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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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2. After reviewing the license carefully, select I accept the terms of the license agreement and click

Next. The Choose Destination Location window appears:

If the location displayed is not where you wish to install the algorithm, click Change to choose a new location and then click Next; otherwise, just click Next.

3. On the Ready to Install the Program window, click the Install button. As the installation proceeds, a status bar shows installation progress. When the installation has completed, the final window appears. If this window asks if you want to reboot your computer, select No, skip this step.

4. Click Finish to exit the installer.

If you upgrade an algorithm to a newer version, the algorithm installer will start with a different welcome page that provides you with three options: Modify, Repair and Remove. Select Repair and follow the instructions of the algorithm installer.

16 IHC Membrane Image Analysis User’s Guide Slide Preparation Slide preparation has a considerable influence on how well image analysis algorithms will perform. Optimizing the slide preparation for image analysis is a good idea to get the most out of automatic image analysis. Keep in mind—an algorithm can only be as good as its input data, the slide. Make sure to control the quality of the glass slides!

Tissue Preparation The tissue preparation is important for scan quality. Folds in the tissue and tissue sections that are too thick will result in blurry images. If working with a blurry slide, preparation of a new slide might be required or the regions affected need to be excluded from the analysis.

Mechanics The mechanics of the slide are important for its scan quality and ease of scanning. The slides should be in clean and good condition—no air pockets under cover slip, no dirt, no fingerprints, no markings, no writing, no extra adhesive, no broken slides, no chips, no scratches, no overhanging cover slip, etc. The tissue ideally should be located in the middle of the slide a distance from the edges of the slide, the label and any other markings. It is helpful for the tissue to be placed consistently in the same location and orientation on the slide. Some of the mechanical problems of a slide can be resolved by cleaning the slide with a cotton tissue or trimming the sides with a razor blade. Permanent problems with a slide may require the preparation of a new slide or excluding the regions affected from the analysis.

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Staining Reproducibility of the IHC stains is important for consistent algorithm performance. The algorithm will be set up for a specific staining process, but it is important to make sure that the variations of the staining process are controlled and eliminated to the greatest extent possible. For this reason, we recommend the use of FDA cleared and approved IHC kits and to employ appropriate morphological studies and controls as specified in the instructions for the IHC kits.

Glass Slide Quality Control It is the responsibility of the lab to verify the quality of the tissue preparation, the mechanics of a slide and the staining.

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The imaging system and its calibration as well as the scanning parameters are critical to transforming a glass slide into a high quality digital slide. Scan slides automatically with “one-touch” operation. Make sure to control the quality of the digital slides!

Imaging System The performance of any image analysis system is very dependent on the quality of the images it processes. Aperio’s ScanScope systems are based on Aperio’s patented line-scanning technology which provides superior high-quality, high-resolution digital slides with virtually no artifacts affecting image analysis.

Calibration Color and intensity reproducibility is important for reliable image analysis. The ScanScope systems provide a high degree of color and intensity reproducibility scan after scan and also from one ScanScope to another. The ScanScope calibrates each slide when it is scanned by performing a white balance (called a “prescan”). This is optimum, as the calibration takes into account the characteristics of each slide and the system at the exact moment of the scan and not at the moment in time when a daily or factory calibration was performed. To assure proper operation of the ScanScope for each scan, the ScanScope analyzes the “prescan” of each slide to determine possible variations of the system, like changing characteristics of the light bulb.

Compression Digital Pathology would not be practical without a lossy data compression of the large digital slide images. With Aperio’s ScanScopes, the user can choose between the standard JPEG and JPEG2000 image compression types and quality factors between 0 and 100.

The IHC Membrane Image Analysis algorithm has been designed to be robust with regard to image compression artifacts. Nevertheless, you should decide on one compression type and one quality factor that will be used for the scanning of all IHC slides.

We recommend compressing the images of IHC slides with the standard JPEG2000 compression type and a quality factor of 70.

Aperio’s ScanScopes provide the capability of applying a sharpening filter while the slides are scanned to provide pathologists with enhanced sharpness of the digital slides. Image analysis algorithms work best on raw imagery; therefore the sharpening filter should be disabled during scanning if the digital slides are intended to be used for image analysis.

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Resolution for IHC Slides Pathologists using a standard microscope typically view IHC slides with a 20X objective which is required to view all relevant cell features for their IHC scoring. The ScanScopes provide slide scanning with 20X and 40X objectives.

The IHC Membrane Image Analysis algorithm is analyzing the same cell features as assessed by the pathologists and therefore requires the same image resolution as used by the pathologists. Although scanning with a 40X objective provides more details, it also requires longer scan times and larger file sizes.

We recommend scanning IHC slides with a 20X objective.

Scanning Parameter Setting It is important that all IHC slides are scanned with the same parameter setting. An IHC scanning parameter set can be set up in the controller XML file to help the operator to make sure that all IHC slides are scanned with the same parameter settings.

Automatic “One-Touch” Scanning Aperio’s ScanScopes can scan slides automatically using “one-touch” operation. The ScanScopes provide a quality factor for each scan from 0 (worst) to 100 (best). Note that a poor quality factor can be caused by poor slide preparation, like dirt, but that the scan of the tissue still might be good—therefore the operator should not rely on the quality factor alone to determine the quality of a scan; he/she may want to use the quality factor as a triage tool to determine which slide he/she wants to review. Poor slide preparation leads to poor image quality which in some cases can be improved by manually scanning the slide; this allows the operator to select the tissue region to be scanned, position the “pre-scan” and position and manually focus the focus points.

Digital Slide Quality Control It is the responsibility of the ScanScope operator to verify that the entire tissue has been scanned and that the entire tissue is in focus.

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The IHC Membrane Image Analysis algorithm needs to be set up for its specific application by tuning its input parameters. Specific applications may have different tissue types, staining processes and/or scoring standards. In the setup, we distinguish between cell feature detection parameters and scoring scheme parameters.

Parameter tuning must be done by a skilled user knowledgeable in image analysis and the biochemistry of the clinical application. If you want help with the setup of the algorithm, please contact Aperio for image analysis services.

Labs use different IHC reagents and kits (for example: Dako and Ventana) with different staining characteristics. Different labs also have slightly different IHC staining processes with different staining characteristics. Aperio’s Image Analysis algorithms provide an automated algorithm training feature that optimizes the algorithm parameters for specific staining characteristics and scoring standards. Keep in mind that to assure the accuracy of the IHC Image Analysis algorithms, the lab needs to follow the quality control instructions recommended in the manufacturer’s labeling of the IHC reagent or kit.

If changes are made in the staining procedure, repeat tuning and validation of the algorithm may be necessary.

Data Sets The tuning of the algorithm should be based on a training data set that is representative for the specific application you want to set up the algorithm for—tissue type, staining process and scoring standard. We recommend using a minimum of twenty slides with about equal distribution in the HER2 score classes: 0, 1+, 2+ and 3+.

Scoring Standard As there is no gold standard for IHC scoring, there are different ways you might want to define a scoring standard for the setup of your algorithm: (1) scores from a panel of three pathologists, (2) a consensus score by a panel of pathologists or (3) a score based on longitudinal studies relating to the clinical outcome.

The following shows an example of how to set up the algorithm with a scoring standard based on three pathologists—the most complex in terms of setting up the training data set, as three pathologists need to score the same slides independently from each other. Scoring standard training requires both a manual read of the glass slide as well as a read of the digital slide.

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Identify Slides for the Training Data Set First, you need to identify slides that you would like to use for the training data set. For example, in a clinical environment where pathologists review digital slides routinely, pathologists could identify appropriate glass slide by the label and digital slides by their Filename and Date. This information is easily accessible in ImageScope when viewing a digital slide by selecting Information… in the Image drop-down menu or by clicking on the information icon. Filename and Date are provided on the Information tab and the label under the Label Image tab.

Manual Read of Glass Slides The digital slide scores and image analysis results need to be related back to manual microscopy scores as those are considered the standard for IHC scoring. Have the three pathologists independently score and record their reads of the corresponding glass slides using a microscope.

Gather Digital Slides for the Training Data Set Navigate to the directory where the digital slides reside. Typically, the ScanScope creates the digital slides after scanning directly in a sub-folder that has the Date as folder name in the D:\Images directory on the ImageServer.

To separate the training data set from the clinical operation, you need to create a copy of the digital slides.

If you use multiple pathologists to provide blind scores for the scoring standard you need to create a copy of the training data set for each pathologist—in our example, we therefore need three copies.

To organize the digital slides for the training data set you could create subfolders in the D:\Images directory. You could have one sub-folder that identifies the specific application and identifies in the folder name the stain, the manufacturer and tissue type (e.g., HER2_Dako_Breast, HER2_Ventana_Breast). In this subfolder you could then have a separate subfolder for each copy of the training data set (e.g., Training1, Training2, Training3).

Organize Training Data Sets in Spectrum Information Manager and Manage Access Rights The administrator (or any user with administrative rights) first makes sure that restricted access rights are set up to protect all data related to the setup of the algorithm and to limit the access to specific data sets to

individual pathologists. Spectrum Information Manager uses a three-step process to set up access rights:

(1) create data groups, (2) grant individual users access to data groups and, (3) associate data to data groups.

We need one data group for each specific application of the algorithm and individual data groups for each participating pathologist. Most likely the data groups for the pathologists have already been created if they are users of Spectrum Information Manager. The administrator logs into Spectrum Information Manager and navigates to the Data Groups view by clicking Data Groups in the Administrative dropdown menu. The administrator can create a new data group by clicking Create Group in the Data Groups view and providing a Data Group Name and Data Group Description. For a specific application data group, use a Data Group Name that identifies the stain, the manufacturer and tissue type and use Algorithm Setup and Validation as Data Group Description.

22 IHC Membrane Image Analysis User’s GuideChapter 6 – Algorithm Setup

Spectrum Information Manager can grant each user specific access rights to any data group. All participating pathologists should be users in Spectrum Information Manager and have full access rights to the specific application data group and their individual data group. Once the setup and/or validation has been completed, access rights to the specific application data groups can be restricted to Read Only or No Access to make sure that no data can be modified. If you want other pathologists or customers to be able to view the data used for the setup and/or validation you can provide them Read Only access rights.

The administrator navigates to the Users view by clicking Users in the Administrative drop-down menu.

To set up a new user, click Create User in the Users view and provide a Login and Password. When you click Edit for a user, the user view will list all defined data groups. The administrator sets the access level for each of the pathologists to Full Control for their own data group and the data group for the setup of the specific application they are participating in. Save settings when done.

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