cytoplasmic user guide
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Cytoplasm Algorithm
Users Guide
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ii Cytoplasm Algorithm Users Guide
Copyright 2012-2013 Aperio Technologies, Inc.
Part Number/Revision: MAN-0220, Revision B
Date: 9 November 2013
This document applies to software versions Release 11.2 and later.
All rights reserved. This document may not be copied in whole or in part or reproduced in any other media without the expresswritten 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 products and services, please visit the Aperio 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 or for other advanced instruction. Aperio 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 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 and SecondSlide are registered trademarks 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;
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
Aperio
1360 Park Center DriveVista, CA 92081
United States
Tel: 866-478-4111 (toll free)
Fax: 760-539-1116
Customer Service:
Tel: 866-478-4111 (toll free)
Email: [email protected]
Technical Support:
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Direct International Tel: 1 (760) 539-1150
US/Canada/Worldwide Email:
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Cytoplasm Algorithm Users Guide iii
Contents
C
HAPTER
1
-
I
NTRODUCTION
....................................................................... 5
Prerequisites ................................................................................................................ 6
Intended Use ............................................................................................................... 6
For More Information ................................................................................................ 6
CHAPTER 2-QUICK REFERENCE.................................................................... 9
Algorithm Input Parameters ................................................................................... 10
General Parameters ............................................................................................... 10
Nuclear Stain Calibration ..................................................................................... 11
Positive Stain Calibration ..................................................................................... 11
3rdStain Calibration .............................................................................................. 11
Nuclear Segmentation .......................................................................................... 12
Cytoplasm Segmentation ..................................................................................... 12
Positivity Thresholds ............................................................................................ 12
Algorithm Outputs ................................................................................................... 13
Algorithm Results .................................................................................................... 13
CHAPTER 3-TUNING THE ALGORITHM......................................................... 15
Before You Start ........................................................................................................ 15
Opening a Digital Slide in Spectrum .................................................................. 15
Selecting the Cytoplasm Algorithm ................................................................... 16
Tuning Parameters ................................................................................................... 17
Calibrating the Nuclear Stain .............................................................................. 17
Calibrating the Positive Stain .............................................................................. 19
Calibrating a 3rd Stain ........................................................................................... 20
Segmenting Nuclei ................................................................................................ 20
Segmenting Cytoplasm ........................................................................................ 21
Setting Positivity Thresholds ............................................................................... 22
Saving the Macro ...................................................................................................... 23
CHAPTER 4-SAMPLE ANALYSIS.................................................................. 25
Running the Cytoplasm Analysis .......................................................................... 25
Interpreting the Results ........................................................................................... 27
Intensity Plot .......................................................................................................... 30
INDEX................................................................................................... 31
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Contents
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Chapter 1 Introduction
6 Cytoplasm Algorithm Users Guide
Prerequisites
The Cytoplasm Algorithm requires that you be using Aperio Spectrum Release
11.2 or later.
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
Intended Use
For research use only. Not for use in diagnostic procedures.
Algorithms are intended to be used by trained pathologists who have an
understanding of the conditions they are testing for in running the algorithm
analysis.
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 ePathology Image Analysis Users
Guidefor assistance.
For More Information
For a quick reference to the Cytoplasm Algorithm, refer to Chapter 2, Quick
Reference on page9.
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Chapter 1 Introduction
Cytoplasm Algorithm Users Guide 7
For details on using the Cytoplasm Algorithm, begin with Chapter 3, Tuning
the Algorithm on page15.Then proceed to Chapter 4, Sample Analysis on
page25.
See theAperio ePathology Image Analysis Users Guidefor information on:
Installing an algorithm
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
Operators Guide.
For details on using ImageScope to view and analyze digital slides and usingannotation tools to select areas of the digital slide to analyze, see the ImageScope
Users Guide.
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Chapter 1 Introduction
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Cytoplasm Algorithm Users Guide 9
2
Quick Reference
This chapter contains a quick reference to all Cytoplasm
Algorithm inputs and outputs. See the following chapters
for details on using the algorithm.
If you are already familiar with using the Cytoplasm Algorithm and need just areminder 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 chapters.
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Chapter 2 Quick Reference
10 Cytoplasm Algorithm Users Guide
Algorithm Input Parameters
Cytoplasm algorithm performance is controlled by a set of
input parameters. When you first bring up the algorithm
window, you see a list of all of the input parameters. Bydefault, the Analysis Stage is set to Report Results, the
stage you will use when you want to run the algorithm,
after you have fine-tuned all of the parameters.
But first you will want to fine-tune the parameters to suit
your analysis needs and the characteristics of your slides,
and then save the algorithm macro with the adjusted
parameters. The next paragraphs give a quick reference
guide to the algorithm parameterssee the following
chapters for more details on using these parameters.
General Parameters
View Widthand View Height Width and
height of processing box in pixels. For most
applications, leave at the default value.
Image Zoom Zoom level to be used. This value
ranges from 1 to zero. 1 is the magnification the
slide was scanned at (for example, 20x) and is full
resolution. A smaller number (lower
magnification) results in faster algorithm run but
less accurate results.
Markup Compression Type This sets the
compression type for the algorithm mark-up
image. Choose better compression if you need the
image for a special purpose.
Compression Quality A higher quality takes
longer and yields larger files. This selection does
not apply to all compression types. For most
applications, leave at the default value.
Classifier If you have classifiers installed on
your Spectrum site, you can select one from this
drop-down list to perform pre-processing on theimage. (Contact your Spectrum administrator for
information on the classifiers available on your site.)
Classifier List If classifiers are available on your Spectrum site, the
classifier you select may allow you to choose classes to use for analysis.
Click the edit icon on the Class List line to see the classes available,
and select one or more classes by selecting the checkbox next to the class.
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Chapter 2 Quick Reference
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Overlap Size Size of the overlap region for each view. This should be
at least as big as the average object size. This is automatically calculated
based on the maximum cell dimension parameter.
Classifier Neighborhood Automatically calculated in microns equal to
the Max Cell Dimension value. Used by classifiers and along withOverlap Size and Max Cell Dimension, make sure cells are only counted
once when they occur in multiple views.
Max Cell Dimension (m) This sets the maximum distance between
two points within a cell in microns. Setting the maximum cell dimension
ensures that cells are only counted once when they occur partially in
multiple views, eliminating view overlap.
Clear Area Intensity The default value is 240 for images scanned by a
ScanScope and defines white balance for the image. This is the reference
for OD (optical density) = 0.
Analysis Stage From this parameter you can select Report Resultswhen you have fine-tuned the parameters and are ready to run the
analysis. Other stages allow you to fine-tune the parameter sets listed
below. See the following chapters for details on tuning the algorithm and
running it.
Nuclear Stain Calibration
After selecting Nuclear Stain Calibrationas the Analysis Stage, you see the
following parameters: Nuclear Stain (Red), Nuclear Stain (Green), and Nuclear
Stain (Blue). Together, these values specify the color of the stain used to mark
nuclei in the sample. The default values are for hematoxylin stain. See
Calibrating the Nuclear Stain on page15 for tips on setting these.
Positive Stain Calibration
After selecting Positive Stain Calibrationas the Analysis Stage, you see the
following parameters: Positive Stain (Red), Positive Stain (Green), and Positive
Stain (Blue). Together, these values specify the color of the stain marking
cytoplasm in the sample. The default values are for the DAB stain. See
Calibrating the Positive Stain on page19 for tips on setting these.
3
rd
Stain Calibration
After selecting 3rdStain Calibrationas the Analysis Stage, you see the followingparameters: 3rdStain (Red), 3rdStain (Green), and 3rdStain (Blue). Together, these
values specify the color of a third stain, if present, used in the slide. Normally,
these values would be zero as we assume you are using two stains, one to
identify nuclei and the other to identify cytoplasm.
See Calibrating a 3rdStain on page20 for tips on setting these.
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Chapter 2 Quick Reference
12 Cytoplasm Algorithm Users Guide
Nuclear Segmentation
After selecting Nuclear Segmentationas the Analysis Stage, you see the
following parameters. These parameters filter and remove unwanted signals by
smoothing (Averaging Radius), removing background (Nuclear Threshold
Type), and removing small nuclei (Min Nuclear Area).
Averaging Radius (m) This specifies the smoothness of the nuclei
image. The larger the radius, the more smooth the image.
Nuclear Segmentation Factor This parameter has a value between zero
and 1. A value of zero uses only the nuclear stain for segmentation, while
nonzero values add in proportionally more of the positive stain.
Nuclear Threshold Type If you choose Adaptive, the algorithm
automatically adjusts to variations in stain intensity to calculate a
threshold. If you choose Manual, you will specify a fixed value.
Min Nuclear Area (m
2
) This specifies the minimum size of nucleararea, and is used to exclude small nuclei and for declustering
neighboring nuclei.
See Segmenting Nuclei on page20 for tips on setting these.
Cytoplasm Segmentation
After selecting Cytoplasm Segmentationas the Analysis Stage, you see the
Cytoplasmic Distance (m) parameter. This defines the maximum allowable
distance for cytoplasm surrounding nuclei.
See Segmenting Cytoplasm on page21 for tips on setting this.
Positivity Thresholds
After selecting Positivity Thresholdsas the Analysis stage, you see the following
parameters: (1+) Threshold, (2+) Threshold, and (3+) Threshold. These define
thresholds for the positive stain.
The scores for average cytoplasm intensity for the selected region are calculated
based on these thresholds.
If you wish greater granularity in your results, you can define additional positive
thresholds.
See Setting Positivity Thresholds on page22 for tips on setting these.
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Cytoplasm Algorithm Users Guide 13
Algorithm Outputs
By clicking the Outputsbutton on the
algorithm parameter window, you can see all
of the outputs that can be shown in youranalysis results in Spectrum.
To remove an output from the analysis
display in Spectrum, clear the checkbox next
to it.
Algorithm Results
The Annotations window shows quantitative
results of the analysis, while the slide image
shows a mark-up image that gives a visual
representation of those results. Most of theresults show threshold scores, defining the
intensity with which the nuclei and cytoplasm
were stained.
For a discussion of the algorithm results, see
Interpreting the Results on page27.
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Chapter 2 Quick Reference
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Cytoplasm Algorithm Users Guide 15
3
Tuning the Algorithm
This chapter discusses how to tune the Cytoplasm
Algorithm parameters to fit your analysis needs and the
characteristics of your digital slides.
The process of creating an algorithm macro involves setting and testing the
algorithm parameters until you have fine-tuned the parameters to give the best
results. You then save the macro to be used for future analyses.
To fine-tune the Cytoplasm Algorithm parameters, select each mode from the
Analysis Stage drop-down list and then use the tuning window to set and test
your parameter values. We recommend going through the tuning parameters in
the order they are listed.
NOTE: Be sure to set the Analysis Stage to Report Resultsbefore running the
final analysisif another Analysis Stage is select when you run the algorithm,
you will see only the results for that tuning stage.
Before You Start
First, be sure that the Cytoplasm Algorithm has been installed both on the
Spectrum server and on your local workstation. See theAperio ePathology Image
Analysis Users Guidefor details on algorithm installation.
Opening a Digital Slide in Spectrum
Cases, specimens and digital slides are managed using Aperios Spectrum. A
pathologist who wants to access a digital slide first needs to log into Spectrum
and navigate to the case and the specimen that shows the list of its associated
digital slides and then open the desired digital slide in ImageScope.
As part of using the Cytoplasm Algorithm, you will be creating an algorithm
macro(a file that contains your algorithm settings). In order to do that, you will
need to be logged into Spectrum as an administrator.
See theAperio ePathology Image Analysis Users Guidefor instructions on opening a
digital slide and creating an algorithm macro.
The instructions inthese chapters
assume you areopening a digital
slide in Spectrum
and analyzing it
directly in theImageScope viewer.
If this is not the case(that is, you are not
using Spectrum, butare opening a digitalslide that is on your
workstation or on aserver directly fromImageScope), the
appearance of someof the ImageScope
windows may differ
slightly, althoughthe Cytoplasm
Algorithm steps willbe the same. See theImageScope Users
Guidefor details on
using digital slidesthat are local or are
on a server.
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Chapter 3 Tuning the Algorithm
16 Cytoplasm Algorithm Users Guide
Once the digital slide is open in ImageScope, you will be able to use the
ImageScope tools to select areas of the digital slide to analyze and use the
Cytoplasm Algorithm to detect and quantify nuclei and cytoplasm.
Selecting the Cytoplasm Algorithm
Once you have selected a digital slide and opened it in ImageScope (as
discussed in theAperio ePathology Image Analysis Users Guide), you see the
digital slide displayed in the main ImageScope window.
For example:
For details on moving around the image, changing its magnification, and
using the drawing tools to select areas of the image, see the ImageScope Users
Guide.
To select the Cytoplasm Algorithm:
1. Go to the ImageScope View menu and select Analysis. The Algorithm
Server Job window displays.
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2. If an algorithm macro for the Cytoplasm Algorithm has not yet been
created, click Create. You must be logged into Spectrum as an
Administrator to use the Createbutton. (If a macro exists and you want
to adjust and test it, select the macro and click Test.)
If you have clicked Create, the Select an Algorithm window appears,which lists all of the algorithms that have been installed.
3. Select the Cytoplasm Algorithm and click Select.
The Algorithms window now displays.
4. Go to the ImageScope View menu and select Annotationsto display the
Annotations window which will display the algorithm analysis results.
When the Cytoplasm Algorithm is shown in the Algorithms window, you can
proceed with fine-tuning the parameters.
Important: To use the algorithm again, you will need to save the algorithm
macro, which consists of all of the settings you have adjusted, Before you closethe Algorithm window, save your settings. See Saving the Macro on page23.
Tuning Parameters
Now you will use the Analysis Stage tuning stages to test and adjust the
algorithm parameters. The default settings for these parameters will give good
results depending on the characteristics of the stains you have used and your
desired results. But to make sure the algorithm works well for your particular
stains and the requirements of your project, follow the steps below.
The default settings assume you are using hematoxylin to stain the nuclei and
DAB to stain cytoplasm. However, you can use this algorithm for otherbiomarkers and tissue types by adjusting the stain color vectors as discussed
below.
We recommend you use the tuning stages in the order in which they are listed in
the Analysis Stage drop-down list.
Calibrating the Nuclear Stain
After selecting Nuclear Stain Calibrationfrom the Algorithm window, you see
the following parameters.
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18 Cytoplasm Algorithm Users Guide
The numbers shown define the color vectors for the nuclear stain used for your
slide. For the best results, you can obtain the correct numbers for the stain you
are using by using the algorithm on a control slide that has only that stain
present. If that is not possible, you can select an area of the digital slide that
contains mostly nuclear stain.Click Tuneand move the tuning window to an area that mostly contains the
nuclear stain.
After a moment, the tuning window displays an image of the stain after
separation, and the Annotations window Tuning Layer displays the values
measured for that stain. This gives a useful visual check that the color is
approximately correct.
To fine-tune the stain color vectors for the nuclear stain, you can enter the values
shown in the Annotations window into the algorithm Nuclear Stain Calibration
parameters.
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Calibrating the Positive Stain
After selecting Positive Stain Calibrationfrom the Algorithm window, you see
the following parameters.
The numbers shown define the color vectors for the cytoplasm stain used for
your slide. For the best results, you can obtain the correct numbers for the stain
you are using by using the algorithm on a control slide that has only that stain
present. If that is not possible, you can select an area of the digital slide that
contains mostly cytoplasm stain.
Click Tuneand move the tuning window to an area that mostly contains the
cytoplasm stain.
After a moment, the tuning window displays an image in which only the brown
stain remains, and the Annotations window Tuning Layer displays the values
measured for that stain.
To fine-tune the stain color vectors for the cytoplasm stain, you can enter the
values shown in the Annotations window into the algorithm Positive Stain
Calibration parameters.
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Calibrating a 3
rd
Stain
After selecting 3rd Stain Calibrationfrom the Algorithm window, you see the
following parameters.
If the nuclear and positive stains are set correctly, there should be very little
shown for the 3rdstain in the tuning window (we assume you are not using a
third stain):
Make sure the values are set to zero if you are not using a third stain.
If you areusing a third stain, you can calibrate it using the same procedure usedin the previous sections on calibrating the nuclear and positive stains.
Segmenting Nuclei
After selecting Nuclear Segmentationfrom the Algorithm window, you see the
following parameters.
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Chapter 3 Tuning the Algorithm
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The tuning window shows the nuclei circled with cytoplasm stain removed. This
gives a visual check that the algorithm is successfully finding nuclei.
Averaging Radius (m) This parameter smoothes out variations in
nuclei size. It corrects improper segmenting of nuclei due to spotty
staining by smoothing pixels and improves nuclear counting accuracy.
Nuclear Segmentation Factor Nuclei can stain imperfectly, picking up
some brown stain. This parameter specifies how much brown stain to
include in nuclei identification. A value of zero gives the strictest
definition of what will be considered nuclei by only considering a stain
vector color of blue. A value of 1 allows both blue and brown stains to be
considered. A value of .2 is a good compromise, allowing a small amount
of brown stain to be included in the definition.
Nuclear Threshold Type Selecting Adaptiveallows the algorithm to
adjust thresholds based on the strength of the staining. Selecting Manual
allows you to enter a fixed value. The Manual setting is not
recommended because it makes the algorithm too specific for this
particular slide rather than making the algorithm useful for a variety of
slides with different staining intensity.
Min Nuclear Area (m2) Sets the minimum area size. When nuclei are
clustered together, decreasing this value allows the algorithm to separate
them.
Segmenting Cytoplasm
After selecting Cytoplasm Segmentationfrom the Algorithm window, you see
the following parameter.
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22 Cytoplasm Algorithm Users Guide
This parameter in microns defines how far from the nucleus the cytoplasm can
be yet still be reported as cytoplasm that surrounds the nucleus. The tuning
window gives you a visual check of this value. In the example below, the blue
areas are nuclei and the yellow areas are the cytoplasm that surround the nuclei.
Adjust the Cytoplasmic Distance parameter until the yellow regions surrounding
the nuclei are the desired size.
Setting Positivity Thresholds
After selecting Positivity Thresholdsfrom the Algorithm window, you see the
following parameters.
The Number of Positive Thresholds defines how many cytoplasm scores you
want to be reported. The default thresholds are 0 (negative), 1 (weak positive), 2
(moderate positive), and 3 (strong positive).
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The individual threshold parameters define how much cytoplasm stain has to be
present to be reported as +1, +2, and so on. The tuning window gives you a visual
check of the different thresholds. For example:
In the tuning window, the following cytoplasm score colors are displayed:
The definitions of these color codes are presented in the final analysis results
displayed in the Annotations window after you run the analysis.
Saving the Macro
After you have tested the algorithm and fine-tuned the parameters, you can save
the algorithm macro. The macro will be used to run the final analyses.
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24 Cytoplasm Algorithm Users Guide
To save the macro (which saves the parameter adjustments you have made):
1. Click Save Macroon the Algorithm window.
You see the Save Macro window:
2. Type a name for the macro and click OK.
Now you will be able to use this macro for future analyses. The macro name will
appear in the Algorithm Server Job window and is also listed on the Spectrum
Macros page.
For a sample analysis using the Cytoplasm Algorithm, see the next chapter.
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4
Sample Analysis
This chapter discusses how to run the Cytoplasm Algorithm
analysis after you have fine-tuned the input parameters as
discussed in the previous chapter and saved an algorithm
macro.
Note: Do not run the algorithm with one of the tuning modes set in the AnalysisStage. In order to see the complete results, select Report Resultsas the Analysis
Stage.
You can analyze slides either using ImageScope or WebScope. For details on
using these digital slide viewers, refer to the ImageScope Users Guideand the
WebScope Users Guide. General information on viewing digital slides, running
batch analyses from Spectrum, and seeing analysis results in Spectrum can be
found in theAperio ePathology Image Analysis Users Guide.
Running the Cytoplasm Analysis
In the previous chapter we fine-tuned the Cytoplasm Algorithm parameters andsaved an algorithm macro using those settings.
In this chapter we will run the algorithm to identify nuclei and the cytoplasm
that surrounds them.
1. Open a digital slide in ImageScope.
2. Go to the ImageScope View menu and select Annotationsto open the
Annotations window where you will see the analysis results.
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26 Cytoplasm Algorithm Users Guide
3. If you will want to analyze a selected region of the slide rather than the
entire slide, use the ImageScope annotation tools to draw an area around
that region. For example, we used the free form drawing pen to select
this region of the slide:
4. Go to the ImageScope View menu and select Analysisto select the
Cytoplasm Algorithm.
5. From the Algorithm Server Job window, select the Cytoplasm Algorithm
macro we saved in the previous chapter.
6. You can now either select Entire Imageto run the algorithm on the
entire digital slide, or you can select Selected Annotationon the
Algorithm Server Job window to analyze just the selected region.
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7. Click Analyze.
Interpreting the Results
Here is a sample of the visual representation of the analysis results:
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2*(%2+) + 3*(%3+) + ... For the usual case where there are 3 thresholds, the
score is between 0 and 300, where 300 represents 100% of cells being 3+.
Cytoplasm: Average Positive Intensity Average intensity of staining
in cytoplasm (cellular average). This is the average of positive cells
only0+ cells are not counted in the average.
Cytoplasm: Percent Positive Cells Percentage of cells having staining
in the cytoplasm.
Nucleus: H-Score A nuclear intensity score derived from the average
intensity of the staining of the nuclei (cellular average) according to the
threshold intervals set in the algorithm macro. For example, if there are
three thresholds defined, this score equals = (%1+) + 2*(%2+) + 3*(%3+) +
... For the usual case where there are 3 thresholds, the score is between 0
and 300, where 300 represents 100% of cells being 3+.
Nucleus: Average Positive Intensity -Average intensity of staining in
nuclei (cellular average). This is the average of positive cells only0+cells are not counted in the average.
Nucleus: Percent Positive Cells Percentage of cells having staining in
the nuclei.
Cytoplasm: Percent (N+) For each cytoplasm threshold defined in the
algorithm, the percentage of cells having staining in the cytoplasm.
Nucleus: Percent (N+) For each nucleus threshold defined in the
algorithm, the percentage of cells having staining in the nuclei.
Number of Cells Total number of cells analyzed.
Percent Colocalized Percentage of cells having staining in both thecytoplasm and nucleus.
Area of Analysis (mm2) Area of the slide that was analyzed.
Cytoplasm Area (mm2)- Total area identified as cytoplasm.
Nuclear Area (mm2)- Total area identified as nuclei.
Beneath the results list is a summary of the Algorithm Inputs containing the
values you set when you created the algorithm macro.
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30 Cytoplasm Algorithm Users Guide
Intensity Plot
If you click the Display Plotsbutton at the top of the Annotations window,
you can display an intensity histogram:
This histogram displays the percentage of cells having staining in the cytoplasm
as a function of intensity (in red) and the percentage of cells having staining in
the nuclei as a function of intensity (in blue).
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Index
algorithm macro, 24
saving, 24
algorithm parameters
3rd stain calibration, 11, 20
cytoplasm segmentation, 12, 21
general, 10
inputs, 10
nuclear segmentation, 12, 20
nuclear stain calibration, 11, 17
outputs, 13
positive stain calibration, 11, 19
positivity thresholds, 12, 22
quick reference, 10
tuning, 17
algorithm results
area of analysis, 29
average positive intensity, 29
cytoplasm area, 29
cytoplasm percent, 29
H-score, 28, 29
nuclear area, 29nucleus average positive
intensity, 29
nucleus percent, 29
nucleus percent positive cells, 29
number of cells, 29
percent colocalized, 29
percent positive cells, 29
plots, 30
analysis stages, 15
Aperio release requirements, 6
histogram, 30
intended use, 6
macro, 24
marking region to analyze, 26
mark-up image, 28
color codes, 28
mark-up image type, 11
monitor requirements, 6
nuclear stain, calibrating, 17
opening image in Specrum, 15
plots, 30
positive stain, calibrating, 19
prerequisites, 6
results, 28
mark-up image, 27
plots, 30
quantitative, 28
visual representation, 27running analysis, 25
sample analysis, 25
segmenting cytoplasm, 21
segmenting nuclei, 20
selecting algorithm, 16, 26
third stain calibration, 20
thresholds, positivity, 22
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Cytoplasm Algorithm Users Guide
MAN-0220 Revision B