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Area Quantification FL

Algorithm

Users Guide

Copyright 2011-2013 Aperio Technologies, Inc.


Part Number/Revision: MAN-0175, Revision B
Date: 1 November 2013
This document applies to software versions Release 11 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:
http://www.aperio.com.

Disclaimers

Use normal care in maintaining and using the Spectrum servers. Interrupting network connections or turning off the Spectrum and
DSR servers while they are processing data (such as when they are analyzing digital slides or generating an audit report) can result
in data loss.
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.

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; 7,116,440; 7,257,268; 7,428,324; 7,457,446; 7,463,761;
7,502,519; 7,518,652; 7.602.524, 7,646,496; 7,738,688 and licensed under one or more of the following U.S. Patents: 6,101,265; 6,272,235;
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Contact Information
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Direct International Tel: 1 (760) 539-1150
US/Canada/Worldwide Email:
support@aperio.com

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Area Quantification FL Algorithm Users Guide

Contents
CHAPTER 1- INTRODUCTION ........................................................................ 5
The Area Quantification FL Algorithm ..................................................................5
Prerequisites ............................................................................................................6
For More Information .............................................................................................6
Intended Use ...............................................................................................................7
CHAPTER 2 - QUICK REFERENCE .................................................................... 9
Algorithm Input Parameters ................................................................................... 11
Setting Thresholds ................................................................................................ 11
Colocalization Parameters ................................................................................... 12
Algorithm Results .................................................................................................... 13
Understanding Intensity Results ........................................................................ 14
Pearson and Overlap Coefficients ...................................................................... 15
CHAPTER 3 - SAMPLE ANALYSIS .................................................................. 17
Sample Mark-up Images ......................................................................................... 20
For More Information .............................................................................................. 22

Area Quantification FL Algorithm Users Guide

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Contents

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Area Quantification FL Algorithm Users Guide

Introduction
This chapter introduces the Area Quantification FL
algorithm. For general information on using an algorithm,
please see the Aperio Image Analysis Users Guide.
The process of analyzing digital images begins with the ScanScope, which creates
digital slides by scanning glass slides. Using Aperio image analysis algorithms to
analyze digital slides provides several benefits:

Increases productivity Computer-based image analysis automates


repetitive tasks and answer questions that are beyond the capabilities of
manual microscopy.

Provides unbiased assessment of the distribution and relationship of one


or more fluorescent biomarkers through out an entire tissue section, as
well as in a user-selected region of interest.

Workflow integration The Spectrum digital pathology information


management software suite integrates image analysis seamlessly into
your digital pathology workflow, requiring no additional work by the
researcher. With the click of a button, the algorithm is executed while
you review the digital slide.

The Area Quantification FL Algorithm


The Area Quantification FL algorithm quantifies multiple fluorescent dyes in the
digital slide and is the professional version of the Positive Pixel Count FL
algorithm. The Area Quantification FL algorithm also supports result plots.
Common applications for this algorithm are:

Area and area fraction analysis (for example, analyzing fluorescence


digital slides showing amyloid plaque, myelin, nuclei, and so on).

Colocalization for gene and protein expression.


This algorithm can be used with fluorescence digital slides created by the Aperio
ScanScope FL. It can also be used on non-Aperio fluorescence images that can be
opened by the ImageScope digital slide viewer. For Aperio ScanScope FL images,
multiple channels can be analyzed with up to three channels per analysis. With

Area Quantification FL Algorithm Users Guide

Chapter 1 Introduction

non-Aperio images, analysis of Red, Green, and Blue (RGB) channels are
supported.

Prerequisites
The Area Quantification FL analysis algorithm requires that you be using Aperio
Release 11 or later. You will use the algorithm to analyze Aperio Fused Image
(AFI) files, which are fused images of the multiple dye channels scanned from
the fluorescent dye-stained slide.
Because Aperio digital slides are by design high resolution and information rich,
for best results you should use a high quality monitor to view them. Make sure
the monitor is at the proper viewing height and in a room with appropriate
lighting. We recommend any high quality LCD monitor meeting the following
requirements:
Display Type:

CRT minimum, LCD (flat panel) recommended

Screen Resolution:

1024(h) x 768(v) pixels minimum, 1920 x 1050 or larger

Screen Size:

15 minimum, 19 or larger recommended

Color Depth:

24 bit

Brightness:

300 cd/m2 minimum, 500 or higher recommended

Contrast Ratio:

500:1 minimum, 1000:1 or higher recommended

recommended.

For More Information


For a quick reference to the Area Quantification FL analysis algorithm, refer to
Chapter 2, Quick Reference on page 9.
For details on using the Area Quantification FL analysis algorithm, go to Chapter
3, Sample Analysis on page 17.
See the Aperio Image Analysis Users Guide for information on:

Installing an algorithm

Creating an algorithm macro

Opening a digital slide to analyze

Selecting areas of a digital slide to analyze

Running the analysis

Exporting analysis results

For details on using the Spectrum digital slide information management system
(for example, for information on running batch analyses), see the
Spectrum/Spectrum Plus Operators Guide.
For details on using ImageScope to view and analyze digital slides and using
annotation tools to select areas of the digital slide to analyze, see the ImageScope
Users Guide.

Area Quantification FL Algorithm Users Guide

Chapter 1 Introduction

Intended Use
For research use only. Not for use in diagnostic procedures.
Algorithms are intended to be used by trained researchers who have an
understanding of the biological characteristics or significance of biomarkers.
Each algorithm has input parameters that must be adjusted by an expert user
who understands the goal of running the analysis and can evaluate the algorithm
performance in meeting that goal.
You will adjust (tune) the parameters until the algorithm results are sufficiently
accurate for the purpose for which you intend to use the algorithm. You will
want to test the algorithm on a variety of images so its performance can be
evaluated across the full spectrum of expected imaging conditions. To be
successful, it is usually necessary to limit the field of application to a particular
tissue type and a specific histological preparation. A more narrowly defined
application and consistency in slide preparation generally equates to a higher
probability of success in obtaining satisfactory algorithm results.
If you get algorithm analysis results that are not what you expected, please see
the appendix Troubleshooting in the Aperio Image Analysis Users Guide for
assistance.

Area Quantification FL Algorithm Users Guide

Chapter 1 Introduction

Area Quantification FL Algorithm Users Guide

Quick Reference
This chapter contains a quick reference to all Area
Quantification FL analysis algorithm inputs and outputs. See
the following chapter for details on using the algorithm.
If you are already familiar with using the Area Quantification FL analysis
algorithm, and need just a reminder of the different algorithm input and output
parameters, please refer to the sections below. For more detailed information on
using the algorithm, see the following chapter.
Use of this algorithm differs from that of similar brightfield algorithms in that
you must tell the algorithm which dyes were used on the slide from which the
digital slide was created. You will also define the minimum and maximum
intensity thresholds of those dyes so that non-specific fluorescence and highly
fluorescent artifacts are excluded from analysis.
Note that you can select whether to plot histograms of the analysis results. (See
the Aperio ePathology Image Analysis Users Guide for more on data plots.)

Area Quantification FL Algorithm Users Guide

Chapter 2 Quick Reference

When creating or testing an algorithm macro, you see the Area Quantification FL
algorithm parameter window:

If you did not open the slide from Spectrum, you dont see the Inputs/Outputs
buttons.

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Area Quantification FL Algorithm Users Guide

Chapter 2 Quick Reference

Algorithm Input Parameters


Area Quantification FL analysis algorithm performance is controlled by a set of
input parameters, which determine many different types of analysis.

Image Zoom Zoom level to be used; a higher zoom results in faster


algorithm run but less accurate results.

These parameters are used if you are using Genie classifiers to preprocess the
image:

Classifier Neighborhood Size (in microns) of the neighborhood to pad


the boundary of each view, as required by the classifier.

Classifier Choose from the list of classifiers (if none are available,
contact the Spectrum Administrator for information on the optional
Genie product).

Class List Pick the classes to retain for further processing.

Setting Thresholds
The intensity threshold values specify the range of intensities to be included in
the analysis. Values that fall outside of these minimum and maximum thresholds
will not be identified as containing the specified dye.
The intensity scale is based on the percentage of the image bit depth or gray
levels representing intensity. For example, a value of 1 represents the maximum
pixel value possible (1024 for an Aperio ScanScope FL image scanned at its
default 10-bit setting). A value of 0.2 represents a pixel value of 0.2 x 1024, or 205.
You can use the Mark-up Image Type drop-down list selections to fine-tune the
threshold values so that the analysis results (and mark-up image) reflect the
structures the research wants to select for consideration.
To fine-tune threshold values:
1.

In the Image Fusion Adjustments window in ImageScope, turn off the


display of all but a single dye.

2.

Draw a small area to analyze.

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Chapter 2 Quick Reference

3.

In the Algorithm parameter window, select the tuning option that


corresponds to the dye you are looking at (that is, if you are viewing Dye
1, choose Tuning All Dye 1).

4.

Run the algorithm with the analysis area selected and Selected
Annotation Layer selected on the Algorithm parameter window.

5.

View the mark-up image in ImageScope.

Do the areas highlighted capture the structures you want to identify with
this dye? If not, adjust the minimum and maximum thresholds until you
are satisfied.

Colocalization Parameters
These parameters are used to specify the dyes you want to measure.
Colocalization refers to the ability to identify pixels in which more than one dye
occurs and to quantify those results, as well as to show the results in a mark-up
image.

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Mark-up Image Type Several different mark-up image types are


available from this drop-down list, including tuning mark-ups used to
fine-tune dye thresholds (see the section above) and different

Area Quantification FL Algorithm Users Guide

Chapter 2 Quick Reference

colocalization options (for example, Dye 2 + 3). The default mark-up


image shows the colocalization of all dyes.

Number of Dyes to Colocalize You can select up to three dyes to


colocalize from this drop-down list.

Dye 1, Dye 2, Dye 3 For up to three dyes, type the names of the
fluorescent dyes you want to quantify. For images created by the
ScanScope FL, these must match the dye channel names shown in the
AFI Image Fusion Adjustments window in ImageScope:

If you are not using an AFI image, the dye channels names wont be
available to you. In this case, you can enter up to three channelsthe
dye names are assumed to be Red, Green and Blue, and you can enter
any combination of these. (Dye names are not case sensitive.

Display Plots Select Yes or No. If you select Yes, you can fine-tune the
histogram by selecting its start value, its end value, and the number of
bins (the number of data points).

Algorithm Results
If you are creating the algorithm macro by opening the digital slide image in
Spectrum, the parameters window contains an Output button to select which
output results will be displayed in Spectrum.

Total Analysis Area (mm2) Total area that is analyzed in squaremillimeters.

Total Stained Area (mm2) Total area that contains all dyes within the
specified thresholds.

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Chapter 2 Quick Reference

For each dye selected, you also see:

Total Area of DYE (mm2) Total area that contains dye within the
specified thresholds.

Percent (DYE) Percentage of pixels that contain the dye within the
specified thresholds.

Intensity (DYE) Average intensity for the pixels that contain the dye
within the specified thresholds.

Understanding Intensity Results


In the case of this algorithm, the colors shown in the mark-up image depend on
the pseudo color associated with the particular dye. For slides created by the
ScanScope, this color was set during scanning based on the wavelength of the
dye. For example, the DAPI fluorochrome is usually shown as blue.
In the paragraphs below, we refer to DYE1, DYE2, and DYE3 as placeholders for
the dyes that appear in your digital slides. In our samples in this guide, these
dyes are:

DYE1 = DAPI
DYE2 = FITC
DYE3 = TRITC

As an example of interpreting the intensity results, the color shown in the


quantitative results for Percent(DYE1, DYE2) is for all pixels that contain both
Dye 1 and Dye 2. This same color in the mark-up image also identifies these
pixels.
The intensities listed under Percent (DYE1+DYE2) in the results give the intensity
of Dye 1 in all areas that contain both Dye 1 and Dye 2 and the intensity of Dye 2
in all areas that contain both Dye 1 and Dye 2. For this type of result, the color
displayed in the results and the mark-up image is formed additively. For
example, if Dye 1 is blue and Dye 2 is green, the pixels that contain both dyes are
displayed as cyan.

Percent (DYE1) Percent of the analyzed area that contains Dye 1.


Shown in the pseudo color for that dye in the mark-up image.

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Intensity (DYE1) Intensity of Dye 1 in areas consisting of only Dye


1.

Percent (DYE1 + DYE2) Percent of the analyzed area that contains Dye
1 and Dye 2.

Intensity (DYE1, DYE1+DYE2) Intensity of Dye 1 in areas


consisting of Dye 1 and Dye 2.

Intensity (DYE2, DYE1+DYE2) Intensity of Dye 2 in areas


consisting of Dye 1 and Dye 2.

Area Quantification FL Algorithm Users Guide

Chapter 2 Quick Reference

Percent (DYE2) Percent of the analyzed area that contains Dye 2.


Shown in the pseudo color for that dye in the mark-up image.

Percent (DYE2+DYE3) Percent of the analyzed area that contains Dye


2 and Dye 3.

Intensity (DYE2, DYE2+DYE3) Intensity of Dye 2 in areas


consisting of Dye 2 and Dye 3.

Intensity (DYE3, DYE2+DYE3) Intensity of Dye 3 in areas


consisting of Dye 2 and Dye 3.

Percent (DYE3) Percent of the analyzed area that contains Dye 3.


Shown in the pseudo color for that dye in the mark-up image.

Intensity (DYE2,DYE2) Intensity of Dye 2 in areas consisting of


only Dye 2.

Intensity (DYE3, DYE3) Intensity of Dye 3 in areas consisting of


only Dye 3.

Percent (DYE1+DYE3) Percent of the analyzed area that contains Dye 1


and Dye 3.

Intensity (DYE1, DYE1+DYE3) Intensity of Dye 1 in areas


consisting of Dye 1 and Dye 3.

Intensity (DYE3, DYE1+DYE3) Intensity of Dye 3 in areas


consisting of Dye 1 and Dye 3.

Percent (DYE1+DYE2+DYE3) Percent of the analyzed area that


contains Dye 1, Dye 2, and Dye 3.

Intensity (DYE1, DYE1+DYE2+DYE3) Intensity of Dye 1 in areas


containing Dye 1, Dye 2, and Dye 3.

Intensity (DYE2, DYE1+DYE2+DYE3) Intensity of Dye 2 in areas


containing Dye 1, Dye 2, and Dye 3.

Intensity (DYE3, DYE1+DYE2+DYE3) Intensity of Dye 3 in areas


containing Dye 1, Dye 2, and Dye 3.

The colors that appear in the Annotations window result pane are the same
colors that appear in the mark-up image. Use the Annotations window as the key
to the mark-up image colors. See the next chapter for several samples of mark-up
images.

Pearson and Overlap Coefficients


Pearson Coefficient and Overlap Coefficient values are calculated for every pair
of dyes based on Pearsons Coefficient and Overlap Coefficient. (Therefore, more
than one dye must be selected to see these outputs.) For information on these
coefficients, refer to:
A Guided Tour into Subcellular Colocalization Analysis in Light Microscopy by S.
Bolte and F. P. Cordelieres (http://www.aomf.ca/pdfs/Bolte06.pdf).
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Chapter 2 Quick Reference

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Area Quantification FL Algorithm Users Guide

Sample Analysis
This chapter shows a sample analysis using Area
Quantification FL. For details on installing an algorithm,
creating an algorithm macro, registering it on Spectrum,
and running an analysis, see the Aperio ePathology Image
Analysis Users Guide.
Follow these steps to use the Area Quantification FL algorithm:
1.

Install the algorithm AreaQuantificationFL_v1_v11.0.0.xxx.exe) on both


your Spectrum server and the workstation on which you will create the
algorithm macro.

2.

From Spectrum, open the Aperio Fused Image (AFI) file of the
fluorescence image you want to analyze in ImageScope.

Note the Image Fusion window, which tells you what fluorescent dyes
were used on the glass slide from which the digital slide was created.

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Chapter 3 Sample Analysis

3.

Go to the View menu and select Analysis. You can either select an
existing FL algorithm macro or click Create to create a new one. If no
macros appears on this window, none have yet been created.
For information on the algorithm parameters to set when creating a
macro, see the preceding chapter. For more information on creating
macros and saving them on Spectrum, see the Aperio ePathology Image
Analysis Users Guide.

4.

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Select an Area Quantification FL algorithm macro, choose whether you


want to analyze the entire digital slide or just an area you have drawn,
and click Analyze.

Area Quantification FL Algorithm Users Guide

Chapter 3 Sample Analysis

5.

Open the ImageScope Annotations window to see the quantitative


results of the analysis.

6.

If you have selected Generate Mark-up image, the ImageScope window


displays the mark-up image. Look at the colors in the Annotations
window to see what color applies to what combination of dyes in the
mark-up image.

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Chapter 3 Sample Analysis

Sample Mark-up Images


The mark-up image gives you a visual way to see the colocalization of dyes at
each individual pixel. The mark-up image created by the Area Quantification FL
algorithm color codes each dye with a unique false color. The false colors of the
individual channels are based on the dye colors used during the scan of the
image.
For dye combinations (Dyes 1 + 2, Dyes 1 + 3, and so on), the analysis combines
the dye colors additively. For example, for our sample digital slide that uses
DAPI (blue) and FITC (green), the color shown for DAPI + FITC is cyan.
The sample below shows the result for the default Mark-up Image Type setting
Colocalized All Dyes.

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Chapter 3 Sample Analysis

The sample below shows the same area of the digital slide with the Mark-up
Image Type set to Colocalized Dyes 1 + 3 only.

This sample shows the mark-up image for pixels that contain all three dyes
(Mark-up Image Type set to Colocalized Dyes 1+2+3 only).

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Chapter 3 Sample Analysis

For More Information


For detailed information on the following topics, see the Aperio ePathology Image
Analysis Users Guide:

Using the analysis tuning window


Selecting areas to analyze
Creating an algorithm macro and registering it on Spectrum
Running an analysis
Exporting analysis results

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Area Quantification FL Algorithm Users Guide

Chapter 3 Sample Analysis

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Area Quantification FL Algorithm Users Guide


MAN-0175, Revision B

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