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1 User Reference Manual CYTO-NUCLEAR IHC QUANTIFICATION [Version 1.0] (Intended for research purposes only)

2 Contents Overview... 3 System Requirements... 4 Compatible File Formats... 4 Input Parameters... 4 Output Parameters... 9 Contact Information... 11

Overview The Indica Labs CytoNuclear IHC Quantification tool is designed to measure both cytoplasmic and nuclear positivity on a cell-by-cell basis.

The second pair of images illustrates a double stain IHC application. Left: Cytoplasmic IHC stained tumor.

3 Overview The Indica Labs CytoNuclear IHC Quantification tool is designed to measure both cytoplasmic and nuclear positivity on a cell-by-cell basis. Furthermore, it is capable of measuring either one or two IHC stains on the same slide. The first pair of images illustrates a cytoplasmic single stain IHC application. The second pair of images illustrates a double stain IHC application. Left: Cytoplasmic IHC stained tumor. Right: Analysis results of cytoplasmic analysis showing negative cells colored (marked blue), weak positive cells (marked yellow), moderaltely positive cells (marked orange), and strongly positive cells (marked red). Left: Pancreas double stained for stained for Insulin and Ki-67. Right: Analysis results showing insulin positive cells (green), Ki-67 positive cells (red), and dual positive cells (cyan).

4 System Requirements The Indica Labs Cyto-Nuclear IHC Quantification tool is designed to integrate seamlessly into the Aperio release 11 platform. This software can be installed to run server-side with the Aperio Spectrum software as well as client side within the Aperio ImageScope software. Both ImageScope and Spectrum should be at version 11.0 or later. Compatible File Formats Since this software can be fully integrated into the Aperio platform it can be used to analyze all image formats that are compatible with Aperio ImageScope and Spectrum. At the time of writing this document, this includes the following image formats: JPG TIFF SVS (Aperio) NDPI (Hamamatsu) MRXS (Zeiss) JP2 CWS (Olympus) Input Parameters The Cyto-Nuclear IHC Quantification algorithm supports a number of input parameters that allow you to configure and fine-tune the algorithm for your particular application. Each of the input parameters is described in this section: Nuclear Stain The nuclear stain (typically haematoxylin) is required to locate cells. This parameter specifies the optical densities of the RGB color components of the nuclear stain. This parameter should consist of three comma separated decimal values each ranging from 0.0 to 1.0. The simplest way to generate these values is to download the free ImageScope EyeDropper Plugin and click on a representative nucleus. The EyeDropper plugin will calculate the RGB OD values for you and copy them to the clipboard.

5 Positive Stain 1 Specifies the RGB OD values that describe the positive IHC stain (usually DAB). This parameter should be used in conjunction with the Stain 1 Localization parameter. The simplest way to generate the values for this parameter is to download the free ImageScope EyeDropper Plugin and click on a representative signal to calculate the RGB OD values. Whenever modifying these values, be sure to test that the stain separation is working properly using the Markup Image parameter to show each stain isolated. Positive Stain 2 Specifies the RGB OD values that describe the positive IHC stain in double stain IHC applications. If yours is a single stain IHC application, then you can set this parameter to 0.0,0.0,0.0 which tells the algorithm that this stain is not set or doesn t exist. But if yours is a double stain application then this parameter should be used in conjunction with the Stain 2 Localization parameter. The simplest way to generate the values for this parameter is to download the free ImageScope EyeDropper Plugin and click on a representative signal to calculate the RGB OD values. Whenever modifying these values, be sure to test that the stain separation is working properly using the Markup Image parameter to show each stain isolated. Stain 1 Localization can be set to Nucleus or Cytoplasm. The algorithm always measures stain 1 in both the nucleus and the cytoplasm separately and both measurements are reported in the outputs. However, this parameter controls which of those two measurements is used for classifying a cell as positive or negative. Furthermore, when this value is set to Nucleus, then stain 1 is also used for detecting cells, in addition to the nuclear stain (usually haematoxylin). Stain 2 Localization can be set to Nucleus or Cytoplasm. The algorithm always measures stain 2 in both the nucleus and the cytoplasm separately and both measurements are reported in the outputs. However, this parameter controls which of those two measurements is used for classifying a cell as positive or negative. Furthermore, when this value is set to Nucleus, then stain 2 is also used for detecting cells, in addition to the nuclear stain (usually haematoxylin). If your application does not use a second positive stain, then you should set Positive Stain 2 to 0.0,0.0,0.0 and then the value of this localization parameter is irrelevant. Minimum Tissue OD An optical density value which allows the algorithm to exclude regions that are not dark enough to be counted as tissue. Cells that

6 reside on the edge of the tissue will be affected by this parameter because their cytoplasmic regions will be restricted from extending beyond the calculated tissue edge. Also, Floating nuclei that are not part of a larger tissue object will be ignored which is not desirable for cell lines and other such non-tissue applications. In those cases, you should set this parameter to 0.0 which means that the algorithm will treat all areas as tissue regardless of the staining. Nuclear Contrast Threshold In order to detect nuclei, the algorithm relies on sufficient contrast between the darker nucleus and the lighter background staining. This value dictates how much contrast is required for nuclear detection. The value ranges from zero to one. Decreasing the value results in fewer nuclei detected, increasing the value results in more. Note, sometimes increasing this value has the unexpected effect of removing nuclei. This occurs because multiple nuclei are joined together and then disqualified because of their large size. Typically this value should not be adjusted far from the default value of 0.5. Minimum Nuclear OD Allows the algorithm to exclude faintly stained objects that are misinterpreted as nuclei. Since nuclei are typically stained darkest with haematoxylin you can set this threshold such that darker objects are true nuclei and lighter objects are not nuclei. The value of this parameter can be a decimal value ranging from 0 (totally white) to 1 (totally black). Values closer to zero will result in more nuclei detected and values closer to 1 will result in only the darkest nuclei being detected. Minimum Nuclear Size Allows the algorithm to exclude detected objects that are too small to be nuclei. The specified value in microns squared should be set to the smallest nucleus size that you d like to detect. Objects smaller than this will not be counted. Maximum Nuclear Size Allows the algorithm to exclude detected objects that are too large to be nuclei. The specified value in microns squared should be set to the largest nucleus size that you d like to detect. Objects smaller than this will not be counted. Maximum Cytoplasmic Radius A cells cytoplasmic region is estimated and drawn in the markup image with a black line that is some distance (pseudoradius) around each nucleus. This parameter controls that distance. Note, in situations where nuclei are close enough together, then this distance is might not be reached, in which case the algorithm will extend the radius only as far as it

7 can before reaching the neighboring cell s cytoplasm. When measuring cytoplasmic positivity everything inside this calculated region excluding the nucleus is measure for each of the positive stains. Decreasing this value has the effect of testing the cytoplasmic positivity closer to the nuclei. Increasing this value has the effect of measuring the cytoplasmic positivity in a wider area around the nuclei. Stain 1 Min OD Weak Specifies the minimum optical density of the Positive Stain 1 that is required to count a cell as Weak Positive for stain 1. The average positive stain OD within a cell s nucleus or cytoplasm (depending on the value of Stain 1 Localization) must be greater than this value to be classified as Stain 1 Weak Positive. Therefore, increasing this value will result in less stain 1 weak positive cells, and decreasing will result in more. Stain 1 Min OD Moderate Specifies the minimum optical density of the Positive Stain 1 that is required to count a cell as Moderate Positive for stain 1. The average positive stain OD within a cell s nucleus or cytoplasm (depending on the value of Stain 1 Localization) must be greater than this value to be classified as Stain 1 moderate Positive. Therefore, increasing this value will result in less stain 1 moderate positive cells, and decreasing will result in more. Stain 1 Min OD Strong Specifies the minimum optical density of the Positive Stain 1 that is required to count a cell as Strong Positive for stain 1. The average positive stain OD within a cell s nucleus or cytoplasm (depending on the value of Stain 1 Localization) must be greater than this value to be classified as Stain 1 Strong Positive. Therefore, increasing this value will result in less stain 1 moderate positive cells, and decreasing will result in more. Stain 2 Min OD Weak Specifies the minimum optical density of the Positive Stain 2 that is required to count a cell as Weak Positive for stain 2. The average positive stain OD within a cell s nucleus or cytoplasm (depending on the value of Stain 2 Localization) must be greater than this value to be classified as Stain 2 Weak Positive. Therefore, increasing this value will result in less stain 2 weak positive cells, and decreasing will result in more. Stain 2 Min OD Moderate Specifies the minimum optical density of the Positive Stain 2 that is required to count a cell as Moderate Positive for stain 2. The average positive stain OD within a cell s nucleus or cytoplasm (depending on the value of Stain 2 Localization) must be greater than this value to be classified

8 as Stain 2 moderate Positive. Therefore, increasing this value will result in less stain 2 moderate positive cells, and decreasing will result in more. Stain 2 Min OD Strong Specifies the minimum optical density of the Positive Stain 2 that is required to count a cell as Strong Positive for stain 2. The average positive stain OD within a cell s nucleus or cytoplasm (depending on the value of Stain 2 Localization) must be greater than this value to be classified as Stain 2 Strong Positive. Therefore, increasing this value will result in less stain 2 moderate positive cells, and decreasing will result in more. Output Image This algorithm allows you to control a variety of output images for tuning purposes and various use cases. During the tuning process, you should test this output to be sure the stains are separating well. You should retest the stain separation for all stains whenever any of the stain inputs are modified. o Nuclear Stain shows the deconvolved stain channel for the nuclear stain. o Positive Stain 1 shows the deconvolved stain channel for positive stain 1. o Positive Stain 2 shows the deconvolved stain channel for positive stain 2. o Stain 1 markup color codes weak, mod, strong cells for stain 1 (useful for setting thresholds and also as final output for single stain analysis). o Stain 2 markup color codes weak, mod, strong cells for stain 2 (useful for setting thresholds) o Dual Stain Markup color codes cells as positive for each stain or dual positive (useful for final output when multiplexing stains) Verbose Outputs In addition to generating summary metrics, this algorithm is capable of generating verbose output which describes the various metrics for each individual cell. This can be a large amount of data for whole slide images so leave this set to None unless you anticipate needing the verbose data. To enable this feature, set this parameter to CSV which will generate a number a CSV (Comma Separated Value) file which can be opened in Excel and most statistical analysis software. Note, on Windows 7, this file is created in C:\ProgramData \ Indica Labs \ Verbose Output \ CytoNuclear_date_time.csv. Please note, ProgramData is a hidden folder so you ll need to type it in the location manually rather than browsing to it. On older operating systems, this file is created in a similar folder structure under a folder

9 called Application Data. Please contact support if you have problems locating this file. Output Parameters This image analysis tool reports a set of results for each distinct analysis region and for the entire digital slide if analyzed. The results include the following output parameters: Total Cells the total number of cells discovered.. % Stain 1 Positive Cells the percentage of the total cells that were positive for stain 1 in the designated stain location (nucleus or This percentage includes all weak, moderate, and strong cells for stain 1. % Stain 1 Positive (weak) Cells the percentage of the total cells that were weakly positive for stain 1 in the designated stain location (nucleus or % Stain 1 Positive (moderate) Cells the percentage of the total cells that were moderately positive for stain 1 in the designated stain location (nucleus or % Stain 1 Positive (strong) Cells the percentage of the total cells that were strongly positive for stain 1 in the designated stain location (nucleus or. % Stain 2 Positive Cells the percentage of the total cells that were positive for stain 2 in the designated stain location (nucleus or This percentage includes all weak, moderate, and strong cells for stain 2. % Stain 2 Positive (weak) Cells the percentage of the total cells that were weakly positive for stain 2 in the designated stain location (nucleus or % Stain 2 Positive (moderate) Cells the percentage of the total cells that were moderately positive for stain 2 in the designated stain location (nucleus or

10 % Stain 2 Positive (strong) Cells the percentage of the total cells that were strongly positive for stain 2 in the designated stain location (nucleus or % Dual Positive Cells the percentage of the total cells that were positive for both stain 1 and stain 2. % Neutral Cells the percentage of the total cells that were neither positive for stain 1, nor stain 2. Avg Stain 1 Nuclear OD average optical density of stain 1 within all nuclei. Note this value is still calculated even if stain 1 is set for cytoplasmic localization. Avg Stain 1 Nuclear OD average optical density of stain 1 within all nuclei. Note this value is still calculated even if stain 1 is set for cytoplasmic localization. Avg Stain 1 Cytoplasmic OD average optical density of stain 1 within all cytoplasmic regions. Note this value is still calculated even if stain 1 is set for nuclear localization. Avg Stain 2 Cytoplasmic OD average optical density of stain 1 within all cytoplasmic regions. Note this value is still calculated even if stain 1 is set for nuclear localization. Avg Cell Area average area of all cells given in microns squared. This area includes both nuclear and cytoplasmic areas combined. Note this value should be used with caution because it is heavily influenced by the input parameter max cytoplasmic radius. Avg Cytoplasmic Area average area of all cells cytoplasmic regions given in microns squared. Does not count nuclear regions. Note this value should be used with caution because it is heavily influenced by the input parameter max cytoplasmic radius. Avg Nucleus Area average area of all cells nuclear regions given in microns squared. Does not include cytoplasmic regions.

11 Total Tissue Area the total area (in microns squared) of tissue area analyzed. Areas that did not exceed the minimum tissue OD are not counted as tissue. Contact Information For technical support and other information, please contact Indica Labs, Inc. Visit: Dial: +1 (505)

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