Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization. Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments. Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images. However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes. Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods. Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins. Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells.

We combined laser-assisted microdissection from H&E-stained paraffin sections, degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR), and comparative genomic hybridization (CGH) to analyse chromosomal imbalances in small tumour areas consisting of 50-100 cells. This approach was used to investigate intratumour genetic heterogeneity in a case of metastatic prostatic adenocarcinoma and chromosomal changes in areas of prostatic intraepithelial neoplasia (PIN) adjacent to the invasive tumour. In four microdissected invasive tumour areas with different histological patterns (acinar, cribriform, papillary and solid) marked intratumour heterogeneity was found by CGH. Recurrent chromosomal imbalances detected in at least two microdissected tumour areas were gains on 1p32-->p36, 2p22, 3q21, 7, 8q21-->q24, 11q12-->q13, 16p12-->p13, 17, 19 and loss on 16q23. Additional chromosomal changes were found in only one of the microdissected areas (gains on 16q21-->q23, 20q22 and losses on 8p21-->p23, 12p11-->q12, 12q21-->q26, 13q21-->q34, 16q12, and 18q22). In PIN, gains on chromosomes 8q21-->q24 and 17 were found in both samples investigated (low and high grade PIN), while gains on chromosomes 7, 11q, 12q, 16p, and 20q and losses on 2p, 8p21-->p23, 12q were found only in one PIN area. Controls to ensure reliable CGH results consisted in CGH analyses of (i) approximately 80 microdissected normal epithelial cells, which showed no aberrations after DOP-PCR and (ii) larger cell numbers (approximately 10(5) or 10(7) cells) of the primary tumour investigated without DOP-PCR and partially displaying the chromosomal imbalances (gain on 16p12-->p13, losses on 2p25, 8p21-->p23, 12p11-->p12, 12q21-->q26, 18q22) found in the small microdissected areas. Microsatellite and FISH analyses further confirmed our CGH results from microdissected cells. The combined approach of laser-assisted microdissection, DOP-PCR and CGH is suitable to identify early genetic changes in PIN and chromosomal imbalances associated with the particular histological patterns of invasive prostatic adenocarcinoma.

Background: For chronic myeloid leukemia, the FISH detection of t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes is an alternative method to bone marrow karyotyping for monitoring treatment. With automation, several drawbacks of manual analysis may be circumvented. In this article, the capabilities of a commercially available automated image acquisition and analysis system were determined by detecting t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes. Methods: Three peripheral blood samples of normal adults, 21 samples of CML patients, and one sample of a t(9;22)(q34;q11) positive cell-line were used. Results: Single nuclei with correctly detected signals amounted to 99.6% of nuclei analyzed after exclusion of overlapping nuclei and nuclei with incorrect signal detection. A cut-off value of 0.84 lm was defined to discrimi-

To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations. Key terms: automatic; quantification; telomere; FISH; microscopy; fluorescence; ALT; senescence Monitoring of telomere length regulation has become an important aspect of stem-cell research, studies on cellular senescence, and cancer research. A number of techniques have been developed to study telomere length in a quantitative manner. The standard procedure is Southern blot hybridization (1). The quantitative fluorescence in situ hybridization (Q-FISH) on metaphase chromosomes allows the quantification of the telomeric FISH signals on individual chromosomes (2-4). Since fluorescence intensity of the telomeric signals was found to be proportional to the size of telomeric repeats, Q-FISH is now widely used (2). For measuring telomere length in interphase nuclei, flow cytometry or fluorescence microscopy can be applied (5-7). Nonadhesive hematopoietic cells seem to be better suited for flow cytometric analyses than solid tumor cells (8,9). Therefore, for the analysis of nonhematopoietic cells, interphase FISH is the method of choice. However, the fluorescence microscopical method to quantify telomere length in interphase nuclei has so far only been performed manually on a restricted number of cells (6,7). To combine the positive aspects of flow cytometric measurements with the ability to quantify individual nuclei by fluorescence microscopical examination, we took advantage of fluorescence-based automatic microscope (FLAME). Thus, all the advantages of interphase measurements, i.e., the analysis of individual cells and the applicability to nonproliferating cells, can be combined with the analyses of statistically relevant cell populations. To develop a reliable method to automatically quantify telomere length in interphase nuclei, we applied a two-color hybridization assay and measured the fluorescence signals with FLAME. We then described the parameters essential for intra-and interexperimental comparisons. We performed intensity measurements in interphase nuclei and compared the results of single channel measurements of the target probe with the results obtained after introducing an internal reference and performing double channel measurements. We validated the quantitative-FISH on interphase nuclei (IQ-FISH) method by measuring telomere lengths of differe...

Objective: To investigate the feasibility of various molecular forms of hemoglobin as markers for fetal nucleated red blood cells (NRBCs). Methods: The presence of epsilon and gamma globin positive NRBCs was investigated in pure fetal blood and in blood from pregnant women before and after chorion biopsy. Maternal samples were enriched for NRBCs by various conventional methods, including limited enrichment by only positive CD71 selection or single density gradient. We searched for fetal cells on slides by automated scanning. Fetal cells were defined by (1) the presence of epsilon or gamma globin and (2) simultaneously by the presence of a Y chromosome signal. Results: 18 of 25 gamma globin positive cells identified in blood samples after chorion biopsy were chromosome Y signal positive, and 1 cell had two X chromosome signals. 263 of 339 epsilon globin positive cells identified in blood samples after chorion biopsy were hybridized with X and Y chromosome probes. None had two X signals, and 249 were Y positive. In blood samples before chorion biopsy, only 1 epsilon globin positive fetal NRBC and no epsilon globin positive maternal NRBCs were found. Conclusions: Epsilon globin may be specific for fetal NRBCs. Only 1 epsilon globin positive fetal cell was identified in 1 of 12 blood samples before chorion biopsy, representing a total of 182 ml of maternal blood. This suggests that most fetal cells found in maternal blood by fluorescence in situ hybridization methods may not be NRBCs.

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Background: The reliable detection and quantification of gene amplifications is crucial to clinical practice. Although there are different detection techniques, the fluorescence in situ hybridization (FISH) method has become highly accepted over past years because it is a reliable, robust, and quick method. Unfortunately, automatic quantification of gene amplification based on fluorescence intensities has not been possible thus far. Because current spot counting methods are reliable only when analyzing low amplification rates, we attempted to establish another method, i.e., to quantify the intensity of different FISH signals using an automatic fluorescence microscopical device on interphase nuclei: interphase quantitative FISH (IQ-FISH).Methods: We quantified the fluorescence intensities of the differently labeled FISH probes (MYCN and D2Z) hybridized to three different neuroblastoma cell lines, six peripheral blood (PB) samples, 10 spiked PB samples, and nine neuroblastoma samples using the Metafer4 system (MetaSystems, Altlussheim, Germany). To obtain the MYCN copy number per cell, the ratio between the fluorescence intensities of the MYCN gene and reference sequence (D2Z) was calculated. For automatic analysis of the HER-2/neu status in tumor cells, labeled FISH probes specific for HER-2/neu and a chromosome 17-specific probe were hybridized to peripheral blood and tumor specimens and analyzed using the automatic device. Results: When measuring the fluorescence intensity per cell for both probe pairs (MYCN/D2Z and HER-2/17p), amplified and non-amplified cells, showed distinct peaks with only little overlap. Whereas normal cells showed a fluorescence ratio peak for MYCN/D2Z between 200 and 800, cells with MYCN amplification clearly exceeded this ratio value (1000 to 25,000). When mixing a varying number of MYCNamplified cells (range 9 -91%) to normal PB, the spiked tumor cells could be identified. Even one neuroblastoma tumor cell in 1000 mononucleated cells could reliably be detected using our device. In neuroblastoma patient samples, non-amplified cells were distinguished from amplified cells. Automatically and manually counted signals gave matching results in amplified and non-amplified samples. HER-2/neuamplified cells were automatically detected in the breast cancer samples analyzed.Conclusion: The automatic measurement of fluorescence signal intensities not only allows a reliable discrimination between non-amplified and amplified cells but also exact quantification of amplified sequences. This is the prerequisite for the following applications: detection of amplified cells in the bone marrow and second-look specimens; comparison between primary and relapse or pre-and post-chemotherapeutic specimens; detection of tumors with focal gene amplification; and quantification of elimination of amplified gene sequences.

In a large-scale catastrophe, such as a nuclear detonation in a major city, it will be crucial to accurately diagnose large numbers of people to direct scarce medical resources to those in greatest need. Currently no FDA-cleared tests are available to diagnose radiation exposures, which can lead to complex, life-threatening injuries. To address this gap, we have achieved substantial advancements in radiation biodosimetry through refinement and adaptation of the cytokinesis-block micronucleus (CBMN) assay as a high throughput, quantitative diagnostic test. The classical CBMN approach, which quantifies micronuclei (MN) resulting from DNA damage, suffers from considerable time and expert labor requirements, in addition to a lack of universal methodology across laboratories. We have developed the CytoRADx™ System to address these drawbacks by implementing a standardized reagent kit, optimized assay protocol, fully automated microscopy and image analysis, and integrated dose prediction. These enhancements allow the CytoRADx System to obtain high-throughput, standardized results without specialized labor or laboratory-specific calibration curves. The CytoRADx System has been optimized for use with both humans and non-human primates (NHP) to quantify radiation dose-dependent formation of micronuclei in lymphocytes, observed using whole blood samples. Cell nuclei and resulting MN are fluorescently stained and preserved on durable microscope slides using materials provided in the kit. Up to 1,000 slides per day are subsequently scanned using the commercially based RADxScan™ Imager with customized software, which automatically quantifies the cellular features and calculates the radiation dose. Using less than 1 mL of blood, irradiated ex vivo, our system has demonstrated accurate and precise measurement of exposures from 0 to 8 Gy (90% of results within 1 Gy of delivered dose). These results were obtained from 636 human samples (24 distinct donors) and 445 NHP samples (30 distinct subjects). The system demonstrated comparable results during in vivo studies, including an investigation of 43 NHPs receiving single-dose total-body irradiation. System performance is repeatable across laboratories, operators, and instruments. Results are also statistically similar across diverse populations, considering various demographics, common medications, medical conditions, and acute injuries associated with radiological disasters. Dose calculations are stable over time as well, providing reproducible results for at least 28 days postirradiation, and for blood specimens collected and stored at room temperature for at least 72 h. The CytoRADx System provides significant advancements in the field of biodosimetry that will enable accurate diagnoses across diverse populations in large-scale emergency scenarios. In addition, our technological enhancements to the well-established CBMN assay provide a pathway for future diagnostic applications, such as toxicology and oncology.