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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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IHC Membrane Image


User’s Guide

©Copyright 2007 Aperio Technologies, Inc.

Part Number/Revision: MAN-0026, Revision B

Date: January 2, 2007

This document applies to software versions Release 8.0 and later.

All rights reserved. This document may not be copied in whole or in part or reproduced in any other media without the

express written permission of Aperio Technologies, Inc. Please note that under copyright law, copying includes translation

into another language.

User Resources

For the latest information on Aperio Technologies products and services, please visit the Aperio Technologies website at:


Disclaimers This manual is not a substitute for the detailed operator training provided by Aperio Technologies, Inc., or for other advanced instruction. Aperio Technologies Field Representatives should be contacted immediately for assistance in the event of any instrument malfunction. Installation of hardware should only be performed by a certified Aperio Technologies Service Engineer.

ImageServer is intended for use with the svs file format (the native format for digital slides created by scanning glass slides with the ScanScope scanner). Educators will use Aperio software to view and modify digital slides in Composite WebSlide (CWS) format.

CAUTION For research and education uses only, not for use in diagnostic procedures. This product has not been approved or cleared as a medical device by the U.S. Food and Drug Administration. The data and images obtained or viewed using this product are not intended for clinical or diagnostic use.

Trademarks and Patents ScanScope is a registered trademark and ImageServer, TMALab, ImageScope, and Spectrum are trademarks of Aperio Technologies, Inc. All other trade names and trademarks are the property of their respective holders.

Aperio products are protected by U.S. Patents: 6,711,283; 6,917,696; 7,035,478; and 7,116,440; and licensed under one or more of the following U.S. Patents: 6,101,265; 6,272,235; 6,522,774; 6,775,402; 6,396,941; 6,674,881; 6,226,392; 6,404,906; 6,674,884;

and 6,466,690.

Contact Information

Headquarters: European Office:

Aperio Technologies, Inc. Aperio 1430 Vantage Court, Suite 106 3 The Sanctuary Vista, CA 92081 Eden Office Park United States Ham Green Bristol BS20 0DD, UK United States of America Tel: 866-

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IHC Workflow

Who This Guide Is Intended For

Intended Use

Algorithm Description


Access a Digital Slide in SIM (Spectrum Information Manager)

Patient Data Confidentiality

Manual Read of a Slide

Image Analysis in ImageScope

Outline Tumor Regions

Run Algorithm

View Results

Final Score

More Information





Data Sets

Cell Feature Detection Parameters

Scoring Scheme Parameters

Validate Algorithm Setup


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This chapter provides an overview of Aperio’s IHC (ImmunoHistoChemistry) Membrane Image Analysis algorithm.

The following chapters of this manual provide step-by-step instructions for a pathologist on how to use the algorithm as well as detailed information on proper slide preparation and scanning, and the installation, setup and performance validation of the algorithm.

IHC Workflow A lab receives a specimen from a biopsy and prepares a block from which an H&E (Haematoxylin and Eosin) slide is prepared. The typical IHC (ImmunoHistoChemistry) workflow starts with the diagnosis of cancer by a pathologist reviewing the H&E slide.

Once the pathologist has determined that a patient has cancer he/she orders an IHC panel. An IHC panel consists of consecutively cut slides from the same specimen block that are then stained with different IHC stains. A typical IHC panel for breast cancer includes the following IHC stains: HER2 (Human Epidermal growth factor Receptor 2), ER (Estrogen Receptor) and PR (Progesterone Receptor). The pathologist then provides a semi-quantitative assessment of the IHC slides to determine the appropriate treatment option (prognostic).

HER2 is a membrane stain from which the pathologist assesses the intensity and completeness of the membrane staining of the tumor cells and determines a score of 0, 1+, 2+ or 3+ for the slide. HER2 is used specifically to assess Herceptin® (Trastuzumab) as a treatment option and to determine the aggressiveness of breast tumors.

ER and PR are nuclear stains from which the pathologist assesses the staining intensity of the nuclei of the tumor cells and determines the percentage of positive stained nuclei as a percentage between 0 to 100% and the average staining intensity of the positive nuclei as a score of 0, 1+, 2+ or 3+. Different labs use different scoring schemes for the nuclear stains. For example, some labs use only the percentage of positive nuclei as a score and use different cut-off thresholds of 1%, 5% or 10% for the interpretation;

other labs use the percentage of positive nuclei and the average staining intensity of the positive nuclei to calculate a combined score like the Allred Score.

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Who This Guide Is Intended For This guide is a reference document that covers all aspects of using the Aperio IHC (ImmunoHistoChemistry) Membrane Image Analysis algorithm. Different steps of your image analysis workflow may involve different people with different skills and responsibilities. Therefore, the chapters in this guide are organized by function so that a particular reader can go directly to the section of interest

to him or her:

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Intended Use The IHC (ImmunoHistoChemistry) Membrane Image Analysis algorithm is intended to be used as an aid to the pathologists for the assessment of IHC HER2 (Human Epidermal growth factor Receptor 2) stained breast tissue slides.

Although the IHC Membrane Image Analysis algorithm has been optimized for IHC HER2 staining on breast tissue, it is a general membrane algorithm that can be used for other tissue types and membrane stains.


For research and educational uses only, not for use in diagnostic procedures. This product has not been approved or cleared as a medical device by the U.S. Food and Drug Administration. The data and images obtained or viewed using this product are not intended for clinical or diagnostic use.

Algorithm Description Prior to running the algorithm, a qualified pathologist needs to use the ImageScope annotation tools to outline a set of tumor-cell only regions that are representative of the tumor.

The IHC Membrane Image Analysis algorithm detects the membrane staining for the individual tumor cells in the selected regions and quantifies the intensity and completeness of the membrane staining.

Tumor cells are individually classified as 0, 1+, 2+ and 3+ based on their membrane staining intensity and completeness. A tumor cell is classified 1+ when there is only partial membrane staining or weak

2 IHC Membrane Image Analysis User’s GuideChapter 1 – Overview

membrane staining. A tumor cell is classified 2+ when there is moderate and complete membrane staining. A tumor cell is classified 3+ when there is intense and complete membrane staining.

Based on the percentages of 0, 1+, 2+ and 3+ classified cell scores, a slide score of 0, 1+, 2+ or 3+ is determined. The score is 3+ if more than 10% of the cells are stained at the 3+ level. The score is 2+ if more than 10% of the cells are stained at the 2+ and higher levels. The score is 1+ if more than 10% of the cells are stained at the 1+ and higher level. The score is 0 if less than 10% of the cells are stained at the 1+ or higher levels.

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Cell3+ A couple of interesting observations can be made from the examples above: cell 1+ and cell 2+ show that cells with and without nuclei are detected, cell 1+ and cell 2+ show that cells with the same membrane stain intensity can be classified differently based on the membrane completeness, and cell 2+ and cell 3+ show that membranes adjacent to a nucleus as well as membranes a distance from the nucleus are detected.

Pathologists perform a complex analysis of stained cell features when scoring IHC slides. Aperio’s IHC Image Analysis algorithms are designed to process the same cell features (nuclei and membrane) and follow the same scoring schemes as the pathologists when assessing an IHC slide under a microscope.

Being able to do this requires high quality digital slides taken at least with a 20X objective (ScanScopes provide 20X and 40X).

As with any image analysis algorithm, the IHC Membrane Image Analysis algorithm must be set up for its specific application by tuning its input parameters. Specific applications may vary in the tissue type, staining process and/or scoring standard. Cell feature detection parameters and scoring scheme parameters are handled separately. The cell feature detection parameters specify cell feature detection thresholds and methods as well as size and shape constraints of cells to distinguish tumor cells from normal, lymphocyte and stroma cells. The scoring scheme parameters specify the staining intensity and membrane completeness thresholds that determine the individual tumor cell classification. The scoring scheme parameters can be trained automatically by presenting the algorithm with a set of digital slides that have been scored according to a scoring standard (for example: by a panel of pathologists or longitudinal studies with clinical outcome).

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). Once a pathologist has outlined the tumor-cell only regions and runs the algorithm, the algorithm provides a markup image and an annotation window as its outputs.

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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+). The annotation window provides the HER2 score, the percentages of 0, 1+, 2+ and 3+ cells and other image analysis statistics. 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. Providing not only the very coarse HER2 score but also the percentages of 3+, 2+, and 1+ cells gives the pathologist a very detailed assessment of the slide and makes it easy for him/her to identify and assess border line cases.

The performance of the IHC Membrane Image Analysis algorithm should be validated following standard laboratory practices for the specific applications before being used for analysis.

The pathologist using the IHC Membrane Image Analysis algorithm should verify its proper operation on each slide analyzed.

Aperio will continue to expand and improve our suite of IHC Image Analysis algorithms, striving to provide you with the tools you need for the standardization and automation of IHC. Please provide us with your feedback on how we can improve our products to better meet your needs (see “Contact Information” on page ii for information on contacting Aperio).

4 IHC Membrane Image Analysis User’s Guide Analysis Step-by-Step This chapter provides step-by-step instructions on how a pathologist would use the IHC Membrane Image Analysis algorithm as an aid for the semi-quantitative assessment of an IHC HER2 (Human Epidermal growth factor Receptor 2) breast tissue slide when reviewing a case via Spectrum Information Manager (SIM).

Access a Digital Slide in SIM (Spectrum Information Manager) Cases, specimens and digital slides are managed using Aperio’s SIM (Spectrum Information Manager). A pathologist who wants to access a digital slide first needs to log into SIM and navigate to the case and the specimen that shows the list of its associated digital slides (H&E, HER2, ER, PR, …).

1. Please ask your SIM administrator for the SIM web-address, your login name, and a password.

2. Open Microsoft Internet Explorer (or another web viewer like Firefox) and enter the SIM webaddress. SIM will provide you with a login page.

3. Log into SIM by entering your login and password and clicking the Login button.

4. Navigate to the list of cases by clicking List all Cases.

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Patient Data Confidentiality Spectrum Information Manager provides a full security system that ensures that users can only access data they are authorized to see, thus protecting patient data from unauthorized access.

Digital slides and related meta-data reside in the Spectrum Information Manager database. Spectrum Information Manager uses an administrator/user structure that restricts security-sensitive actions to an

authorized administrator. The administrator can:

Set up users with passwords. Users must log into Spectrum Information Manager with their user name and password and be authenticated before they can access data.

Set up data groups that restrict access to specific users.

Set users’ access permissions to grant them full access, read-only access or no access to the defined data groups.

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Manual Read of a Slide Traditionally, the pathologist reads the IHC glass slide manually using a microscope. Aperio provides a digital pathology workflow in which the pathologist can do a manual read of the digital slide.

A good monitor is a recommended investment for any pathologist who uses a computer monitor for the manual read of a digital slide.

We recommend any high quality LCD monitor meeting the following minimum requirements:

Display Type: LCD Screen Resolution: 1680(h) x 1050(v) pixels Screen Size: 24” Color Depth: 24 bit Brightness: 300 cd/m2

Contrast Ratio: 500:1

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