Rozprawy doktorskie: „Flow cytometers”

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Bausch, Cornelius Sebastian [Verfasser]. "Tubular coplanar waveguides for the use in radio-frequency microfluidic flow cytometers / Cornelius Sebastian Bausch". München : Verlag Dr. Hut, 2016. http://d-nb.info/1103872044/34.

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Le, Lann Lucas. "Elaboration d'une procédure standardisée d'harmonisation des données de cytométrie en flux dans le cadre d'études multicentriques Multi-center harmonization of flow cytometers in the context of the European “PRECISESADS” project, in Autoimmunity Reviews 15(11), November 2016". Thesis, Brest, 2019. http://www.theses.fr/2019BRES0048.

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L'objectif de la thèse est d'assurer la comparabilité des données de cytométrie générées au cours d'une étude multi-centrique.Ce travail de thèse s'inscrit dans le cadre du projet PRECISESADS. Ce projet européen cherche à reclassifier les maladies auto-immunes systémiques en utilisant une approche dite "omique" pour obtenir des signatures biologiques. Cette approche regroupe des outils comme la génomique, la protéomique ... et lacytométrie en flux. Le nombre important d'individus inclus dans le projet rend obligatoire l'utilisation d'outils informatiques pour automatiser l'analyse des milliers de fichiers de cytométrie obtenus au cours des cinq années du projet. Les fréquences des populations cellulaires extraites des fichiers sont comparables entre les centres mais l'intensité médiane de fluorescence (IMF) des molécules étudiées ne l'est pas, malgré une phase de normalisation des données. La cause de cette non-comparabilité est une combinaison d'un effet lot et d'un effet centre. Ces deux effets peuvent être corrigés à l'aide de coefficients spécifiques. La normalisation et la correction des effets lot et centre par l'élaboration de script R et sous python, permettent d'obtenir des IMF comparables entre les centres. Au final, ce travail de thèse a permis d'établir une nouvelle procédure standardisée utilisable dans tous les projets multi-centriques prospectifs d'analyse de données de cytométrie en flux
The aim of this thesis is to ensure the comparability of flow cytometry data within the context of multi-center studies. This thesis work is part of the PRECISESADS project. This European project seeks to reclassify the systemic autoimmune diseases using "omic" data to find useful biological signatures. This encompasses tools like genomic, proteomic ... and flow cytometry. The inclusion of numerous individuals in the project make the use of informatics tools a must for the analysis automation of the thousands flow cytometry files obtained during the 5-years period of the project.Cell populations frequencies extracted from the files are comparable between centers but that is not the case for the median of fluorescence intensities (MFI) of the studied molecules, despite a normalization step. The origin of this incomparability is due to a combination of a batch effect and a center effect. Those two effects can be corrected with specific coefficients. The normalization and correction of both batch and center effect thanks to the elaboration of new R script and python script allow the production of comparable MFI between centers. Overall, this thesis work established a new standardized procedure, efficient for any multi-center projects of flow cytometry data analysis

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Venkatesh, Mukung C. (Mukund Chakravarthy). "Optimization of the mini-flo flow cytometer dc by Mukund C. Venkatesh". Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/40009.

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Davey, Hazel Marie. "Flow cytometry of microorganisms". Thesis, Aberystwyth University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309050.

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Novak, John P. (John Peter) 1957. "Development of the in vivo flow cytometer". Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/30331.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2004.
Includes bibliographical references.
An in vivo flow cytometer has been developed that allows the real-time detection and quantification of circulating cells containing fluorescent proteins or labeled with fluorochrome molecules in live animals, without the need to extract blood samples. A stationary laser beam is focused by a cylindrical lens to a slit of light that is then demagnified and focused across a blood vessel by an achromat and microscope objective. Fluorescent cells are excited one by one as they flow through the excitation laser light slit, creating a burst of fluorescence whose width is inversely proportional to their velocity. The fluorescence signal is detected through a confocal slit aperture using a photomultiplier tube. The analog signal from the photomultiplier tube is then digitized, filtered, and recorded as a function of time onto a computer. Computer programs post- process the data for the presence of cell signal, as well as various aspects of the cell signal such as height, width, and temporal location of the signal peak. Two in vivo flow cytometers have been built: a single-slit, single-color system and a two- slit, two-color system. The single-slit, single-color system provides excitation at 632 nm, and the two-slit, two-color system provides excitation at 632 nm and 473 nm. The two- slit, two-color system can operate in several different modes: single-slit at 632 nm or 473 nm, double-slit at 632 nm or 473 nm, and double-slit with one excitation slit at 632 nm and the other at 473 nm.
(cont.) Thus far, the single-slit, single-color system has been used to study the circulation kinetics of different prostate cancer and leukemia cell lines with different metastatic potential, as well as the effect of different host environments (i.e., mouse versus rat). In addition, the device has been used to develop a new in vivo labeling method of white blood cells that does not result in significant depletion of the labeled cells, allowing for the possibility of autoimmune and transplant rejection studies. The two-slit, two-color system is being used to track two different cell populations, or one cell population labeled with two different markers, one of which can be the green fluorescent protein.
by John P. Novak.
Ph.D.

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Wållberg, Fredrik. "Flow cytometry for bioprocess control". Licentiate thesis, KTH, Biotechnology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-1736.

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During bio-technical processing it is important to monitorbiological parameters such as cell growth, viability andproduct formation. Many of the analyses traditionally used areslow to perform and provide only average data for thepopulation. Flow cytometry is a laser-based technique, whichmeasures physical properties of a cell in a flowing stream, ata rate of several thousand cells per second. It offers theprospect of an at-line, multi-parameter analysis of individualmicroorganisms in a population.

In this project several methods for at-line measurements ofbioprocesses were developed such as protocol's for measuringcell concentration, viability and product formation. Theprimary focus was on prokaryotic organisms (E. coli) but eukaryotic organisms (P. pastoris) were included.

The possibility to use volumetric cell counting to measurecell concentration (cell number) was evaluated. It was shownthat the method was applicable for high cell density processesof bothE. coliandP. pastoris.

The combination of Bis- (1,3-dibutylbarbituric acid)trimethine oxonol (depolarised membranes) and propidium iodide(loss of membrane integrity) as fluorescent markers was usefulto measure viability at-line of cells in high cell densityprocesses. The protocol was shown to be reproducible forE. coliandP. pastoris.

The viability staining was used to study the kinetics ofweak organic acids (food preservatives). The protocol provideddata about cell functions such as membrane depolarisation andloss of membrane integrity caused by introducing weak organicacids to shake flask cultures ofE. coli.

Labeling inclusion bodies with fluorescent antibodiesprovided a method, which could specifically monitor theincreased accumulation of recombinant promegapoetin proteinwith process time. This technique was further developed forintracellular staining by application of a permeabilising stepbefore labeling with antibodies. Staining of inclusion bodiesdirectly inside permeabilised cells gave information about thedistribution of protein expression in the cell population.

In conclusion, flow cytometry provides an at-line, singlecell technique for measurement of several biological parametersin bioprocesses.

Key words: flow cytometry, Partec PAS, propidium iodide(PI), bis- (1,3-dibutylbarbituric acid) trimethine oxonol(BOX), Alexa fluor 488, bioprocess,E. coli,P. pastoris, inclusion body, food preservatives,viability, membrane potential

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Helou, Michael [Verfasser]. "Magnetic Flow Cytometry / Michael Helou". Kiel : Universitätsbibliothek Kiel, 2018. http://d-nb.info/1169132596/34.

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Stewart, Justin William. "Photonic Crystal-Based Flow Cytometry". Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5396.

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Photonic crystals serve as powerful building blocks for the development of lab-on-chip devices. Currently they are used for a wide range of miniaturized optical components such as extremely compact waveguides to refractive-index based optical sensors. Here we propose a new technique for analyzing and characterizing cells through the design of a micro-flow cytometer using photonic crystals. While lab scale flow cytometers have been critical to many developments in cellular biology they are not portable, difficult to use and relatively expensive. By making a miniature sensor capable of replicating the same functionality as the large scale units with photonic crystals, we hope to produce a device that can be easily integrated into a lab-on-chip and inexpensively mass produced for use outside of the lab. Using specialized FDTD software, the proposed technique has been studied, and multiple important flow cytometry functions have been established. As individual cells flow near the crystal surface, transmission of light through the photonic crystal is influenced accordingly. By analyzing the resulting changes in transmission, information such as cell counting and shape characterization have been demonstrated. Furthermore, correlations for simultaneously determining the size and refractive indices of cells has been shown by applying the statistical concepts of central moments.

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Sobek, Daniel. "Microfabricated fused silica flow chambers for flow cytometry". Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/10262.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1997.
Includes bibliographical references (leaves 107-116).
by Daniel Sobek.
Ph.D.

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Collins, Gary Stephen. "Multivariate analysis of flow cytometry data". Thesis, University of Exeter, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324749.

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Shooshtari, Parisa. "Computational techniques for flow cytometry : the application for automated analysis of innate immune response flow cytometry data". Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42179.

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Flow cytometry (FCM) is a technique for measuring physical, chemical and biological characteristics of individual cells. Recent advances in FCM have provided researchers with the facility to improve their understanding of the tremendously complex immune system. However, the technology is hampered by current manual analysis methodologies. In this thesis, I developed computational methods for the automated analysis of immune response FCM data to address this bottleneck. I hypothesized that highly accurate results could be obtained through learning from the patterns that a biology expert applies when doing the analysis manually. In FCM data analysis, it is often desirable to identify homogeneous subsets of cells within a sample. Traditionally, this is done through manual gating, a procedure that can be subjective and time-consuming. I developed SamSPECTRAL, an automated spectral-based clustering algorithm to identify FCM cell populations of any shape, size and distribution while addressing the drawbacks of manual gating. A particularly signi cant achievement of SamSPECTRAL was its successful performance in nding rare cell populations. Similarly, in most FCM applications, it is required to match similar cell populations between di erent FCM samples. I developed a novel learning-based cluster matching method that incorporates domain expert knowledge to nd the best matches of target populations among all clusters generated by a clustering algorithm. Immunophenotyping of immune cells and measuring cytokine responses are two main components of immune response FCM data analysis. I combined the SamSPECTRAL algorithm and cluster matching to perform automated immunophenotyping. I also devised a method to measure cytokine responses automatically. After developing computational methods for each of the above analysis components separately, I organized them into a semi-automated pipeline, so they all work together as a uni ed package. My experiments on 216 FCM samples con rmed that my semi-automated pipeline can reproduce manual analysis results highly accurately both for immunophenotyping and measuring cytokine responses. My other main contributions were correlation analysis of intracellular and secreted cytokines, and developing a formula called GiMFI to improve measuring functional response of cytokine-producing cells using ow cytometry assay.

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Condrau, Marc Anton. "Time-resolved fluorescence measurement in flow cytometry /". [S.l.] : [s.n.], 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10267.

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Porter, Jonathan David. "Applications of flow cytometry to bacterial ecology". Thesis, University of Liverpool, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385323.

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Heiden, Thomas. "Clinical fluorescence cytometry : improvements to preparation methods and instrumentation /". Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3648-X/.

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Herrala, S. (Sauli). "Level set clustering in a flow cytometry dataset". Master's thesis, University of Oulu, 2016. http://urn.fi/URN:NBN:fi:oulu-201604191509.

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Pro Gradu -työn tarkoituksena oli testata kuinka hyvin tasopuumenetelmä soveltuu virtaussytometria-aineiston klusterointiin, ja verrata sitä k-lähimmän naapurin menetelmään. Tasopuumenetelmää myös parannettiin lisäämällä Nelder-Mead (NM) -optimointimenetelmä yhteen työvaiheeseen, jotta laskenta-ajat pysyisivät hallinnassa. Virtaussytometriassa mitataan yksittäisia soluja ja niiden ominaisuuksia, kun ne kulkevat laserin läpi. Ennen mittausta näyte on käsitelty fluerosoivalla aineella, jolloin solu hohtaa valon aallonpituuksia kulkiessaan laserin läpi. Nykyaikaiset laitteet pystyvät mittaamaan satoja tuhansia soluja ja kymmeniä aallonpituuksia kerralla. Tälläisen aineiston käsittely perinteisillä menetelmillä on erittäin raskasta. FlowCAP-konsortio perustettiin testaamaan kuinka hyvin erilaiset klusterointi- ja luokittelumenetelmät soveltuvat sytometria-aineiston käsittelyyn. Tässä gradussa käytetään FlowCAP II -kisasta “akuutti myelooinen leukemia” -aineistoa, joka sisältää 43 sairastunutta ja 359 tervettä verrokkia. Henkilön luokitteleminen terveisiin ja sairaisiin tehtiin tukivektorikoneella tasopuumenetelmästä tai k-lähimmän naapurin menetelmästä saatuja klustereita hyödyntäen. NM-menetelmällä saatiin selvä ajallinen hyöty ilman suurempaa menetystä tarkkuudessa, koska NM-menetelmä pääsi aina hyvin lähelle alkuperäisen algoritmin tulosta. Tasopuumenetelmä itsessään ei ollut k-lähimmän naapurin menetelmää parempi. Tämä johtui osaksi siitä, että variaatio tasopuumenetelmän tuloksien välillä oli hyvin suuri. Ero tasopuumenetelmän ja k-lähimmän naapurin menetelmän välillä oli kuitenkin pieni.

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Helstad, Amanda. "Application of Flow Cytometry for Slow Sand Filters". Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-157760.

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This project investigated the bacteria in water entering and leaving the slow sand filters at Ringsjö Water Works using flow cytometry. The purpose was to explore the possibility of utilising flow cytometry as a monitoring method for optimising water production using slow sand filters. Data describing the bacterial community in water was collected over seven weeks and analysed with FlowJo, flow cytometric image comparison and Minitab. The total cell count, intact cell count and the percentage of high nucleic acid bacteria were analysed. These parameters were highly dependent on scraping events, water entering the filters and season. The results indicated that flow cytometry has great potential for use as a monitoring method, although more data should be collected to establish expected trends and secure baseline values for routine comparisons.

Fördröjning av publikation fram till 31 december 2020.

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Buldini, Barbara. "Flow cytometry application in hematological malignancies of childhood". Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425984.

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The PhD research work was performed, for the first part (1 year) at the Pediatric Haematology-Oncology Department, Fondazione IRCCS Policlinico San Matteo, Pavia University and for the second part (2 years) at the Pediatric Haematology-Oncology Department, Padova University, two excellent setting for a specialized training in pediatric haematology-oncology. The PhD program was targeted in both a clinical and laboratory research experience in order to perform a translational research on pediatric patients affected by a wide range of hematological disorders, both malignant and non-malignant. The efforts were coordinated to study the biology and therapy of pediatric Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS).

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Huang, Ming-Chieh. "Silicon microfabricated device for non-sheath-flow cytometer-based chemical analysis and microchannel flow sensing /". Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/5870.

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Wu, Jianglai. "A light sheet based fluorescence imaging flow cytometer for phytoplankton analysis". HKBU Institutional Repository, 2014. https://repository.hkbu.edu.hk/etd_oa/36.

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Monitoring phytoplankton species composition and their abundance are routine tasks in marine ecological research and environmental monitoring. As phytoplankton populations are highly heterogeneous in terms of size, morphology, and most significantly, their abundance can change drastically in a very short time, it is extremely difficult to quantify and monitor them and there are demands on the instrumentation. Conventional optical microscopy and flow cytometry are the main tools to enumerate and identify phytoplankton, but they have a compromise between spatial information and acquisition speed. While imaging flow cytometry has the potential to integrate the benefit of high spatial resolution from optical microscopy and the advantage of high throughput from flow cytometry, two intrinsic blur sources, motion blur and out-of-focus blur, prevent imaging flow cytometers from obtaining high spatial resolution images with high throughput. To address these limitations, in this work, a novel light sheet based fluorescence imaging flow cytometer has been proposed, constructed, and tested for phytoplankton analysis. Both 2D and 3D imaging mode of the light sheet based fluorescence imaging flow cytometer have been investigated. In the 2D imaging mode, the instrument can screen untreated costal water samples at a volumetric throughput up to 1 ml/min. The instrument demonstrated shows a high immunity to motion blur, and all-in-focus fluorescence images are captured with a lateral resolution of 0.75 ± 0.06 µm for a wide size range ~ 1 µm to ~ 200 µm that includes pico-, nano-and microphytoplankton. This is made possible by suppressing the out-of-focus blur using thin light sheet illumination and image deconvolution, and by precluding the motion blur with a unique flow configuration. With these abilities, the instrument demonstrated has high potential as a practical field instrument for monitoring phytoplankton. In the 3D imaging mode, the instrument can scan a large number of phytoplankton cells in a short time with spatial resolution as achieved by light sheet microscopy. The lateral resolution is 0.81 ± 0.07 µm, and axial resolution in terms of FWHM of the axial scattering PSF is 1.42 ± 0.15 µm. The volumetric throughput of the instrument is 0.5 µl/min. This is benefitted from the improvement that 3D images can be acquired without the need of sample immobilization, in contrast to existing 3D imaging approaches, such as confocal fluorescence microscopy. Preliminary results from untreated coastal water samples and cultured samples show promising potentials of the instrument for phytoplankton monitoring and scientific research.

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Meyer, Michael [Verfasser]. "Applications of aptamers in flow cytometry assays / Michael Meyer". Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2014. http://d-nb.info/1059512718/34.

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Alexander, R. G. "Flow cytometry and cell sorting in plant genetic manipulations". Thesis, University of Nottingham, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356016.

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Spurgeon, Benjamin E. J. "Phosphoproteomic analysis of platelet signalling cascades by flow cytometry". Thesis, University of Hull, 2014. http://hydra.hull.ac.uk/resources/hull:10543.

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The activation of blood platelets is a critical haemostatic response that serves to prevent haemorrhage, but unregulated platelet activation is associated with arterial thrombosis. Endothelial-derived inhibitors prostacyclin (PGI₂) and nitric oxide (NO) activate protein kinase-mediated signalling cascades to regulate platelet function and prevent vascular thrombosis. These signalling cascades involve a number of complex protein phosphorylation reactions, which regulate different aspects of platelet function. Dissecting the signalling events that regulate platelet function could facilitate the development of novel antiplatelet agents. Intraplatelet protein phosphorylation is commonly measured by immunoblotting, which is not conducive to whole blood analysis and therefore may not provide an accurate representation of signalling events in a (patho)physiological context. Therefore, the major aim of this thesis was to develop methodologies that could examine platelet signalling events in a more physiological context. In particular, we wanted to develop methodologies that could evaluate the ability of PGI₂ to modulate blood platelet activity. Using whole blood flow cytometry, PGI₂ was found to inhibit platelet fibrinogen binding and P-selectin expression, two independent markers of platelet activation. The inhibition of platelet function by PGI₂ corresponded with increased phosphorylation of proteins known to be targeted by PGI₂-mediated signalling cascades including vasodilator-stimulated phosphoprotein (VASP). In the next series of experiments, we developed an assay to evaluate these signalling events in whole blood. This phosphoflow assay was sensitive enough to accurately and reproducibly detect subtle dose- and time-dependent changes in protein phosphorylation in whole blood that were consistent with immunoblotting protocols with washed platelets. The application of fluorescent barcoding protocols to this assay enabled the simultaneous staining and acquisition of 24-96 samples in a single analysis tube. To exploit the high-throughput nature of the method and demonstrate its value as a drug discovery platform, we screened a library of 70 prostaglandins for their ability to stimulate intraplatelet VASP phosphorylation. The screen revealed three previously uncharacterised molecules that stimulated cAMP formation, induced VASP phosphorylation, and inhibited platelet aggregation. Because whole blood samples could be processed after cold storage, the method could be performed on samples obtained at remote locations such as clinical sites. To this end, we showed that the method could be used to measure signalling events in patients with polycystic ovary syndrome (PCOS), an endocrine disorder associated with platelet dysfunction. We envisage that the method will be useful for basic scientists, clinicians, and pharmacologists seeking novel therapies.

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Autret, Arnaud. "Modular neural networks for analysis of flow cytometry data". Thesis, University of South Wales, 2003. https://pure.southwales.ac.uk/en/studentthesis/modular-neural-networks-for-analysis-of-flow-cytometry-data(49f3349b-e86a-4bfb-a689-c853323b6f2d).html.

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In predicting environmental hazards or estimating the impact of human activities on the marine ecosystem, scientists have multiplied the need for sample analysis. The classical microscopic approach is time consuming and wastes the talent and intellectual abilities of trained specialists. Therefore, scientists developed an automated optical tool, called a Flow Cytometer (FC), to analyse samples quickly and in large quantities. The flow cytometer has successfully been applied to real phytoplankton studies. However, analysis of the data extracted from samples is still required. Artificial Neural Networks (ANNs) are one of the tools applied to FC data analysis. Despite several successful applications, ANNs have not been widely adopted by the marine biologist community, as they can not possible to change the number of species in the classification problem without retraining of the full system from scratch. Training is time consuming and requires expertise in ANNs. Moreover, most ANN paradigms cannot cope effectively with unknown data, such as data coming from new phytoplankton species or from species outside the scope of the studies. This project developed a new ANN technique based on a modular architecture that removes the need for retraining and allows unknowns to be detected and rejected. Furthermore, the Support Vector Machine architecture is applied in this domain for the first time and compared against another ANN paradigm called Radial Basis Function Networks. The results show that the modular architecture is able to effectively deal with new data which can be incorporated into the ANN architecture without fully retraining the system.

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BRANCHI, FEDERICA. "THE ROLE OF FLOW CYTOMETRY IN COMPLICATED CELIAC DISEASE". Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/553600.

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Background Refractory celiac disease (RCD) is defined as persistence or recurrence of clinical symptoms of malabsorption and histologic signs of villous atrophy despite at least 1 year of strict adherence to a gluten-free diet. Two different entities of RCD have been described: RCD I shows a polyclonal pattern of intraepithelial lymphocytes (IEL), while in RCD II a monoclonal transformation of IEL can be identified (usually detected by means of TCR clonality analysis). Patients with RCD II have a poorer prognosis and a high risk of development of enteropathy-associated T-cell lymphoma (EATL), so that it has recently been proposed to consider RCD II as a form of low-grade intraepithelial lymphoma (pre-EATL). Recently, flow cytometric analysis of isolated intestinal lymphocytes has been introduced as new diagnostic modality for the detection of aberrant intestinal lymphocytes (ILs) and as a stronger predictor of EATL development than gene clonality analysis. Aims The aims of this study were (i) to evaluate the presence of aberrant ILs in a cohort of non-celiac, celiac and RCD patients by means of flow cytometry, (ii) to verify whether there is an association between clinical characteristics of RCD patients and presence of aberrant ILs, (iii) to compare different ILs detection strategies with the purpose of validating a simpler strategy than the ones currently proposed and (iv) to evaluate whether flow cytometric analysis of aberrant ILs could accurately identify RCD II/pre-EATL patients in our cohort. Methods Flow cytometry analysis of intestinal lymphocytes isolated from duodenal biopsy specimens from RCD, uncomplicated CD patients and controls was performed. Several lymphocyte markers (CD3, CD4, CD8, CD7, CD103, TCRγδ) were applied in order to identify aberrant ILs, defined by means of several gating strategies including cytCD3+surfCD3-CD7+ and surfCD3-CD7+CD103+. Percentages of aberrant ILs as well as clinical characteristics in different patients groups were compared. Results A total of 130 flow cytometry assays were performed on 109 patients, including 42 controls, 21 active CD, 16 CD on GFD and 30 RCD. RCD patients were initially subgrouped according to the presence (TCRclon+, n=17) or absence (TCRclon-, n=13) of TCR clonality: the presence of elevated aberrant ILs was compared between the two subgroups, with elevated ILs detectable exclusively in the RCDclon+. Patients with elevated ILs also showed a significantly more severe malabsorption (assessed by a composite score). A cut-off of 11% aberrant ILs for the most reliable strategy allowed to identify 2 patients with EATL, as well as a small group of high-risk RCD (5/30) that were classified as RCDII/pre-EATL. According to low aberrant ILs levels, the other RCD patients (21/30) were classified as RCD I/low-risk RCD. However, this technique proved negative in 2 cases of overt EATL and was not able to correctly identify as “high-risk RCD” one patient with ulcerative jejunitis (who later developed a EATL), as well as one patient in whom a diagnosis of gamma-delta T-cell lymphoma was made. In the setting of RCD, aberrant ILs assessment by means of flow cytometry showed a Specificity of 100% but a Sensitivity of 67% for the detection of pre-EATL/EATL Alternative, simpler gating strategies for aberrant ILs showed similar accuracy to the principal strategy, however these results need further validation. Conclusion In routine clinical practice, flow cytometry for the assessment of aberrant ILs could prove a simple and accurate predictor for high-risk RCD. However, its use as a diagnostic strategy to classify patients into RCD I (low risk) and RCD II/pre-EATL could lead to missing cases of RCD patients with elevated risk. In order to prevent the consequences of false negative results, a multifaceted diagnostic approach taking TCR clonality and clinical manifestations (i.e. malabsorption) into account could maximize accuracy.

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Farahat, Nahla Mohamed Gamal. "Minimal residual disease in acute leukaemia by quantitative flow cytometry". Thesis, Institute of Cancer Research (University Of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244275.

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Jepras, Robert Ian. "Applications of photon correlation spectroscopy and flow cytometry to microbiology". Thesis, Open University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.290872.

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Want, Andrew James. "Physiological studies on bacterial fermentations using multi-parameter flow cytometry". Thesis, University of Birmingham, 2010. http://etheses.bham.ac.uk//id/eprint/1048/.

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Two staining protocols were formulated that enabled the detection of cellular stress at the single-cell level for Bacillus cereus. Both DiOC6(3) and RedoxSensor Green™ can be employed to detect perturbations in the energetic status of the cell at concentrations of 0.30 \mug.mL-1 and 3.0 \muM respectively. These methods can be employed for sensitive analysis of bacteria of both industrial and clinical interest. Flow cytometry was used throughout this work in order to assess the quality of recombinant Escherichia coli populations present within an agitated bioreactor. It was demonstrated in shake-flask culture that the cells could be grown to moderate cell densities (OD600nm 7 25) whilst producing measurable levels of antibody fragment. Despite being described in a patent which claims invention of a 100 % effective repression system (Hodgson et al., 2006), there was extensive evidence of promoter leakiness. Fab production was usually synonymous with cellular breakdown, however, a strategy based on simultaneous feeding and induction, before the exhaustion of the primary carbon source, yielded the highest concentration of Fab, 105 mg.L-1, with more than 50 mg.L-1 successfully targeted to the extracellular environment. Unlike all the previous cultures, this attainment also preceded the breakdown in the cellular structure.

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Veale, Margaret Fiona. "Activated immune cells : 1H-NMR spectroscopy and flow cytometry studies". Thesis, The University of Sydney, 1996. https://hdl.handle.net/2123/27547.

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Proton nuclear magnetic resonance (lH-NMR) spectroscopy has been used to study immune cell activation. One-dimensional and two—dimensional 1H-NMR spectra of activated immune cells are dominated by signals arising from elevated levels of isotropically tumbling (ie., NMR-visible) mobile lipid. An increase in the level of mobile lipid compared with that seen in resting cells has been observed in a variety of activated immune cells. The appearance of the mobile lipid is associated with activation and can be induced by a variety of stimuli. However, the origin and function of the neutral lipid resonances in 1H-NMR spectra of activated immune and other cell types has been unresolved. I propose that the mobile lipids arise from phosphatidylcholine (PC) cycling.

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Hedblom, Sofia. "Detection and quantification of fetal hemoglobin in blood using flow cytometry". Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-176636.

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Analytical methods used clinically in Sweden for detection and quantification of fetal hemoglobin (HbF) in maternal blood are either the microscopic method Kleinhauer Betkes test (KBT) or high performance liquid chromatography. A more modern alternative to detect and quantify HbF+ erythrocytes is flow cytometry. The aim of this project was therefore to evaluate the commercial kit "Fetal Cell Count kit" using flow cytometry. The kit used two antibodies; one directed against the specific γ-chain of HbF protein and the other directed against the intracellular enzyme carbanhydrase (CA), which is found in all erythrocytes in adults. The resulting data showed good precision, sensitivity and linearity. A reference interval based on male blood donors was determined to <0.1 % HbF+ erythrocytes and <1.3 %F-cells. The kit is well suited to detect and quantify F-cells. It could be used as a important tool to follow-up patients withβ-thalassemia and sickle cell anemia. However the kit was not as useful for detection and quantification of HbF+ erytrocytes in fetomaternal hemorrhage induced by Rhimmunization.

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Hall, Jerome Lynn. "Optical Properties of Marine Phytoplankton: A Study in Multiparameter Flow Cytometry". NSUWorks, 1989. http://nsuworks.nova.edu/occ_stuetd/364.

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Batch cultures of the cyanobacteria Synechococcus spp. (strains 48 Syn and 2346) and Porphyridium cruentum (Rhodophyta) were grown in nitrogen-limited (N:P=4:l) and phosphorus-limited (N:P=50:l) seawater media. Optical properties, including particle size (forward angle light scatter), particle granularity (right angle light scatter) and relative mean channel red (> 600 nm) and green (510-550 nm) fluorescence were measured for 10 days using a flow cytometer. Dissolved nitrogen (nitrate), phosphorus (phosphate), cell abundance and chlorophyll concentrations were also measured. Results indicated that phosphorus-limited cultures yield higher chlorophyll concentration, fluorescence and granularity (right angle scatter) values than did nitrogen-limited cultures. Comparison of these samples with a preliminary investigation shows nutrient-rich cultures (N:P=4:l, N:P=50:l) have larger particle size and higher fluorescence values than relatively nutrient-poor cells cultured in f/20 media. Secondary (R2) populations have been determined for all samples, either by light scatter or fluorescence anomalies. Particularly notable was Synechococcus 2346 (phosphorus-limited) which exhibited a secondary population characteristic for more than half of the experiment. Highly fluorescent particles are suggested as either formative daughter cells, cellular “clumping" or a cellular optical response to batch culture turbidity; these particles have a profound influence on the relative refractive index of the culture with time. Flow cytometric analysis can be an effective tool in the determination of not only differences in the optical properties and fluorescent signatures of various cyanobacterial strains, but also of population variation within a single strain.

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Lam, Yung-chun Nelson, i 林勇進. "Annual distribution of phytoplankton in Tolo Harbour: a flow cytometry approach". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B3124192X.

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Deans, Gordon Taylor. "The role of flow cytometry and image analysis in colorectal carcinoma". Thesis, Queen's University Belfast, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334468.

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Anvarian, Amir Hossein Pour-Taghi. "Physiology of Escherichia coli in orange juice : applications of flow cytometry". Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/5653/.

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Flow cytometry (FCM) was utilized for monitoring the physiology of \(E.\) \(coli\) cells in orange juice (OJ) as well as a model orange juice (MOJ). Compared to FCM, plate counts highly underestimated the true number of viable cells in OJ. As a part of this study, the effects of the change in major components of OJ on viability of the cells in OJ and MOJ was investigated using FCM. Increase in ascorbic acid and amino acid concentrations of MOJ improved both the culturability and FCM viability of the cells. FCM was also employed for studying the effects of OJ clarification on viability of \(E.\) \(coli\) in OJ. Although, reduction in cloud content of OJ increased the number of healthy cells, however, the removal of cloud particles of larger than 0.7 μm appeared to increase the antimicrobial efficacy of particles of smaller than 0.7 μm. The effects of washing E. coli cells with available chlorine, H\(_2\)O\(_2\) and organic acids on their subsequent viability in OJ was also investigated. While increase in concentration of sanitizers resulted in a significant reduction in healthy populations, the total number of viable cells either remained constant or increased particularly in case of H\(_2\)O\(_2\)-washed cells.

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34

Bliss, Jonathan G. "Rapid detemination of antimicrobial susceptibility using gel microdroplets and flow cytometry". Thesis, Massachusetts Institute of Technology, 1990. http://hdl.handle.net/1721.1/12797.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Whitaker College of Health Sciences and Technology, 1991.
Includes bibliographical references (leaves 226-233).
by Jonathan G. Bliss.
Ph.D.

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35

Shimada, Toshihide. "Detection of IgA binding site by flow cytometry with fluorescent microspheres". Kyoto University, 1992. http://hdl.handle.net/2433/168720.

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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第5141号
医博第1403号
新制||医||540(附属図書館)
UT51-92-K331
京都大学大学院医学研究科病理系専攻
(主査)教授鈴木康弘, 教授内田温士, 教授杉山武敏
学位規則第4条第1項該当

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36

Lam, Yung-chun Nelson. "Annual distribution of phytoplankton in Tolo Harbour a flow cytometry approach /". Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B22718874.

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Simone, Lucchesi. "Computational flow cytometry for characterizing the immune response in vaccine studies". Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1095903.

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Flow cytometry is a powerful technique used to quantify the expression of multiple extracellular and intracellular molecules on single cells, allowing the phenotypic and functional characterization of cell populations. When many parameters are investigated simultaneously, it is not feasible to analyze all the possible bi-dimensional combinations of marker expression with classical manual analysis and the use of advanced automated tools to process and analyze high-dimensional data sets becomes necessary. To overcome this problem, novel computational techniques have been developed in the recent years, and computational flow cytometry has become a novel discipline useful for providing a set of tools to analyze, visualize and interpret large amounts of cell data in a more automated and unbiased way. The present thesis is focused on the automated analysis of high-dimensional flow cytometric data to characterize, in an unbiased and data-driven way, the B and T cell immune responses elicited in the mouse model by different vaccine formulations. An in depth investigation of the automated tools currently available for the analysis of multiparametric flow cytometric data, discussing the advantages and the limits of the most commonly used algorithms was also conducted (Chapter 3). The different B cell subsets elicited by immunization with or without the vaccine adjuvant CAF01 were characterized by automated analysis employing the FlowSOM clustering approach (Chapter 4). The computational analysis allowed to identify different B cell populations, including plasmablasts, plasma cells, germinal center B cells and their intermediate subsets. Among these reactivated cells, the frequency of plasma cells was significantly higher in lymph nodes of mice immunized with the adjuvanted formulation compared to antigen alone. Integration of clustering and dimensionality reduction approaches allowed also the identification of a sub-population of germinal center memory B cells, that could not be identified with a single automated tool (Chapter 5). The polyfunctional activity of antigen-specific CD4+ T cells was analyzed in mice immunized with heterologous prime-boost vaccine formulations. The automated analysis allowed to visualize clusters of cells producing different patterns of cytokines, according to the different adjuvants used for priming and boosting, expanding the knowledge on the adjuvant role in modulating the T helper effector function (Chapter 6). In conclusion, the computational approach has allowed to characterize in an unbiased way the B and T cell responses following immunization with different vaccine strategies, detecting clusters of cells that would be hardly identified with traditional analysis. Automated tools address many needs associated with high-dimensional datasets analysis and these results strengthen their use in vaccination studies.

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38

Martini, V. "CLINICAL PATHOLOGICAL FEATURES OF CANINE HEMATOPOIETIC NEOPLASMS ASSESSED VIA FLOW CYTOMETRY". Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/232394.

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Flow cytometry (FC) is an advanced diagnostic technique widely used in human medicine to confirm, classify and stage hematopoietic neoplasms (HNs): it supplies an objective evaluation of the size and internal complexity of the cells and identifies the antigenic pattern expressed by each cell, which is specific for cellular lineage and maturative stage. FC is presently spreading also in veterinary medicine and is mostly used to confirm the diagnosis and determine the immunophenotype of canine HNs. Aim of this study was to apply FC to the study of specific clinical pathological features of canine HNs. In particular, we focused on three points: 1) staging of diffuse large B-cell lymphoma. We validated a FC technique to assess peripheral blood and bone marrow infiltration by neoplastic cells and proved its prognostic value. 2) antigen aberrancies. We investigated the prognostic role of specific antigen aberrancies in canine lymphomas and found the dogs with CD4+CD8+ T-cell lymphomas had a poorer prognosis compared to other T-cell aberrant lymphomas. In addition, we highlighted an important diagnostic role for antigen aberrancies in canine small clear cell lymphoma. 3) CD44 expression. We proved that CD44, which has a role in the pathogenesis and dissemination of many human and canine neoplasms, is expressed at different degrees on neoplastic cells from different canine HNs, with acute leukemias showing the highest degree of expression. Overall, our results suggest that FC can be a useful tool not only to confirm the diagnosis and assess the immunophenotype of canine HNs, but also to assess clinical pathological aspects and additional prognostic parameters specific for the neoplastic subtype identified.

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39

Pitzler, Christian Verfasser], Ulrich [Akademischer Betreuer] [Schwaneberg i Andrij Z. [Akademischer Betreuer] Pich. "Novel flow cytometer-based platforms for directed evolution / Christian Pitzler ; Ulrich Schwaneberg, Andrij Pich". Aachen : Universitätsbibliothek der RWTH Aachen, 2015. http://d-nb.info/1130590496/34.

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Welsh, Joshua. "Flow cytometer optimisation and standardisation for the study of extracellular vesicles as translational biomarkers". Thesis, University of Southampton, 2016. https://eprints.soton.ac.uk/410614/.

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Background: The term ‘extracellular vesicles’ (EVs) encompasses a range of vesicles. These include apoptotic vesicles (1000-300nm), microvesicles (30-1000nm), exosomes (~30-120nm) and retrovirus like vesicles (90-100nm). EVs have been linked to promising diagnostic, and therapeutic potentials. Their characterisation is poorly understood due to the lack of resolution and standardisation in detection equipment currently used. Aims & Methods: In this thesis, I have developed methods for flow cytometer (FCM) resolution quantification, improvement, and standardisation. This involved building, testing and validating FCM optical models for EV analysis standardisation, and optimising FCM settings and protocols to increase resolution and decreasing variation in results. I then tested the benefits of these optimisations on EV analysis, which involved comparing optimised to non-optimised EV analysis protocols utilising clinical samples. Finally, EVs potential as translational biomarkers in non-alcoholic fatty liver disease (NAFLD) was investigated, employing the previously developed protocols in this thesis. Results: FCM optimisations combined with a novel fluorescent assay resulted in a validated modelling technique, that allows diameter of EVs in plasma samples to be approximated using their scatter power, and separation of microvesicles, apoptotic vesicles, and residual platelets. Comparison of EV optimised to non-optimised protocols showed the FCM optimisation protocol to have increased EV absolute count reliability, and lower variation between results, when compared to a non-optimised FCM analysis protocol. Upon applying these methods to a biobank of clinical samples from individuals with NAFLD, novel insights were gained between the association of platelet-, endothelial-, and leukocyte-derived EVs in the progression of the disease. A clinically relevant finding being leukocyte EVs showing potential as a diagnostic marker of liver fibrosis severity.

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41

Pitzler, Christian [Verfasser], Ulrich [Akademischer Betreuer] Schwaneberg i Andrij Z. [Akademischer Betreuer] Pich. "Novel flow cytometer-based platforms for directed evolution / Christian Pitzler ; Ulrich Schwaneberg, Andrij Pich". Aachen : Universitätsbibliothek der RWTH Aachen, 2015. http://d-nb.info/1130590496/34.

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42

Owen, Henry R. "Use of monoploid solanum phureja in cell and tissue culture techniques for potato improvement". Diss., This resource online, 1987. http://scholar.lib.vt.edu/theses/available/etd-07282008-135528/.

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Mulhearn, Ben. "Identifying immune biomarkers to predict treatment response to biologic drugs in rheumatoid arthritis". Thesis, University of Manchester, 2018. https://www.research.manchester.ac.uk/portal/en/theses/identifying-immune-biomarkers-to-predict-treatment-response-to-biologic-drugs-in-rheumatoid-arthritis(c311fc8c-4239-444a-9912-ddd4fde5f7fa).html.

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Rheumatoid arthritis (RA) is a chronic, heterogeneous, autoimmune disease that causes inflammation of synovial joints leading to pain, stiffness and swelling. If left untreated, RA results in irreversible joint destruction and long term disability. Initial treatment with glucocorticoids and other immunosuppressive agents suppresses inflammation. However, many of these drugs are not well-tolerated due to extensive side effects or are simply ineffective. The discovery of tumour necrosis factor-α (TNF) as a key mediator of inflammation in RA led to the development of monoclonal anti-TNF antibody therapy. Since then, other biologic drugs targeting immune pathways have been developed for RA, including interleukin-6 (IL-6) blockade, B cell depletion, and T cell co-stimulation blockade. Not all patients will respond to their first biologic drug and currently there is no way to predict which patient will respond to each different class of drug. Generally, 3 – 6 months are required to determine clinical efficacy, during which time joint inflammation proceeds. Therefore, discovering biomarkers to predict treatment response is a research priority. Biologic drugs target immune pathways. As single cell technology advances and has increasing capacity to identify subtle changes in many cell subsets, I hypothesise that studying the blood immune cell landscape will define cellular biomarker profiles relevant to each individual patient’s disease.

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44

Cullen, Matthew John. "The use of flow cytometry in the diagnosis of the Myelodysplastic Syndromes". Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/13713/.

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The Myelodysplastic Syndromes (MDS) are a biologically and clinically heterogeneous group of bone marrow haematopoietic cell disorders that result in ineffective haematopoiesis. Unlike most forms of haematological malignancy, the diagnosis of MDS remains heavily reliant on subjective morphological interpretation which can result in inaccurate and missed diagnoses. The use of flow cytometric immunophenotyping offers a potential solution to aid in the diagnosis of MDS, and numerous flow cytometric scoring schemes have been already been proposed and tested. However, most flow cytometric scoring schemes are user-defined, with simple schemes lacking diagnostic sensitivity, whilst the more comprehensive schemes may be unfeasible to implement in a large-scale diagnostic setting. The use of machine learning classifiers offered a more subjective approach to the use of flow cytometric data. Therefore, we have tested a series of classifiers both by combining simple immunophenotypic and demographic features, and by utilising a 2 tube-immunophenotyping panel which contained a large array of numerical and immunophenotypic attributes which had been identified as being abnormal in MDS patients. We have shown that machine learning classifier-based approaches could reproducibly identify patients with definite abnormalities in MDS, and those with normal haematopoietic populations in non-diagnostic, reactive conditions. The classifiers further offered the ability to aid in the triage of patients unlikely to be MDS by providing the basis to a diagnostic confidence score. The application of multiple classifiers also identified a grey-area of MDS patients who were consistently misclassified and who may prove to be challenging to diagnose by flow cytometry, due to an absence of aberrant immunophenotypic features. Finally, we have also shown that a combination of immunophenotyping and targeted gene mutation analysis provides the potential to identify non-diagnostic cases which may progress to MDS. It is in a combination of these two techniques where the future of MDS diagnosis may lie.

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Björklund, Elisabet. "Multiparameter flow cytometry and minimal residual disease in patients with acute leukemia /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-624-3.

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Levering, Wilfridus Henricus Bernardus Maria. "External quality assessment in flow cytometry educational aspects and trends toward improvement /". [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2007. http://hdl.handle.net/1765/10602.

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Zare, Habil. "Automatic analysis of flow cytometry data and its application to lymphoma diagnosis". Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/39660.

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Flow cytometry has many applications in clinical medicine and biological research. For many modern applications, traditional methods of manual data interpretation are not efficient due to the large amount of complex, high dimensional data. In this thesis, I discuss some of the important challenges towards automatic analysis of flow cytometry data and propose my solutions. To validate my approach on addressing real life problems, I developed an automatic pipeline for analyzing flow cytometry data and applied it to clinical data. My pipeline can potentially be useful for improving quality check on diagnosis, assisting discovery of novel phenotypes, and making clinical recommendations. Furthermore, some of the challenges that I studied are rooted in more general areas of computer science, and therefore, the tools and techniques that I developed can be applied to a wider range of problems in data mining and machine learning. Enhancement to spectral clustering algorithm and proposing a novel scheme for scoring features are two examples of my contributions to computer science that were developed as part of this thesis.

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48

Tu, Ran [Verfasser]. "Flow cytometry based screening systems for directed evolution of proteases / Ran Tu". Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2010. http://d-nb.info/1035032872/34.

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Karlsson, Mathilda. "Can the proliferative ability of chicken cardiomyocytes be assessed using flow cytometry?" Thesis, Linköpings universitet, Biologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-128890.

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The study of the formation of new cardiac muscle cells during postnatal development is a relatively new field. During fetal development, new cells are formed as the heart grows. However, the proliferative ability of postnatal cardiomyocytes is still debated. While several studies have been made on mammals, less is known about the chicken cardiac cells and their postnatal proliferation. As almost all previous studies have used microscopy-based cell counting methods, there has been some limitations on accuracy and amounts of cells that could be counted. The aim of this study is to develop a method for using flow cytometry to analyze proliferative ability of chicken cardiomyocytes and to investigate if any postnatal proliferation exists. For this study, 4 weeks old Red Junglefowl (Gallus gallus) chickens were used for isolating cardiomyocytes. In addition, 19 days old Red Junglefowl embryos were used to asses if a longer incubation time would yield a higher number of proliferative cells. Cells were stained using a commercial EdU imaging kit and analyzed using flow cytometry and imaging flow cytometry. The produced results could not be used for determining the proliferative ability of the cardiomyocytes, but provides crucial information for possible method improvements. In conclusion, this study has laid important groundwork for future studies on the proliferative ability of chicken cardiomyocytes.

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COZZI, MARZIA. "FLOW CYTOMETRY FOR THE DIAGNOSIS AND THE CHARACTERIZATION OF CANINE LYMPHOPROLIPHERATIVE TUMORS". Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/580991.

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ABSTRACT Flow cytometry (FC) is a diagnostic technique in continuous development and, to date, it plays a fundamental role in human medicine for the diagnosis and the classification of hematopoietic tumors. Several studies state that the information obtained from the FC analysis in addition to defining the immunophenotype of the tumor cells, hence the origin of the neoplasia, can help to predict its biological behavior (Swerdlow et al., 2016). FC has thus become a simple and objective method to characterize hematopoietic tumors in order to obtain also valuable prognostic information. In veterinary medicine, FC is increasingly adopted for the diagnosis, staging, and monitoring of hematopoietic tumors, but despite advances in the generation and validation of antibodies for the use in dogs, the characterization of such neoplasms remains challenging (Wilkerson et al., 2005; Comazzi and Gelain, 2011). The aim of this doctoral project is to describe some less frequent subtypes of lymphomas and leukemia of the dog via FC, in order to define its biological behavior and to investigate whether there is any variable of prognostic value among all the factors analyzed. For this purpose, four studies will be illustrated; the first is a retrospective work, aiming to evaluate pre-analytical factors that may affect the diagnostic utility of FC in samples of lymph node aspirates. The work included 987 cases selected in the period 2009-2015, in which a lymph node aspirate was sent to our laboratory with suspect of lymphoproliferative disease. In order to define any possible bias affecting the outcome of the FC diagnosis, the variables analyzed were related to the animal (breed, sex, age), related to the operator (year, season, method of delivery to the laboratory, referring veterinarian) and related to the sample (type of material, cell concentration, presence of cytological slides, presence of artifacts). Of the factors considered, the sample cellularity and the presence of dead cells were the ones that most influenced the possibility of obtaining an adequate diagnosis. FC was, however, conclusive in almost all the samples, that were characterized by good quality and adequate sampling conditions. The study focused on TZL, a peculiar canine lymphoma with an indolent behavior, aimed to characterize this entity from a clinical and pathological point of view. The first phase of the work was retrospective, with the aim of describing clinical presentation and outcome of 51 cases selected between 2009 and 2014. The second phase of the study was aimed at clarifying the origin of the peculiar CD45 negative T-immunophenotype (Martini et al., 2013; Seelig et al., 2014); specifically, we confirmed the absence of the surface protein by means of two different techniques (flow cytometry and immunohistochemistry) and verified whether the transcript and the gene encoding the protein were present. The results confirmed that this type of lymphoma has indolent behavior with long survival times, despite being often diagnosed at the V stage of the disease. Furthermore, we can note that the origin of the phenotypic aberration is probably attributable to transcription factors, given the absence of transcription associated with the presence of the corresponding genomic tract. The objective of the third study was focused on the description of the biological behavior of nodal-type marginal lymphoma (nMZL). Although in literature it is classified as indolent lymphoma, some publications reported cases with a rather aggressive behavior (Flood-Knapik et al., 2012; Valli et al., 2013; Aresu et al., 2015; Marconato et al., 2015). Clinical information was collected from 35 retrospectively selected nMZL cases, with complete staging and standardized therapies. In our cohort, this lymphoma did not show and indolent behavior, with a generalized involvement and with short survival times, almost overlapping with the high-grade diffuse B-cell lymphoma (DLBCL). Thanks to the results of this work, discussions could be opened on the correct therapeutic approach. The latest study aimed to evaluate new antigens for the diagnosis and stratification of patients with CLL-B. The expression of the ZAP70 and CD38 markers in human medicine is closely related to the progression of the disease, with much lower survivals in patients with expression over specific thresholds (Rossi et al., 2010; Sulda et al., 2012). In the present project, these markers were evaluated for the first time in 37 blood samples of dogs with chronic B-cell leukemia, together with CD25 and ki67. Clinical data of the cases were obtained and survival analysis finally revealed that ZAP70 is a potential prognostic marker, providing bases for further studies with larger case studies and standardized therapy. The results of my doctoral project confirm that FC is a good technique for the study of the clinical-pathological aspects of lymphoproliferative tumors of dogs, and provides useful information to complete the biological profile of these tumors, also laying the foundations for future investigations on the usefulness of the proposed new markers.

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