The Pathology Company. Cytoplasm Algorithm. User s Guide

Transcription

1 The Pathology Company Cytoplasm Algorithm User s Guide MAN-0220, Revision C 1 December 2014

2 Cytoplasm Algorithm User s Guide This document applies to eslide Manager Release 12.2 and later. Copyright Notice ÌÌCopyright Aperio. All rights reserved. LEICA and the Leica logo are registered trademarks of Leica Microsystems IR GmbH. Aperio is a registered trademark of Leica Biosystems Imaging, Inc. in the USA and other countries. Customer Resources ÌÌFor the latest information on Leica Biosystems Aperio epathology products and services, please visit Disclaimers ÌÌUse normal care in maintaining and using Aperio epathology servers. Interrupting network connections or turning off the servers while they are processing data (such as when they are analyzing eslides or generating an audit report) can result in data loss. ÌÌThis manual is not a substitute for the detailed operator training provided by Leica Biosystems Imaging or for other advanced instruction. Leica Biosystems Imaging 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 Leica Biosystems Imaging Service Engineer. ÌÌImageServer is intended for use with eslides created by scanning glass slides with the scanner. Educators will use Aperio epathology software to view and modify eslides in Composite WebSlide (CWS) format. Patents ÌÌAperio epathology 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; , 7,646,496; 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 Leica Biosystems Imaging, Inc. Headquarters Customer Support General Information Leica Biosystems Imaging, Inc Park Center Drive Vista, CA USA Tel: +1 (866) (toll free) Direct International Tel: +1 (760) US/Canada Tel: +1 (866) (toll free) Direct International Tel: +1 (760) US/Canada/Worldwide TechServices@LeicaBiosystems.com US/Canada Tel: +1 (866) (toll free) Direct International Tel: +1 (760) epathology@leicabiosystems.com 2 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

3 Contents 1 Introduction... 5 Intended Use...5 Prerequisites...6 Installing the Algorithm...6 For More Information Quick Reference... 7 Algorithm Input Parameters...7 General Parameters...7 Nuclear Stain Calibration...8 Positive Stain Calibration rd Stain Calibration...8 Nuclear Segmentation...8 Cytoplasm Segmentation...9 Positivity Thresholds...9 Algorithm Outputs...9 Algorithm Results Tuning the Algorithm Before You Start...10 Opening an eslide in eslide Manager...10 Selecting the Cytoplasm Algorithm...10 Tuning Parameters...12 Calibrating the Nuclear Stain...13 Calibrating the Positive Stain...13 Calibrating a 3 rd Stain...14 Segmenting Nuclei...14 Segmenting Cytoplasm...15 Setting Positivity Thresholds...16 Saving the Macro...17 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

4 Contents 4 Sample Analysis Running the Cytoplasm Analysis...18 Interpreting the Results...19 Intensity Plot...21 Index Symbols Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

5 1 Introduction This chapter introduces the Cytoplasm Algorithm. For general information on using an algorithm, please see the Aperio epathology Image Analysis User s Guide. When a pathologist or researcher needs to measure protein expression within immunohistochemisty (IHC) stained tissue using brightfield microscopy, the tissue is illuminated with visible white light and protein expression is typically observed with diaminobenzidine (DAB), a brown stain. In most cases, the darker the stain the greater the protein expression. Counterstains such as Hematoxylin, a blue stain, are applied for morphologic detail of the surrounding tissue to help study the tissue and draw conclusions. The Cytoplasm Algorithm completes the Aperio cell-based analysis module for analyzing IHC-stained tissues. Together with the membrane and nuclear algorithms, all major sub-cellular compartments can now be analyzed on the Aperio epathology Platform. The Cytoplasm Algorithm by default is set to analyze DAB staining intensity and provide the percentage of cells containing stain within in the nucleus and the cytoplasm. The intensity for the positive stain is divided into four score classes with the ability to add additional classes (up to 10 total). In addition, the algorithm provides an H-Score, a colocalized result and a histogram for the IHC staining of the cytoplasm and nuclei. The ability to differentiate between staining in the two sub-cellular compartments is an important feature of the Cytoplasm Algorithm. Translocation between the cytoplasm and the nucleus is a common method to regulate the activity of proteins in the cell, especially transcription factors and cell cycle proteins. Under circumstances where certain cellular pathways are disrupted, such as cancer, sub-cellular localization of these proteins may be altered. Therefore, pathologists or researchers often evaluate the respective localization of these proteins in the nucleus or the cytoplasm in order to gain insight into the status of cells and tissues. The Cytoplasm Algorithm can facilitate this analysis. 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. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

6 Chapter 1: Introduction If you get algorithm analysis results that are not what you expected, please see the Troubleshooting information in the Aperio epathology Image Analysis User s Guide for assistance. Prerequisites The Cytoplasm Algorithm requires that you be using Aperio eslide Manager Release 11.2 or later. Because Aperio eslides 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 requirements recommended in the Aperio epathology System Requirements. Installing the Algorithm In most cases you install the algorithm on the eslide Manager server only. (In fact, Technical Services may install the algorithm for you on your server.) This is because you typically fine-tune and save the algorithm parameters on the eslide Manager server. For the rare case that you need to fine-tune the algorithm parameters on your local workstation or by using a local image, refer to the Aperio epathology Image Analysis User s Guide for installation and use instructions. For More Information For a quick reference to the Cytoplasm Algorithm, refer Chapter 2: Quick Reference on page 7. For details on using the Cytoplasm Algorithm, begin with Chapter 3: Tuning the Algorithm on page 10. Then proceed to Chapter 4: Sample Analysis on page 18. See the Aperio epathology Image Analysis User s Guide for information on: ``Installing an algorithm ``Opening an eslide to analyze ``Selecting areas of an eslide to analyze ``Running the analysis ``Exporting analysis results For details on using eslide Manager (for example, for information on running batch analyses), see the eslide Manager Operator s Guide. For details on using ImageScope to view and analyze eslides and for information on using annotation tools to select areas of the eslide to analyze, see the ImageScope User s Guide. 6 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

7 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 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 chapters. Algorithm Input Parameters Cytoplasm algorithm performance is controlled by a set of input parameters. When you first begin to tune the algorithm parameters (see the next chapter), you see a list of all of the input parameters. By default, 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 parameters see the following chapters for more details on using these parameters. General Parameters ``View Width and 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 eslide Manager site, you can select one from this drop-down list to perform pre-processing on the image. (Contact your eslide Manager administrator for information on the classifiers available on your site.) `` Classifier List If classifiers are available on your eslide Manager 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. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

8 Chapter 2: Quick Reference ``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 with Overlap Size and Max Cell Dimension, make sure cells are only counted once when they occur in multiple views. ``Max Cell Dimension (µm) 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 an Aperio scanner 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 Results when 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 Calibration as 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 page 13 for tips on setting these. Positive Stain Calibration After selecting Positive Stain Calibration as 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 page 13 for tips on setting these. 3 rd Stain Calibration After selecting 3 rd Stain Calibration as the Analysis Stage, you see the following parameters: 3 rd Stain (Red), 3 rd Stain (Green), and 3 rd Stain (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 3rd Stain on page 14 for tips on setting these. Nuclear Segmentation After selecting Nuclear Segmentation as 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 (µm) This specifies the smoothness of the nuclei image. The larger the radius, the more smooth the image. 8 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

9 Chapter 2: Quick Reference ``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 (µm 2 ) This specifies the minimum size of nuclear area, and is used to exclude small nuclei and for declustering neighboring nuclei. See Segmenting Nuclei on page 14 for tips on setting these. Cytoplasm Segmentation After selecting Cytoplasm Segmentation as the Analysis Stage, you see the Cytoplasmic Distance (µm) parameter. This defines the maximum allowable distance for cytoplasm surrounding nuclei. See Segmenting Cytoplasm on page 15 for tips on setting this. Positivity Thresholds After selecting Positivity Thresholds as 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 page 16 for tips on setting these. Algorithm Outputs By clicking the Outputs button on the algorithm parameter window, you can see all of the outputs that can be shown in your analysis results in eslide Manager. To remove an output from the analysis display in eslide Manager, 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 the results 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 page 19. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

10 3 Tuning the Algorithm This chapter discusses how to tune the Cytoplasm Algorithm parameters to fit your analysis needs and the characteristics of your eslides. 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 Results before running the final analysis if 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 on the eslide Manager server. See Installing the Algorithm on page 6 for information on algorithm installation. Opening an eslide in eslide Manager Cases, specimens and eslides are managed using eslide Manager. A pathologist who wants to access an eslide first needs to log into eslide Manager and navigate to the case and the specimen that shows the list of its associated eslides and then open the desired eslide 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 eslide Manager as an administrator. See the Aperio epathology Image Analysis User s Guide for instructions on opening an eslide and creating an algorithm macro. Once the eslide is open in ImageScope, you will be able to use the ImageScope tools to select areas of the eslide to analyze and use the Cytoplasm Algorithm to detect and quantify nuclei and cytoplasm. Selecting the Cytoplasm Algorithm Once you have selected an eslide and opened it in ImageScope (as discussed in the Aperio epathology Image Analysis User s Guide), you see the eslide displayed in the main ImageScope window. 10 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

11 Chapter 3: Tuning the Algorithm 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 User s Guide. To select the Cytoplasm Algorithm: 1. Go to the ImageScope View menu and select Analysis. The Algorithm Server Job window displays. a. To test an existing macro for this algorithm and adjust its parameters, click the Cytoplasm algorithm macro in this window to select it and click Test. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

12 Chapter 3: Tuning the Algorithm b. If a macro doesn t already exist, to create a new macro click Create. (If the Test or Create buttons are not enabled, you may not have permissions to create or modify algorithm macros; contact your eslide Manager administrator for assistance.) From the Select Algorithms window, click the Cytoplasm algorithm to select it and click Select. The Algorithms window displays when you are creating or testing a macro, and a tuning window also automatically opens on the eslide image in ImageScope. You can adjust the parameters in the Algorithms window as discussed below and move the tuning window on the image to see a markup image of the results. 2. Go to the ImageScope View menu and select Annotations to 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 close the Algorithm window, save your settings. See Saving the Macro on page 17. 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 other biomarkers 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. 12 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

13 Chapter 3: Tuning the Algorithm Calibrating the Nuclear Stain After selecting Nuclear Stain Calibration from the Algorithm window, you see the following parameters. 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 eslide that contains mostly nuclear stain. 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. Calibrating the Positive Stain After selecting Positive Stain Calibration from the Algorithm window, you see the following parameters. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

14 Chapter 3: Tuning the Algorithm 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 eslide that contains mostly cytoplasm stain. (If using a control slide, you will need to save the macro to save the adjustments you have already made, open the new slide, and then test the macro again to open it for new adjustments.) 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. Calibrating a 3 rd Stain After selecting 3rd Stain Calibration from 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 3 rd stain 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 are using a third stain, you can calibrate it using the same procedure used in the previous sections on calibrating the nuclear and positive stains. Segmenting Nuclei After selecting Nuclear Segmentation from the Algorithm window, you see the following parameters. 14 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

15 Chapter 3: Tuning the Algorithm 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 (µm) 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 Adaptive allows 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 (µm 2 ) Sets the minimum area size. When nuclei are clustered together, decreasing this value allows the algorithm to separate them. Segmenting Cytoplasm After selecting Cytoplasm Segmentation from the Algorithm window, you see the following parameter. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

16 Chapter 3: Tuning the Algorithm 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 Thresholds from 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). 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. 16 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

17 Chapter 3: Tuning the Algorithm 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. To save the macro (which saves the parameter adjustments you have made): 1. Click Save Macro on the Algorithm window. 2. When the Save Macro window appears, type a name for the macro and specify the data group it will be associated with and click OK. (Only eslide Manager operators who have permission to use the macro s data group will be able to use the macro.) 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 eslide Manager Macros page. For a sample analysis using the Cytoplasm Algorithm, see the next chapter. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

18 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. Important: Do not run the algorithm with one of the tuning modes set in the Analysis Stage. In order to see the complete results, select Report Results as the Analysis Stage. You analyze slides using ImageScope. Refer to the ImageScope User s Guide for details. For general information on running batch analyses from eslide Manager, and seeing analysis results in eslide Manager, see the Aperio epathology Image Analysis User s Guide. Running the Cytoplasm Analysis In the previous chapter we fine-tuned the Cytoplasm Algorithm parameters and saved 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 an eslide in ImageScope. 2. Go to the ImageScope View menu and select Annotations to open the Annotations window where you will see the analysis results. 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 Analysis. 5. From the Algorithm Server Job window, select the Cytoplasm Algorithm macro we saved in the previous chapter. 18 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

19 Chapter 4: Sample Analysis 6. You can now either select Entire Image to run the algorithm on the entire eslide, or you can select Selected Annotation on the Algorithm Server Job window to analyze just the selected region. 7. Click Analyze. Interpreting the Results Here is a sample of the visual representation of the analysis results: The Annotations window shows the quantitative results, and a color key that allows you to interpret the intensity scores in the visual mark-up image. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

20 Chapter 4: Sample Analysis For example: The dark red in the mark-up image is defined in the Annotations window as Cytoplasm Percent (3+), identifying areas that are intensely stained for cytoplasm. Cytoplasm results are color-coded in yellow/orange/red, and nuclear results are color coded in blues. The color-coded list provides the following information in addition to a key to the mark-up image: ``Cytoplasm: H-Score A cytoplasmic intensity score derived from the average intensity of the staining of the cytoplasm (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+. ``Cytoplasm: Average Positive Intensity Average intensity of staining in cytoplasm (cellular average). This is the average of positive cells only 0+ 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 only 0+ 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. 20 Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

21 Chapter 4: Sample Analysis ``Percent Colocalized Percentage of cells having staining in both the cytoplasm and nucleus. ``Area of Analysis (mm 2 ) Area of the slide that was analyzed. ``Cytoplasm Area (mm 2 ) Total area identified as cytoplasm. ``Nuclear Area (mm 2 ) 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. Intensity Plot If you click the Display Plots button 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). Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

22 Index A algorithm macro 17 saving 17 algorithm parameters 3rd stain calibration 8, 14 cytoplasm segmentation 9, 15 general 7 nuclear segmentation 8, 14 nuclear stain calibration 8, 13 positive stain calibration 8, 13 positivity thresholds 9, 16 tuning 12 algorithm results area of analysis 21 average positive intensity 20 cytoplasm area 21 cytoplasm percent 20 H-score 20 nuclear area 21 nucleus percent 20 nucleus percent positive cells 20 number of cells 20 percent colocalized 21 percent positive cells 20 plots 21 Aperio release requirements 6 H histogram 21 I intended use 5 M macro 17 marking region to analyze 18 mark-up image 19 color codes 19 mark-up image type 8 monitor requirements 6 N nuclear stain, calibrating 13 O opening image in Specrum 10 P plots 21 positive stain, calibrating 13 prerequisites 6 R results 20 mark-up image 19 plots 21 quantitative 20 visual representation 19 running analysis 18 S sample analysis 18 segmenting cytoplasm 15 segmenting nuclei 14 selecting algorithm 10, 18 T third stain calibration 14 thresholds, positivity Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc. 2014

23 Symbols ÌÌThe following symbols may appear on your product label or in this user s guide: Manufacturer Date of manufacture (year - month - day) European Union Authorized Representative In vitro diagnostic device Serial number Relative humidity range Storage temperature range Electronic and electrical equipment waste disposal The exclamation point within an equilateral triangle is intended to alert you to the presence of important operating and maintenance (servicing) instructions. Le point d exclamation dans un triangle équilatéral vise à avertir l utilisateur qu il s agit d instructions d utilisation et d entretien importantes. The lightning flash with arrowhead symbol within an equilateral triangle is intended to alert you to the presence of uninsulated dangerous voltage within the product s enclosure that may be of sufficient magnitude to constitute a risk of electric shock to persons. Le symbole de l éclair avec la pointe de flèche dans un triangle équilatéral vise à avertir l utilisateur que le boîtier du produit présente une «tension dangereuse» non isolée d une amplitude suffisante pour constituer un risque d électrocution. The flat surface with waves symbol within an equilateral triangle is intended to alert you to the presence of hot surfaces which could cause burn damage. Le symbole d une surface plane et de vagues dans un triangle équilatéral vise à avertir l utilisateur de la présence de surfaces chaudes qui peuvent causer des brûlures. The UV lamp within an equilateral triangle is intended to alert you to the presence of UV light within the product s enclosure that may be of sufficient magnitude to constitute a risk to the operator. La lampe UV dans un triangle équilatéral vise à avertir l utilisateur de la présence de rayonnement UV dans le boîtier du produit qui peut être d une amplitude suffisante pour constituer un risque pour l utilisateur. Cytoplasm Algorithm User s Guide, Revision C Leica Biosystems Imaging, Inc

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