cytoplasmic user guide

8/10/2019 Cytoplasmic User Guide

1/32

Cytoplasm Algorithm

Users Guide


2/32

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:

US/Canada Tel: 1 (866) 478-3999 (toll free)

Direct International Tel: 1 (760) 539-1150

US/Canada/Worldwide Email:

[email protected]


3/32

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


4/32

Contents

iv Cytoplasm Algorithm Users Guide


5/32


6/32

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.


7/32


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.


8/32




9/32


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.


10/32

Chapter 2 Quick Reference


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.


11/32



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.


12/32



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.


13/32



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.


14/32




15/32


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.


16/32

Chapter 3 Tuning the Algorithm


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.


17/32



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.


18/32



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.


19/32



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.


20/32



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



21/32



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.


22/32



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


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).


23/32



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.


24/32



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.


25/32


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.


26/32

Chapter 4 Sample Analysis


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.


27/32



7. Click Analyze.

Interpreting the Results

Here is a sample of the visual representation of the analysis results:


28/32


29/32



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.


30/32



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).


31/32


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


32/32

Cytoplasm Algorithm Users Guide

MAN-0220 Revision B

cytoplasmic user guide

Documents