Report of the Third Hungarian cell analysis conference: 16–18 May 2002, Budapest, Hungary

Abstract

This conference was a successful continuation of previous conferences such as the Hungarian Cell Analysis Conference, Budapest, 1998 and 2000, and the ISAC-Sponsored International Conference for Flow Cytometry and Image Analysis, Epona, 1999. The Cell Analysis Section of the Hungarian Biophysical Society organized all four conferences. The popularity of the conference was hallmarked by the large number of participants (close to 300). The form of the conference proved to be very attractive: In the mornings, experts from specific fields delivered scientific lectures. In addition to Hungarian scientists, such as Margit Balázs, Gyula Hadlacky, Béla Molnár, László G. Puskas, János Szöllösi, and György Vereb, experts from abroad significantly raised the scientific standards of the conference. The international speakers included Peter Adorjan, Francis Mandy, Abe Schwartz, Howard M. Shapiro, and the authors of this introduction. In the afternoons, practical demonstrations were presented, ranging from basic techniques to state-of-the-art instrumentation. Practical training included cell culturing; mechanical cell separation for flow cytometry; methods for detection of cell proliferation; detection of apoptosis; fluorescence microscopy, image acquisition, and processing; confocal laser microscopy; fluorescence in situ hybridization; magnetic cell separation; magnetic mRNA isolation; real-time and traditional polymerase chain reaction; and detection of mutation. During the conference the attendees were able to choose six sessions of 11 topics. These demonstrations were headed by one of the speakers and sponsored by commercial companies that provided special kits and/or instruments to ensure a stable basis for successful practical training. An impressive selection of commercial companies provided contacts and improved awareness of advanced analytical cytology for the participants. The purpose of the conference was multifaceted. It served as a forum for presentations from Hungarian scientists, and, with the active participation of foreign experts, it opened a window to state-of-the art methods and techniques. The conference also facilitated the research cooperation between well-established scientists and Ph.D. graduate students, with the major purpose of the conference being education. The average age of the attendees was approximately 30 as more than 50% of the participants were Ph.D. students. Graduate students were able to earn credit points toward their final examination. The venue for the conference was the Medical University of Semmelweis in Budapest, the capital of Hungary. The university has great traditions, with its name reflecting the rich history of scholastic activity in Budapest. It was over 150 years ago that Ignaz Semmelweis completed his classic epidemiologic study in Budapest, studies that led to effective prophylaxis against childbed fever that still stands as the hallmark of evidence-based studies in epidemiology. First prize: Generation and Characterization by Flow Cytometry of Dendritic Cells, by Gizella Veszely, János Fent, Ágnes Nagy, and Furész József, Department of Pathophysiology, Institute for Health Protection of HDF, Budapest. Second prize: Hemocyte-Specific Molecular Markers in the Hematopoiesis and Innate Immunity of Drosophila melanogaster, by István Nagy, Éva Kurucz, and István Andó, Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged. Third prize: Astrocytes Support the Neuronal Differentiation of Neuroectodermal Progenitor Cells, by Vanda Szlávik, Zsuzsanna Környei, and Emília Madarász, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest. On the following pages, the reader will find the rich program of the Third Hungarian Cell Analysis Conference through the lecture and poster abstracts. METHYLATION BASED CLASS PREDICTION USING SUPPORT VECTOR MACHINES Péter Adorján, Fabian Model, Alexander Olek, Christian Piepenbrock Epigenomics AG Kastanienalle 24 10435 Berlin Germany Molecular portraits, such as mRNA expression or DNA methylation patterns, have been shown to be strongly correlated with phenotypical parameters. These molecular patterns can be revealed routinely on a genomic scale. This means that the several hundreds or thousands of variables are measured in parallel in a single experiment. The major goal of these experiments is to identify those genes whose expression or methylation pattern correlates strongly with the investigated tissue classes because these genes have a crucial importance for diagnostic or pharmaceutical development. Here we demonstrate novel machine learning techniques to visualize and interpret these high dimensional microarray data sets. In order to perform a methylation based class prediction we use the well known support vector machine algorithm. This algorithm has shown outstanding performance in several areas of application and has already been successfully used to classify mRNA expression data. The major problem of all classification algorithms for methylation and expression data analysis alike is the high dimension of input space compared to the small number of available samples. Although the support vector machine is designed to overcome this problem it still suffers from these extreme conditions. Therefore feature selection is of crucial importance for good performance and we give special consideration to it by comparing several methods on our methylation data. The data set consists of cell lines and primary tissue obtained from patients with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). A total of 17 ALL and 8 AML samples were included. The methylation status of these samples was evaluated at 81 CpG dinucleotide positions located in CpG rich regions of the promoters, intronic and coding sequences of 11 genes. These were randomly selected from a panel of genes representing different pathways associated with tumor genesis. Our results clearly demonstrate that microarray based methylation analysis combined with supervised learning techniques can reliably predict known tumor classes. Classification results were comparable to mRNA expression data and our results suggest, that methylation analysis should be applied to other kinds of tissue. Well-documented tissue samples with patient history can be obtained only as archived specimens. This strongly limits the amount and number of tissues available for expression analysis. The methylation approach has the potential to overcome this fundamental limitation: through the mere fact that the stable DNA is the object of study, extraction of material is possible form archived samples. This enables the examination of methylation patterns in large numbers of archived specimen with comprehensive clinical records and removes one of the major limitations for the discovery of complex biological processes by statistical means. Contact: peter.adorjan@epigenomics.com APPLICATION OF FLUORESCENCE IN SITU HYBRIDIZATION IN THE DIAGNOSIS OF MALIGNANT DISEASES Margit Balázs, Andrea Treszl, Róza Ádány University of Debrecen, Medical and Health Science Center, Department of Preventive Medicine, Debrecen, Hungary Genetic alterations of malignant diseases or premalignant lesions are in many cases associated with the prognosis of the disease. Identification of chromosomal alterations that are involved in the initiation and progression of the malignant process may allow not only the better prediction and monitoring of the disease but it can lead to the development of new therapeutic strategies. Conventional banding analyses are not easy to perform because metaphase chromosomes of sufficient quality and quantity are often difficult to obtain from many solid tumors. Over the last decade, fluorescence in situ hybridization (FISH) has become a powerful and essential technique to detect chromosome copy number changes and structural alterations in metaphase and interphase cells. It can be used in different field of biology, including the study of chromatin organization, gene mapping, karyotype analysis, radiation dosimetry and clinical diagnosis of malignant diseases. For the detection of numerical and structural chromosome alterations several different types of probes are now commercially available. DNA probes recognizing repeat sequence targets, such as alphoid and satellite DNA, mostly present in the centromeric and telomeric regions, are used routinely to detect chromosome aneuploidy. Locus-specific probes are usually collections of one or a few cloned DNA sequences ranging from just one or less than one kb to over 1 Mb and are applied to study gene amplifications and deletions. For targets of much larger scale, from chromosome bands up to the entire genomes, more complex mixtures of DNA sequences are used as probes. With the availability of an increasing number of spectrally distinct fluorophores it is possible to visualize all chromosomes with different colors using painting probes for all 11chromosomes in one experiment. This multiplex-FISH (M-FISH) technique relies on digital image analysis and allows the rapid detection of numerical and structural alterations of metaphase chromosomes obtained from tumor cells. Another methodological breakthrough in FISH technology is comparative genomic hybridization (CGH). CGH has the advantage that allows the tumor genome to be screened for copy number changes without the need to obtain metaphase spreads from the tumor cells, chromosome copy number alterations can be detected and mapped throughout the tumour genome in a single hybridization. The CGH technique is based on dual color, competitive FISH and is performed using differentially labeled test DNA obtained from tumor cells (e.g. green fluorescence) and reference normal DNA (e.g. red fluorescence) co-hybridized to normal human chromosomes (counterstained with a blue fluorescent DNA specific dye). If over-represented or amplified sequences are present in the test DNA, that region of the normal chromosomes will hybridize increased amount of tumor DNA and will result in an increase of the green to red fluorescence intensity ratio. Under-represented or deleted regions will be represented by the decrease in the green to red ratio on the normal chromosomes. The different FISH techniques have been applied for many solid and haematological tumor types, cell lines and archival materials to characterize chromosomal alterations. The use of FISH methods in molecular pathology will be of great value in the early detection of malignant lesions and monitoring the effect of different therapies in cancer. (OTKA 32587, ETT587/2000) ARTIFICIAL CHROMOSOMES IN GENE THERAPY Gyula Hadlaczky Institute of Genetics, BRC, Hungarian Academy of Sciences, H-6726 Szeged, Temesvari krt. 62, Hungary Satellite DNA-based artificial chromosomes (SATACs) can be generated by induced de novo chromosome formation, in cells of different mammalian species including humans. These artificially generated stable accessory chromosomes are composed of predictable DNA sequences and they contain defined genetic information. Human Satellite DNA-based artificial chromosomes (SATACs) developed in our laboratory represent a potential non-integrating vector with megabasepair size carrying capacity. SATACs may serve as stable neutral platform for persistent or controlled expression of therapeutic gene(s). Generation of SATACs in a reproducible manner from predictable DNA sequences. Over the recent years, a number of different mouse, mouse/hamster, hamster, hamster/human, and human SATACs were made. Large-scale purification of SATACs by flow cytometry with fluorescence activated dual laser-beam cell sorter (FACS). Stable transfer of SATACs into different cells (mouse, hamster bovine, human) and embryos (mouse, bovine) while at the same time preserving their structural integrity and function. FACS purified SATACs have successfully been delivered to recipient cells by microinjection, microcell-mediated mitotic chromosome transfer, with cationic lipids and dendrimers, sonoporation, direct chromosome uptake, etc. Generation of transgenic animals with purified SATACs and germline transmission of these mammalian artificial chromosomes have been demonstrated. Tissue specific expression of a therapeutic gene from SATACs in transgenic animals (mouse). Based on the above achievements, in the long term, carefully designed artificial chromosomes may play an important role in human germline gene therapy, while in the short term they offer great potential for ex vivo somatic gene therapy. The author is the Founding and Chief Scientist of Chromos Molecular Systems Inc., Burnaby, Canada. FLOW CYTOMETRIC FLUORESCENCE LIFETIME ANALYSIS OF NUCLEIC ACID BINDING FLUOROCHROMES Harry A. Crissman, H. Helen Cui, John A. Steinkamp Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA A new dimension has been added to multiparameter flow cytometric analysis through development of the Los Alamos Phase Sensitive Flow Cytometer with capabilities for performing fluorescence lifetime measurements as well as conventional FCM measurements. Monitoring the changes in the absolute lifetime value of the probe yields information relating to the changes in molecular conformation and the functional activity of the molecular target. Lifetime values also provide unique signatures for resolving the emissions of multiple fluorochrome labels with overlapping spectra, thereby increasing the number of fluorochrome combinations using a single excitation source. Lifetime analysis of cells stained with different nucleic acid-binding fluorochromes revealed several other unique observations and demonstrated the accuracy of the PS-FCM methodology. Our lifetime studies provided the discrimination of DNA and dsRNA based on differences in the lifetime value of either PI or EB bound to the respective nucleic acids. Differences in lifetime values relate to the differences in the structure of the nucleic acid complexes, as well as the dissimilarities in the dye-intercalation into DNA or dsRNA. Similar lifetime data were obtained with fluorescent chemotherapeutic agents, including ellipticine and adriamycin, thereby allowing, potentially, for discriminating and quantitating binding of these drugs to either DNA or RNA. Bivariate profiles of lifetime versus DNA content, obtained from analysis of EB stained, HL-60 cell populations induced into apoptosis showed a 3.0 ns reduction in the lifetime of EB bound to apoptotic cells compared to the non-apoptotic subpopulation. DNA content and lifetime analysis revealed a unique subpopulation of human skin fibroblasts cells in very early S phase with a significantly reduced EB-lifetime. Multiparameter DNA content, EB lifetime and immunofluorescent antibody analysis of cyclin D and cyclin E levels in asynchronous HSF cells demonstrated that the subpopulation of cells contained elevated levels of both cyclin D and cyclin E, characteristic of cells in very early S phase. Following release of synchronized cells from G1/S phase, the subpopulation entered mid-S phase with EB lifetime values elevated above G1 phase cells and a progressive increase in EB lifetime was noted as cells proceed to the G2/M phase. These studies demonstrate applications of lifetime measurements for the analysis of the binding of different fluorochromes to DNA or RNA in single cells. Data also show applications of lifetime measurements for monitoring changes in chromatin structure associated with cell cycle progression, cellular differentiation, or DNA damage, as in the early stages of apoptosis. Potential modifications of the PS-FCM will provide for simultaneous measurement of multiple lifetimes, thereby enhancing detection and quantitation of fluorescent compounds, including chemotherapeutic agents, bound to multiple subcellular complexes in viable cells. Supported by the US Department of Energy and the Los Alamos National Flow Cytometry Resource (NIH p41-RR011315) and NIH grant R01 CA92632. T-CELL SUBSET ENUMERATION: PAST, PRESENT AND FUTURE Ferenc Mandy National HIV Immunology Laboratory, Health Canada, Ottawa, ON, Canada Flow cytometry impacted HIV disease monitoring more than any other clinical condition. Therefore, it seems appropriate to review the evolution of immunophenotyping in the context of following the fight against AIDS over the past 20 years. Contrary to some of the original expectations, it was AIDS, not some frequently performed oncological test that was responsible for the massive and rapid worldwide mobilization of flow cytometers into clinical immunology laboratories. In 1981, reports appeared from various parts of the USA about young gay men who had unusual immunosuppression manifesting as opportunistic infections. Soon it was discovered that the hallmark of this new disease, acquired immunodeficiency syndrome (AIDS), was a decrease in numbers of CD4 T-cells in peripheral blood. Clinical flow cytometers handle 5 or 6 distinct parameters: forward scatter (FS), side scatter (SS), and three or four fluorescent light (FL) signals. The two light scatters are intrinsic parameters that define morphological features of leukocytes: size and granularity, respectively. The FL parameters measure extrinsic attributes, such as identification of surface antigens; via fluorescent scattering from fluorochromes coupled to monoclonal antibodies (MAbs). Fluorescein isothiocyanate (FITC) is the most universal fluorochrome. The second common dye is, R-phycoerythrin (PE). Both dyes excite at 488 nm. As third and fourth dyes, both natural and man-made tandem dyes are utilized. Currently, simultaneous four-color immunophenotyping is the advanced clinical method. Multi-color application is accomplished either by adding a fourth PMT for the detection in the far red (Beckman Coulter), or by adding an additional laser as well as a fourth PMT (BD Biosciences). The dye APC is used as the fourth fluorochrome with the red diode laser that emits at 635 nm. In the quest to eradicate the AIDS pandemic, perhaps the next generation of multi-laser, multi-parameter instruments will accelerate discoveries in cellular immunology and that in turn will lead to breakthroughs in the fundamental understanding of events in adaptive and innate immunity. It is predicted that cytometers will continue to shrink in size and cost. That engineers that develop instruments to comply with the visions incorporating cytomics will also design low cost instruments to deal with the realities of the resource poor parts of the globe. RARE CELL ANALYSIS AND MULTIFUNCTIONAL EVALUATION BY CHIP TECHNOLOGIES Béla Molnár, Orsolya Galamb, Ferenc Sipos, Zsolt Tulassay Cell Analysis Lab. II. Dept. of Medicine, Semmelweis University, Budapest, Hungary, H-1088 Background: Chip and array technologies are used more and more for the multifunctional analysis of pathologically altered cells. Today there are DNA methylation and polymorphism chips, mRNA expression and protein chips commercially available. The required cell amount for a single chip analysis is changing from 10 000 to 200 000 cells. The focused interest of clinical researchers in rare cell applications is limited by the discrepancy between the amount of isolable cells (from 1 to several thousands) and the RNA, DNA or protein amount that is necessary for reliable array analysis. Aims: Evaluation of the available multiplication techniques for the increase of the rare cells' cell component amount for routine chip analysis. Results: Basically one can choose between different methods. These can include the increase of the amount of the isolated cells, the multiplication of the isolated cell number by culturing or the multiplication of the isolated cell components. The first alternative is the application of increased amount of sample volume for rare cell isolation. Isolation efficiency can be enhanced in the case of magnetic isolation using increased amount of magnetic antibodies. The isolated cells can be cultured, too. However in this case the cell function can be altered due to different culturing conditions. The RNA amplification can be performed by T7 RNA amplification protocol. DNA amplification can be performed by using RAP-PCR (random access primed PCR), DOP-PCR (degenerate oligonucleotid primer PCR) or PEP-PCR (primer extension protocol PCR) techniques. This way not only DNA, but cDNA can be amplified, too. The amount of available proteins isolated from in vivo cells cannot be multiplied according to our knowledge, until now. In single cases, after the determination of the protein structure, in vitro translation systems can be used. For the increase of the chip sensitivity tyramin signal amplification protocol can be used. Discussion: Today the commercially available chip arrays require intermediate amplification techniques for rare cell applications. Further technological improvements are required in the chip sensitivity and standardization efforts are necessary to have comparable results from different laboratories. FABRICATION AND APPLICATION OF DNA-MICROARRAYS László G. Puskás DNA-chip Laboratory, Biological Research Center, Hungarian Academy of Sciences, Szeged, P.O. Box 521, H-6701, Hungary Large-scale and simultaneous measurement of gene expression using hybridization of complex probes, representing the active genes of a certain biological sample to arrays of cDNA fragments or oligonucleotides is becoming a widely used technique in functional molecular biology. Using DNA-microarrays or DNA-chips, global gene expression changes of diverse physiologic and pathologic states, single nucleotide polymorphisms or mutations can be followed. In recent years, this analysis is based on hybridization of fluorescent labeled probes prepared from mRNA, total RNA, or DNA obtained from diverse biological samples to microarrays having complementary sequences as targets on their surfaces. In the case of transcriptome analysis for each hybridization usually a mixture of two fluorescent labeled probes is applied onto a cDNA-microarray, where one labeled probe is obtained from a control (untreated or unaffected) and the other is from a treated or affected sample. This direct comparative hybridization method allows a quantitative comparison of the relative abundance of individual sequences. Of great interest recently has been the potential application of microarray technology to follow the effects of disease-inducing elements, determine new disease subclasses, and predict the outcome of drug treatment based on exclusively the gene expression pattern of the patient. To obtain appropriate amounts of RNA for standard labeling techniques, milligrams of tissue or millions of cells are needed, or alternatively one can apply different sample or signal amplification methods. We developed a novel amplification technique, which combines PCR amplification and in vitro transcription to obtain high-quality RNA for labeling starting from micrograms of total RNA. We have also investigated the reproducibility, reliability and sensitivity of the method. To monitor the gene expression of homogenous sample isolated from a patient or other clinical sample appropriate separation techniques, such as fluorescent activated cell sorter have to be used to obtain pure cell population as starting material. In combination of these methods precise and valuable information can be gained on differentiation, malfunction and disease progress arisen from specific cell types. QUANTITATIVE IMMUNOFLUORESCENCE BY FLOW CYTOMETRY Abe Schwartz Center for Quantitative Cytometry, San Juan, PR 00919 USA One of the major goals of quantitative immunofluorescence is to determine the average number of specific receptors on a cell population. Flow cytometry is a powerful tool that enables the operator, through multi-parameters, to select cell populations and measure the fluorescence signal arising from antibodies or other probes bound to that population. iF = [geΩϵϕ∫Q(λ)s(λ)T(λ)dλ]c These factors can all be removed from consideration by the introduction of a fluorescence unit that is independent of the instrument, environmental and molecular properties. This unit is Molecules of Equivalent Soluble Fluorochrome (MESF). As implied by the name, this unit indicates the intensity of the standard or sample relative to the intensity of a gravimetric primary solution of the same fluorochrome. Solutions and/or particle suspensions may be calibrated in MESF units against such a primary solution using spectrofluorometry, and in turn used as standards, so long as the following criteria are observed: The excitation and emission spectra of the standards have to match those of the sample. The environmental response of the fluorochrome of the standards must match those of the sample. The standards and the sample must be run on the same instrument at the same settings. Care must be taken since the number of receptors on a cell may not be reflected by measure the number of receptors available to bind to a particular monoclonal antibody due to steric hindrance may affect antibody binding, as well as binding stoichiometry where additional Scatchard analysis may be required. In summary, MESF units allow for determinations of the number of antibodies binding to cells independent of the instrument and the environment that provides a unique tool for comparison of data across different laboratories and extended periods of time. THE EVOLUTION OF CYTOMETRY Howard M. Shapiro 283 Highland Avenue, West Newton, MA 02465-2513, USA; e-mail: hms@shapirolab.com Until the mid-20th Century, determining whether cells were present in a specimen, how many there were, what kinds of cells were represented and what their functional characteristics might be required that a human interpret a microscope image. The same tasks remain for modern cytometers, specialized microscopes in which technology improves on what could be obtained “by eye” alone. From the 1930′s on, Caspersson developed microspectrophotometers to quantify abnormalities in DNA, RNA, and protein content in tumors based on UV absorption. The first applications of flow cytometry, beginning in the 1940′s, were to counting and sizing cells in liquid suspension and aerosols, using measurements of light scattering or electrical impedance (Coulter principle). By the 1960′s, investigators were attempting to automate analysis of the Papanicolaou smear and the differential white blood cell count. Kamentsky concluded that limitations of existing hardware and software made it impossible to develop a practical high-resolution scanning instrument; he built a flow cytometer, with a dedicated computer, that could measure as many as four parameters. The problem of isolating cells for identification and further analysis was solved in the mid 1960′s, when Fulwyler and Kamentsky, respectively, demonstrated droplet deflection-based and fluidic cell sorters. By the late 1960′s, fluorescence measurements using reagents such as nucleic acid dyes and labeled antibodies were providing information about a large variety of cellular constituents. Groups led by Göhde, Herzenberg, Kamentsky, and Van Dilla developed apparatus for fluorescence flow cytometry and sorting that was produced commercially in the early 1970′s. A flow cytometric differential leukocyte counter also reached the market at this time. Herzenberg's fluorescence-activated cell sorter attracted immunologists, who used the instrument to separate various cells of the immune system. The technology aided in the development of monoclonal antibodies as reagents in the 1980′s, and was made immensely more powerful thereby. As it became practical to simultaneously measure multiple antigens in or on a single cell, additional fluorescent labels, including new dyes, phycobiliproteins, and tandem conjugates were developed. By the mid-1980′s, bench-top analyzers with improved optics, which could make sensitive measurements using air-cooled lasers, began to appear. Their adaptation in research and clinical laboratories was catalyzed by the emergence of AIDS, in which CD4+ lymphocyte counts provided valuable prognostic information. Analysis of tumor DNA content by flow cytometry also became clinically important. The application of multiparameter measurement and analysis was greatly facilitated by developments in personal computers. Flow cytometers were also used to identify and sort individual human chromosomes and sperm, and methods for measurement of physiologic parameters, such as membrane potential, intracellular calcium content, and pH were developed. By the mid-1990′s, it was recognized that the Aequorea green fluorescent protein (GFP) and related proteins, produced after cotransfection with other genes of interest, could act as reporters, indicating whether transfection had been successful in an individual cell. Today, practical diode and solid state sources at wavelengths ranging from the UV to the infrared make even multibeam, multiparameter flow cytometers smaller, less power-hungry, and less expensive, enabling them to be used for a wider range of applications. Kamentsky's Laser Scanning Cytometer, an instrument that uses low resolution imaging to extract much of the same information from cells as is now conventionally obtained by flow cytometry. Even simpler static cytometry apparatus has been described to facilitate CD4+ T-cell counting in resource-poor countries heavily affected by the HIV epidemic. Flow cytometry has taken the differential count beyond the wildest dreams of the pioneers in the field, enabling precise analysis of normal and abnormal cells from the blood, bone marrow, and immune system. Image analyzing systems have been approved for automated screening of Papanicolaou smears. The range of particles amenable to cytometric analysis and sorting now runs from single molecules to multicellular organisms, and the pace of progress is