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«IHC Membrane Image Analysis User’s Guide ©Copyright 2007 Aperio Technologies, Inc. Part Number/Revision: MAN-0026, Revision B Date: January 2, ...»

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Stand-alone image analysis systems that work as an adjunct to manual microscopy create extra work for the lab technicians and the pathologists. Aperio’s IHC Image Analysis algorithms are well integrated into the Digital Pathology workflow provided by Aperio’s ScanScope systems and Spectrum software suite.

With the touch of a button, the IHC Image Analysis algorithm can be used while you view the digital slide.

The pathologist views the IHC digital slide in ImageScope. The pathologist might want to view the corresponding H&E slide as well to determine the tumor regions. ImageScope allows viewing the H&E and IHC digital slides simultaneously.

7. Open the digital slide in ImageScope by clicking on its thumbnail in the specimen digital slide list. ImageScope will open and display the digital slide.

8. Use the zooming and panning features of ImageScope to view the digital slide.

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Slide Quality Control Note that the quality of the manual read of a slide and the IHC Image Analysis depends on the laboratory following the quality control instructions recommended in the labeling of IHC assays.

The staining process can yield artifacts that need to be assessed properly by the pathologists. Typical artifacts to consider are: normal epithelium staining, heterogeneous staining, background staining, cystoplasmic staining as well as edge, thermal and crush artifacts. Staining artifacts should be considered carefully and regions with staining artifacts may need to be excluded from the manual read and IHC Image Analysis.

The IHC Membrane Image Analysis algorithm is sensitive to excessive cytoplasmic staining and tissue being out of focus. It is the responsibility of the pathologist to know the limitations of the algorithm and to make sure that only good quality digital slides are used for the IHC Membrane Image Analysis algorithm.

If slides have poor quality, it might be necessary to prepare a new slide or to re-scan the slide.

9. The pathologist assesses the presence of any staining artifacts, in particular cytoplasmic staining, and requests the preparation of a new slide if excessive cytoplasmic staining is observed.

10. The pathologist assesses the focus of the digital slide and requests a re-scan of the slide if the tissue in the tumor regions is out of focus.

It is recommended that the pathologist follows the appropriate instructions in the specific IHC assay and associated scoring guide to perform the manual read of the slide.

11. The pathologist determines the HER2 score based on his/her manual read of the slide.

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Image Analysis in ImageScope

There are three simple steps involved in performing the IHC Membrane Image Analysis on a digital slide:

(1) outline tumor regions, (2) run algorithm, and (3) view results.

Typically, a pathologist performs all three steps in ImageScope after he has done the manual read of the slide. To accommodate different workflows, this process can be broken into separate distinct steps in which the algorithm can be executed from Spectrum Information Manager (batch processing).

Outline Tumor Regions It is up to the pathologist to identify the tumor regions and outline a representative set of tumor-cell only regions for the algorithm to analyze. The pathologist needs to make sure he properly identifies invasive cancer (vs. carcinoma in situ) and does not include regions with staining artifacts in the outlined tumor regions. For the selection of the tumor regions it is important to appropriately (same ratio of tumor cells) represent the heterogeneity of staining of a tumor by providing a sufficiently large number of tumor cells (if possible between 15 and 20 regions and a minimum of about 1000 tumor cells) to maximize robust analysis results. The IHC Membrane Image Analysis algorithm provides only a limited capability to identify tumor cells (vs. normal, lymphocyte or stroma cells); therefore the pathologist should only outline tumor-cell only regions in the tumor regions.

12. The pathologist uses the mouse with the pen tools in ImageScope to outline a representative and extensive set of tumor-cell only regions to be analyzed by the IHC Membrane Image Analysis algorithm. Regions can be outlined anywhere in the slide and at any magnification level (images are processed at 20X).

Here is the list of pen tools in ImageScope (commands written in italic are preferred for fast, single-hand


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Note that the pen tools are not persistent—for each region you want to outline you need to click first on the pen tool of your choice and then outline the region.

Note that if you interrupt the drawing of a region, ImageScope will close the region by connecting the end points (not visible). If this is not what you wanted, then you should delete the current outlined region by clicking on the Delete key and starting over again with this region.

To delete any and not only the last region, you need to open the Annotations window by selecting Annotations Window in the View drop-down menu, select Layer 1 in the Layers area by clicking Layer 1 and selecting the region you want to delete in the Layer Regions area by clicking on the region number.

The selected region will be displayed by ImageScope in the middle of the digital slide view with a black line in the middle of the colored region outline. To delete the selected region, click on the red X icon in the Layer Regions area.

The entire slide is important for the IHC Image Analysis. A prior selection of fields for the image analysis limits introduces bias to the analysis. Aperio’s IHC Image Analysis algorithms do whole slide processing.

The entire digital slide is available for IHC Image Analysis.

Run Algorithm The pathologist uses the IHC Membrane Image Analysis algorithm as a macro, which means that none of the input parameters can be modified by the user. The input parameters have been locked down after successful setup of the algorithm.

The IHC Membrane Image Analysis algorithm should only be applied for the specific application it is set up for. Using an algorithm for a different application, like a different tissue type or including stroma and lymphocyte cells in the tumor regions, can yield meaningless results.

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13. To initiate an analysis job, the pathologist selects the Algorithm Server Job window by going to the View menu and selecting Analysis.

14. The pathologist selects the appropriate algorithm macro for this specific application by clicking on the macro name.

A lab can create multiple macros of the same IHC Membrane Image Analysis algorithm for different applications (for example for HER2 reagents from different vendors like Dako and Ventana).

15. The pathologist chooses if the algorithm should generate a markup image by selecting the Generate Markup Image check box (default: checked).

16. The pathologist starts the execution of the algorithm by clicking the Analyze button.

Digital slides are large images and the analysis of large images requires considerable processing resources. Lack of resources can create problems such as a time delay or lag in the viewing of the digital slides by the pathologist if the images are analyzed on his/her computer. Aperio provides “server-side” processing for its IHC Image Analysis algorithms so that the images are processed where they reside, thereby eliminating the need for moving the images over the network and using the processing resources on the pathologist’s computer. Aperio’s “server-side” analysis also provides a scaleable computer-farm solution that allows easily adding computer resources to the server to meet expanding analysis needs.

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View Results After the analysis is complete, results of the algorithm analysis can be reviewed in the Annotations window.

17. The pathologist views the Annotations window by selecting Annotation Window in the View drop-down menu.

The processing of the image is completed when a new layer (Layer 2 in the illustration above) appears as a child layer to Layer 1 in the Layers area. Layer 1 contains the tumor region outlines provided by the pathologist. Layer 2 contains the algorithm results. To view the algorithm results, select Layer 2.

18. The pathologist selects Layer 2 by clicking Layer 2 in the Layers area.

The IHC Membrane Image Analysis algorithm provides the following results:

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(1) Slide’s HER2 score and percentages of 0, 1+, 2+ and 3+ cells as well as other image analysis statistics (based on all the cells in all the outlined tumor regions) displayed in the Layer Attributes area.

(2) Regions’ HER2 score and percentages of 0, 1+, 2+ and 3+ cells as well as other image analysis statistics for each outlined tumor region are individually displayed in the Layer Regions area.

The other image analysis statistics include: average membrane intensity as intensity value 0 to 255, percentage and number of cells with complete membrane staining, number of 3+, 2+, 1+ and 0 cells and total number of cells.

(1) Markup Image The pathologist can view the image of the digital slides with or without the markup image.

The markup image highlights the detected cell features (black = nuclei and membrane) and the membrane staining which is color-coded according to the cell classification (blue = 0, yellow = 1+, orange = 2+, red = 3+).

19. The pathologist can view a specific outlined tumor region that corresponds to a region in the Layer Regions area by clicking on the Region number.

20. The pathologist views the digital slide with or without the Markup Image by clicking on the eye icon (without) (with markup image) in the Layers area.

Algorithm Quality Control IHC image analysis is an aid to the pathologists. It is the responsibility of the pathologist to verify the proper operation of the algorithm.

It is important that the pathologists feel comfortable with the system but identify when the system might have failed (quality control). Aperio’s IHC Image Analysis algorithms make it easy for the pathologists to follow what the algorithms are doing and how they determined the scores. By providing meaningful output of the different processing steps to which the pathologist can relate intuitively, the same cell features and same scoring scheme used for manual scoring, specifically: (1) a markup image of the detected cell features and classification of each cell (0, 1+, 2+ and 3+) and (2) the calculated percentages of cells per cell class from which the scores are calculated directly.

The pathologist should review the markup image for each of the outlined tumor regions and make sure that the algorithm properly detected and classified the membrane staining for the tumor cells.

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21. The pathologist reviews the markup images of the outlined tumor regions and verifies the proper operation of the IHC Membrane Image Analysis algorithm.

If there are regions where the algorithm did not perform properly, then those regions need to be excluded from the analysis. To delete regions from the analysis those regions need to be deleted from Layer 1 and the analysis needs to be re-run on Layer 1 creating a new child layer with the Image Analysis results. You may want to delete the old Image Analysis result layer Layer 2 before re-running the analysis. Deleting regions may require adding new regions to make sure that the set of tumor regions used for the analysis is representative for the tumor. If it is impossible to outline a representative set of tumor regions where the algorithm performs properly, then the IHC Membrane Image Analysis should not be used for this particular slide. Please let us know if you have slides on which the IHC Membrane Image Analysis unexpectedly does not work.

Final Score The IHC Membrane Image Analysis is intended to be used as an aid to a pathologist. It is the responsibility of the pathologist to provide the final score based on his/her qualitative assessment and the quantitative information provided by the IHC Membrane Image Analysis algorithm.

22. The pathologist determines the final HER2 score.

New scoring schemes like the H-Score have been introduced that are based on the same calculated percentages of cells per cell class as the traditional IHC scores, but provide a finer quantization. Aperio’s IHC Image Analysis algorithms provide those calculated percentages of cells per cell class as well and thereby support new scoring schemes including the H-score.

23. When you are done with ImageScope, please exit (go to the File menu and select Exit).

24. When you are done with reviewing cases in SIM, please log out from SIM (go to the Logout menu) and exit Microsoft Internet Explorer (go the File menu and select Exit).

More Information The IHC Membrane Image Analysis algorithm is typically used from within ImageScope, but can also be used from Spectrum Information Manager or TMALab (Tissue Micro Array).

For more details on how to run Aperio algorithms, see:

Spectrum Information Manager Operator’s Guide.

Spectrum Information Manager Administrator’s Guide.

ImageScope User’s Guide.

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This chapter provides instructions on how to install the IHC Membrane Image Analysis algorithm.

Installing an algorithm is a simple matter of using Windows Explorer to navigate to the location of the algorithm installer file and double-clicking the file name. This file may be given to you on a CD or your network administrator may have placed it on your network.

You need to install the algorithm on your ImageServer and locally on the desktops of all users who want to be able to modify the algorithm parameters and set up the algorithm. Make sure you have installed the same version of the algorithm on the server and on all desktops.

Follow the instructions of the algorithm installer. After starting the algorithm installer, you see the

welcome window:

1. On the main installer screen, click Next. A window displaying the Aperio user license appears.

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