densitoquant 1.15.1 user’s guide · densitoquant 1.15.1 user’s guide the software is provided...

DQ1.15.1-UG-EN Rev1

DensitoQuant1.15.1

User’s Guide

April 17, 2012Rev. 1

DensitoQuant 1.15.1 User’s Guide

ContentsDisclaimer....................................................................................................................................3

Character Formats and Symbols...................................................................................................5

Terms and Abbreviations..............................................................................................................6

1 About DensitoQuant and Measurement Visualization...............................................................71.1 DensitoQuant in the 3DHISTECH Product Family......................................................................71.2 Overview of DensitoQuant........................................................................................................8

1.2.1 DensitoQuant Algorithm..................................................................................................................................81.2.2 Annotations, Fields, and Objects in DensitoQuant .........................................................................................81.2.3 MISP Files.......................................................................................................................................................101.2.4 Using a MISP File on Other Slides...................................................................................................................101.2.5 DensitoQuant Measurements........................................................................................................................11

2 Installation of DensitoQuant 1.15.1 Bundled with Pannoramic Viewer 1.15.2.........................122.1 Prerequisites of the Installation...............................................................................................122.2 Licensing...................................................................................................................................122.3 Installing Pannoramic Viewer and Application Modules.........................................................13

3 Working with DensitoQuant....................................................................................................213.1 Before you start.......................................................................................................................213.2 Creating Annotation for DensitoQuant....................................................................................223.3 Creating DensitoQuant Measurement.....................................................................................223.4 Exporting Measurement Data..................................................................................................263.5 Running Batch Process in DensitoQuant.................................................................................26

Index..........................................................................................................................................28

References.................................................................................................................................29

April 17, 2012 – Rev. 1 3DHISTECH Ltd. 2(29)


DisclaimerCopyright © 2001-2012 3DHISTECH Ltd.All rights reserved.

Intellectual property related to the 3DHISTECH products are as follows:

EP 1,789,831; US 7,663,078; EP 2,024,772; JP 2009-512685; US 12/301,781; EP 1,644,767; JP 2007-516451; US7,327,515; EP 1,994,398; JP 2008-557835; US 12/281,566; U0700210; US 6,246,785; HU0900142; HU0700404; PCT/HU2007/000065; EP 08762668.5; US 12/663,537; PCT/AT2006/000492; JP 2008-542552; EP 06817469; US 12/095,545; PCT/AT2006/000493; EP 06817470.5; US 12/095,596; HU0900741; US 61/264,732; US 11/826,752; P0401870; PCT/IB2005/050344; US 11/662,976; HU0700409; US 12/042,050; US 77/636,144 and 007502032.

The above mentioned granted patents, granted utility model, registered trademarks, pending international and national patent applications and the other corresponding IP are the sole properties of the 3DHISTECH Ltd.

3DHISTECH Ltd. is not liable for damage of whatever nature (including, but not limited to, general or specific damage, indirect damage, consequential damage or incidental damage, including the results of the analysis of the digitized slides, for example, change of health status related to erroneous diagnosis from the digitized slide(s)) that stems from or is associated with use of Product, digitized slides, quality of staining, quality of stained slides, quality of used method of staining. 3DHISTECH Ltd. assumes no responsibility for the functionality and fault-free condition of your “application programs” (Workflows, VBA macros, Commander scripts).

CAUTION

For research and education uses only, not for use in diagnostic procedures. This product has not been approved or cleared as a medical device by the U.S. Food and Drug Administration, nor any other regulatory authority. The data and images obtained or viewed using this product are not intended for clinical or diagnostic use.

For performance evaluation only, the performance characteristics of this product have not been established.

Third-Party Disclaimer (LibTIFF – TIFF Library and Utilities)

3DHISTECH Ltd. is not liable for damage of whatever nature (including, but not limited to, general or specific damage, indirect damage, consequential damage or incidental damage, including damage resulting from lost profits, interruption of business, loss of business information, pecuniary loss or similar damage) that stems from or is associated with the incorporated part of this Product that is made by Silicon Graphics Inc.



THE SOFTWARE IS PROVIDED “AS IS” AND WITHOUT WARRANTY OF ANY KIND, EXPRESSED, IMPLIED OR OTHERWISE, INCLUDING WITHOUT LIMITATION, ANY WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE.

IN NO EVENT SHALL SAM LEFFLER OR SILICON GRAPHICS BE LIABLE FOR ANY SPECIAL, INCIDENTAL, INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND, OR FOR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS OF USE, DATA OR PROFITS, WHETHER OR NOT ADVISED OF THE POSSIBILITY OF DAMAGE, AND ON ANY THEORY OF LIABILITY, ARISING OUT OF OR IN CONNECTION WITH THE USE OR PERFORMANCE OF THIS SOFTWARE.

Further Information

For the latest information on 3DHISTECH products and services, please visit our website at the following URL: http ://www.3dhistech.com .

Company Address: 29-33 Konkoly-Thege Miklos street1121 Budapest – HUNGARY


http://www.3DHISTECH.com/

http://www.3DHISTECH.com/

DensitoQuant 1.15.1 User’s Guide Character Formats and Symbols

Character Formats and SymbolsThe following character formats and symbols are used in this User’s Guide:

Example Abbreviation or term that is explained in section Terms and Abbreviations (page 6).

Example Words or characters that appear on the screen. These include field names, screen and window titles, push-buttons and menu names, paths or options.

Keys on the keyboard. For example, function keys (such as F11) or the Ctrl+M key combination.

Example Cross-references to other documents or sections within this document.

Warning! Indicates that you need an additional module to use the feature or that there are prerequisites for the task.

Important!Contains an important piece of information or a recommendation. The application will work if you choose not to follow the recommendation but its performance might be less than optimal.

Note: Contains a suggestion about using the application in some other way or to some interesting purpose.


DensitoQuant 1.15.1 User’s Guide Terms and Abbreviations

Terms and AbbreviationsAnnotation A particular detail of a digitized slide. DensitoQuant can perform measurements

on annotations.

Digital slide / Virtual slide Slide digitized with a Pannoramic scanner microscope, for example Pannoramic SCAN. Consists of an MRXS file and a folder with the same name as the MRXS file.

Field In DensitoQuant: an annotation.

In TumorBoard: a grid cell.

Field-specific measurement

A measurement that is related to the entire annotation or the selected area of the annotation.

DensitoQuant algorithm A segmentation and measuring algorithm run under Pannoramic Viewer on slides digitized with Pannoramic microscopes.

IHC Immunohistochemistry

TumorBoard An optional module for Pannoramic Viewer. It allows you to perform (semi)quantitative histopathological examinations of IHC-stained slides, digitized with Pannoramic scanners.

TumorBoard project A set (or subset) of slides stored in the same folder for batch evaluation.

Layer A group of cell compartments matching the same stain and size criteria. For example, you can configure DensitoQuant to detect the nuclei and the cytoplasms in an annotation as two layers based on how the stain dyed them.

Measurement DensitoQuant can perform two types of measurements:

• field-specific (related to the annotation)

• object-specific

MISP Microscopic Image Segmentation Profile

A DensitoQuant segmentation and measuring algorithm is stored in a MISP file.

MRXS File extension of a digitized Pannoramic slide.

Object A cell compartment (for example the nucleus), cell or tissue that is relevant for the immunohistochemical analysis.

Object-specific measurement

A measurement that relates to the objects found in the annotation, for example its area or perimeter.

Segmentation Identification of cells or cell compartments based on their different reactions to the immunohistochemical staining.


DensitoQuant 1.15.1 User’s Guide 1 About DensitoQuant andMeasurement Visualization

1 About DensitoQuant and Measurement VisualizationDensitoQuant is an embedded module under Pannoramic Viewer. It extends the basic application and offers a framework to configure, perform and visualize immunohistochemical measurements on the virtual slides scanned and digitized with Pannoramic microscopes or on other virtual slides converted to MRXS file format.

DensitoQuant measures the density of immunostain based on the colors of cell compartments from immunohistochemical stains.

1.1 DensitoQuant in the 3DHISTECH Product Family

The following picture shows how DensitoQuant extends Pannoramic Viewer and how it fits into the 3DHISTECH product family.

1. Slides are digitized with Pannoramic microscopes, for example, Pannoramic SCAN 150. For more details, see Pannoramic SCAN 150 1.15 User’s Guide. A digitized slide consists of an MRXS file and a folder with the same name as the MRXS file.

2. Digitized slides are stored either on a local hard disk of your computer or on a dedicated server (for example, Pannoramic CaseCenter) for storing virtual slides.

3. Virtual slides stored locally can be viewed directly in Pannoramic Viewer. If a slide is stored on a CaseCenter server, it can be opened in Pannoramic Viewer or via Teleconsultation



from the CaseCenter web interface. Local slides are opened directly with Pannoramic Viewer.

4. DensitoQuant allows you to perform measurements on digital slides and to export measurement data.

5. Digital slides, including slides that store DensitoQuant measurements, can be shared via PathoNet (www.pathonet.org). They can also be used in E-School as training and/or exam materials.

1.2 Overview of DensitoQuant

You perform two types of task in DensitoQuant:

• adjust detection settings of the algorithm that create measurements and calculates scores for each selected annotation.

• export measurement results.

1.2.1 DensitoQuant Algorithm

DensitoQuant algorithm parameters can be set for each annotation on a slide. The information needed for the algorithm is not saved on the slide but in a Microscopic Image Segmentation Profile (MISP) file, which can be later used to evaluate other annotations on the same slide or on other slides. Measurement result are saved directly to the slide.

Note: Since DensitoQuant allows you to make measurements on virtual slides, and digital image processing can cause loss of quality, please, always make sure that the measurements are acceptable to you.

1.2.2 Annotations, Fields, and Objects in DensitoQuant

DensitoQuant uses annotations. If you want to perform measurements on the entire digitized area of the slide, you need an annotation that covers the entire area.

You can select any of the annotations created in Pannoramic Viewer, including annotations containing DensitoQuant measurements.


http://www.pathonet.org/


Note:

You can add a caption and a description to an annotation. If you want to see a piece of information, for example a short comment, to be visible in the gallery that you can export from DensitoQuant, add it to the caption. Only the annotation caption is exported, the description is not.

Create unique captions to make annotations distinguishable, thus the measurements can be easily identifiable. For more information on creating and managing annotations, see section 4.9 Working with Annotations in Pannoramic Viewer 1.15.2 User’s Guide.

The following image shows a virtual slide treated with IHC stains. There are two annotations on the slide, which are possible targets for DensitoQuant analyses.

Annotation types:

• field: user-defined annotation (rectangle, polygonal/freehand)

• objects: (nuclei or membrane tissues): algorithm-defined annotation in the field



If you work with TMA slides, Pannoramic Viewer and DensitoQuant handles spots as annotations, since the TMA application is able to create annotations around the spots with the Spotfinding function.

1.2.3 MISP Files

DensitoQuant algorithms are saved in MISP files that store the following information:

• color tolerance and pixel intensity values

1.2.4 Using a MISP File on Other Slides

You can reuse a profile stored in a MISP file on other slides from the same batch (for example, slides from the same specimen) without modifications. You can also use the same algorithm on slides from other specimens with the same staining.

You might need to adjust the profile to ensure that differences in the specimen or stain quality do not affect the result.



1.2.5 DensitoQuant Measurements

DensitoQuant creates field-specific measurements: sums up pixel number, density, and ratio. The following data are displayed in the Field Statistics panel (see section 3.3 for more information):

No. Total pixels

No. Negative pixels

No. Weak-Positive pixels

No. Moderate-Positive pixels

No. Strong-Positive pixels

Mean Density of all pixels

Mean Density of Negative pixels

Mean Density of Weak-Positive pixels

Mean Density of Moderate-Positive pixels

Mean Density of Strong-Positive pixels

Ratio of Negative pixels to total pixel number

Ratio of Weak-Positive pixels to total pixel number

Ratio of Moderate-Positive pixels to total pixel number

Ratio of Strong-Positive pixels to total pixel number

Ratio of of all pixels to total pixel number


DensitoQuant 1.15.1 User’s Guide 2 Installation of DensitoQuant 1.15.1Bundled with Pannoramic Viewer

1.15.2

2 Installation of DensitoQuant 1.15.1 Bundled with Pannoramic Viewer 1.15.2DensitoQuant is not a stand-alone application. Because it runs under Pannoramic Viewer, you have to install Pannoramic Viewer before installing the application.

2.1 Prerequisites of the Installation

Prerequisites Description

Hardware Minimum:

Pentium P4, 3GHz, 1GB RAM

Recommended:

Pentium C2D (DualCore) 2 GHz, 2GB RAM

Operating system Microsoft Windows XP Professional SP3 32-bit or Windows 7 64-bit, EN/HU

Disk space Pannoramic Viewer 1.15.2 bundled with DensitoQuant 1.15.1

Minimum: 300MB

Recommended: 500MB if using Windows XP and prerequisites need to be installed

2.2 Licensing

You receive a product-specific license when purchasing any of the 3DHISTECH applications.

Floating license can be used for running applications within a LAN (Local Area Network) segment. The dongle key must be plugged into a computer that can be accessed any time (switched on and continuously operating) and on which a free Pannoramic Viewer has been installed.

Important!

The license server using the dongle and the connecting clients have to be within the same local network (LAN).

Contact 3DHISTECH support at [email protected] if you want to extend the license, as in this case some configuration of the license server and of the clients is required.



1.15.2

2.3 Installing Pannoramic Viewer and Application Modules

1. Launch the 3DHISTECH_Apps_1_15_1_9_RTM.exe file that you received on a 3DHISTECH installation disc or downloaded in a compressed file, then follow the instructions in the installation wizard.

NOTE: If the wizard finds a previous version of the application, it has to be uninstalled first. See steps 12-16.

2. Set the license mode. If necessary, select install with HASP dongle, otherwise select a license file.



1.15.2

3. Click Browse to locate and open the license file.

4. Accept the End-User License Agreement, and click Next.



1.15.2

5. Select the components you want to install.

6. The installer automatically downloads the installers of the missing prerequisites.

If you need help with installing the prerequisites, contact the 3DHISTECH Support at [email protected].

7. Click Install to install prerequisite software, then follow the instructions of the installation wizard.


mailto:[email protected]


1.15.2

8. When all the prerequisites have been installed, click Install to continue with the installation of the selected components of the 3DHISTECH application package.

9. A progress bar and an information box is displayed, showing the status of the installation procedure.



1.15.2

10. Leave the Show User's Guide option checked to open the folder containing the digital copy of this User's Guide. Click Finish to close installation wizard.

11. If the installer cannot find Adobe Reader on your computer, the below dialog box appears.

Click Yes, and the official Adobe website will be opened in your browser where you can download the Adobe Reader installation file from.



1.15.2

12. If there is an older version of the applications installed on your computer, you have to uninstall them first.

13. If the uninstall wizard window opens, click Next.



1.15.2

14. The wizard automatically detects the folder of the previously installed applications. Click Uninstall to proceed.

15. When the uninstallation of Pannoramic Viewer and its modules is complete, click Finish to close the wizard.



1.15.2

16. After the previously installed applications are removed, click Next to launch the installation wizard.


DensitoQuant 1.15.1 User’s Guide 3 Working with DensitoQuant

3 Working with DensitoQuant

3.1 Before you start

DensitoQuant works on selected annotations. You can run other algorithms on several annotations by selecting all the annotations you need before you start the algorithm.

Important!

DensitoQuant algorithms utilize the computer’s hardware (mainly the CPU and the memory). The speed of the DensitoQuant evaluation depends on the hardware configuration and performance, the size and the number of the measured annotations, and the quantity of detected objects.

During detection process, displaying of the detected objects and relating parameters depends on the memory capacity of the computer; in extreme cases measurement process can be foiled because of insufficient RAM when analyzing the whole slide.

To ensure accurate results and to reduce computation time:

• Make the training annotation as small as possible. The annotated areas should not be larger than 300×300 µm (1:1 magnification).

• When you create a free-hand annotation, follow the outline of the area as closely as possible.

• Select the annotation area appropriately, for example, select an area that is rich in tumor cells instead of connective tissue.

Note:

If you need to make measurements on the entire specimen, simply select the whole slide.



3.2 Creating Annotation for DensitoQuant

To create annotations for DensitoQuant, follow the steps below:

1. Open the slide in Pannoramic Viewer. For more information on how to open slides in Pannoramic Viewer, see section 4 Working with Pannoramic Viewer in Pannoramic Viewer 1.15.2 User’s Guide.

2. Create the annotation on the area of your interest:

I. Select the annotation type (rectangle or free-hand) and cover the area of your interest with the annotation.

For information on navigating and zooming on the slide, see section 4.5 Viewing slides in Pannoramic Viewer 1.15.2 User’s Guide.

II. In the Create or Edit Annotation window add a title to the annotation, enter a comment, determine the background color of the title and the outline color of the annotation. You can either select from predetermined colors or define custom colors.

Annotation descriptions are visible only in Pannoramic Viewer.

3. Repeat step 2 for each area on which you want to run the algorithm.

3.3 Creating DensitoQuant Measurement

Whenever you create a MISP file, you train DensitoQuant. In the DensitoQuant Wizard you mark sample objects (you put a color marker/virtual stain on them) and select filters.

After you set the basis for the object identification, you can run DensitoQuant on any number of annotations of this slide, it will detect the objects by color according to your settings.

Note:

To view the existing annotations and existing measurements on the slide, click INFO in the slide toolbar and then select Show Annotations or Show Measurement Annotations from the shortcut menu.



Important!

Select Show Measurement Data from the Viewer’s INFO menu to display detailed measurement information according to annotations only when the analysis process is finished, otherwise the slide must be reopened.

1. Open the annotated slide on which you want to run DensitoQuant in Pannoramic Viewer.

2. Launch the Image Analysis Modules menu by clicking the icon in the toolbar, then select DensitoQuant in the IHC applications section of the window.

Click Delete Measurements to clear all the previously saved measurement data related to the slide.

DensitoQuant leaves the slide open. If more than one slide is open when you launch the Wizard, it always uses the active one.



3. In the Available window panel, select the annotation(s) (in case of TMA slides, the spot number) on which you want to run the algorithm, and move them to the Selected panel by clicking > or >>. If you need the whole slide to be processed, select the Process whole Slide option.

NOTE: If you call the DensitoQuant application through a TMA project, the Annotation Selection window is NOT displayed, and all the spots will automatically be selected for measurement.

4. After clicking the Start button, the main application window appears:



In the Detection field of the main window, you can set tolerance values and Score levels.

By activating the Toggle Mask you will be able to make real-time adjustments, also measurement results can be seen in the Field Statistics area.

When adjusting tolerance values (detection tolerance of blue and brown pixels) the current value is shown above the slider.

Drag and move the gap between Score level bars to set pixel intensity according to score levels.

Field Statistics Lists measurement results

Copies measurement data to clipboard

Segmentation Profile (MISP file) can be saved, loaded, or the default can be restored.

Navigate between annotations with the arrow buttons, or measure them all in case the desired detection values are set.

Detection results are displayed in the Field Statistics panel of the preview window. For the detailed list of measurement data, see section 1.2.5 .

5. The Field Statistics list contains information based on the actual preview – when zooming in the annotation (in the preview) the application automatically updates these values.

To lock the screen, so that dragging the displayed preview image in a way that other areas of the slide (beyond the boundaries of the annotation) cannot be seen, activate the Navigation Lock function at the bottom left corner of the preview screen.

Note, that this still makes zooming in and out within the annotation and navigation between annotations available for you.

6. You can finalize the profile creation by saving the MISP file by clicking Save, or quit the wizard by closing the window. If the profile is not saved when exiting the window, you receive an error message.

Click No to confirm exit without measuring all the selected annotations.



3.4 Exporting Measurement Data

To export DensitoQuant measurement results do the following:

1. If the Field Statistics function is activated, copy measurement data to clipboard by clicking

the button.

2. Create a new or open an existing text or CSV file in Notepad, Word, or Excel.

3. Paste (Ctrl+V) the data into the file.

4. Save the file as a text or CSV file.

3.5 Running Batch Process in DensitoQuant

Pannoramic Viewer allows you batch processing a set of slides. You can find the batch processing icon at the bottom section of the tree-view panel on the left side of the main window.

1. Select a slide or folder in the tree-view panel.

2. Click the button to the item to the list.

3. Remove a slide from the list by clicking the button.

4. If you want to upload slides to the CaseCenter server, click the button.



5. Click the button when finished, and the following window appears:

6. Select the slides you want DensitoQuant to work with and specify annotations and MISPs for which they should be analyzed.

7. Click the Add button to add the slide to the Batch Processing List.

8. Activate Export Setting options if necessary.

9. Click Batch Processing when finished.


DensitoQuant 1.15.1 User’s Guide Index

Index

AAnnotation.............................................................................................................................................21, 27

BBatch Process...............................................................................................................................................26

CCaseCenter...........................................................................................................................................7, 8, 26Computation time........................................................................................................................................21

FField...............................................................................................................................................................5

LLayer...............................................................................................................................................................6

MMeasurement...............................................................................................................................................21MISP....................................................................................................................................6, 8, 10, 22, 25, 27

SSegmentation.......................................................................................................................................6, 8, 25Segmentation Profile............................................................................................................................6, 8, 25

VVirtual slide.........................................................................................................................................6, 7, 8, 9Virtual stain..................................................................................................................................................22


DensitoQuant 1.15.1 User’s Guide References

References

Pannoramic Viewer 1.15.2 User’s Guide

TumorBoard 1.15.1 User’s Guide


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