Mezinárodní konference Analytical Cytometry V


Vyšlo v časopise: Čas. Lék. čes. 2009; 148: 604-609
Kategorie: Abstrakta

Olomouc, 5.–8. září 2009

Ve dnech 5. až 8. září 2009 se v Olomouci konala mezinárodní konference Analytical Cytometry V. Konferenci organizovala Česká společnost pro analytickou cytometrii ve spolupráci s Ústavem experimentální botaniky AV ČR, v.v.i., Olomouc, Biofyzikálním ústavem AV ČR, v.v.i., Brno, Univerzitou Palackého v Olomouci, Českou imunologickou společností, Českou hematologickou společností ČLS J. E. Purkyně a Českou společností alergologie a klinické imunologie.

Česká společnost pro analytickou cytometrii byla založena v roce 2001 a sdružuje odborné pracovníky zabývající se aplikací průtokové cytometrie v základním teoretickém a aplikovaném klinickém výzkumu (www.csac.cz).

Již v pořadí V. konference byla výjimečná v tom, že se poprvé podařilo sloučit část základního výzkumu a část klinickou, která dosud probíhala samostatně v rámci Dnů průtokové cytometrie v Nemocnici Na Homolce. Konference se uskutečnila v Olomouci, v nádherných prostorách Uměleckého centra Univerzity Palackého – Konviktu pod záštitou rektora Univerzity Palackého prof. RNDr. L. Dvořáka, CSc.

Odborné přednášky byly rozděleny do dvou částí. První den byl věnován plenárním přednáškám, které byly prezentovány zahraničními přednášejícími. Témata byla různorodá pokrývající širokou škálu biologického a rostlinného výzkumu stejně jako klinických aplikací. Přednášející představují absolutní špičku ve svém oboru a tomu odpovídala i odborná úroveň jejich sdělení. V. Ouředník konferenci zahájil plenární přednáškou Per aspera ex astra – the cell from a cosmic perspective“, ve které nesmírně poutavým způsobem prezentoval nejmodernější poznatky historie vzniku kosmu a života na Zemi. Následovaly přednášky zahraničních přednášejících: D. Basiji: Emerging Clinical Applications of Imaging Flow Cytometry, S. Boettchera: Multicolor flow cytometry for classification and minimal residual disease follow-up in B cell malignancies, DH. Busche: Stem-cell like plasticity of distinct subsets of antigen-specific CD8+ T cells, Ger van den Engheho:Application of flow cytometry and cell sorting in marine microbiology, T Horna:Development of cellular assays for automated microscopy to measure multiple parameters by single cell high content analysisL. Rauové: Mechanisms underlying thrombosis in heparin induced thrombocytopenia, J. Ouřednikové:Since when was genius found respectable or honesty in science, A. Tomera: Flow cytometric analysis of platelet activation process – in vitro and ex vivo a T. Horna: Development of cellular assays for automated microscopy to measure multiple parameters by single cell High Content Analysis.

V dalších dvou dnech pak proběhly prezentace 47 odborných sdělení, jejichž autoři byli vyzváni organizátory k prezentaci. Paralelně probíhaly sekce s tématy pokrývající metodologickou oblast průtokové cytometrie, aplikaci do rostlinného nebo experimentálního klinického výzkumu. Byly prezentovány práce zahrnující metodologie třídění buněk, digitální mikroskopie a příbuzných nejmodernějších optických metod včetně obrazové analýzy a využití DNA čipů. Klinická část probíhala ve dvou sekcích. V první byly prezentovány práce týkající se postavení T regulačních lymfocytů a jejich uplatnění v současné diagnostice. Přednášky S. Blažíčkové, T. Eckschlagera, V. Krále se věnovaly úloze T regulačních lymfocytů u autoimunitních a nádorových onemocnění, L. Sedláčková prezentovala možnosti průtokové cytometrie v diagnostice potravinových alergií. Druhá část byla věnována zejména oblasti hematoonkologie, kde pracoviště ve FN Motol s představiteli O. Hrušákem, E. Mejstříkovou a T. Kalinou představuje mezinárodní špičku v této oblasti.

Na závěr konference byly vybrány nejlepší prezentace formou přednášky a formou posterů, jejichž autoři obdrželi finanční odměnu.

Úroveň konference byla na velice vysoké odborné úrovni a organizátory potěšil pozitivní ohlas z úst zahraničních i domácích účastníků. Ti ocenili prvotřídní organizátorské schopnosti týmu doc. J. Doležela, nádherné prostředí konání konference – Uměleckého centra Palackého Univerzity – Konviktu a především velice vysokou odbornou úroveň prezentovaných přednášek a prací. Příští konference se bude konat za 2 roky v Praze.

MUDr. Miroslav Průcha, Ph.D.

Oddělení klinické biochemie, hematologie a imunologie Nemocnice Na Homolce

Roentgenova 2, 150 30 Praha 5

e-mail: miroslav.prucha@homolka.cz

Per aspera ex astra - the cell from a cosmic perspective

Ouředník V.

Department of Biomedical Sciences, Iowa State University, Ames, Iowa, USA; e-mail: voured@iastate.edu and Center for Astrophotography, Alpine Astrovillage Lue-Stailas, Val Muestair, Switzerland; e-mail: aav@alpineastrovillage.com

A living cell is more complex than any inanimate matter created in the Universe. This presentation summarizes, in general terms, our present knowledge of the major events that led to the emergence of life during cosmic evolution. We will see how the initial abundance of hydrogen and helium in space became progressively “contaminated” with traces of heavy elements, the prerequisites for future planet formation and appearance of organic molecules.

Already towards the end of the first billion years of Earth’s existence, these molecules were used to produce the most essential building blocks of life: amino acids and nucleic acids. Leading from the simplicity of an early, radiation-dominated cosmos to the complexity (and increasing entropy) of inanimate matter and the appearance of life, Nature’s path appears to logically connect the latter to the rest of the expanding material Universe.

Fig. 1. Massive stars (10–30 solar masses) have been essential for the appearance of heavy elements beyond iron in the Universe. Their large stellar winds and the ending of their life in form of supernovae have seeded the interstellar space with all the elements in the Periodic Table for billions of years. The famous Crab Nebula, imaged here by the Hubble Space Telescope, is the remnant of a supernova explosion observed in the constellation of Taurus by Chinese astronomers in the year 1054 AD. The nebula is about 6500 light years away and spans about 10 light years. Its filaments are still expanding at a speed of at least 1000 km/sec and the central neutron star (3 × 10<sup>9</sup> tons/cm<sup>3</sup>!) is a pulsar rotating about 30 times each second. Image credit: NASA/ESA
Fig. 1. Massive stars (10–30 solar masses) have been essential for the appearance of heavy elements beyond iron in the Universe. Their large stellar winds and the ending of their life in form of supernovae have seeded the interstellar space with all the elements in the Periodic Table for billions of years. The famous Crab Nebula, imaged here by the Hubble Space Telescope, is the remnant of a supernova explosion observed in the constellation of Taurus by Chinese astronomers in the year 1054 AD. The nebula is about 6500 light years away and spans about 10 light years. Its filaments are still expanding at a speed of at least 1000 km/sec and the central neutron star (3 × 10<sup>9</sup> tons/cm<sup>3</sup>!) is a pulsar rotating about 30 times each second. Image credit: NASA/ESA

Since when was genius found respectable or honesty in science

Ouředník J.

Department of Biomedical Sciences, Iowa State University, Ames, USA; e-mail: joured@iastate.edu and Center for Astrophotography, Alpine Astrovillage Lue-Stailas, Val Muestair, Switzerland;e-mail: aav@alpineastrovillage.com

Ethical concepts and principles, such as honesty, integrity, trust, accountability, respect, confidentiality, and fairness, play a key role in shaping research conduct within science. Ethical conduct is important to foster collaboration, cooperation, and trust among scientists, to advance the goals of research, to fulfill scientists’ social responsibilities, and to avoid or minimize damaging scandals resulting from unethical or illegal behavior.

Research always takes place within a social context. Economical and political interests as well as social, cultural, and religious values influence scientific goals, resources, and practices. Money plays a crucial role in deciding whether a particular problem will be studied, who will study it, how it will be studied, and even whether the results will be published.

The talk discusses motivations for becoming a scientist and possible temptations that can lead to scientific dishonesty. Presented will be also some particular cases of irresponsible behavior in science and how they negatively influenced the integrity of the investigations in specific scientific areas, including biomedical and stem cell research.

Emerging clinical applications of imaging flow cytometry

Basiji D.

Amnis Corporation, 2505 Third Ave., Suite 210, Seattle, WA 98121, USA; e-mail: basiji@amnis.com

Imaging flow cytometry is now in use at over 50 basic and clinical research institutions and has been employed in over 70 scientific publications. The technology images cells directly in flow, combining the information content of microscopy (12 images per cell) with the speed (1000 cells per second) and population statistics of flow cytometry. The technology is being used in multiple clinical areas, including the detection of minimal residual disease via high throughput FISH, immune monitoring pre- and post-transplant for the informed modulation of immunosuppressive therapy, and the detection, enumeration, and functional analysis of exceedingly rare circulating endothelial cells. This presentation will include extensive data from these studies and the novel clinical applications that are emerging.

Application of flow cytometry and cell sorting in marine microbiology

van den Engh G.

VP Advanced Cytometry, BD Cell Analysis, 12730 28th Ave NE, Seattle, WA 98125, USA; e-mail: engh@cytopeia.com

In recent years, flow cytometry has been proven to be a useful tool in oceanography for measuring pico-plankton. Use of flow cytmetry has led to significant discoveries. Carbon fixation by cyanobacteria in the tropical oceans is much more important than previously thought. It has been discovered that cyanobacteria also play a role in nitrogen fixation. These findings force us to reevaluate the importance of marine microorganisms in the global carbon cycle.

The light scattering and fluorescent properties of Pico-plankton are very different form the mammalian cells that are usually analyzed in flow cytometry. Much can be done to improve the instruments for oceanographers. Improvements in both instrument design and the manner in which phytoplankton is measured will be described. Results obtained on a recent research cruise to the arctic will be presented. It is expected technological improvements that are inspired by oceanography will also lead to improvements in biomedical instrumentation.

Flow cytometry as a proteomics tool

Lund-Johansen F.

Dept. of Imunology, Rikshospitalet-Radiumhospitalet, N-0027 Oslo, Norway; e-mail: fridtjof.lund-johansen@medisin.uio.no

Elements from DNA microarray analysis such as sample labeling and microspotting of capture reagents have been successfully adapted to multiplex measurement of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from western blotting and resolved protein size as a second parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble western blots with bands of antibody reactivity across the size separation range (670–10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label was measured by high speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal reference of specificity.

Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins and cdk-inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes.

Flow cytometric analysis of platelet activation: basic and clinical application

Tomer A.

Ben-Gurion University of the Negev, Faculty of Health Sciences; Blood Bank and Transfusion Medicine, Soroka Medical Center, Beer-Sheva, Israel; e-mail: atomer@bgu.ac.il

Flow cytometry is a useful technique for the study of platelet activation process in vitro and ex vivo. Using activation specific markers, flow cytometric analysis can demonstrate the a) activation of the major membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor (integrin αIIbß3); b) release reaction, by the expression of P-selectin (CD62p), the α-granule GP translocated to the platelet surface membrane following activation, and c) platelet procoagulant activity, as demonstrated by the binding of annexin A5 protein to the membrane anionic-phospholipids – the prothrombinase-complex (prothrombin, factors V and X) binding sites – exposed on the surface of activated platelets.

During coronary angioplasty (PTCA), blood samples obtained from five patients demonstrated the activation of platelet GP IIb/IIIa and the expression of P-selectin. Later studies showed the degree of activation to be correlated with the rate of vascular re-occlusion.

Thirty-three sickle cell disease patients, eleven during pain episodes and twenty-two during periods without pain, showed increased platelet activation that was markedly increased during pain episodes. Moreover, patients with pain episodes showed a highly activated platelet microparticles (3–30%) with markedly increased binding of annexin A5. The platelet procoagulant activity was associated with increased plasma markers of thrombin generation and fibrinolytic activity.

Sixty-eight women with pregnancy-related hypertension (PRH) demonstrated significantly increased expression of p-selectin compared to normal pregnant controls that was further enhanced during preeclampsia. Platelet procoagulant activity was significantly increased in women with PRH compared to normals, followed by marked augmentation during preeclampsia. The platelet procoagulant activity was associated by the appearance of platelet microparticles (5–30%) expressing high binding (100-fold) of annexin A5. The flow cytometric findings were corroborated by elevated plasma markers of thrombin generation, thrombin-anti-thrombin complex and prothrombin fragment F1.2., and with the pregnancy outcome.

Using flow cytometric techniques, a rapid, sensitive and specific functional method was developed for the diagnosis of heparin-induced thrombocytopenia, by demonstrating in vitro activation of normal platelets by patients’ sera in the presence of pharmacological concentrations of heparin.

We conclude that flow cytometry may be a useful tool for the studies of mechanisms inducing in vivo and in vitro platelet activation and for the detection of ongoing in vivo prothrombotic activity.

Mechanisms underlying thrombosis in heparin induced thrombocytopenia

Rauova L.

Department of Pediatrics, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; e-mail: lubica@email.chop.edu

Unfractionated heparin remains the drug of choice for a significant number of medical indications where rapid and reversible anticoagulation is necessary or desired. A serious complication of treatment with heparin is heparin induced thrombocytopenia (HIT), an autoimmune disorder distinguished by low platelet counts, paradoxically associated with a high risk of limb- and life-threatening thrombosis. The reason for development of thrombosis has not been well defined and consequently the effectiveness of therapy is incomplete and may be complicated by bleeding. An array of approaches, including a key role for flow cytometry, has been employed to elucidate the molecular mechanisms of HIT. HIT is caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAG). The pathogenically important PF4/GAG complexes are of very high molecular weight and form over a remarkably narrow molar range of reactants. The unique feature of HIT antibodies, in contrast to other autoimmune thrombocytopenic antibodies, is their ability to activate platelets and other vascular cells. Following activation, platelets release large amounts of chemokines, including PF4 and RANTES. These two chemokines have been shown to cooperate to enhance the arrest of monocytes on (activated) endothelium and to lead to more surface binding of PF4. Subsequent interaction of HIT antibodies with PF4 bound to the monocyte surface leads to expression of tissue factor in monocytes that causes accelerated membrane-dependent coagulation. An explosive feed forward, prothrombotic loop is further enhanced by tissue factor-bearing microparticles formed as a consequence of cellular activation. Cessation of heparin is necessary but not sufficient therapeutic intervention and HIT can also occur in patients days after withdrawing heparin. At this juncture, not only is there no heparin remaining, but also the targeted platelets are likely to have been cleared. At this point, the procoagulant state is most likely driven by activated monocytes, which according to our preliminary studies have persistent surface HIT antigen at low PF4 levels. Our model of the role of platelet and monocyte surface PF4/GAG complexes in the pathogenesis of HIT, depicted in Figure 1, accounts for the seemingly paradoxical occurrence of thrombocytopenia and thrombosis, and why the procoagulant state persists after heparin has been discontinued and thrombocytopenia has resolved. It also suggests that steps proximal to the generation of thrombin, including antigen formation on cell surfaces, and Fc receptor-mediated platelet and monocyte activation, upstream of thrombin formation, may be more rational targets for intervention than administration of thrombin inhibitors.

Fig. 1. Model of the role of platelet and monocyte surface PF4/GAG complexes in the pathogenesis of HIT
Fig. 1. Model of the role of platelet and monocyte surface PF4/GAG complexes in the pathogenesis of HIT

Stem-cell like plasticity of distinct subsets of antigen-specific CD8+ T cells

Busch DH.

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstr. 30, 81675 Munich, Germany; e-mail: dirk.busch@lrz.tum.de

It is well accepted that CD8+ T cells play a pivotal role in providing protection against infection with intracellular pathogens and some tumors. In many cases protective immunity is maintained for long periods of time (immunological memory). Over the past years, it has become evident that in order to fulfill these multiple tasks, distinct subsets of effector and memory T cells have to be generated. Until today, however, only little is known about the underlying mechanisms of subset differentiation and the timing of lineage fate decisions. In this context, it is of special importance to determine at which level of clonal expansion functional and phenotypical heterogeneity is achieved. Different models for T cell subset diversification have been proposed; these differ mainly in the time point during priming and clonal expansion (prior, during, or beyond the first cell division) when differentiation programs are induced. Recently developed single-cell adoptive transfer technology has allowed us to demonstrate that individual precursor cells still bear the full plasticity to develop into a plethora different T cell subsets. This observation targets the shaping of T cell subset differentiation towards factors that are still operative beyond the first cell division. These findings have important implications for vaccine development, as the modulation of differentiation patterns towards distinct subsets could become a powerful strategy to enhance the efficacy and quality of vaccines. Furthermore, our findings indicate that adoptive T cell immunotherapies might be possible to perform with very low cell numbers, which could facilitate the transfer of this promising approach to a variety of different clinical applications.

References

1. Stemberger C, Huster H, Koffler M, Anderl F, Schiemann M, Wagner H, Busch DH. A single naive CD8+ T cell precursor can develop into diverse effector and memory subsets. Immunity 2007; 27: 985–997.

2. Stemberger C, Neuenhahn M, Gebhardt FE, Schiemann M, Buchholz VR, Busch DH. Stem cell-like plasticity of naēve and distinct memory CD8(+) T cell subsets. Semin Immunol 2009; 21: 62–68.

Multicolor flow cytometry for classification and minimal residual disease follow-up in B cell malignancies

Böttcher S.

Second Department of Medicine, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany; e-mail: s.boettcher@med2.uni-kiel.de

The advent of multicolor flow cytometry, now allowing to simultaneously assess eight and more fluorochromes using routine bench top analyzers continues to change our approach to many hematological diseases. High speed and broad availability, regarded as the main advantages of flow cytometry for decades, are now supplemented with improved precision and sensitivity. On the other hand, those novel developments also increase the complexity of flow cytometric approaches. Therefore, careful design of standardized multicolor panels and appropriate standardized techniques for staining and instrument set-up as well as novel analysis tools are mandatory to maximally exploit the newly emerging possibilities of flow in hemato-oncology.

B cell malignancies belong to the most common diseases assessed in clinical flow cytometry. They can be used as a particularly well-suited examples on how the novel technical developments will influence our approaches to hematological diseases at initial diagnosis and for follow-up.

Multicolor flow can improve the distinction between B lineage Non-Hodgkin lymphoma entities. The EuroFlow consortium (coordinator: Prof. Dr. J .J. M. van Dongen, Rotterdam) developed an eight-color staining protocol for fast differential diagnosis of B lineage Non-Hodgkin Lymphoma subtypes using flow cytometry only (Böttcher et al., 2009a). The protocol comprised antigens associated with B cell maturation, markers known to be associated with particular B-cell malignancies, integrins, and chemokine receptors. The development of the panel utilized principal component analysis algorithms integrated into Infinicyt software to identify the markers with highest power to separate any two B-NHL entities. In a step-wise testing procedure using more than 150 B-NHL cases novel markers (e.g. CD200) were introduced into the panel to improve the differential diagnosis. On the other hand, redundant markers could be deleted using the same analysis tools. The combination of the final panel version and INFINICYT software now allows the reliable differential diagnosis of most B-NHL diseases using a 24 antigen immunophenotype. The EuroFlow consortium currently acquires several hundreds of well-characterized B NHL cases to be used as templates for semi-automated diagnosis of mature B cell malignancies.

Modern flow cytometry can also be applied to sensitively assess treatment responses after therapy in B lineage malignancies. These measurements of minimal residual disease (MRD) are currently gaining interest as for many diseases a multitude of treatment options are becoming available. MRD measurements can be used to tailor therapy for individual patients. In chronic lymphocytic leukemia and acute lymphoblastic leukemia multicolor MRD flow already has demonstrated its clinical significance to predict time to disease progression. Besides MRD flow, several PCR methods are currently in use for MRD analysis in B cell malignancies. Comparative analyses to labor-intensive ASO primer real-time quantitative PCR in CLL proved that 4 color MRD flow is equally effective as PCR for MRD assessments with sensitivities of up to 10-4. Interestingly, this even was the case in patients who received rituximab-containing regimens (Böttcher et al., 2009b). Accordingly, MRD measurements using flow were of great significance for progression free survival in both arms of a randomized clinical trial that recruited patients treated with chemotherapy with or without the monoclonal antibody CD20.

It is expected that the same algorithms that were applied to improve differential diagnosis between B-NHL malignancies will also be highly valuable to design 8 color protocols that distinguish normal B cells from B cell malignancies. Those protocols will be applicable to malignancies with heterogenous immunophenotype, such as mantle cell lymphomas. This is an effort currently undertaken by the EuroFlow consortium.

References

1. Böttcher S, Rawstron A, Flores J, Lucio P, de Tute R, Mendonca A, Lecrevisse Q, Kalina T, Kneba M, van Dongen JJM, Orfao A. Improved differential diagnosis between WHO-defined mature B-cell malignancies using integrated 8-color flow cytometry and novel software for multivariate analysis of immunophneotypic data. Haematologica 2009; 94 (s2): 273 abstract n. 0670.

2. Böttcher S, Stilgenbauer S, Busch S, Brüggemann M, Raff T, Pott C, Fischer K, Fingerle-Rowson G, Döhner H, Hallek M, Kneba M, Ritgen M. Standardized MRD flow and ASO IGH RQ-PCR for MRD quantification in CLL patients after rituximab-containing immunochemotherapy – a comparative analysis. Leukemia 2009 (in press).

A new complex epigenetic marker: the replication-coupled, cell cycle dependent, dual modification within the histone tail

Fidlerová H.1, Kalinová J.1, Blechová M.2, Velek J.2, Raška I.1

1Charles University in Prague, First Faculty of Medicine and Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Albertov 4, 128 01 Prague 2, Czech Republic; e-mail: iraska@lf1.cuni.cz

2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nam. 2, 166 10 Prague 6, Czech Republic

We shall describe an evidence for the reversible, cold-dependent detection of the epitope (referred to as epiC), recognized by a monoclonal anti-actin antibody in diploid human fibroblast cell nuclei and mitotic chromosomes. With a help of the detailed 3D confocal and cross-correlation analysis along with biochemical and immunochemical experiments, we shall show that the epiC positivity appears on the newly replicated chromatin domains with some delay (of about 1 h) with respect to their DNA replication and then persists on the newly replicated chromatin until next early G1 phase, during which it disappears. However, while this epiC positivity is detected in the early replicated chromatin domains, it is not found in the mid/late replicating domains either during S phase, or at any other cell cycle phases. The unique spatio-temporal pattern of epiC positivity suggests that some replication-coupled modulation of early replicated chromatin domains, which could be involved in transfer/maintenance of epigenetic information on transcriptionally competent part of genome, is detected. Indeed, in contrast to the actin epitope, epiC consists of different amino acid sequence within histone H4 tail carrying two postranslational modifications and is a new complex epigenetic histone marker that might represent a global marker related to the timing decision point. The relevance of epiC marker in the transfer/maintenance of epigenetic information on the transcriptionally competent part of the genome will be dealt with.

This work was supported by the Czech grants 304/06/1691, LC535, MSM0021620806 and AV0Z50110509.

Development of cellular assays for automated microscopy to measure multiple parameters by single cell High Content Analysis

Horn T.

European Technology Center, BD Biosciences, Becton Dickinson AG, Binningerstr. 94, CH- 4123 Allschwil, Switzerland; e-mail: thomas_horn@europe.bd.com

High-content imaging is an automated process that begins with image acquisition and concludes with data analysis. This process requires fully automated microscopy that integrates image analysis and subsequent transfer of the numerical data into statistical representations such as graphs, heat maps and tables. The main focus of this technology lays on transferring automatically acquired images into meaningful data.

The power of high-content imaging resides in the ability to measure not only fluorescence intensity and morphological changes, but also temporal and spatial dynamics. This technology allows also to perform multiplexed assays that simultaneously measure and analyze a large number of such cellular parameters. However, multiplexing is often restricted due to reagent, assay, and instrumentation limitations. BD Biosciences is addressing this by developing specific instrumentation, assays, reagents, and software that enable such multiplexing. We will highlight the capabilities of the BD Pathway™, such as kinetic and confocal imaging, and the impact these features have on high-content imaging. In particular, we will address several challenging applications including stem cells and neurobiology. In addition, we will discuss the development of a line of BD™ Bioimaging Certified reagents that have direct impact on the ability to multiplex.

To facilitate the use of antibodies for imaging applications, BD Biosciences embarked on a screening process to evaluate a large library of monoclonal antibodies for utility in bioimaging applications. The library of antibodies included specificities that recognized proteins involved in cell signaling, cell cycle, apoptosis, cancer and neurobiology. Antibodies were tested using 3 cell lines and several fix/perm methods that are relevant to high-content imaging applications on automated platforms. Specific criteria regarding signal to noise, sub-cellular localization and other important parameters were used to qualify reagents. Two distinct Bioimaging Certified reagent product lines resulted from this antibody screen. One is a continually expanding collection of currently greater than 200 unlabeled primary antibodies that have been shown to have general utility in bioimaging applications. The other is a subset of these reagents that have been further developed into primary conjugated antibodies to facilitate multiplexed high content analysis with monoclonal antibodies. These reagents, combined with an automated imaging platform, will enable the rapid development of novel cell-based assays.

To enable live-cell assays we developed an environmentally controlled on-stage chamber with an integrated pipetting system in combination with an automated fluorescent microscope for imaging of individual cell cultures in multi-well plates. The broad range of assay types include nuclear protein translocations, live/dead cell detection, intracellular calcium dynamics and various measures of the apoptotic cascade like Caspase-3, JC-1 distribution and nuclear fragmentation. For another parameter, DNA strand break count we demonstrated the effect of utilizing our confocal imaging technology to improve the quality of the data by achieving higher z’-scores. The use of our Neurite Outgrowth and Angiogenesis algorithms will demonstrate the capacity to detect differentiation and morphological changes depending upon exposure to various stimuli. Furthermore new applications such as intracellular bacterial count, cell cycle analysis and the multiplexing of intracellular calcium concentration measurements with mitochondrial membrane potential assessment will be discussed.

Our data will show that live-cell imaging of kinetics and endpoint parameters together with high-resolution confocal microscopy is a powerful tool for exploring cellular events in multidimensional space and time.


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Adiktologie Alergologie a imunologie Angiologie Audiologie a foniatrie Biochemie Dermatologie Dětská gastroenterologie Dětská chirurgie Dětská kardiologie Dětská neurologie Dětská otorinolaryngologie Dětská psychiatrie Dětská revmatologie Diabetologie Farmacie Chirurgie cévní Algeziologie Dentální hygienistka

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