automated slide analysis

1: Automated Slide Analysis In the following presentation I will recall some basic aspects of automatic scanning in cytogenetics. I will begin with the application of metaphase finding and at the end I will briefly touch rare cell scanning for the assessment of minimal residual disease. I will show you metaphase finding and rare cell scanning software in action. Those of you who are interested to see a more detailed demonstration with not only the computer but also the new Axioplan2 Imaging microscope in action are cordially invited to visit our booth in the exhibit area any time during the conference. In the following presentation I will recall some basic aspects of automatic scanning in cytogenetics. I will begin with the application of metaphase finding and at the end I will briefly touch rare cell scanning for the assessment of minimal residual disease. I will show you metaphase finding and rare cell scanning software in action. Those of you who are interested to see a more detailed demonstration with not only the computer but also the new Axioplan2 Imaging microscope in action are cordially invited to visit our booth in the exhibit area any time during the conference.

Slide 2:There are two situations that call for automatic scanning because manual scanning is tedious, time consuming and error-prone: A) very few objects are available for analysis and it is essential to find as many as possible, ideally all of them. This may be the case in tumor cytogenetics, where a slide may contain only very few metaphases or in the assessment of the occurrence rate of residual tumor cells or micrometastases in bone marrow or peripheral blood. B) if a statistical analysis of many objects has to be performed. Examples are aberration scoring in metaphases eg. in toxicology or radiation biology, counting of FISH spots in interphase nuclei, and detection of co-localisations indicating a particular translocation (e.g. brc/abl probes for the detection of the Philadelphia chromosome in chronic myeloid leukemia).There are two situations that call for automatic scanning because manual scanning is tedious, time consuming and error-prone: A) very few objects are available for analysis and it is essential to find as many as possible, ideally all of them. This may be the case in tumor cytogenetics, where a slide may contain only very few metaphases or in the assessment of the occurrence rate of residual tumor cells or micrometastases in bone marrow or peripheral blood. B) if a statistical analysis of many objects has to be performed. Examples are aberration scoring in metaphases eg. in toxicology or radiation biology, counting of FISH spots in interphase nuclei, and detection of co-localisations indicating a particular translocation (e.g. brc/abl probes for the detection of the Philadelphia chromosome in chronic myeloid leukemia).

Slide 4:What are the main components of the scanning system? A motorized microscope, the Axioplan2 mot is required that needs to be equipped with a motorized scanning stage. The standard stage used has a capacity of 8 slides. The microscope is controlled by a computer that does also digitize and analyze the images taken by the CCD camera. For fluorescence scanning the CCD camera needs long time integration capability and the microscope needs to be equipped with a motorized filter turret or excitation filter wheel. What are the main components of the scanning system? A motorized microscope, the Axioplan2 mot is required that needs to be equipped with a motorized scanning stage. The standard stage used has a capacity of 8 slides. The microscope is controlled by a computer that does also digitize and analyze the images taken by the CCD camera. For fluorescence scanning the CCD camera needs long time integration capability and the microscope needs to be equipped with a motorized filter turret or excitation filter wheel.

Slide 7:The fundamental concept of slide scanning is shown on this slide: A microscope slide is scanned in a systematic pattern at relatively low optical magnification. The magnification is a compromise between number of fields to scan and thus speed on one hand and the resolution on the other hand. It is important that the captured fields do slightly overlap in order to avoid gaps which could lead to missing objects. Depending on the specific application and the objects of interest appropriate criteria or classifiers have to be used to detect the objects of interest. To detect metaphases, the software has to analyze the morphology and to find object clusters with a certain number and shape of individual objects. The coordinates of the found objects, measured object features as well as gallery images are recorded during the scan. The fundamental concept of slide scanning is shown on this slide: A microscope slide is scanned in a systematic pattern at relatively low optical magnification. The magnification is a compromise between number of fields to scan and thus speed on one hand and the resolution on the other hand. It is important that the captured fields do slightly overlap in order to avoid gaps which could lead to missing objects. Depending on the specific application and the objects of interest appropriate criteria or classifiers have to be used to detect the objects of interest. To detect metaphases, the software has to analyze the morphology and to find object clusters with a certain number and shape of individual objects. The coordinates of the found objects, measured object features as well as gallery images are recorded during the scan.

Slide 12:Once the scan has been completed, the image gallery is used to reject false positives and to mark objects that seem suitable for analysis (shown here for metaphases captured in transmitted light). With a single mouse click any object can be relocated under the microscope if necessary. For analysis, automatic relocation of the preselected objects is usually done at high optical magnification to capture images of maximum resolution.Once the scan has been completed, the image gallery is used to reject false positives and to mark objects that seem suitable for analysis (shown here for metaphases captured in transmitted light). With a single mouse click any object can be relocated under the microscope if necessary. For analysis, automatic relocation of the preselected objects is usually done at high optical magnification to capture images of maximum resolution.

Slide 13:Once the scan has been completed, the image gallery is used to reject false positives and to mark objects that seem suitable for analysis (shown here for metaphases captured in transmitted light). With a single mouse click any object can be relocated under the microscope if necessary. For analysis, automatic relocation of the preselected objects is usually done at high optical magnification to capture images of maximum resolution.Once the scan has been completed, the image gallery is used to reject false positives and to mark objects that seem suitable for analysis (shown here for metaphases captured in transmitted light). With a single mouse click any object can be relocated under the microscope if necessary. For analysis, automatic relocation of the preselected objects is usually done at high optical magnification to capture images of maximum resolution.

23: Feasibility Study: Automatic HER-2/neu assessment in human breast cancer tissue specimen HER-2/neu (c-erb2) amplification is a prognostic factor in critical stage II, node-positive patients (chemotherapy, Herceptin� treatment: yes/no) HER-2/neu probe: Vysis PathVysion� Kit Detect FISH signals in formalin-fixed, paraffin-embedded breast cancer tissue sections 

24: HER-2/neu : CEP17 ratio: ? 2 HER-2/neu : CEP17 ratio: > 2 not amplified amplified Crititical: HER-2/neu : CEP17 ratio between 1.8-2.2 

Slide 26:Analysed FOVs area search (left), position list search (right). Analysed FOVs area search (left), position list search (right).

Slide 27:Sample #2-C6 with tiles.Sample #2-C6 with tiles.

Slide 28:Sample #6-D1, poor hybridisation quality (�spot background�).Sample #6-D1, poor hybridisation quality (�spot background�).

35: Semi-automatic assessment of FISH signal scoring is feasible in tissue sections The combination of interactive definition of ROIs and �Tile sampling� is a robust approach to overcome the problems associated with FISH in tissue sections Clinical trials for FDA approval are on their way (finalized 2Q 2004). Preliminary results: overall error rate: approx. 2 % Systematic error: 0 % About 20 � 30 % of slides are automatically rejected due to �poor� quality

Slide 40:There are two situations that call for automatic scanning because manual scanning is tedious, time consuming and error-prone: A) very few objects are available for analysis and it is essential to find as many as possible, ideally all of them. This may be the case in tumor cytogenetics, where a slide may contain only very few metaphases or in the assessment of the occurrence rate of residual tumor cells or micrometastases in bone marrow or peripheral blood. B) if a statistical analysis of many objects has to be performed. Examples are aberration scoring in metaphases eg. in toxicology or radiation biology, counting of FISH spots in interphase nuclei, and detection of co-localisations indicating a particular translocation (e.g. brc/abl probes for the detection of the Philadelphia chromosome in chronic myeloid leukemia).There are two situations that call for automatic scanning because manual scanning is tedious, time consuming and error-prone: A) very few objects are available for analysis and it is essential to find as many as possible, ideally all of them. This may be the case in tumor cytogenetics, where a slide may contain only very few metaphases or in the assessment of the occurrence rate of residual tumor cells or micrometastases in bone marrow or peripheral blood. B) if a statistical analysis of many objects has to be performed. Examples are aberration scoring in metaphases eg. in toxicology or radiation biology, counting of FISH spots in interphase nuclei, and detection of co-localisations indicating a particular translocation (e.g. brc/abl probes for the detection of the Philadelphia chromosome in chronic myeloid leukemia).

Slide 41:There are two situations that call for automatic scanning because manual scanning is tedious, time consuming and error-prone: A) very few objects are available for analysis and it is essential to find as many as possible, ideally all of them. This may be the case in tumor cytogenetics, where a slide may contain only very few metaphases or in the assessment of the occurrence rate of residual tumor cells or micrometastases in bone marrow or peripheral blood. B) if a statistical analysis of many objects has to be performed. Examples are aberration scoring in metaphases eg. in toxicology or radiation biology, counting of FISH spots in interphase nuclei, and detection of co-localisations indicating a particular translocation (e.g. brc/abl probes for the detection of the Philadelphia chromosome in chronic myeloid leukemia).There are two situations that call for automatic scanning because manual scanning is tedious, time consuming and error-prone: A) very few objects are available for analysis and it is essential to find as many as possible, ideally all of them. This may be the case in tumor cytogenetics, where a slide may contain only very few metaphases or in the assessment of the occurrence rate of residual tumor cells or micrometastases in bone marrow or peripheral blood. B) if a statistical analysis of many objects has to be performed. Examples are aberration scoring in metaphases eg. in toxicology or radiation biology, counting of FISH spots in interphase nuclei, and detection of co-localisations indicating a particular translocation (e.g. brc/abl probes for the detection of the Philadelphia chromosome in chronic myeloid leukemia).