areaquantificationfl user guide

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Area Quantification FL

Algorithm

Users Guide


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Contents

iv Area Quantification FL Algorithm Users Guide


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Area Quantification FL Algorithm Users Guide 5

1

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


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Chapter 1 Introduction

6 Area Quantification FL Algorithm Users Guide

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 AperioRelease 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

recommended.

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

For More Information

For a quick reference to the Area Quantification FL analysis algorithm, refer to

Chapter 2, Quick Reference on page9.

For details on using the Area Quantification FL analysis algorithm, go to Chapter

3, Sample Analysis on page17.

See theAperio Image Analysis Users Guidefor 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.


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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 theAperio Image Analysis Users Guidefor

assistance.


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2

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

theAperio ePathology Image Analysis Users Guidefor more on data plots.)


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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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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 thestructures 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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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 beavailable 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 SelectYes orNo. 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 Outputbutton to select whichoutput results will be displayed in Spectrum.

Total Analysis Area (mm2) Total area that is analyzed in square-

millimeters.

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

specified thresholds.


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For each dye selected, you also see:

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


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


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 inyourdigital 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 bothDye 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.

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.




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

only Dye 2.

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

2 and Dye 3.







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.





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 areascontaining 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 Microscopyby S.

Bolte and F. P. Cordelieres (http://www.aomf.ca/pdfs/Bolte06.pdf).


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3

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 Createto 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 creatingmacros and saving them on Spectrum, see theAperio ePathology Image

Analysis Users Guide.

4. 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.


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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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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 theindividual 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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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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For More Information

For detailed information on the following topics, see theAperio 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

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