Analysis Of Rare Cells Research Articles

Abstract 1553: Redefining CTCs: Detection of cytokeratin-negative circulating tumor cells (CTCs)

Abstract Introduction: There is growing evidence that CTCs display an epithelial-mesenchymal transition phenotype. Current enrichment techniques rely upon epithelial markers for capture (anti-EpCAM antibodies and cytokeratin [CK]) and may miss important populations of circulating tumor cells. We sought to develop a new method for identification of CTCs. Methods: After IRB approval, blood from patients with ovarian or colorectal carcinoma was collected. Cell Enrichment and Extraction (CEE) technology, a microfluidic-based device using an antibody cocktail targeting epithelial and mesenchymal markers, was utilized for the capture and analysis of rare cells in blood. We enumerated cells that were CK+ and/or contained complex aneuploidy by fluorescent in situ hybridization (FISH). Blood samples from healthy volunteers served as a negative control. Fresh frozen tumor samples were analyzed on a subset of patients to search for concordance of molecular alterations with CTCs. An orthotopic ovarian cancer model was used to evaluate effects of chemotherapy on CTCs. Results: Enumeration of CK+ cells identified an average of 1 and 2 cells per 10 mL blood in ovarian and colorectal cancer patients, independent of stage or tumor grade. Enumeration did not correlate with serum tumor marker levels. The majority of CK+ cells had complex aneuploidy. Ovarian cancer patients had equal numbers of CK- and CK+ complex aneuploid cells (p=1.0). A three-fold increase in CK- over CK+ complex aneuploid cells was noted in patients with colorectal cancer, although not statistically significant (p<0.19). The total number of complex aneuploid cells did not correlate with serum tumor markers. Similar patterns of complex aneuploidy were identified in patients’ primary tumors as were seen in their CK- cells from blood, indicating that the cells identified in circulation were indeed CTCs. No CK+ or complex aneuploid cells were found in healthy volunteers. In the orthotopic mouse model, compared to tumor-bearing controls, chemotherapy treatment initially lead to a 17-fold increase in apoptotic appearing CTCs (p<0.02) followed by a 15-fold decrease in CTCs (p<0.03), which also reflected tumor response. Discussion: Here, we have developed and characterized a novel and robust method for detection of CTCs. The power of this approach lies in its ability to detect CTCs with either an epithelial or EMT-positive phenotype. Our findings suggest current enrichment techniques may be missing an important population of cells. Complex aneuploid CK- cells challenge the current definition of a CTC and merit further development in clinical settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1553. doi:10.1158/1538-7445.AM2011-1553

Abstract 3159: BRAFV600E mutation analysis in circulating tumor cells

Abstract Background: The concept of “liquid biopsy” refers to analysis of rare cells in the blood such as Circulating Tumor Cells (CTC) and represents a powerful tool for molecular characterization of tumors for which biopsies are not available. Because of a low frequency of CTCs, assay sensitivity for mutational analysis of CTCs needs to be below 1%. An activating mutation in the serine/threonine kinase BRAF (V600E) is present in up to 70% of melanomas and 10% of colon carcinomas. Herein, we developed and validated a highly-sensitive allele-specific PCR (AS-PCR) assay to detect the presence of the BRAFV600E mutation in CTCs. Methods: Genomic DNA isolation was optimized for rare cell analysis. AS-PCR for detection of BRAFV600E mutation was developed and tested, using cell spiking experiments to define assay sensitivity, limit of detection, assay precision and specificity. Use of the CTC BRAFV600E AS-PCR assay was demonstrated in melanoma and colon cancer patient blood. Results: Performance of the BRAFV600E AS-PCR was tested using gDNA extracted from HT-29 colon cancer cells heterozygous for BRAFV600E, and BRAFwild-type MDA-MB-231 breast cancer cells. Assay sensitivity and limit of detection for determination of BRAFV600E in a background of BRAFwt was tested by spiking HT-29 cells into 50,000 MDA-MB-231 cells at following ratios: 5000 (5% mutant DNA copies), 500 (0.5%), 50 (0.05%), 10 (0.01%), 5 (0.005%) and 0 (0%). The assay limit of detection was found to be 0.01% or 5 BRAFV600E mutant copies in a wt background. Precision was demonstrated by repeating assay sensitivity testing at the limit of detection on three different days. The performance and specificity of the BRAF AS-PCR assay in enriched CTCs was further verified using HT-29 and MDA-MB-231 cells spiked into donor blood and isolated by EpCAM-based CellSearch® CTC Profile kit, which we have previously shown to yield a higher number of CTCs than the standard CellSearch® CTC enumeration kit. Finally, CTC BRAFV600E AS-PCR assay performance was demonstrated in patients with colon cancer as well as melanoma, in which CTCs were isolated using a previously developed melanoma immunomagnetic enrichment panel. Conclusions: We have developed an AS-PCR assay for detection of the BRAFV600E mutation in CTCs. Validation testing demonstrated that the assay detects 0.01% or 5 copies of BRAFV600E DNA in a wild-type background. Testing melanoma and colon cancer patient blood specimens demonstrated feasibility of the BRAFV600E mutation analysis using liquid biopsy. Further concordance testing between tumor biopsies and blood specimens is ongoing and will establish whether liquid biopsies are a suitable alternative for identifying patients for whom a particular targeted therapy may or may not be effective based on genetic status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3159. doi:10.1158/1538-7445.AM2011-3159

Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip

We demonstrate lensless fluorescent microscopy over a large field-of-view of ~60 mm(2) with a spatial resolution of <4 µm. In this on-chip fluorescent imaging modality, the samples are placed on a fiber-optic faceplate that is tapered such that the density of the fiber-optic waveguides on the top facet is >5 fold larger than the bottom one. Placed on this tapered faceplate, the fluorescent samples are pumped from the side through a glass hemisphere interface. After excitation of the samples, the pump light is rejected through total internal reflection that occurs at the bottom facet of the sample substrate. The fluorescent emission from the sample is then collected by the smaller end of the tapered faceplate and is delivered to an opto-electronic sensor-array to be digitally sampled. Using a compressive sampling algorithm, we decode these raw lensfree images to validate the resolution (<4 µm) of this on-chip fluorescent imaging platform using microparticles as well as labeled Giardia muris cysts. This wide-field lensfree fluorescent microscopy platform, being compact and high-throughput, might provide a valuable tool especially for cytometry, rare cell analysis (involving large area microfluidic systems) as well as for microarray imaging applications.

Small but mighty: How the MACS®‐technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years

Today, magnetic cell sorting and flow cytometric cell sorting both are state-of-the-art technologies, with a plethora of applications in biology and biomedicine. Both technologies have their stand-alone applications, but they also perfectly complement each other, in particular for the analysis and isolation of fragile and rare cells. The technological evolution from simple magnets and steel wool separation columns to sophisticated instrumentation and automated procedures has paved the way for magnetic cell separation in the scientific community. This review will focus on applications of magnetic cell sorting with commercially available paramagnetic MACS(R)-nanoparticles (Miltenyi Biotech, Bergisch-Gladbach, Germany) that have allowed unprecedented approaches to the exploration of the immune system in basal and clinical research of humans and rodents. Thus, during the last 20 years this technology has been continuously developed so that the widest array of leukocyte subsets, stem cells, and connective tissue cells can be addressed by MACS(R)-conjugated antibodies directed against cell-specific surface antigens or secreted cytokines for research and clinical applications.

Microsystems for the Capture of Low-Abundance Cells

Efficient selection and enumeration of low-abundance biological cells are highly important in a variety of applications. For example, the clinical utility of circulating tumor cells (CTCs) in peripheral blood is recognized as a viable biomarker for the management of various cancers, in which the clinically relevant number of CTCs per 7.5 ml of blood is two to five. Although there are several methods for isolating rare cells from a variety of heterogeneous samples, such as immunomagnetic-assisted cell sorting and fluorescence-activated cell sorting, they are fraught with challenges. Microsystem-based technologies are providing new opportunities for selecting and isolating rare cells from complex, heterogeneous samples. Such approaches involve reductions in target-cell loss, process automation, and minimization of contamination issues. In this review, we introduce different application areas requiring rare cell analysis, conventional techniques for their selection, and finally microsystem approaches for low-abundance-cell isolation and enumeration.

Lensless wide-field fluorescent imaging on a chip using compressive decoding of sparse objects

We demonstrate the use of a compressive sampling algorithm for on-chip fluorescent imaging of sparse objects over an ultra-large field-of-view (>8 cm2) without the need for any lenses or mechanical scanning. In this lensfree imaging technique, fluorescent samples placed on a chip are excited through a prism interface, where the pump light is filtered out by total internal reflection after exciting the entire sample volume. The emitted fluorescent light from the specimen is collected through an on-chip fiber-optic faceplate and is delivered to a wide field-of-view opto-electronic sensor array for lensless recording of fluorescent spots corresponding to the samples. A compressive sampling based optimization algorithm is then used to rapidly reconstruct the sparse distribution of fluorescent sources to achieve ~10 µm spatial resolution over the entire active region of the sensor-array, i.e., over an imaging field-of-view of >8 cm2. Such a wide-field lensless fluorescent imaging platform could especially be significant for high-throughput imaging cytometry, rare cell analysis, as well as for micro-array research.

Abstract 1162: RNA expression analysis of rare tumor cells by laser-mediated lysis

Abstract Molecular analysis of live rare cells, such as circulating tumor cells (CTCs) is challenging. Current strategies include antibody-mediated capture and size selection and are limited in the purity and recovery of the cells of interest. We have developed a novel approach for rare cell analysis based on the specific lysis of targeted cells in a background of a vast majority of other cell using a precisely targeted laser pulse. Instantly lysed cells release their contents into the medium, including mRNA, providing a method to analyze mRNA content in selected cells. Using a high throughput plate cytometer equipped with lasers for in situ cell manipulation (LEAP Cell Processing and Purification Workstation), we have analyzed RNA from individual tumor cells in a model of CTCs consisting of carcinoma cells spiked into human PBMCs. A 5-gene panel was measured by RT-PCR from single tumor cells and was detected in 94% of the cases with 0% false positives. RNA analysis of single tumor cells via laser-mediated lysis represents a new principle for the analysis of rare cells, as the specificity of analysis is dependent on imaging and laser targeting only and free of any limitations imposed by an antibody- or size-dependent purification strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1162.

Automatic retrieval of single microchimeric cells and verification of identity by on‐chip multiplex PCR

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.

Microfluidic stochastic confinement enhances analysis of rare cells by isolating cells and creating high density environments for control of diffusible signals.

Rare cells can be difficult to analyze because they either occur in low numbers or coexist with a more abundant cell type, yet their detection is crucial for diagnosing disease and maintaining human health. In this tutorial review, we introduce the concept of microfluidic stochastic confinement for use in detection and analysis of rare cells. Stochastic confinement provides two advantages: (1) it separates rare single cells from the bulk mixture and (2) it allows signals to locally accumulate to a higher concentration around a single cell than in the bulk mixture. Microfluidics is an attractive method for implementing stochastic confinement because it provides simple handling of small volumes. We present technologies for microfluidic stochastic confinement that utilize both wells and droplets for the detection and analysis of single cells. We address how these microfluidic technologies have been used to observe new behavior, increase speed of detection, and enhance cultivation of rare cells. We discuss potential applications of microfluidic stochastic confinement to fields such as human diagnostics and environmental testing.

A prospective pilot study of circulating endothelial cells as a potential new biomarker in portal hypertension

Peripheral circulating endothelial cells (CEC) have been proposed as a prognostic marker in cardiovascular diseases. Cirrhosis and portal hypertension are associated with vascular injury yet little is known about CEC count in these conditions. Therefore, we evaluated CEC count in patients with cirrhosis, and correlated it with markers of portal hypertension/disease severity. Fifteen patients with cirrhosis/portal hypertension and 15 matched controls were prospectively recruited for study participation. An automated rare cell analysis system was used to enumerate CEC from peripheral blood and correlated with clinical features. Median CEC levels were significantly higher in patients with cirrhosis as compared with controls (median [interquartile range (IQR)]; cirrhosis: 73.7 cells/4 ml [53.7-140.3]; controls: 28.7 cells/4 ml [21-58.7]; P=0.021). Ratio of CEC to platelet count (CEC/PC) also distinguished patients with cirrhosis from controls (IQR; cirrhosis: 0.723 [0.396-1.672]; controls: 0.126 [0.103-0.333]; P<0.001). Receiver operator characteristic analysis revealed that CEC cut-off of 42 cells/4 ml showed sensitivity of 87% and specificity of 74% for differentiating cirrhosis from controls (AUC: 0.74), while CEC/PC ratio at 0.21 showed sensitivity of 100% and specificity of 73% (AUC: 0.89). Furthermore, CEC/PC index was significantly elevated in patients with hepatic decompensation as defined by Child B/C (P<0.05). The intra- and interobserver variability correlation coefficients for CEC measurement were 0.9989 and 0.9986 respectively. Median CEC count and CEC/PC ratio are significantly elevated in patients with cirrhosis, with CEC/PC also increased in patients with decompensated cirrhosis. These data provide rationale for larger validation studies to assess if CEC may have prognostic utility in patients with cirrhosis and portal hypertension.

Sorting of Leishmania-bearing dendritic cells reveals subtle parasite-induced modulation of host-cell gene expression

Once in the mouse skin, Leishmania (L) amazonensis amastigotes are hosted by professional mononuclear phagocytes such as dendritic cells (DCs). When monitored after parasite inoculation, the frequency of amastigote-hosting DCs is very low (<1%) in both the skin and skin-draining lymph nodes. Therefore, we designed and validated an efficient procedure to purify live amastigotes-hosting DCs with the objective to facilitate quantitative and qualitative analysis of such rare cells. To this end, a L. amazonensis transgenic parasite expressing DsRed2 fluorescent protein was generated and added to mouse bone marrow-derived DC cultures. Then, a high speed sorting procedure, performed in BSL-2 containment, was setup to pick out only DCs hosting live amastigotes. This study reveals, for the first time, a unique transcript pattern from sorted live amastigotes-hosting DCs that would have been undetectable in unsorted samples. It was indeed possible to highlight a significant and coordinated up-regulation of l-arginine transporter and arginase2 transcripts in Leishmania-hosting DCs compared to un-parasitized DCs. These results indicate that arginine catabolism for polyamine generation is dominating over L-arginine catabolism for NO generation. In conclusion, this approach provides a powerful method for further characterisation, of amastigote-hosting DCs in the skin and the skin-draining lymph nodes.

PO8-184 CIRCULATING STROMAL COLONY-FORMING UNITS AND CORONARY ATHEROSCLEROSIS

To develop a reliable technique to enrich for rare cells in blood suspensions using only negative selection steps including a flow-through immunomagnetic cell separations system and by optimizing variables normally encountered during such enrichment processes.A human breast cancer cell line was cultivated and spiked at a ratio of 1 cancer cell to 105 total leukocytes in buffy coat or 1 cancer cell to 108 total cells in whole blood samples. The final, optimized process consisted of: a red cell lysis step, immunomagnetically staining leukocytes with an anti-CD45 PE, anti- MACS sandwich, immunomagnetic sorting using a flow-through system (QMS), and a final cell analysis step using either an automated cell counter, filtration, and visual counting or a cytospin analysis.The final, optimized process produced a final enrichment of the rare cancer cells of 5.17 log10 and an average, final recovery of 46%. It should be noted that a negative depletion protocol was used (i.e., no labeling of the rare cancer cells was used).To the authors' knowledge, no examples in the literature exist of a 5.17 log10 enrichment of cancer cells in human blood using a negative depletion protocol. The closest example is a 4 log10 enrichment in which two positive magnetic cell separation steps were used (none were used in this study). Ongoing studies are investigating further modifications of the precommercial, prototype flow-through immunmagnetic separation system to increase both the enrichment and recovery rate. However, even at current performance levels, the presented process could significantly improve visual and molecular analysis of rare cells in blood.

Endothelial cells in peripheral blood of healthy subjects and patients with metastatic carcinomas

A lack of standardized assays and consensus of cell definition has lead to a wide variation in the reported range of circulating endothelial cells (CECs). An automated rare cell analysis system was used to enumerate nucleated, CD146+/CD105+/CD45- CECs in 4 mL of blood. Recoveries of spiked HUVECs were linear over a range of 0-1,241 cells (R2>or=0.99) with recoveries of >or=70% at each spike level. Correlation coefficient values for interoperator variability and duplicate sample variation were (R2=0.99 and 0.90), respectively. Correlation of CEC counts between tubes 1-2 and 2-3 drawn from the same subject in sequence differed (R2=0.48 and 0.63, respectively). The normal CEC reference range established in 249 healthy donors was 1-20 CECs/mL blood. CEC counts were significantly higher in the 206 metastatic carcinoma patients (P<0.0001). CECs can be accurately and reproducibly enumerated in blood and are elevated in metastatic carcinomas compared with healthy donors. Phlebotomy procedures can affect endothelial cell counts.

Enrichment of rare cancer cells through depletion of normal cells using density and flow-through, immunomagnetic cell separation

To develop a reliable technique to enrich for rare cells in blood suspensions using only negative selection steps including a flow-through immunomagnetic cell separations system and by optimizing variables normally encountered during such enrichment processes. A human breast cancer cell line was cultivated and spiked at a ratio of 1 cancer cell to 10(5) total leukocytes in buffy coat or 1 cancer cell to 10(8) total cells in whole blood samples. The final, optimized process consisted of: a red cell lysis step, immunomagnetically staining leukocytes with an anti-CD45 PE, anti- MACS sandwich, immunomagnetic sorting using a flow-through system (QMS), and a final cell analysis step using either an automated cell counter, filtration, and visual counting or a cytospin analysis. The final, optimized process produced a final enrichment of the rare cancer cells of 5.17 log(10) and an average, final recovery of 46%. It should be noted that a negative depletion protocol was used (i.e., no labeling of the rare cancer cells was used). To the authors' knowledge, no examples in the literature exist of a 5.17 log(10) enrichment of cancer cells in human blood using a negative depletion protocol. The closest example is a 4 log(10) enrichment in which two positive magnetic cell separation steps were used (none were used in this study). Ongoing studies are investigating further modifications of the precommercial, prototype flow-through immunmagnetic separation system to increase both the enrichment and recovery rate. However, even at current performance levels, the presented process could significantly improve visual and molecular analysis of rare cells in blood.

Automated detection of immunofluorescently labeled cytomegalovirus-infected cells in isolated peripheral blood leukocytes using decision tree analysis.

Cytomegalovirus (CMV) infection continues to be a major problem for immunocompromised patients. Detection of viral antigens in leukocytes (antigenemia assay) is widely used for the diagnosis of CMV infection and for guiding antiviral therapy. The antigenemia technique, contingent upon the manual microscopic analysis of rare cells, is a laborious task that is subject to human error. In this study, we combine automated microscopy with artificial intelligence for reliable detection of fluorescently labeled CMV-infected cells. Cytospin preparations of peripheral blood leukocytes were immunofluorescently labeled for the CMV lower matrix phosphoprotein (pp65) and scanned in the Rare Event Imaging System (REIS), a fully automated image cytometer. The REIS detected potential positive objects and digitally recorded 49 measured cellular features for each identified case. The measurement data of these objects were analyzed by the See5 decision tree (DT) algorithm to ascertain whether they were true-positive detections. The DT was built from the measurement data of 2,047 true- and 2,028 false-positive detections, collected from 32 patient samples. By designating misclassifications of false-negatives three times more costly, the 10-fold cross-validation sensitivity, specificity, and misclassification error of the assay was 94.3%, 56.2%, and 25%, respectively. The method was also validated using an independent test set of 21 patient samples, in which similar results were obtained. To our knowledge, this study represents the first attempt to improve the accuracy of rare event image cytometry through the implementation of artificial intelligence methodology. Results suggest that cost-sensitive decision tree analysis of digitally measured cellular features vastly improves the performance of rare event image cytometry.

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

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 Balazs, Gyula Hadlacky, Bela Molnar, Laszlo G. Puskas, Janos Szollosi, and Gyorgy 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 Balazs and Attila Tarnok) 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, Janos Fent, Agnes Nagy, and Furesz Jozsef, 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 Istvan Nagy, Eva Kurucz, and Istvan 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 Szlavik, Zsuzsanna Kornyei, and Emilia Madarasz, 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.

Precious cells contain precious information: strategies and pitfalls in expression analysis from a few cells.

Expression analysis, often encompassed in the term "functional genomics," is the link between physiology and molecular biology. Often, specific physiological changes in plant development are due to a limited number of genes, expressed exclusively in very few cells of an organ or organism. Compounding the situation, these physiological changes may also be transient. Therefore, searching for the responsible genes, though exciting and necessary to understand important processes, is hindered primarily by the scarcity of "precious" cells in the desired physiological state. Used judiciously, molecular methods such as reverse transcription polymerase chain reaction (RT-PCR), microarray analysis, or subtractive hybridization allow analysis of rare or special cells. Each of these methods has its advantages and pitfalls. Working with precious cells entails special biological strategies to avoid excessive work in obtaining the data and misinterpretation of it. To illustrate the logic and methods involved in working with precious cells-tissues, we describe how subtractive hybridization followed by expressed sequence tag (EST) sequencing can be used to search for a few genes specific to a few available cells.

Commercial high speed machines open new opportunities in high throughput flow cytometry (HTFC)

Two recent events have opened a new domain of flow cytometry applications which we term high throughput flow cytometry (HTFC). The release of a commercial high speed sorter in 1994 placed HTFC within the reach of anyone who could buy one of the new machines and not just the handful of advanced laboratories worldwide that had custom built their own high speed sorters. The advent in 1999 of HTFC analysis capabilities of 100 000 cells/s marks the second stage in this enabling of HTFC. We describe the technical basis of HTFC. The commercial high speed sorters measure cells in dead-times three to six times shorter than conventional machines. They can sort with high yield and high purity at rates from 25 000 to 60 000 cells/s, depending on their settings, mainly by virtue of their use of high drop creation rates 100 000 drops/s or more. Finally, one series can analyse the measured cells at rates exceeding these sort-rates and at least six times faster than conventional sorters could. The performance of the systems made by the three manufacturers can be readily assessed for single laser systems. Comparison becomes difficult for multiple beam machines, due to requirements for multi-beam sampling for each cell and due to the demands of fluorescence compensation between signals from one laser and between signals from two or three lasers. Applications are described in the field of rare cell analysis and isolation as well as from sorting of abundant cell populations.