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 Bala´zs, Gyula Hadlacky, Be´la Molna´r, La´szlo´ G. Puskas, Ja´nos Szo¨llo¨si, and Gyo¨rgy 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 stateof-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. At the conference both the lectures and the posters proved to be of high quality, demonstrating the high scientific standards of the community.

 The topics of the posters ranged from oncology (basic and applied research) to signal transduction, genetics, fertility research, and investigation in lower organisms such as Drosophila and earthworm. The methods applied were varied and highly sophisticated. Of 29 poster presentations, three prizes were awarded. Originally the poster awards committee (Margit Bala´zs and Attila Ta´rnok) was supposed to give only one award, but the decision was not easy because of the large number of excellent posters. With the financial help of commercial companies, the committee was able to present three poster awards instead of one. These awards were: First prize: Generation and Characterization by Flow Cytometry of Dendritic Cells, by Gizella Veszely, Ja´nos Fent, A´gnes Nagy, and Fure´sz Jo´zsef, 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 Istva´n Nagy, E´va Kurucz, and Istva´n Ando´, 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 Szla´vik, Zsuzsanna Ko¨rnyei, and Emı´lia Madara´sz, 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 Pe´ter Adorja´ n, 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 2 APPLICATION OF FLUORESCENCE IN SITU HYBRIDIZATION IN THE DIAGNOSIS OF MALIGNANT DISEASES Margit Bala´zs, Andrea Treszl, Ro´za A´ da´ ny 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 (MFISH) 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 overrepresented 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) 3 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). To become a potential vector for use in gene therapy, SATACs have already passed several hurdles: 1. 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. 2. Large-scale purification of SATACs by flow cytometry with fluorescence activated dual laser-beam cell sorter (FACS). 3. 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. 

4. Generation of transgenic animals with purified SATACs and germline transmission of these mammalian artificial chromosomes have been demonstrated. 5. 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. 4 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 PSFCM 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.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.