonspecialists to effect a renaissance in our understanding of microbial heterogeneity. PMID:8987359

  20. Flow Cytometry-Based Methods to Characterize Immune Senescence in Nonhuman Primates.

    PubMed

    Meyer, Christine; Haberthur, Kristen; Asquith, Mark; Messaoudi, Ilhem

    2015-01-01

    Flow cytometry is an invaluable technique that can be used to phenotypically and functionally characterize immune cell populations ex vivo. This technology has greatly advanced our ability to gain critical insight into age-related changes in immune function, commonly known as immune senescence. Rodents have been traditionally used to investigate the molecular mechanisms of immune senescence because they offer the distinct advantages of an extensive set of reagents, the presence of genetically modified strains, and a short lifespan that allows for longevity studies of short duration. More recently, nonhuman primates (NHPs), and specifically rhesus macaques, have emerged as a leading translational model to study various aspects of human aging. In contrast to rodents, they share significant genetic homology as well as physiological and behavioral characteristics with humans. Furthermore, rhesus macaques are a long-lived outbred species, which makes them an ideal translational model. Therefore, NHPs offer a unique opportunity to carry out mechanistic studies under controlled laboratory conditions (e.g., photoperiod, temperature, diet, and medications) in a species that closely mimics human biology. Moreover similar techniques (e.g., activity recording and MRI) can be used to measure physiological parameters in NHPs, making direct comparisons between NHP and human data sets possible. In addition, the outbred genetics of NHPs enables rigorous validation of research findings that goes beyond proof of principle. Finally, self-selection bias that is often unavoidable in human clinical trials can be completely eliminated with NHP studies. Here we describe flow cytometry-based methods to phenotypically and functionally characterize innate immune cells as well as T and B lymphocyte subsets from isolated peripheral blood mononuclear cells (PBMC) in rhesus macaques. PMID:26420709

  1. Using flow cytometry to estimate pollen DNA content: improved methodology and applications

    PubMed Central

    Kron, Paul; Husband, Brian C.

    2012-01-01

    Background and Aims Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility. Methods The method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species. Key Results Data quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %. Conclusions The high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry. PMID

  2. Microsatellite and flow cytometry analysis to help understand the origin of Dioscorea alata polyploids

    PubMed Central

    Nemorin, A.; David, J.; Maledon, E.; Nudol, E.; Dalon, J.; Arnau, G.

    2013-01-01

    Background and Aims Dioscorea alata is a polyploid species with a ploidy level ranging from diploid (2n = 2x = 40) to tetraploid (2n = 4x = 80). Ploidy increase is correlated with better agronomic performance. The lack of knowledge about the origin of D. alata spontaneous polyploids (triploids and tetraploids) limits the efficiency of polyploid breeding. The objective of the present study was to use flow cytometry and microsatellite markers to understand the origin of D. alata polyploids. Methods Different progeny generated by intracytotype crosses (2x × 2x) and intercytotype crosses (2x × 4x and 3x × 2x) were analysed in order to understand endosperm incompatibility phenomena and gamete origins via the heterozygosity rate transmitted to progeny. Results This work shows that in a 2x × 2x cross, triploids with viable seeds are obtained only via a phenomenon of diploid female non-gametic reduction. The study of the transmission of heterozygosity made it possible to exclude polyspermy and polyembryony as the mechanisms at the origin of triploids. The fact that no seedlings were obtained by a 3x × 2x cross made it possible to confirm the sterility of triploid females. Flow cytometry analyses carried out on the endosperm of seeds resulting from 2x × 4x crosses revealed endosperm incompatibility phenomena. Conclusions The major conclusion is that the polyploids of D. alata would have appeared through the formation of unreduced gametes. The triploid pool would have been built and diversified through the formation of 2n gametes in diploid females as the result of the non-viability of seeds resulting from the formation of 2n sperm and of the non-viability of intercytotype crosses. The tetraploids would have appeared through bilateral sexual polyploidization via the union of two unreduced gametes due to the sterility of triploids. PMID:23912697

  3. Detection of reticulated platelets in whole blood of rats using flow cytometry.

    PubMed

    Pankraz, Alexander; Ledieu, David; Pralet, Dominique; Provencher-Bolliger, Anne

    2008-09-01

    As opposed to erythropoiesis, which is regularly assessed in the peripheral blood of animals by reticulocyte count, thrombopoiesis is rarely assessed in assays that detect immature platelets in the peripheral blood. An assessment of recent thrombopoiesis is feasible with the analysis of reticulated platelets in the peripheral blood via flow cytometry, but rarely performed. The aim of this study was to establish an assay for the detection of reticulated platelets in whole blood of rats via flow cytometry, using a two-color staining method with a platelet-specific antibody (CD61-PE) and thiazole orange to detect RNA-containing platelets. Platelets were detected in K3EDTA-anticoagulated, paraformaldehyde-fixed samples, using a CD61-PE antibody as well as a gate specific for the light scatter properties of platelets. The intra-assay coefficient of variation varied between 3.6% and 8.3% (n=6 animals). The stability of the assay was determined by storing blood prior to staining, storing stained samples for up to 2h at room temperature, and by diluting the blood prior to analysis with autologous plasma to create samples with artificial anemia and thrombocytopenia. Only samples stored at room temperature prior to analysis showed a significantly lower percentage of reticulated platelets. Percentage of reticulated platelets in the reference population (n=41 rats) was 10.0+/-1.3% reticulated platelets (mean+/-SD; min=6.2%; max=12.5%). These data show that the detection of reticulated platelets in whole blood of rats using a platelet-specific antibody is feasible. This test presents a minimal-invasive method to assess thrombopoiesis in rats that can be used for example in preclinical toxicological studies.

  4. Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples.

    PubMed

    Azad, Ariful; Rajwa, Bartek; Pothen, Alex

    2016-01-01

    We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations' characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group of homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are

  5. Use of flow cytometry for analysis of phage-mediated killing of Enterobacter aerogenes.

    PubMed

    Verthé, Kristof; Verstraete, Willy

    2006-09-01

    In this study, the use of flow cytometry to analyze phage-mediated killing of Enterobacter aerogenes under varying conditions of temperature and nutrient availability was assessed. Bacteriophage UZ1, specific for an E. aerogenes strain, was applied at a multiplicity of infection (MOI) of 1 and 1000 to a Teflon surface, artificially infected with its host at a level of 4.5 log cells. After incubation for 20 h, bacteriophages were quantified using the soft agar layer method. For the quantification of bacterial cells, plate counting and flow cytometric analysis of live/dead stained cells were performed in parallel. At an MOI of 1, phage treatment was successful only after incubation under nutrient-rich conditions at 37 degrees C: E. aerogenes cells were not detected and a tenfold increase in phage UZ1 was observed. At a MOI of 1000, no E. aerogenes cells could be cultured after incubation at 37 and 4 degrees C. However, flow cytometric analysis revealed that lysis did not occur at 4 degrees C but was achieved during subsequent plate culture. In conclusion, the use of flow cytometry enabled identification of culture-based bias during plate culture. The flow cytometric assay used in this study proved to be rapid, as this culture-independent method does not require lengthy incubation periods post-sampling. The bacteriophage-mediated killing of E. aerogenes cells on Teflon surfaces indicated that disinfection of E. aerogenes with bacteriophage UZ1 can be successful when high MOIs are achieved, while at low multiplicities of infection conditions favorable for phage replication are required.

  6. Microfluidic fluorescence in situ hybridization and flow cytometry (µFlowFISH)

    PubMed Central

    Liu, Peng; Meagher, Robert J.; Light, Yooli Kim; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P.; Hazen, Terry C.; Singh, Anup K.

    2011-01-01

    We describe an integrated microfluidic device (µFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(VI) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The µFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed; and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp. strain RCH2 that are involved in Cr(VI) reduction and immobilization. Combined labeling and detection efficiencies of 74–97% were observed in experiments with simple mixtures of cultured cells confirmed specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of µFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford Site. We were able to monitor the numbers of Pseudomonas sp. with only 100–200 cells loaded into the microchip. The µFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

  7. Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

    PubMed

    Ribera, Jordi; Zamora, Lurdes; Juncà, Jordi; Rodríguez, Inés; Marcé, Silvia; Cabezón, Marta; Millá, Fuensanta

    2013-07-25

    In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society. PMID:23894019

  8. Multispectral Imaging Broadens Cellular Analysis

    NASA Technical Reports Server (NTRS)

    2007-01-01

    Amnis Corporation, a Seattle-based biotechnology company, developed ImageStream to produce sensitive fluorescence images of cells in flow. The company responded to an SBIR solicitation from Ames Research Center, and proposed to evaluate several methods of extending the depth of field for its ImageStream system and implement the best as an upgrade to its commercial products. This would allow users to view whole cells at the same time, rather than just one section of each cell. Through Phase I and II SBIR contracts, Ames provided Amnis the funding the company needed to develop this extended functionality. For NASA, the resulting high-speed image flow cytometry process made its way into Medusa, a life-detection instrument built to collect, store, and analyze sample organisms from erupting hydrothermal vents, and has the potential to benefit space flight health monitoring. On the commercial end, Amnis has implemented the process in ImageStream, combining high-resolution microscopy and flow cytometry in a single instrument, giving researchers the power to conduct quantitative analyses of individual cells and cell populations at the same time, in the same experiment. ImageStream is also built for many other applications, including cell signaling and pathway analysis; classification and characterization of peripheral blood mononuclear cell populations; quantitative morphology; apoptosis (cell death) assays; gene expression analysis; analysis of cell conjugates; molecular distribution; and receptor mapping and distribution.

  9. IMAGES, IMAGES, IMAGES

    SciTech Connect

    Marcus, A.

    1980-07-01

    The role of images of information (charts, diagrams, maps, and symbols) for effective presentation of facts and concepts is expanding dramatically because of advances in computer graphics technology, increasingly hetero-lingual, hetero-cultural world target populations of information providers, the urgent need to convey more efficiently vast amounts of information, the broadening population of (non-expert) computer users, the decrease of available time for reading texts and for decision making, and the general level of literacy. A coalition of visual performance experts, human engineering specialists, computer scientists, and graphic designers/artists is required to resolve human factors aspects of images of information. The need for, nature of, and benefits of interdisciplinary effort are discussed. The results of an interdisciplinary collaboration are demonstrated in a product for visualizing complex information about global energy interdependence. An invited panel will respond to the presentation.

  10. Flow Cytometry: A Promising Technique for the Study of Silicone Oil-Induced Particulate Formation in Protein Formulations

    PubMed Central

    Ludwig, D. Brett; Trotter, Joseph T.; Gabrielson, John P.; Carpenter, John F.

    2010-01-01

    Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also utilized to investigate the effects of silicone oil emulsions on the stability BSA, lysozyme, abatacept or trastuzumab formulations containing surfactant, sodium chloride or sucrose. To aid in particle characterization, the fluorescence detection capabilities of Flow cytometry were exploited by staining silicone oil with BODIPY® 493/503 and model proteins with Alexa Fluor® 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. Addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations. PMID:21146492

  11. Two-photon, two-color in vivo flow cytometry to noninvasively monitor multiple circulating cell lines

    NASA Astrophysics Data System (ADS)

    Tkaczyk, Eric R.; Zhong, Cheng Frank; Ye, Jing Yong; Katnik, Steve; Myc, Andrzej; Thomas, Thommey; Luker, Kathryn E.; Luker, Gary D.; Baker, James R., Jr.; Norris, Theodore B.

    2007-07-01

    We have developed a new two-photon system for in vivo flow cytometry, thereby allowing us to simultaneously quantify different circulating populations in a single animal. The instrument was able to resolve minute-by-minute depletion dynamics of injected fluorescent microspheres at finer time scales than conventional flow cytometry. Also observed were the circulation dynamics of human MCF-7 and MDA-MB-435 breast cancer cells, which have low and high metastatic potential, respectively. After co-injection of both cell types into mice, markedly greater numbers of MCF-7 cells were present in the circulation at early time points. While low metastatic MCF-7 cells were cleared from the vascular system within 24 hours, detectable numbers of metastatic MDA-MB- 435 cells in the circulation remained constant over time. When we replace the commercial (80-MHz) NIR excitation laser with a reduced-repetition-rate (20-MHz) mode-locked oscillator, the signal is enhanced four-fold, enabling superior detection in blood of cell lines expressing fluorescent proteins tdTomato and mPlum (crosslabeled with DiI and DiD). Detection sensitivity versus incident laser power is understood in terms of detected event photon count distribution, which can be predicted with simple fluorophore distribution assumptions. The technique of two-color, two-photon flow cytometry greatly enhances the capabilities of ex vivo flow cytometry to investigate dynamics of circulating cells in cancer and other important diseases.

  12. Rapid staining and enumeration of small numbers of total bacteria in water by solid-phase laser cytometry

    NASA Technical Reports Server (NTRS)

    Broadaway, Susan C.; Barton, Stephanie A.; Pyle, Barry H.

    2003-01-01

    The nucleic acid stain SYBR Green I was evaluated for use with solid-phase laser cytometry to obtain total bacterial cell counts from several water sources with small bacterial numbers. Results were obtained within 30 min and exceeded or equaled counts on R2A agar plates incubated for 14 days at room temperature.

  13. Flow cytometry and K-mer analysis estimates of the genome sizes of Bemisia tabaci B and Q (Hemiptera: Aleyrodidae)

    PubMed Central

    Guo, Li T.; Wang, Shao L.; Wu, Qing J.; Zhou, Xu G.; Xie, Wen; Zhang, You J.

    2015-01-01

    The genome sizes of the B- and Q-types of the whitefly Bemisia tabaci (Gennnadius) were estimated using flow cytometry (Drosophila melanogaster as the DNA reference standard and propidium iodide (PI) as the fluorochrome) and k-mer analysis. For flow cytometry, the mean nuclear DNA content was 0.686 pg for B-type males, 1.392 pg for B-type females, 0.680 pg for Q-type males, and 1.306 pg for Q-type females. Based on the relationship between DNA content and genome size (1 pg DNA = 980 Mbp), the haploid genome size of B. tabaci ranged from 640 to 682 Mbp. For k-mer analysis, genome size of B-type by two methods were consistent highly, but the k-mer depth distribution graph of Q-type was not enough perfect and the genome size was estimated about 60 M larger than its flow cytometry result. These results corroborate previous reports of genome size based on karyotype analysis and chromosome counting. However, these estimates differ from previous flow cytometry esti