Signal Analysis

Automation of FISH Spot Counting

Unattended and automated analysis of fluorescence signals, signal fusions, and spot patterns.

The analysis of fluorescence signal patterns / FISH spots in cells or cell nuclei is the basis for many assays in hematology and cancer genetics. With the automated fluorescence signal analysis system based on the Metafer platform (MetaCyte), these patterns are analyzed automatically, precisely, and reproducibly. Results are stored together with the cell or nuclei images, and they can be displayed in the image gallery, in histograms and scatter plots, or as a summary table in one of Neon's beautiful reports.

The system's ultra-flexible architecture allows for addressing principally any imaging task. Signal channels can be combined to an assay, and the images obtained in each channel can be processed individually. Morphology of target cells, nuclei, or other objects can be precisely defined with a number of cell selection parameters. More than 300 features of objects (e.g., of area, shape, intensity, and signal distribution) can be measured. The results of such a measurement can recursively be used to trigger other features, so that extremely sophisticated analyses can be done for each single image. All the parameters are then stored within a classifier, setting the scoring standard for many future experiments. Once created, a classifier can be selected with some simple mouse clicks or even by triggers from outside (e.g., from a LIMS or a bar code).

The powerful tools of Metafer help providing image analysis solutions in many fields of work. Toxicologists, microbiologists, pathologists, hematologists, and many other researchers and clinicians worldwide rely on the outstanding characteristics of the system.

Rapid Scoring

Metafer does a lot more than simply counting signals: for example it is also able to identify and count fusions of signals from different color channels. In this way the software has the capability to analyze all signal patterns found in samples hybridized with MetaSystems Probes locus-specific probes. If it comes to automation of signal analysis, it often appears that human scorers pre-select cells they can easily interpret, while tending to ignore the difficult-to-interpret cells. The automated scanning system provides unbiased interpretation, scoring many cells that are often ignored via manual spot counting. Therefore, many labs are used to working with very low abnormal cut off values for positivity during manual observation. As a result of these discrepancies between automated and manual scoring strategies, people spend significant time to correct automated results, or to reject cells they do not like. With Metafer this can be different: MetaSystems created Rapid Scoring (RS), a new method of analyzing FISH signals which combines manual scoring strategies with the advantages of automated scoring. RS uses Metafer to automatically score cell signal patterns. All analyzed cells are displayed in a gallery. Signal patterns are summarized in a convenient graph and a table. Immediately after the scan technicians can begin categorizing cells with doubtful results. Categories are assigned to each cell using an external keypad, and new categories (groups of cells) can be added any time. An empty scoring sheet displayed left of the automated results dynamically displays the manual results while the categorization is proceeding. An enlarged image of the unprocessed cell in question facilitates evaluation of signal patterns.

Tissue FISH

Metafer is very strong in the analysis of FISH patterns in single cell nuclei, but it can do a lot more than this. Fluorescence signals in tissue sections, for instance, can be easily analyzed with the dedicated software interface of Metafer.

First of all, multiple options for pre-scans can be used to provide sample overviews. Pre-scans could even be done on a separate sample with a subsequent, H&E stained tissue section. Regions of interest can be marked in the tissue viewing software, and a convenient tool to edit tissue micro array (TMA) patterns facilitates high-throughput analyses.

Once images of selected regions in the tissue or the TMA are acquired automatically, they can be loaded into the dedicated user interface. Cells of interest are quickly selected with pen or mouse. On selection all FISH signals in the selected cell/nucleus are automatically analyzed. Analysis can encompass counting of signals, but also the detection of fused signals, a quantification of intensities, the identification of split signals and much more. Results for each nucleus are shown together with a gallery image, thus offering the possibility to immediately check and correct the data. The configurable data histogram is updated with each nucleus that is added to the gallery. After finishing analysis, data can be summarized and printed.

Though nucleus selection is done very conveniently in the software interface, special algorithms are available for automated nucleus segmentation. Pre-segmented nuclei can quickly be included in the analysis using a pen tip or a mouse click. A toolkit of dedicated image processing operations offers image enhancement prior to analysis. It is possible to quickly switch between the original and the processed image. Due to this functionality, Metafer has been successfully applied to the diagnosis of solid tumors, no matter whether based on conventional tissue sections or on TMA preparation.

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