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HistoQuest 3.0 - TissueGnostics

1. u Region 006 _ Region 006 ac C Timelapse Regions bA Region 036 Figure 12 Expand Collapse Time Regions buttons Description section Depending on the type of item selected in the Select Region section a different description will be displayed in the Description section see image below Page 24 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Description Experiment Demo Project has 8 slides Experiment Demo _Project has 5 regions E a d Experiment Demo Project has 1 TMA block Description for Experiment Demo_Project has 25 TMA spots Experiment Comment Descripton Slide Slide 1 has 5 regions Comment Description for Slide Description Region Region 001 information Acquired Yes Description for FOV matrix size is 6x 6 Reg ion Number of images 31 Objective 70x Figure 13 Description section Import TissueFAXS HistoFAXS regions When Import TissueFAXS HistoFAXS regions option is checked the imported regions from TissueFAXS HistoFAXS will keep the original names and will be treated as ROIs preserving the same shape as in TissueFAXS If this option remains unchecked only the sample without the ROIs will be imported Page 25 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com Subregions SAAN a AAG N ALR SAAS ALALILA LS IAIA Oi IA A AA O NN CE PERE EE
2. Image Width 1600 px Image Height 1200 px Pixel Width 0 25 um Pixel Height 0 25 um Do you know the objective that was used to acquire these images Objective Figure 54 Import Folder Wizard Step 2 Advanced Settings The user has three possibilities to enter the size of the FOV for more information please see Chapter 4 1 2 of this document The Objective value can also be modified by the user 4 2 3 Step 3 Choose Markers All HistoQuest projects must have a set of defined markers in order to analyze the provided images This step is identical to the fourth step of the Import TissueFAXS HistoFAXS project wizard and it is used to choose the markers for the current project For a more detailed explanation see Chapter 4 1 3 of this document The user will be able to choose e Segmentation Method e Strategy for events in ROI e Color Separation Method cannot be changed By removing all markers by pressing Remove All Markers fn i lf the user loads a profile the segmentation method and the color separation method ote button the user will be able again to choose for the project any marker he wants Page 52 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Choose Markers Select markers from the existing ones or create new ones to use in this HistoQuest project E Choose Images What markers do you want to use in your project E HistoQuest Project E Choose Markers What
3. a Videos iL Computer B Windows C coy Work D cow Personal E a Data 2 FF tiu Network File name Figure 40 Load Profile from Existing Project browsing for the project Page 43 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 7 Load Profile from Existing Project or Load Profile and Diagrams from Existing frote Project options can be activated only when creating the project you will not be able to use them once the project was already created The diagrams will be imported from the first sample of the selected project Load Profile from Existing Project and Load Profile and Diagrams from Existing Project options require a license UjReauirements __ Load Profile requires a license for Load Marker Profile or Save Marker Profile Add Marker In any new HistoQuest project the user can operate with one of the existing markers but he can also create a new marker e Toadd anew marker press Add Marker button then type a name and optionally a description After the creation of the project the respective marker will receive the color separation method the segmentation method and the strategy for events in ROI properties Hematoxylin Description Hematoxylin stain is one of the most commonly used stains in histology Figure 41 Add Marker Dialog 7 The name is mandatory vf Note The name must not coincide with the name of an ex
4. HistoQuest contains modules that were developed with Visual C so this redistributables kit is required If you have installed previous pre release versions of Visual C 2005 or Visual Studio 2005 such as Beta 1 Beta 2 or Community Technical Preview CTP builds then you must uninstall these versions via Add Remove Programs in Control Panel before installing the final released version voi Note For further information about this package please visit the Microsoft homepage Page 11 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 2 5 Intel Performance Primitives 5 3 Intel Integrated Performance Primitives Intel IPP is a software library that increases performance of Intel s latest microprocessors Incorporating these functions into the HistoQuest software will ensure that the potential of your Intel based PC can be fully exploited for analysis of your samples 2 6 MARX Dongle Drivers HistoQuest uses a hardware based security solution from MARX Software Security When purchasing HistoQuest you will also receive an USB dongle that must be inserted in an USB port In order to successfully use the dongle you must install the corresponding driver for the operating system 2 System Requirements 2 1 Hardware Requirements Minimum e Intel Pentium 4 Processor 3 GHz e 16GB e USB Port for dongle e 100 GB available hard disk space Single monitor system with a resolution of 1024x
5. Figure 21 HistoQuest group of ROIs from TissueFAXS HistoFAXS categories c 975 mm 4 fee Figure 22 HistoQuest group of ROIs from TissueFAXS HistoFAXS categories d TMA Projects For TMA projects the user can create samples for Page 31 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Each TMA spot e Each TMA block e Each TMA slide For the Create Sample for each TMA spot HistoQuest will create a sample from each TMA spot in the project Create Sample For each TMA spot Create Sample For each TMA block Create Sample For each TMA slide Figure 23 Create Sample for each TMA spot HO oe TE W o0 a 2 oe8 090900 Figure 24 TMA spot in TissueFAXS Page 32 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Age oy ERED ail Oei deml ui 1 TEI T Figure 25 TMA spot in HistoQuest For the Create Sample for each TMA block HistoQuest will create a sample from each TMA block in the project Create Sample For each TMA spot Create Sample For each TMA slide Figure 26 Create Sample for each TMA block Page 33 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual o w E T EZ 22 Be Bee maa Figure 28 TMA block in HistoQuest Page 34 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Create Sample For each TMA spot Create Sample For each TMA spot Crea
6. LAE Figure 296 Markers toolbar Gain by increasing this parameter the gray image associated to the shade becomes brighter Hematoxylin Ki 6 DAB Multiple Reference Shades SOY xX f CAEL len lam loas loann Neen 30 00 o0 10 00 Figure 297 Shades Gain parameter Sigma by increasing this parameter more hues close to the shade are taken into consideration and thus represented in the gray image Hematoxylin ki 67 DAB Multiple Reference Shades E y lt B ee ee je e Sigma BiAA E 0 00 1000 00 Figure 298 Shades Sigma parameter Page 197 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Gauss represents the amount of blurring to be performed on the gray representation of the shade By increasing this parameter more blurring will be performed on the specific shade Hematoxylin ki 67 DAB Multiple Reference Shades SRY xX Ca CE Figure 299 Shades Gauss parameter Color Space This is a non editable field it only shows in which color space the shade was computed 7 When you modify a value of a parameter for a single shade you can apply this note modification for the respective parameter to all shades by pressing F3 from your keyboard Avoid picking background shades or such shades which are very close to the background Each shade added to a marker is by default a measurement shade Segmentati
7. Layout Common Fixed horizontal number of diagrams with the size PE Fixed vertical number of diagrams with the size ZANR Scattergrams Flow Layout with the size all i Small Range Limit Histograms Cu toff Decimals Cutoff Quantile Value Figure 455 Diagram Options Common dialog For Scattergrams the user will be able to set the Point Style and the Point Size Page 296 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Histograms Figure 456 Diagram Options Scattergrams settings dialog e For Scattergrams the user will be able to set the Bins Count the Style and the Color of bins Page 297 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Common PE Scattergrams Histograms Figure 457 Diagram Options Histograms settings dialog Sr Another way to obtain the same dialog is Tools Options Diagram ote For a detailed explanation of these issues please see Chapter 17 3 Page 298 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 14 Forward and Backward Connection Two of the most powerful tools in HistoQuest are Forward and Backward Connection They realize the identification of events between diagrams raw data and the corresponding images from each marker IE E Figure 458 Forward and Backward connection Region Viewer Page 299 of 351 Internal www tissuegnostic
8. Point Generation The detected sample is highlighted by a contour which consists of a specific number of connected points With these two parameters the contour fitting can be adjusted e Distance the initially detected region will be inflated with the specified number of pixels in order to entirely include the edges of the tissue and a small surrounding area e Max Step this parameter represents the maximum distance between two generated points The smaller the distance the more accurate the contour of a given region will be detected Tissue Advanced Settings Poink Generation a ds 2 _ _ l 1000 Figure 167 Advanced Setting Point Generation tab Show ROI interior This option available in the region contextual menu will highlight on the sample the interior of the selected ROI This feature will be useful when a freedrawn shape is very complex and it is difficult to asses its interior area Right click on the desired region and choose from the contextual menu Show Interior Region name Page 124 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual ROI 09 o o005 mm 4 J i Copy to Clipboard Save Figure 169 Region with highlighted interior Automatically display properties for selected ROI When clicking inside a ROI on the sample the name of the respective ROI will be highlighted in the project tree and the Project Items properties control wil
9. SF Se E K port TissueFAXS HistoFAXS Proje Fle Choose TissueFAXS HistoFAXS Project Select the TissueFAXS HistoFAXS project file from which you want to import regions in this HistoQuest project E Choose TissueFAXSHistoFAXS Project Where is your TissueFAXS HistoFAXS project HistoQuest Project F Projects TissueFAXS Demo_Project Demo_Project aqproj Choose Markers OE Demo_Project OW slide 1 EO Region 001 j Region 001 SO Timelapse Regions _ Region 001 Timelapse 2 of 3 uF Region 001 Timelapse 3 of 3 Select acquired regions Custom Select Select Only Standard Regions Select Only Time Regions Collapse Time Regions Expand Time Regions Description Region Region 001 Timelapse 1 of 3 information Acquired Yes FOV matrix size is 6 x 6 Number of images 36 Objective 20x FOV Size 0 54mm x 0 40mm TMA Grouping Create Sample for each TMA spot Import TissueFAXS HistoFAXS regions Import TissueFAXS HistoFAXS logical groups Import TissueFAXS HistoFAXS subregions Create Group of ROIs from TissueFAXS HistoFAXS categories Note Your project will have 2 samples TISSUEGN Figure 8 Import TissueFAXS HistoFAXS Project Step 2 You can also see which regions of the TissueFAXS HistoFAXS project can be imported in HistoQuest and select the desired ones You can find here the description for e The experiment number of slides regions TMA blocks TMA spo
10. Total Scanned Area 0 120000 mm Total Exduded Area 0 000000 mm Events Count 1 05 Use Illumination Correction No Cache Built Yes Comment Figure 111 Project Items Properties Control Sample e Excluded FOVs Count the number of FOVs excluded from the analysis when using exclusion areas or the flags e Total Scanned Area the initially scanned area of the tissue e Total Excluded Area the excluded area from the analysis when using exclusion areas or the flags Columns Count FOVs Count Exduded FOVs Count 0 000976 mm 0 Figure 112 Project Items Properties Control ROI When a region is selected in the viewer its properties are displayed in the Project rote Items Properties Control Sample Colon 1 Number of ROIs 2 Area 0 001689 mm Events Count Not computed Figure 113 Project Items Properties Control Group Page 90 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Exdusion Area 02 Coloni 0 000358 mm Figure 114 Project Items Properties Control Exclusion Area j _ The Events Count of a Group will not be displayed until it is loaded in the detail ote _ window or when the corresponding raw data is loaded in the Raw Data window The Events Count of a Group will always be Not Computed if the group is not loaded in the detail or raw data When the strategy for events in ROI is Crop events to ROI s shape and the group contains overlapping regions even if the
11. Backward Connection for gate In the image above the last window illustrates in red the selected events and in green the unselected ones Page 304 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Without a license the user won t be able to use Backward Connection W Requirements View only events from input gates When asking for backward connection from a diagram with input gates only input gates events should be displayed by default on the images This option is available in Tools Options General Backward Connection To enable the option mark the corresponding checkbox i Tice N a O T a N E aa E HistoQues UpTtions General emen 1 Backward Connection p ae i Show all fields of view f ET Figure 465 View only events from input gates option 14 3 2 Histograms By accessing the contextual menu of the histograms a set of operations for backward data will be displayed when choosing View Backward Data View Backward Data For Bin View Backward Data For Left Quadrants View Backward Data For Right Guadrants Figure 466 Histogram Backward Data context menu e View Backward Data for Bin shows the backward data for the events belonging to a bin e View Backward Data for Left Right Quadrants shows the backward data of the events placed on any of the two quadrants created by the vertical axis of the cutoff When accessing any of the options for back
12. Group Auboscale Mo t Scaling type Linear Current Region Group Figure 404 Scattergram Parameters control box When Autoscale is set on Yes value the user can propagate this setting to different regions Scaling type Linear Current Region Group Hematoxylin Area urn Current Sample and its Regions 0 All Samples and their Regions 160 Current Region Group Auboscale Mo t Scaling type Linear Current Region Group Figure 405 Scattergram Parameters control box Autoscale parameter The Scaling type parameter changes the scaling from linear to logarithmic and from logarithmic to linear The changes can be propagated to different regions Page 266 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Hematonytli 10 Logarithmic Scaling type Ke DAB Sum Intensity z0 40 Hematoxylin Mean Intensity DAB Sum Inten Hematoxylin Mean Intensity Ki6 GAB Sum Inten 10090 Linear Scaling oo KIF DAB s Intensity i 20 40 Hematoxylin Mean Intensity Figure 406 Scattergram Parameters Scaling Type parameter Logarithmic left and Linear right e Set Cutoff on option will display a menu in which the user will be able to set new values for one axis of the cutoff Also the cutoff can be propagated from this dialog In order to perform the propagation the axes of the different diagrams should be the same Current
13. Manual Correction Without a license the user will not be able to apply manual correction Also in the raw data dialog the user will not be able to delete events If the user does not have a license for a certain feature HistoQuest will display an informative message Your license does not allow you to import or add TFAXS HistoFaxs projects For more information contact your local distributor A list of authorized distributors is ae here Figure 530 Example of License Message Page 348 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 19 Memory Issues Every time HistoQuest needs to load the events for a project item the memory is checked to see if there is enough memory available to perform that action If the memory is insufficient an explanatory message is presented to the user according to the situation Below there is a list of situations that might generate memory related issues e Loading a project item in detail window if events cannot be loaded the project item is still opened The image results are available but the diagrams are empty Printing contains a warning message e Loading Compare Set the compare set is opened even if events cannot be loaded but the diagrams are empty A warning message is shown e View Raw Data this option requires more memory and it can happen more often that the actual events cannot be displayed The form is still opened but with no events a warning me
14. Show annotations in browser Measurement Units Diagram System Data Figure 188 Set ROI Color The Delete Results options will delete the data of all regions in a group If one of the ROIs from a Group contains results a warning message will appear Figure 189 Delete Results warning message Page 135 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Show in Detail Window Analyze Analyze Unprocessed FOVs Rename Remove Add to Existing Group Add to New Group Copy Propagate ROI Figure 190 Delete Results option from context menu 4 When the strategy for events in ROI is Crop events to ROI s shape the groups of rote _ ROls will not contain any events In this case events count value will be 0 and the z diagrams will be empty Rename group with propagation By giving a name to an existing group the respective name will be propagated to all samples if marking the Propagate Group to all Samples checkbox What is the name of the new group Propagate Group to all Samples The Group will be propagated only on the samples where it doesn t already exist Figure 191 Rename group with propagation Import Groups of ROIs Import Groups of ROIs option can be found in the toolbar of the Region viewer and also in the toolbar of the Detail Window Press Import Groups of ROIs button in order to open Import Export Groups of ROIs
15. Use the keys on the keyboard e Zoom out use the Zoom out button of the Region Viewer The zoom out is performed centered Use the keys on the keyboard e Change zoom level The current level of zoom is displayed and it can be changed by typing a new value in the edit box of the Region Viewer pos e View best fit This option allows the user to display the region so that it fits in the display area use the Best Fit button P of the Image Viewer use the B key on the keyboard e View original size Page 76 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual This option allows the user to display the region in its original size as it was acquired zoom level 100 use the View original size button 1 1 of the Region Viewer use the O key on the keyboard e Zoom to a specified area of the image Select a rectangular area of the image with the mouse to draw a rectangle keep the right mouse button pressed This option will zoom centered so that the specified region fits into the display area Objective Buttons For your convenience HistoQuest offers a selection of objective lens magnifications frequently used in histology and pathology for viewing immunohistochemical samples In order to observe a sample as it would look like with a 10x objective lens just press the 10x button to see it at the magnification of a 63x objective lens press the 63x
16. e Event Label Count e Area Sum By pressing the right mouse button on one of the already existing column headers the user will visualize a menu from which he will be able to modify the Raw Data window ajl Sort Ascending Sort Descending Clear Sorting z all Group By Box S42 column Chooser J Best Fit Se Filter Editor Best Fit all columns Figure 390 Raw Data context menu Page 257 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Adding Other Parameters in the Raw Data grid By default the Raw Data window displays only one parameter for the events of a region In order to add more parameters the user has two options e By selecting Column Chooser from the right mouse button menu on the existing column headers ail Sort Ascending Sort Descending al Group By Box S Column Chooser c Best Fit Sr Filter Editor Best Fit all columns Figure 391 Adding more Parameters context menu a By selecting Column Chooser from the right mouse button menu on the empty column header Group By Box S Column Chooser SY Filter Editor Best Fit all columns Figure 392 Adding more Parameters context menu b The user will drag the desired parameter name and drop it on the grid header Page 258 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Customization Hematoxylin Area Hematoxylin Compactness Hematoxylin Eccentri
17. e Master marker Area e Non master marker Area 11 4 3 Raw Data Columns Configuration For this segmentation method the Raw Data window will display the following default columns e Region of Interest e Field of View e Event Label Page 230 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Master marker Area e Non master marker Area 11 4 4 Statistics Configuration The Statistics for the Total Area Measurements segmentation method will display the following columns e For Total Area Measurements with count used in computing the percents X Sum i e sum value for the parameter shown on the x axis Y Sum i e sum value for the parameter shown on the y axis e For Total Area Measurements with area of a marker used in computing the percents X Sum i e sum value for the parameter shown on the x axis Y Sum i e sum value for the parameter shown on the y axis Percent Area of percent area of chosen marker relative to the total area of the marker Percent Area of Area Measurement Unit percent area of chosen marker relative to the total area of the Sample ROI Group statistics O OOOO O Gs FT uacrant Hematoxyin Area CD3 Fast Red A w C2 es wooo i ses eo w ham ems wo o w eo owa O feo oa EEA E u o aom es o ae o Hooo a p eooo en o Ja es sangre Figure 360 Total Area Measurements Statistics section 11 5 Configurable statistics
18. e Z Position the position to use from the Z stack If there is only one Z position the spin edit will only have the value O zero If the image has a single focus plan the Z Position from the wizard will be disabled Now press Next 4 3 2 Step 2 HistoQuest Project This is the same Project Info dialog as in the wizards for importing a TissueFAXS HistoFAXS project or an image including Basic and Advanced Settings Default values are filled for Project Name and Project Folder If the Project Folder does not exist the user will be asked if he wants to create it Page 56 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Basic Setings Advanced Settings What s the name of the project 2008 08 06_13 19 50 Project Description Where will the project be stored F Projects HistoQuest 2008 08 06_ 13 19 50 If the project folder does not exist do you want to create it Create Project Folder Copy images to project folder or leave images in their folder Figure 61 Import from Hamamatsu NanoZoomer Step 2 Basic Settings If you do not want to use the default project folder provided by HistoQuest you can add the project name you desire and HistoQuest will create this project folder if you check Create Project Folder A The fully qualified hiqproj file name must be less than 260 characters and the project J Note folder name must be less than 248 characters If the storage folder diffe
19. By choosing this option the Manage Gates dialog will be displayed bo he Hematosylin Mean Intensity Ki 7 DAB Mean Inten i Sample 02 a Positive Ki67 DAB be Sample 03 Sample 04 i Sample 05 Double click on a gate to display the corresponding scattergram Figure 499 Manage Gates dialog This dialog will contain all gates created on the scattergrams of the samples The user will be able to configure the gates for more details please see Chapter 13 3 1 and also delete them 17 3 3 Statistics Report The Statistics Report tool shows the different parameters of particular regions in a tabular configuration Page 326 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Project Items samples GE Slide 4 Region 037 jH Slide 4 Region 037 er Groups sa Group Ol 00 Slide 4 Region 302 Slide 4 Region 302 C Region 302 sal Groups 2D Slide 4 Region 303 p slide4 Region 303 Region 303 5d Groups EO side4 Regon oe Slide 4 Region 306 C Region 306 sal Groups Sla Slide 5 l5lide 5 O TMA Block 00071 a01 L TMA Block 00071 aoz TMA Block 00071 a03 nDO TMA Block 00071 a04 O TMA Block 00071 a05 LO TMA Block 00071 B01 Oma Block 00071 B02 Tm Block 00071 B03 O TMA Block 00071 B04 n C TMA Block 00071 BOS o l Fill Report Data Clear Report Data Drag a column header here
20. Changing these settings will not affect opened project Analyze Project Beep after Finish C Shut down after Finish Figure 502 Options Menu General Analysis dialog Any change performed in these fields will be displayed as default when creating new projects P Th Note Backward Connection set the number of FOVs to be shown in backward connection Page 328 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual General Backward Connection Show all fields of view EJ Show only fields of view View only events from input gates Figure 503 Options Menu General Backward Connection settings box Task Manager the number of concurrent running tasks will be set and also the number of threads assigned to a task By using the parallel computing the speed of analysis can be increased a If the result of the multiplication between the maximum number of concurrent tasks and vf note the number of threads for a task equals the number of cores in the PC e g quad core CPU s have 4 cores working in parallel the overall performance of the computer for OTHER tasks and applications might become very poor You may either leave the computer working on the HistoQuest analysis only or select a number of concurrent task at least one lower than the number of cores in your computer In case the result of the respective multiplication overpasses the number of cores of the PC there will be a
21. Options Menu Diagram Common settings box e Layout Fixed horizontal number of diagrams sets the number of diagrams displayed on a row Fixed vertical number of diagrams sets the number of diagrams displayed on a column Flow Layout the number of diagrams displayed on a row depends on the size of the diagrams and also the size of the Results tab With the size sets the dimensions of a diagram Automatic represents the default value for a diagram The other values are expressed in pixels Automatic mu iFern Fi Aukomatiz Figure 515 Diagram layout Size e Display Show Grid by checking this option a grid will be displayed on each diagram Page 337 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Display _ Show Grid Cutoff Color Figure 516 Options Menu Diagram Display Hematosylin Sum Intensity Hematoxylin Sum Intensity 0 0 10000 20000 30000 D 10000 20000 30000 Hematoxylin Sum Intensity Hematoxylin Sum Intensity Figure 517 Options Menu Diagram Show Grid disabled left and Show Grid enabled right Cutoff Color here you can set the color of the cutoff e Cutoff Decimals here you can set the number of decimals for a cutoff value e Small Range Limit here you can set the threshold value for an axis e Cutoff Quantile Value the events population according to a percent given by the user for more details please see Chapter 13 3 1 If
22. _ Show grid lines for Samples imported from TissueFAXS HistoFAXS or Hamamatsu NanoZoomer Ca _ Show name only for selected ROI Automatzaton _ Indude user name in annotations contents 4 l Show annotations in browser Measurement Units System Data Figure 214 Exclusion Area settings By right clicking on the Exclusion Area a contextual menu will appear Page 149 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com View Event Data Remove Exclusion Area OL Copy to Clipboard Save Figure 215 Exclusion Area contextual menu e View Event Data see Chapter 13 2 2 Remove Exclusion Area if choosing this option a message box will appear asking you to confirm the deletion of the selected item Figure 216 Remove Exclusion Area message box e Copy to Clipboard Displayed Image Displayed Image With Data Figure 217 Copy to Clipboard menu e Save Displayed Image as Displayed Image With Data as Figure 218 Save menu Page 150 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual If an exclusion area has FOVs that are excluded from the analysis its area will not be note affected But if the exclusion area has removed FOVs meaning without tissue the area will be affected When the Strategy for events in ROI is not set to Crop events to ROI s shape then on the non master marker event
23. each click on the separated image adds a new shade to the selected marker a Color bh si 7 Picker ie vz Marker Hematoxylin T a r m e TEF Figure 288 Picking Shades e Click again on the Color Picker button after all desired shades were chosen Separated option For a more precise selection choose Options from the Separated menu shown above A dialog will appear containing a set of specific clustering parameters for the Separated option Color Space Afinity Method Maximum Iterations Minimum Percent Figure 289 Clustering parameters dialog The Clustering is a process that separates into groups the color populations and it uses the following parameters e Color space it specifies the color model used for performing color separation It contains the following color models RGB color space it is not indicated to choose this color model because lighter hues of different colors might be grouped together HSV and HSL color spaces these two color models have the advantage that in clustering hues of different intensities are grouped together LAB and LUV color spaces are the most indicated in clustering since they are closer to human perception Page 193 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Color Space AFinity Method Maximum Iterations Minimum Percent Figure 290 Clustering parameters Color Space parameter e Affinit
24. intensities TssveFAxSplus is used for the siandardizaton of issue analysis in combination Login Version 3 0 User Name Administrator Password hoe ooh Copyright 2006 2011 TissueGnostics GmbH All rights reserved Figure 2 Login Panel Page 14 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Loading HistoQuest takes several seconds During this process of program initialization a splash screen is displayed until the application is loaded and ready to be used TISSUE Wc HMEDICAL BIA TEE H FOtwu TIO H S e o OAN HISTO ce TISSUE ANALYSIS SOFTWARE CE ATTENTION CONS UCIT OPERATING INSTRUCTIONS FOR USE Figure 3 Splash Screen 3 2 Startup window After the splash screen disappears the main window of the application is shown In order to use the full HistoQuest functionality either a new project must be created or an existing one must be loaded Having a project is mandatory otherwise the user is not able to select any images for the analysis nor choose any markers and perform analysis All this information is embedded in a project Only one project can be opened at a time and there are four available possibilities to perform this operation Page 15 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Open an existing HistoQuest project TissueFAXS HistoFAXS Import a TissueFAXS HistoFAXS project Import images from a folder
25. n Background Threshold Default Appl Figure 331 Automatic Background Threshold Background Threshold e Use Nuclei Mask By selecting Nuclei mask the area of the binary objects in the nucleus mask is also used aS measurement mask in all other channels Page 216 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Yarious Shapes 1 0 Use Ring Mask Yes j Interior Radius 6 67 um Exterior Radius 13 33 um Use Identified Cell Mask Yes fe Max Growing Steps 26 67 um 7 Skip Steps 0 um Use Nuclei Mask Automatic Background Th Background Threshold Figure 332 Use Nuclei Mask note By pressing Reset Parameters button all the parameters settings modified by the user will be restored to their default values 11 2 2 Diagrams Configuration For this segmentation method the Results tab of the detail window will display the following default diagrams set Scattergrams e Master marker Mean Intensity Master marker Area e Master marker Mean Intensity Non master marker Mean Intensity Histograms e Master marker Mean Intensity e Non master marker Mean Intensity 11 2 3 Raw Data Columns Configuration For this segmentation method the Raw Data window will display the following default columns e Region of Interest e Field of View e Event Label e Master marker Mean Intensity e Non master marker Mean Intensity 11 2 4 Statistics Configuration The Sta
26. 000000 000001 35 195490 0 000000 000001 31 150300 0 000000 000001 32 984570 0 000000 ooo0o1 38 818420 0 000000 000001 31 375000 0 000000 000001 32 966670 0 000000 000001 39 084870 0 000000 000001 26 500000 0 000000 ooo001 32 287500 0 000000 ooo001 29652480 o 000000 000001 30 397730 0 000000 000001 62 281800 0 000000 000001 27 879700 0 000000 000001 52 750480 0 000000 000001 59 558410 0 000000 ooo001 34 810890 0 000000 000001 29 395260 0 000000 000001 68 276600 0 000000 AYG 51 74 ss Awale Export To Excel Export To PDF Export To TSW Delete Events view Backward Data Exit Backward Connection D Mm A Pa Tee eee OF OD OO ws A A Be A MN e O Po w oo e Figure 386 Raw Data window e View Raw Data option on the right mouse button menu on the Results tab on the detail window Page 254 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Manage Diagrams Diagram Options Manage Gates 40 a0 Hematoxilyn Mean Intensity Figure 387 View Raw Data contextual menu e By accessing Project View Raw Data a Raw Data window will open where the region for which the data shall be displayed can be selected by pressing twice the left mouse button on the region name in the left list of the window Page 255 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Raw Data Sampe tml Raw Data for gt gt Sample 01 showing 41966 even
27. 1 2 Chapter 6 2 and Chapter 6 3 EE 4 E Figure 105 Shapes Manipulation Toolbar 5 8 Show Hide Shape This button is also common for ROIs Annotations and Exclusions If the checkbox is marked for Show Hide ROIs for example all the ROIs on the sample will be visible if it is not marked all ROIs will be hidden This setting will not be saved if you restart HistoQuest l Coloni a e e W a lt m E Show Hide ROIs va Show Hide Annotations P wa a ba SR inl i Show Hide Exclusion Regions r es ion a i a b g po L 7 Ys i a Cna Figure 106 Show Hide Shape button Page 86 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 5 9 Edit Mode Button The Edit Mode button allows the user to modify the shape and the position of ROIs and Annotations e To modify the size of a FOV find the resize points on the contour of the ROI then drag them by holding pressed the left mouse button until you obtain the desired size ROIs of any shape have resize points Figure 107 Modify ROIs Using keyboard controls to move ROIs You can use the lt keyboard controls in order to move a ROI to a new position For the rectangular and circular ROIs use CTRL key in combination with and controls in order to increase or decrease the dimension of the ROI 5 10 Analyze Button The Analyze button OF starts the analysis for the loaded project item This
28. 2 Figure 9 Regions to be imported to HistoQuest Regions can be selected one by one simply check the box associated with the region by the slide they are defined on check the box associated with the slide and available regions of it will be selected or by project check the box associated with the TissueFAXS HistoFAXS project and all regions from all slides will be selected Slides that do not contain acquired regions cannot be selected This page also provides descriptions of all items of the imported project A user can check if a region was acquired how many images it contains how many regions are defined on each slide and how many slides are in the project as previously mentioned in this chapter Maybe not all shown regions can be imported to HistoQuest and this step specifies those that are allowed A region that was not acquired in TissueFAXS cannot be used so this limitation is shown by a special font and a special representative image as seen below Regions which can be imported F OAR RE 006 oP Region 036 which cannot be imported Page 22 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 10 Items description A preview of each region and slide is displayed in the right part of the window if available The regions that are listed in red are not acquired Select acquired regions section This section gives three selection options Select acquired region
29. 40 A2 VMN OT ORION acre pacers cee ase rtd E ae iota poised vas E desea nse aerate ewan E E 47 A a ia lit 5 97616 21 F 9 Aen er ne enn sr tr one one A ene ee eee er eee 47 4 2 2 Step 2 HistoQuest PIO CCl issecnotcnsien ncceceuenestenn ce nnnceejas satdansten vee vee noscueicncieseciceanneeiexaieh ne ddaoneueexeiennesiaecounedaveonenterew dese 50 4 2 3 Step 3 Choose Markers seisseen ee eee eee eee 52 4 3 Import Images from a Hamamatsu Nanozoomer File ccc ccecccceececeeceeeeeceeeeeeeeeseeeeseeeeaeeeseeeeseueeseeeeseeesaeeees 53 4 3 1 Step 1 Choose Hamamatsu NanoZoomer file ccc cccceeccceeeeceeeeceeeeseeeeae cece ceseueeseeeeseeeseeeeseueeseeetaes 54 A32 Z 6 1 PrO Cl ne ee a ene cen eee eee eee 56 Aa Ep aa 9 810 e111 72 9 cee on a eee ee eee ee eee 58 AA MMPOM IMAGES TOM a Mirax TING sccncusezseciuddencesandenacswaneosesheuveadsnedaaeetont geecewebencasirweeec ea aa aana aa iaa Naaa 60 4A 1 Step L Choose Mirax Me eee oe ee ee oe nee ee eee ee eee 60 4A 2 2 wg 00 17 GC ee tc a ee ee eee eee 63 4 4 3 Step 3 Choose IMIAIK ONS ecicds sede ses Qcedaceiedestissudoicapeelsnseeteeedensredseuictutec seated eacistesedencoudsodeeteledebesabesstscetendaet 64 4 5 Open HistoQuest PIOIC Cl iczissaccestpceescaveceandcnssucesateceneeaesrun esate suesdengeetscbueseneconasunseeiecenadanusetsediicuabiestexeiceasanseniedaee 66 ATE Glad cad 9 ee ee ere E 69 AT BAAN AIAN S
30. Annotations note Annotations can also be created on the ROI detail window Annotations management can be done from the Project Items or directly on the sample In the Project Items panel select desired annotation and right click on its name in order to display a contextual menu with the following options Rename give a different name to the annotation Remove delete annotation Create ROI from Annotation create a ROI from the selected annotation Create Exclusion Area from Annotation create an exclusion area from the selected annotation Edit opens Edit Annotation dialog For the Line Annotations the options Create ROI from Annotation and Create fote Exclusion Area from Annotation will not be available Page 141 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Slide 1 Region 001 Simple ROIs EG Exdusion Areas L Exclusion Area 02 a Rename ile Annotation O02 L Annotation 03 Remove Create ROI from Annotation Create Exclusion Area from Annotation Edit By right clicking inside an annotation directly on the sample you will get a context menu where you can edit the annotation content create a ROI or an Exclusion Area or delete the annotation rh a Figure 202 Annotation contextual menu If choosing the Edit annotation entry from the annotation contextual menu the following dialog will open Page 142 of 351 Internal www
31. Background Threshold a Post Processing Order Remove Merge Virtual Channel no Remove Labels Mo Post Processing Order Remove Merge UseMergingRules Remove Labels gt Max Combined Area 135416 67 um Use Merging Rules 3 Max Involved Compactness 0 9 z Group Max 4 i Min Resulted Compactness 0 6 Figure 343 Use Merging Rules Parameters If you set the value equal to or higher than 4 for Group Max parameter a warning vf Note message will appear in the parameter box displayed in red Use Merging Rules Yes j Max Combined Area 225 29 ym Max Involved Compactness 0 9 4 ee Min Resulted Compactness Processing might take too long Default Figure 344 Group Max parameter warning message e Virtual Channel you can choose to run the segmentation on the original nuclei image or on a virtual channel If you choose no the segmentation algorithm runs on the nuclei channel Page 223 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual arious Shapes 7 0 Nuclei Size 5 Discrimination Area 1 Discrimination ray 1 Automatic Background Threshold Wo Background Threshold 25 Post Processing Order Remove Labels nuclear Use Merging Rules cytoplasmatic Figure 345 Virtual Channel no If you choose nuclear the virtual channel is built by mixing the nuclei channel with the other channels Yarious Shapes 2 0 Muclei Size 5 Discrimination Area 1 Discrimination Gray 1 Automatic B
32. Backward Connection This function establishes a connection between a single dot or a group of dots e g in a gate or a quadrant and their original location in the source images and visualizes where certain cells or cell populations are located in the tissue sample By double clicking on a row in the Raw Data grid that event will be highlighted in the Project Viewer The event will be highlighted in the diagrams and also in the Region Viewer To stop the highlighting click anywhere on the Region Viewer or just close the Raw Data dialog 14 3 Backward Data In order to access backward connection the user should obtain backward data These data can be obtained from both types of diagrams scattergrams and histograms accessing the different options for this operation 14 3 1 Scattergrams Accessing the contextual menu of the scattergrams a set of operations for backward data will be displayed when choosing View Backward Data View Backward Data Around This Point View Backward Data For Left Quadrants View Backward Data For Right Guadrants View Backward Data For Upper Quadrants View Backward Data For Lower Quadrants View Backward Data For UL Quadrant View Backward Data For UR Quadrant View Backward Data For LL Quadrant View Backward Data For LR Quadrant Figure 462 Scattergram Backward Data context menu e View Backward Data Around this Point shows the backward data of the events on a small area around the mouse curs
33. DAB Me OP LL Hematosylin Mean Intensity Ki 67 DAB Me LA gt Hermatoxyvlin Mean Intensity Ki 67 DAB Me a UL gt Ki 67 DAB Mean Intensity Hematoxylin Me a UR gt Ki 67 DAB Mean Intensity Hematoxylin Me gt KI 6 DAG Mean Intensit Figure 435 Histograms Properties Dialog By pressing the Configure button on each histogram 3S a dialog will be displayed where these parameters can be set e Anew Name for a histogram _ The name of a histogram should be unique In case you want to introduce an already vf note existing name an error message will appear Figure 436 Histogram name error message e The Input Gates created by the cutoffs and gates from the other diagrams Choosing Input Gates for diagram Page 284 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Choosing some Input Gates for a diagram will show in the diagram only those events from the Input Gates If more input gates are selected the union of the events from the input gates is considered as input from the diagram Not all gates can be selected as input gate for a diagram Gates defined on that diagram and gates that could create interdependencies between diagrams are not allowed to be selected they are marked by this red sign amp in the list instead of checkbox Actions to be performed within a histogram In each histogram the following actions a
34. Demo Project Images Uv Add Subfolder Images Exclude _higtmp folder a TT amp SY Images 9 7 2010 9 7 2010 SHY Slide 1 9 7 2010 9 7 2010 GF Region 001 9 7 2010 9 7 2010 oY TimeRegion 4 9 7 2010 9 7 2010 V Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 W Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Z Demo Project Region 001 TimeRe 9 7 2010 E Demo Project Region 001 TimeRe 9 7 2010 Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 V Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Note Your project will have 6 samples Figure 51 Add Subfolder Images option Advanced Settings Page 49 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Basic settings Advanced settings Do you want to filter out some images Images Filtering Include only the following extensions The file name should contain the following pattern use and wildcards Figure 52 Import Folder Wizard Step 1 Advanced Settings You can select what type of images will be imported by using the extension based filter available in the Advanced Settings tab In the Images Filtering list mark the checkboxes corresponding to the desired extensions 7 All image files within a single folder will be considered as belonging to the same vf note sample If you have images that belong to different samples you have two options 1
35. Figure 68 Import Mirax Project Step 1 b At this point the following data is available Image Map the preview of the region as it is stored in mrxs file J e The size of extracted images choose between 1024x1024 or 2048x2048 e The format of the extracted images choose between png jog or bmp e The zoom level choose between 7 7 1 2 1 4 1 8 1 16 1 32 1 64 Mirax slide information Sample size in pixels the size of the Mirax slide expressed in pixels Sample size in millimeters the size of the Mirax slide expressed in millimeters Pixel size the size of the pixels that form the Mirax slide Physical magnification the magnification used to acquire the Mirax slide Scanned FOVs count how many tiles of 256x256 pixels contains the Mirax project Export information e Sample size FOVs the dimension of the FOV matrix corresponding to HistoQuest input region e FOV size the size of the Field of View to be used in HistoQuest expressed in millimeters An If you change the size and the zoom level of the extracted images the Sample size ote value and the FOV size value from the Export information section will automatically update Page 62 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Now press Next 4 4 2 Step 2 HistoQuest Project This is the same Project Info dialog as in the wizards for importing a TissueFAXS HistoFAXS project or an image including Basic and Advanced Se
36. Hamamatsu NanoZoomer Import images from a Hamamatsu NanoZoomer file Import images from a Mirax file Figure 4 Start Window e To create a new project the user must select Import TissueFAXS Project HistoFAXS Import Folder Import Hamamatsu NanoZoomer or Import images from a Mirax file from the start up window A proper wizard will appear and the user will be prompted to enter the project data He can also start these wizards from the File menu For more details please see Chapter 4 e Toopen an existing project the user will choose Open Existing Project from the start up window or the Open option from the File menu or press the Open button from the main toolbar Pen i In order to open an existing project you can also use the CTRL O key combination Q TIP from your keyboard Jn When creating a new project the first sample is automatically loaded in the main ns bai image viewer Page 16 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 4 Projects When creating a project if the name of the path exceeds Windows limits the user will receive a warning message This means the path to the project is too long note Also the path to the images must not exceed above mentioned limits Regarding projects storage Avoid storing projects on Desktop My Documents or in any other folder located on the system drive usually C If the drive is nearly at its full capacity the system
37. Region roup O Current Sample and its Regions O All Samples and their Regions Cancel Figure 407 Scattergrams Set Cutoff control box e View Backward Data will display the menu containing all possible options for backward data on a scattergram For more details please see Chapter 14 2 e Copy to Clipboard creates a copy of a scattergram which allows the user to paste it into another software application e g MS Power Point e Save as will Save a scattergram as an image file to a selected drive The user will be able to choose the name and the extension of the newly created image file Configure Scattergram Page 267 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Y Axis Marker Marker Hematoxylin Hematoxylin l Parameter Parameter Mean Intensity Area Mame Hematoxylin Mean Intensity Hematoxylin Area Input Gates Ta T Da a UR gt Hermatoxyvlin Mean Intensity Ki 67 DAB Mean Intensity Wa LL gt Hematoxylin Mean Intensity Ki 67 DAB Mean Intensity Waj LR gt Hermatox lin Mean Intensity Ki 67 DAB Mean Intensity ol UL gt Ki 67 DAB Mean Intensity Hematoxylin Mean Intensity Waj UR gt Ki 67 DAB Mean Intensity Hematoxylin Mean Intensity LL 2Ki 67 DAB Mean Intensity Hematoxylin Mean Intensity Number of Shown Evenks 0 100 100 Figure 408 Scattergram Properties dialog By pres
38. TissueFAXS projects hence the Import Folder option cannot be used for a TMA project e To open a TMA project using the Open Existing Project option follow the steps described in Chapter 4 4 e Touse the Open TissueFAXS Project option for a TMA project follow the steps described in Chapter 4 4 T For Open TissueFAXS Project the user must choose the type of TMA sample vf note grouping according to his needs Create Sample for each TMA spot Create Sample for each TMA block Create Sample for each TMA slide Page 73 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Descrip tion Experiment Demo_Projec has slides Experiment Demo_Project has 6 regions Experiment Demo Project has 1 TMA block Experiment Demo_Project has 25 TMA spots Comment TMA Grouping Create Sample for each TMA spot Import Tissu Import Tissu Create Sample for each TMA block Pee ee eah THA Se Create Group of ROIs from TissueFAXS HistoFAXS categories Note Your project will have 3 samples Figure 84 Choose TMA Grouping combo box Page 74 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 5 Region Viewer The Region Viewer is the component used to visualize the entire sample and also the ROIs and the Groups To open the Region Viewer the user must double click on the name in the Project Items or double click on the sample opened in the main form A map is provided th
39. TissueGnostics oa TISSUE GNOSTICS ol Taborstrasse 10 2 8 A 1020 Vienna Austria C MEDICAL amp BIOTECH SOLUTIONS Figure 1 Product labels Serial number Dz WEEE symbol separate disposal of electrical and electronic equipment atal C CE mark Manufacturer Notice regarding anti virus applications e All the TissueGnostics products are shipped with the Kaspersky Anti Virus in order to ensure optimal functionality e Therefore TissueGnostics only guarantees for Kaspersky Anti Virus to work with its products e The user may decide to use other anti virus software only upon own responsibility e Any problem that arises due to other anti virus software installed will void TissueGnostics warranty and every support or service action required by the client in order to restore any of TissueGnostics damaged instruments will be billed to the customer Page 6 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 1 Introduction Introduction to histology Histology is a fundamental basis both in routine diagnostics and pathology as well as basic research Over the preceding decades an ever increasing amount of specific molecular markers has been established which help pathologists clinicians and researchers to appropriately characterize diseases and understand molecular interdependencies For such a characterization however a simple look and conclude investigation is not sufficient For many ma
40. Yalue Maximum value For Hematoxylin Mean Intensity 300 Apply to Current Region troup C Current Sample and its Regions O All Samples and their Regions Figure 430 Histograms Set Max Value for control box e Parameters displays a dialog where it is possible to set max value for the axes make Autoscale and set the scaling type for both axes of the histogram and propagate the new values to the diagrams from other regions containing similar parameters Page 281 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Sj Hematoxylin Sum Intensity j Auboscale No Current Sample and its Regions Scaling type Linear All Samples and their Regions a lee Current Region Group Autoscale No i Scaling type Linear Current Region Group Figure 431 Histogram Parameters control box When Autoscale is set on Yes value the user can propagate this setting to different regions Hematoxylin Sum Inkensity Linear Current Sample and its Regions 0 All Samples and their Regions 12 Current Region Group Autoscale Mo i Scaling type Linear Current Region Group Figure 432 Scattergram Parameters control box Autoscale parameter The Scaling type parameter changes the scaling from linear to logarithmic and from logarithmic to linear The changes can be propagated to different regions Page 282 of 351 Internal www tissuegnostics com HistoQuest 3 0
41. after the merging process Page 212 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual arious Shapes 1 0 Nuclei Size 5 Yarious Shapes 1 0 Discrimination Area 1 Nuclei Size Discrimination Gray 1 Discrimination Area l Automatic Background Threshold Yes Discrimination Garay l virtual Channel na Automatic Background Threshold Yes Post Processing Order Remove Merge virtual Channel na Remove Labels Mo Post Processing Order Remove Merge Use Merging Rules Remove Labels No Max Combined Area 135416 67 um j Max Involved Compactness 0 9 Group Max 4 i Min Resulted Compactness 0 6 Figure 323 Use Merging Rules Parameters lf you set the value equal to or higher than 4 for Group Max parameter a warning message will appear in the parameter box displayed in red Use Merging Rules Yes Max Combined Area 225 29 um z Max Involved Compactness 0 9 ny ie Min Resulted Compactness Processing might take too long Default Figure 324 Group Max parameter warning message Non Master Marker Parameters The non master channels are segmented using an especially designed algorithm which starts from the segmentation obtained on the master channel A series of configurable features are available for this process e Use Ring Mask if you select this a ring mask around the master channel detected objects is created and included in the segmentation result Yarious Shap
42. analyzed with a total number of 2 FOVs The following regions will be analyzed Coloni ROI 08 1 FOVs Colonin ROI 10 1 FOVs Beep after finish _ Shut down after finish Figure 372 Analyze Summary dialog By marking the Beep after finish option HistoQuest will announce the finish of the analysis process by repeated beep sounds If you check this option at the end of the analysis you will also receive the following message Page 242 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Analysis done Figure 373 Analysis done message By marking the Shut down after finish option the computer will automatically shut down after the analysis is done l CAUTION 12 1 4 Group of ROIs Analysis There are two ways to analyze a group of ROIs Make sure no other applications are running that will require the computer to stay on power when you select the Shut down after finish option e Analyze from the right mouse button menu on the group s name from the Project Items Analyze Unprocessed FOVs Rename Remove Change color Delete Results Propagate Group to all Samples Figure 374 Analyze Group of ROIs from contextual menu e Analyze button on the toolbar of the group detail window if the group was loaded rote Analyzing a group means analyzing any ROI included in it When the Strategy for events in ROI is set to Crop events to ROI s shape all the gr
43. and or Ki 67 DAB taking into account also nuclei with strong Ki 67 DAB and low Hematoxylin expression arious Shapes 1 0 Muclei Size 5 Discrimination Area 1 Discrimination ray 1 Automatic Background Threshold Yes virtual Channel Post Processing Order Remove Labels nuclear Use Merging Rules Figure 320 Virtual Channel Parameter e Post Processing Order Parameter specifies the order of execution for the following operations Remove Labels and Merge Labels Yarious Shapes 1 0 Nuclei Size 5 Discrimination Area 1 Discrimination ray 1 Automatic Background Threshold Yes Virtual Channel Post Processing Order Remove Merge Remove Labels Remove Merge Use Merging Rules Merge Remove Figure 321 Post Processing Order Parameter Page 211 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual If there are very large detected labels the amount of required memory will also increase significantly Vi Note e Remove Labels Parameters removes the objects that fulfill certain criteria i e too small Smaller Than all objects with the area smaller than the specified value will be removed from the segmented image Larger Than all objects with the area larger than the specified value will be removed from the segmented image Weaker Than all objects with the mean intensity lower than the specified value will be removed from the segmented image Stronger Than all objects with the me
44. annotation and choosing the Edit Annotation option from the menu that appears Page 143 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual An ankm ar 0 0009 mm2 Figure 204 Edit Annotation command in the mouse context menu J a dialog will be displayed Please insert annotation content below Figure 205 Edit Annotations dialog This dialog contains a set of options that allow the user to manage the annotations It also contains the Visibility checkboxes for every annotation by checking the checkbox for an annotation the annotation will be visible on the sample When closing HistoQuest the Visibility settings will not be saved Page 144 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Edit annotation directly on the sample An annotation can also be edited directly on the image viewer it can be renamed its content can be changed or it can be removed To edit an annotation you must use the little black framed square on the contour of the annotation If pressed this little square will display an edit box where you can operate the following actions a Annotation 03 iT k a a ae i PE n 7 Figure 206 Edit annotation directly on the sample e Edit the name of the annotation write the desired name in the upper left corner of the box then press Enter key to save the changes e Write a comment in the space below the nam
45. been selected as part of a mixture cannot be selected anymore as being a mixture of another marker and itself ror HistoQuest x Cannot set this marker bo be mixture because other markers use it as pure marker Figure 309 Mixture marker warning message Without a license the user won t be able to use Single Reference Shade method l Requirements Measure reactivity in different cellular compartments It is possible to measure reactivity in different cellular compartments when using the automatic color separation single reference shades option to copy the non master channel including the shade in order to make a virtual channel in other words analyze with same shades but with a different mask Page 204 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 11 Segmentation Methods HistoQuest offers unique and proprietary algorithms for recognition of individual cells in a tissue context An algorithm is a sequence of mathematically realized processing steps applied to an image or part of an image with the aim of improving image quality and or segmenting relevant objects in this case cells in an image 1 Discriminates background from specific cell objects 2 Recognizes individual cells where does one cell end and the neighboring cell begin 3 Measures the marker intensity of each cell as well as other parameters related to intensity texture and morphology The starting point
46. block or slide a TMA license is required il Requirements In the lower part of the Choose Regions dialog a message regarding the status of the selected samples will always be displayed 1 When you have between one and ten samples Note Your project will have 5 samples 2 When no sample is selected Warning You must select at least one region 3 When you have more than ten samples Warning Your project will have x samples TMA Grouping Create Sample for each TMA block Import TissueFAXS HistoFAXxS regions Import TissueFAXS HistoFAXS logical groups Import TissueFAXS HistoFAXS subregions Import TissueFAXS HistoFAXS regions Import TissueFAXS HistoFAXS logical groups Import TissueFAXS HistoFAXS subregions Warning You must select at least one region TMA Grouping Create Sample for each TMA block Import TissueFAXS HistoFAXS regions Import TissueFAXS HistoFAXS logical groups Import TissueFAXS HistoFAXS subregions i Warning Your project will have 14 samples Figure 33 Status of the selected samples examples of displayed messages 4 1 2 Step 2 HistoQuest Project To perform this step of the Import TissueFAXS HistoFAXS project wizard the user must provide identification information for the HistoQuest project he is about to create HistoQuest Project step of the wizard has two tabs Basic Settings and Advanced Settings Basic Settings Page 37 of 351 Internal www tissuegnostic
47. button can be disabled in certain situation for example if the project item is already in the task manager For more details please see Chapter 12 5 11 View Raw Data Button EE The View Raw Data Button opens the Raw Data dialog please see Chapter 13 2 5 12 Easy drawing tool This tool helps the user define as fast as possible multiple similar shapes like ROIs and Annotations i e define 50 Tumor regions with minimal number of clicks Page 87 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Coloni Se 5 a Blk 7 Figure 108 Regions Easy Tool button Press Easy ROI button and a dialog will appear called New Regions In this dialog you will select e The type of region to be added ROI or Annotation e The desired shape rectangular elliptic freedrawn custom line only for annotations e The group where the new shape will belong choose one of the existing groups or create a new one Without closing the New regions dialog start drawing desired shapes on the sample Close the dialog only when you have finished drawing Tumor 02 _ 0 0136 mm Elliptic Line FreeDrawn Normal Adjacent Ti Ez 0 0255 mm 2 Normal Adjacent Tissu e O02 0 0277 mm 4 Figure 109 Using Regions Easy Tool feature Page 88 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 5 13 Options Button ke The Options Button opens the Image Viewer Optio
48. cache is build the sample will be displayed in best fit mode Page 101 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual The steps to be followed are the same like those when a project is firstly created with the Import TissueFAXS HistoFAXS Project option except the fact the user will not be allowed to change the project name project folder and markers All types of regions of the current project can be visualized in the Project Items panel of the Project Browser Project Browser Compare Sets f Images Compare Sets i Processed 1 out of 1 FOVs Simple ROIs Region of Lit L ROI 02 Processed 1 out of 1 FOVs Interest ROO Processed 1 out of 1 FOVs een 7 ROLO4 Processed 1 out of 1 FOVs Group 0i P ROLO3 OO i ROLO4 7 zal Exdusion Areas Exclusion ber Annotations Olea Annotation 01 Figure 130 Project Items panel of the Project Browser For projects obtained with the Import Folder Wizard a default sample is created that takes the name of the imported folder This sample contains the images from the folder arranged in a matrix For projects obtained with the Import TissueFAXS HistoFAXS Project Wizard the application creates a sample for every brightfield TissueFAXS HistoFAXS region of interest selected The name of such a region will be a combination of the slide name and the region of interest name provided by the TissueFAXS HistoFAXS project In thi
49. choosing the images a sample can be divided in two types e Sample with related images this sample is the HistoQuest equivalent of a region of interest from a brightfield TissueFAXS HistoFAXS Hamamatsu NanoZoomer project or Mirax project The HistoQuest projects obtained by importing regions from a TissueFAXS HistoFAXS project can contain multiple samples WjRequirements NanoZoomer files and Mirax files a license is required For creating samples from TissueFAXS projects or from Hamamatsu The user can add many more samples after the creation of the project using the Add TissueFAXS HistoFAXS Project option By pressing the Add Samples from button from the HistoQuest toolbar the following menu will appear lt A Note TissueF AXS HIstoF AAs Folder Hamamatsu NanoeZoomer Mirax Figure 129 Add Samples from menu e Sample with unrelated images this sample comes from importing images from a folder In this case the HistoQuest project will only have one sample The user can add more samples after the creation of the project with the first sample using the Add Samples from option The steps to be followed are the same like those when a project is firstly created with the Import Folder option except the fact the user will not be allowed to change the project name project folder and markers for more details please see Chapter 4 1 Af Note After opening a sample in the main viewer and the
50. configurable Each marker defined for the current project will have a set of parameters related to the segmentation method The set of parameters is different from one method to the other The master marker also has different parameters than the rest of the markers These parameters are displayed on the lower part of the Marker Browser The current segmentation method name is also displayed Page 207 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual In the figure below a comparison is shown of the default parameters for a master marker left side and a non master marker right side for the Various Shapes 2 0 method arious Shapes 2 0 Nuclei Size z Discrimination Area 1 arious Shapes 2 0 Discrimination Gray 1 Use Ring Mask Ves Automatic Background Threshold Yes p Interior Radius i Threshold Range 5 255 A Exterior Radius 7 Virtual Channel no Use Identified Cell Mask No Post Processing Order Remove Merge Use Nuclei Mask Mo Remove Labels Mo Automatic Background Th Yes Use Merging Rules Mo i Threshold Range 5 255 Figure 312 Various Shapes 2 0 Default parameters for a master marker left and a non master marker right In the figure below the common control for master and non master markers for the Total Area Measurements method is shown Total Area Measurements Automatic Background Threshold Lower Upper Figure 313 Total Area Measurements 11 2 Various Shapes 1 0 His
51. e Elliptic iQ For the elliptic ROIs after its shape is chosen from the shape manipulation toolbar the user has to press down the left mouse button in the middle of the interest area and drag until the circular shape of the ROI will surround the desired region Then the mouse button is released tt Si p E ROIA i A m mri 5 UO U2 mm ty 5 as Figure 141 Elliptic ROI e Custom vl In order to create the polygon shape of a custom ROI the user has to select it from the shape toolbar Each leg of the polygon is defined by two left mouse clicks one at the beginning and one at the end When all legs of the desired polygonal shape have been defined right click the mouse in order to finish the custom ROI and the ROI will be automatically closed yh oy gt fe 0 0034 mm 2 S boo eoh ee Figure 142 Custom ROI e Freedrawn EB he shape of the freedrawn ROI has to be chosen from the ROI manipulation toolbar To begin defining the shape the user has to press down the left mouse button and keep it pressed while the entire area is surrounded The shape is defined by the track of the mouse Releasing the mouse button closes the shape Page 109 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Fae Figure 143 Freedrawn ROI By pressing any of the four buttons described above in order to create a ROI it will not note remain selected For each ROI you want to create you must first
52. for the markers Add Rename Remove and Remove All System Data i Markers Tee tr a g a Figure 526 Options menu System Data Markers dialog e Profiles All the existing profiles and their properties and contents are displayed in a list The following operations are available for the profiles Rename Remove and Remove All Page 342 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual System Data Profiles DE Hematoxylin Yes Ki 6 DAB Figure 527 Options menu System Data Profiles dialog 17 4 Help Menu Figure 528 Help menu Contents here you can access the electronic version of the HistoQuest user manual a Please note the electronic version of HistoQuest user manual is provided in PDF vf note format therefore you should have installed Adobe Reader on your work station About this option will display a splash screen containing the main information about the HistoQuest version in use and also the Disclaimer section Page 343 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual TISSUE W GNostics MeEGOIiC AL 5 Bio Te amp ctH S OLUTIO H HISTO QUEST TISSUE ANALYSIS SOFTWARE CE JA ATTENTION CONSULT OPERATING INSTRUCTIONS FOR USE TissueFAKSpIUS is 2a micoscopehased cel analysis system for cel in oyome paral en ections and TMAS It came of the software modules TissvefAxs TissueQuest and wW
53. is a oy in at Pe ae ie Region OOF EJ 47 mira 2 1 Figure 18 HistoQuest Categories Create Group of ROIs from TissueFAXS HistoFAXS categories When Create Group of ROIs from TissueFAXS HistoFAXS categories option is checked the HistoQuest project will open them The imported categories from the acquisition module will keep the original name and will be treated as groups of ROls Page 29 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Project Browser Samples Compare Sets E Slide 4 Region 007 Simple ROIs z Category A 1 Category A 2 Category B 3 b Category B 4 a Category A f Category A 1 A Category A 2 Veet OTT Category B 3 Not processed Not processed Not processed Not processed Not processed Not processed Figure 20 HistoQuest group of ROIs from TissueFAXS HistoFAXS categories b Internal Page 30 of 351 www tissuegnostics com HistoQuest 3 0 User Manual Project Browser Samples Compare Sets Images Compare Sets Project Items Slide 4 Region 007 Not processed Simple ROIs f Region 007 Not processed 2 Category A 1 Not processed Category A 2 Not processed bo Category B 3 Not processed 2 Category B 4 Not processed a Category A Category A 1 a Category A 2 Category B Category B 3 a Category B 4 Group 01 Category B 3 Category B 4 Exdusion Areas
54. is a bar of a frequency distribution in which the frequency of one measurement parameter is represented by the height of the bar and the class interval by the width In other words a histogram tells us how many cells express how much of a given marker As any graphical chart it is based on classes in HistoQuest these classes meaning sets of events e g with similar color density It may appear like bins line style and line style with fill histogram above the diagram area In addition by pressing the right mouse button on a histogram a contextual menu like the one below will appear Page 280 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Auboscale on Hematoxylin Mean Intensity Auboscale On Axis Set Max Value For Hematoxylin Mean Intensity Set Max Value For Axis Parameters Sek Cutoff Delete Cutoff Copy Cutoff Paste Cutoff View Backward Data Copy To Clipboard Save S Figure 429 Histogram contextual menu e Autoscale on option available for any of the axes will set the maximum value of a chosen axis to be the maximum value of a parameter to the affected axes e Set Max Value for option will allow setting a new maximum value for an axis From the same menu the newly introduced value can be propagated to diagrams belonging to other regions In order to perform the propagation the axes of the different diagrams should be the same Set Max
55. is looking for the images in the following folder F Projects HistoQuest Colon Colon Please contact the support team at support tissuegnostics com to help you with this issue Source Open sample Figure 77 Missing images warning message box 4 6 Project Properties In the Project Properties all relevant settings for the current project are stored and may be subject to change fn Changing the Project Properties might require reanalyzing your samples ote In the main toolbar choose Project Project Properties The following dialog will appear Page 69 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Name Description Location F Projects HistoQuest Colon1 Segmentation Method Various Shapes 2 0 Strategy for events in ROJ Select only events entirely in ROI Color Separation Method Single Reference Shade FOV Size FOV Width mm FOV Height mm Image Width 1600 px Image Height 1200 px Pixel Width 0 25 um Pixel Height 0 25 Figure 78 Project Properties dialog For each project the user can modify the properties anytime 1 2 3 PEREN W Requirements The name of the project is mandatory Optionally a short description of the project can be realized Provide the location where the project will be stored Use the Browse button to choose the desired location Choose one of the Segmentation Methods Various Shapes1 0 Various Shapes 2
56. it might be possible that the High Outlier was decreased too much e K by increasing this parameter lighter shades will not be considered for the detection process fn ie Single Reference Shade works only for staining protocols with two or three markers yf NO e View Gray Image view Gray Image the user will see the computed gray image for the corresponding shade representing the abundance map of the selected marker Page 201 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 305 View Gray Image e View Color Image the user will see the color image for the corresponding shade This shows the color component from the original image only for the selected marker Page 202 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 306 View Color Image Hematoxylin only e Intensity the user has the possibility to set the intensity which will be used in the transformed gray value image to display the reference color shade Weaker or stronger color densities will be displayed by lower or higher gray values respectively The value in the edit box represents the abundance value associated with the pure color of the selected marker Maximum allowed value is 255 This value must be set so that the gray image has a good contrast and does not have saturated areas i e larger areas with value 255 It is recommended to be set so that a strong positive area has a valu
57. least two input items are required ote e The Side by Side option will build similar diagrams for each region in order to compare them e For Overlay section the data from all regions is represented in different colors on a single diagram The New Compare Set dialog will appear Internal Page 154 of 351 www tissuegnostics com HistoQuest 3 0 User Manual ew Compare Set ah ee Compare Set Name Gland Compare Set Input Side By Side Diagrams Overlay Diagrams Regions o O ON i Sample 01 i m x Unselect All e Sample 01 _ Tumor Gland 1 Invert Selection P Tumor Gland 2 O Small Gland 1 Expand all C Small Gland 2 Call All ee Normal Gland 1 OO Normal Gland 2 Tumor Glands Cd Small Glands Normal Glands EL Sample o2 _ Sample oz C Region 002 HLI Sample 03 P Sample 03 Move Up BuU Sample 04 _ Sample 04 Figure 225 New Compare Set dialog First the user can set the name of the new Compare Set Then he will have to check at least two regions Input regions buttons selects all the regions in the Input section unselects any previous selection reverses a previous selection expands the tree panel items makes all items visible collapses the tree panel items hides all items changes the position of a region to an upper one changes the position of a region to a lower one 7 2 Side by Side Diagrams By c
58. registration points simultaneously by using the black frame you Mote can move them only if they are all entirely inside the sample If they are outside the sample you can only rotate the frame Example of synchronized visualization of processed images Figure 126 Images Compare Sets original images Page 99 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual eT a a FP ee ee ee Tie oP a Be it Hi RA Bel bisp fe 5 J a d a EET sO 10722 Vil COA Region 006 icine e ee fa ies sa as ee tee ie 2 ogee linepa eF a Tue setts st ei oP ae he Pr iy al k ii a AR IEE ae ie a 10722 Vil CO3 Region 008 10722 vil andeccenensoernn k fee re Ei Ere i i ae 7 F i g TAR ar ote of 8 ae halla 7 malia Aan b T aE ee ee a amp i T L y a Te al oe epu al Parle ae Seapine a i Phas Ka Figure 128 Images Compare Sets synchronized images If using synchronization feature the next time you will open the project the samples will remain synchronized Page 100 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 6 Project Items 6 1 Samples and ROIs 6 1 1 Samples The analysis in HistoQuest is performed on immunohistochemically stained tissue samples A sample is a set of color images the user wants to analyze using the methods provided in HistoQuest According to the method of
59. s contextual menu on the sample The operation of transforming an annotation into a ROI or an exclusion area can be realized by pressing the right button of the mouse on the desired annotation directly on the sample then choosing the Create ROI Exclusion area from Annotation name option T Annotation 08 i 5 00022 mm Edit Annotation f Create ROI from Annotation 08 Create Exclusion Area from Annotation 08 Remove Annotation 08 Figure 223 Create ROI Exclusion Area from Annotation directly on the sample note Being a measurement tool a line annotation cannot be transformed into other shapes Page 153 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 7 Compare Sets A Compare Set represents a set of diagrams used to compare at least two project items in order to facilitate user s work when performing the comparative analysis The sets of diagrams to be compared are presented in two ways side by side and overlay depending on individual research needs The user will set the type of diagrams scattergrams and histograms the markers and the parameters 7 1 Create Compare Sets In order to create a Compare Set the user must press the Compare Sets tab header on the Project Browser then access New ME Project Browser Compare Sets Images Compare Sets Samples New Edit Delete Delete All Figure 224 Compare Sets tab header fn To create a compare set at
60. samples ROIs or groups and their corresponding analysis in the case of serial sections by automatic synchronized display To use Images Compare Sets choose Images Compare Sets tab from the Project Browser then press New A dialog called New Images Compare Set will open where you must check the desired items for image comparison Project Browser ca ee Samples Compare Sets Images Compare Sets 9 A eis Ne edit Delete Delete All j Ww imaoe Compare Set az aoe x Image Compare Set Name Image Compare Set 05 Trout a Project Browser Compare Sets Images Compare Sets CE 15187 C03 Regon 001 teen New Edit Delete Delete All HE 13147 CDS Region 001 na 0 rors Image Compare Set 04 w UOT 15 o Input 0 Ror 17 an 13187 CD53 Region O01 ROI 15 i 13187 CD45 Region 002 Region 002 ROT 24 HO aa 26 C Ror 27 O Rora HE Group 02 C Group a3 EC 13187 C45 Regon 002 C 13187 C045 Region 002 l C Regian 002 O Ror 02 0 2 0 2 iL Group 02 Bil C Group 03 Figure 117 Creating Images Compare Sets At this point the Image Compare Set viewer will automatically open Page 92 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual eee 13187 CD3 Region 001 Regan OO A 0 0014 mmi Ae e Show registration toolbar is wd shows hides a toolbar used to synchronize multiple similar tissues rm nag
61. t be able to compute area for regions of interest exclusions area and annotations Also he won t be able to compute distance for line annotation TMA The user won t be able to create samples from TMA blocks and from TMA slides Also a license is needed to create projects from TissueFAXS projects Nuclear Measurement Mask The user won t be able to use nuclear measurement mask Also a license to use Various Shapes is needed Page 347 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual lf the user owns a predefined number of uses the counter will be incremented only if the analysis for each FOV succeeds Ring Measurement Mask The user won t be able to use ring measurement mask Also a license to use Various Shapes is needed If the user owns a predefined number of uses the counter will be incremented only if the analysis for each FOV succeeds Identified Cell Mask The user won t be able to use identified cell mask Also a license to use Various Shapes is needed If the user owns a predefined number of uses the counter will be incremented only if the analysis for each FOV succeeds Region EasyTool The user won t be able to use region easy tool Also a license to define the regions of interest or annotations is needed If the user owns a predefined number of uses the counter will be incremented after each use Measure in DifferentCompartments Copy Channel The user won t be able to copy channels
62. t check the check box the Send Error button will be disabled A Product Improvement Program pA TissueGnostics Product Improvement Program Agreement 1 About TissueGnostics Product Improvement Program Focusing on innovation TigsueGnostics TS is constantly involved in understanch Le antigipating customer needs in order to create l A fogscts an dgsolutighs The LG Broduct laproy e anh a Figure 508 HistoQuest Product Improvement Agreement Send Error button effectively sends the message to your support team Hide Details lt lt button by pressing this button you can display or hide the data list related to the error HistoQuest encountered an error x Error description Generated test exception 7 Gomess dose Read Pnet Improvement Program Terms Figure 509 HistoQuest error dialog hidden details e Submit Report this option effectively sends your message to the support team Page 332 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Submit report Report description Pe O Report details _ Tagree Send Report Read Product Improvement Program Terms Figure 510 HistoQuest Submit report dialog e Enable Verbose Logging this option provides detailed data for diagnoses e Register hiqproj file extension it creates a link between HistoQuest and the existing hiqproj projects in order to automatica
63. the Project Items Show Show in Detail Window Analyze Analyze Unprocessed FOVs Rename Remove Remove All ROIs Remove All Groups Remove All Exclusions Remove All Annotations Paste Build Cache Delete Cache Delete Results Figure 364 Analyze All ROIs from context menu Page 236 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e By pressing the Analyze button 47 4lyze from the sample viewer You will have to select the desired ROIs in the list Fap Select Input Please select the project items you wish to analyze E Select Input Summary Samples Se _ Coloni _ Coloni F ROI 08 _ Analyze Only Unprocessed FOV Figure 365 Analyze menu from the Sample Viewer for ROIs For the Analyze Unprocessed FOVs option from the right mouse button menu on the J note ROIs name on the Project Items please see Chapter 6 1 2 Strategy for events at ROI borders To access the Strategies for events at ROI borders the user must go to Project Project Properties The events within a ROI can be computed and displayed in three different ways Select events entirely or partially in ROI Select only events entirely in ROT Crop events to RT s shape Figure 366 Strategies for events at ROI borders Page 237 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Select only events entirely in ROI only the events placed entirely in t
64. the Region Viewer The map provided has a set of parameters size opacity location These parameters are saved by the application To change the default settings for the map just right click on the map and choose Configure Map from the displayed menu Figure 89 Configure Map The configuration window that appears has three pages e Opacity allows the user to change the degree of transparency for the map by using a slider Opacity Size Location Transparent Opaque Figure 90 Configure Map Opacity control e Size allows the user to change the size of the map By default the value of this parameter is Medium Page 78 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Very small Small Medium i Very big Figure 91 Configure Map Size control e Location allows the user to change the corner of the Region Viewer in which the map should be displayed top left top right bottom left bottom right and the indentation from the closest border By default the map is displayed in the bottom left corner C Top left corner O Top right corner Bottom right corner s Bottom left corner Figure 92 Configure Map Indentation control and Location radio buttons Page 79 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 5 3 Ruler Figure 93 Ruler The Ruler is a software feature which shows the physical dimensions of a current viewing size A physi
65. the higher the probability is of that pixel to belong to the respective reference shade and the higher the resulting gray value of that pixel will be in the transformed image This means however if a color shade is selected as reference shade which represents a low positive marker value a pixel representing this low positive shade will be represented by a high gray value as it posses a high probability to belong to that low positive shade If such a shade is used for measurement of marker intensity the low positive pixel will be transformed into a strong positive pixel In order to avoid this false transformation the Measure checkbox must be checked ONLY for color shades which represent a strong positive color shade Therefore HistoQuest offers alternative methods for cytometric quantification and comparative measurement of marker density Page 190 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Hematoxylin Figure 282 Marker Browser tabs The shades of a marker if any are displayed in the Marker Browser There will be a separate page for each marker existing in the project For any new marker or an already existing marker added in the New Project Wizard the Marker Browser will be empty it does not contain any shades and its parameters are the default ones Hematoxylin Multiple Reference Shades Figure 283 Empty Marker Browser For using these empty markers in t
66. the result of the segmentation arious Shapes 1 0 Nuclei Size Discrimination Area Discrimination ray Automatic Background Threshold virtual Channel Post Processing Order Remove Labels Use Merging Rules Figure 318 Discrimination Gray Parameter e Automatic Background Threshold specifies if automatic detection of the background level should be used by selecting Yes or can be set manually by selecting No and providing the desired value Page 210 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Yarious Shapes 1 0 Huclei Size Discrimination Area Discrimination ray Automatic Background Threshold Virtual Channel Post Processing Order Remove Labels Use Merging Rules Figure 319 Automatic Background Threshold Parameter e Virtual channel you can choose to run the segmentation on the original nuclei image or on a virtual channel The nuclear virtual channel is built by mixing the nuclei channel with the other channels This mixing is computed in a way that takes into account the amount of markers from all channels This is especially beneficial for nuclear markers i e Ki 67 In such cases the strong positive Ki 67 nuclei might have a low content of counterstain Hematoxylin The approach of searching nuclei only by evaluating Hematoxylin might fail for the strong positive cells In contrast the approach of the nuclear Virtual channel searches for cells that have Hematoxylin
67. they will be removed If a profile was already loaded other markers existing or new can be added Loading a profile or loading a profile from an existing project changes the current segmentation method color separation method and the strategy for events in ROIs lf choosing Load Profile no other marker can be added to the profile s configuration If a profile is loaded the markers the segmentation method and the color separation method cannot be changed from here Load Profile from Existing Project HistoQuest gives the possibility to use a profile from another project that already exists Figure 39 Load Profile from Existing Project dropdown box There are two possibilities e Load Profile from Existing Project choose this option to use a profile from an existing project e Load Profile and Diagrams from Existing Project when you import only the profile the newly created project will have its diagrams set by default If you wish you can choose to load a profile and also the diagrams from an existing project Choose one of these two options then use the browser in order to select the project that contains desired profile and diagrams and load it by pressing Open button 3 Computer Data 2 F Projects HistoQuest Colonl 4 Search Coloni Organize New folder am E3 Documents Name Date modified al Music eae E Pict L Colenl higpro 3 2 2011 3 36 PM HIQPROJ File Pictures
68. use Save Marker Profile a license is required ll Requirements Profile with name Ki67 D46 Hematoxylin already exists Do you want to replace it s contents Figure 272 Overwriting Profile Message box 9 2 2 Load Profile from Project You can load a configuration of markers from a different project while creating the current project or later on Page 185 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Load Marker Profile Manage Profiles Figure 273 Load Profile from Project option 9 2 3 Load Marker Profile In order to load a profile within a project access the Load Marker Profile button from the profiles menu select the desired profile from the list and click Load Adding a new profile or a marker to the loaded profile will delete any previously fv obtained data To use Load Marker Profile a license is required ij Requirements Profle en m Hematoxylin ial i K 67 DAB m Colon H Ki 67 DAB Various Shapes 2 0 Single Reference Shade Select only events entir m E Prostate H Ki 67 DAB Various Shapes 2 0 Single Reference Shade Select only events entir Figure 274 Load Profiles dialog To visualize the Description field for a profile click the corresponding cell in the dialog Page 186 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 275 Profile description visualizati
69. warning message General Task Manager Maximum number of concurrent tasks Humber of threads For a task Note This change will take place after restarting HistoQuest Warning Total number of threads exceedes the number of processor cores Your system might experience slow operation Figure 504 Options Menu General Task Manager Warning message on a computer equipped with a processor with 2 cores on a quad core Skin in this section the user can change the skin appearance of the graphical user interface of HistoQuest Page 329 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual General Skin Skin C Caramel 9 Liquid Sky O Money Twins 3 London Liguid Sky 9 Lilian O Glass Oceans O The Asphalt World O Stardust 3 iMaginary O Office 2007 Blue O Black Office 2007 Black Blue O Office 2007 Silver Coffee TissueGnostics Orange Figure 505 Options Menu General Skin settings box Support in this section the user should provide the required data in order to receive support from TissueGnostics General Support Name Doo O From Address Oo k Submit Options Support Address suppor t tssuegnostcs com SMTP Server mail tissuegnostics to com 587 Auto Save Report Test Connection _ Submit Report _ Open Reports Folder _ Enable Verbose Logging Send Saved Reports Figure 506 Options Me
70. will look the same on the original image If the ROI s is are pasted through Images Compare Sets within the same compare set the propagated ROI s will look the same on transformed images Page 111 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Drag and Drop ROls In the project items tree a ROI can be added to an existing group by using drag and drop feature Project Browser Not processed Not processed i ROL 04 Tay i ROI OS Figure 148 Drag and drop ROI to group Edit ROIs In order to edit ROIs use the Edit Mode button For more details please see Chapter 5 8 Buttons e The Show Shapes button choose Show Hide ROls option to show and hide the ROI shapes on the sample For more details please see Chapter 5 8 e Create Group of ROIs button it is used to create groups of ROIs If several ROIs shall be analyzed together then groups of ROIs can be created A group of ROIs will create one group result where the data cells and measurement values are all put together Several groups of ROIs may be created and used for comparison e g tumor area versus normal adjacent tissue See also Chapter 0 Q One ROI can be member of more than one group TIP You can also create a group of ROIs by selecting directly on the sample two or more ROls then right clicking to access the contextual menu and pressing Create Group of ROIs option Page 112
71. 0 or Total Area Measurements Choose how events shall be treated at the boarder of a ROI Choose one of the two available Color Separation Methods Adjust the FOV Size To be able to modify the Segmentation Method and the Color Separation Method a license is required Adjusting the FOV size Manually Page 70 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual FOV Size Predefined FOV Width mm FOV Height an Image Width 1600 px Image Height 1200 px Pixel Width 0 25 um Pixel Height 0 25 Figure 79 FOV Size manually e Predefined select from a list of predefined values FOV Size Predefined FOV Width Image Width Objective 10x CMount 0 5x Top Port PixeLINK 1600x1200 Objective 20x CMount 0 5x Top Port PixeLINK 1600x1200 Pixel Width Objective 40x Oil CMount 0 5x Top Port PixeLINK 1600x1200 Objective 63x Oil CMount 0 5x Top Port PixeLINK 1600x1200 Hamamatsu Nanofoomer Objective 20x 1600x1200 Objective Hamamatsu NanoZoomer Objective 20x 1280x1280 Hamamatsu NanoZoomer Objective 20x 1280x1024 Hamamatsu NanoZoomer Objective 20x 1024 1024 Figure 80 FOV Size predefined e User predefined values the user can enter in the list his own predefined values press the button enter a name for the new template in the Name field Page 71 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual FOY Size Templates
72. 00 52 750480 59 558410 34 6810690 29395260 6g rogni AYG 51 74 nn Export To Excel Export To PDF Export To TSW Delete Events view Backward Data Exit Backward Connection m Region OF Interest Field OF View Event Label Hematoxilyn Mean Intensity ki 67 DAB Mean Intensity 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 AYG 1 06 Close This is a feature that allows you to visualize the following numerical values for each of the columns from the raw data window You can access this menu by pressing the right mouse button on the area below any of the columns Page 256 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Sum the sum of all values of the column Min the lowest value of the column Max the higher value of the column Count the number of existing values per column Average the average value for the entire column Sum Min Max i ge 5 w Count gt t Average Mone Figure 389 Summary Row contextual menu By default the Raw Data window will display a grid with the following summary row functions For Various Shapes 1 0 and Various Shapes 2 0 segmentation methods e Event Label Count e Mean Intensity Average For Total Area Measurements segmentation method
73. 2 Events Region OF Interest ROl 10 Processed 4 out of 4 Fons 4 FOYS 4081 Events Sample Sample Oz Mot processed 145 Fos Figure 497 Project Summary Analyze this option performs the same action as the Analyze button for more details please see Chapter 5 10 from the current manual Build Cache by choosing this option HistoQuest will build cache for a samples View Raw Data this option performs the same action as the View Raw Data Button it opens the Raw Data dialog for more details please see Chapter 13 2 from the current manual Delete Cache by choosing this option HistoQuest will delete cache for all Project Items Delete Image Results by choosing this option HistoQuest will delete image results for a Project ltems Delete Project s Results by choosing this option HistoQuest will delete the results for all Project Items Finalize Analysis check uncheck the finalization status for more details please see Chapter 4 7 from the current manual Page 325 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 17 3 Tools Menu Manage Diagrams Manage Gates Statistics Report Options Figure 498 Tools menu 17 3 1 Manage Diagrams Manage Diagrams option will display a dialog in which the user will be able to create configure delete and propagate diagrams For more details please see Chapter 13 3 3 from the current manual 17 3 2 Manage Gates
74. 3 33 pm Use Identified Cell Mask Exterior Radius fe Max Growing Steps Interior Radius Re eee ee pee ee Pea a EXCEPIO F d d Ws fi Skip Steps PEUDE ECDC EEC Use Nuclei Mask Automatic Background Th Haa L 13 330 um 666 GO Default Apply Figure 327 Use Ring Mask Parameter Exterior Radius e Use Identified Cell Mask By selecting Identified cell mask as measurement strategy the nuclei are used as seeds e g starting points of image analysis and cell recognition This algorithm searches for specific staining patterns in the other channels of one field of view and causes binary mask objects to grow along those specific structures Page 214 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual arious Shapes 1 0 Use Ring Mask Yes j Interior Radius 6 67 um e Exterior Radius 13 33 um T FiFi E R eee i el E E IE Ic ENIneO Le Mask Max Growing Steps 2 Skip Steps Use Nuclei Mask Automatic Background Th Yes Figure 328 Use Identified Cell Mask Maximum Growing Steps The user may limit this growth process on demand by setting this parameter Yarious Shapes 1 0 Use Ring Mask Yes be Interior Radius 6 67 Hm i Exterior Radius 13 53 um Use Identified Cell Mask Yes ll 26 67 um Skip Steps Max Growing Steps Use Nuclei Mask Automatic Background Th ee OO O O O O O O O O O S E S Max Growing Steps Quinn 2
75. 36 830650 40 794970 31 535690 30 7543590 36 220660 a4 295920 33 0 6050 424099190 46 3705 70 39022420 Seer AE 3 439516 12 544220 Doli ro 14 316560 16 666670 15 318440 10 449540 15 990340 az 146670 15503500 12 452740 13 504170 13 805190 15 776120 10379600 9 466666 13 994570 19 695120 14 627160 11 918030 12 867920 15 118920 10 201520 11 906400 16 608330 11 204190 WOLF rte 17 813250 20 712500 11 720000 15500000 A 4 547170 280 AvG 40 90 Export To Excel Export To PDF Export To CSW Delete Events View Backward Data Exit Backward Connection Figure 461 Forward and Backward connection Raw Data Window View Backward Data displays only the events belonging to a gate or cutoff quadrant for which backward connection was demanded From Raw Data you can perform backward connection only for filtered events Exit Backward Connection displays the entire event list of a region 14 1 Forward Connection By double clicking on an event on the marker images that event will be highlighted in the Raw Data dialog The event will be highlighted in the diagrams and also in the Raw Data window To stop the highlighting click anywhere on the Region Viewer or just close the Raw Data dialog Without a license the user won t be able to use Forward Connection J Requirements i Page 301 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 14 2
76. 6 6 Hm 333 LT O Defaut Appl Figure 329 Use Identified Cell Mask Max Growing Steps Skip Steps in order to reach the marker staining some additional growing steps might be required In this case the growing process will not start immediately at the border of the nucleus but outside at a distance specified by this parameter Page 215 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual arious Shapes 1 0 Use Ring Mask Yes j Interior Radius 6 67 um i Exterior Radius 13 33 pm Use Identified Cell Mask Max Growing Steps ae Skip Steps Use Nuclei Mask i Automatic Background Th Oum Hri 333 33M Default Appl Figure 330 Use Identified Cell Mask Skip Steps e Automatic Background Threshold the threshold level for the cell mask can be computed automatically by selecting Yes or can be set manually by selecting No and providing the desired value You can estimate the value of the background by using the mouse and enabling the RGB tool You can see the value of the gray level by holding the mouse above an area with the background This is a good starting point in estimating the best value for the background threshold Yarious Shapes 1 0 Use Ring Mask Yes Interior Radius 6 67 Hm Exterior Radius 13 33 pm Use Identified Cell Mask Yes Max Growing Steps 26 67 um F Skip Steps 0 uri Use Nuclei Mask Mo Automatic Background Th Mo 25 Background Threshold kd
77. 6 67 um Exterior Radius 13 33 um Use Identified Cell Mask Yes Max Growing Steps 26 67 Lm 2 Skip Steps 0 um Use Nuclei Mask Automatic Background Th No Background Threshold 25 Figure 259 Copy Marker Parameters control a Copy the Following marker settings Shades _ Segmentation Parameters From marker Ki6 D46 To the markers checked below Te a Copy _OfF_Ki67 DAB Page 179 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 260 Copy Marker Parameters control b e Copy the following marker settings indicates which parameters are to be copied from a marker to another e From marker here you choose the source marker e To the markers checked below here you choose the destination marker The segmentation parameters will not be copied between master and non master J Note N markers but only between non master channels Copy Marker Settings Copy the Following marker settings Shades Segmentation Parameters From marker Ki6 D46 To the markers checked below Te Figure 261 Copy Marker Parameters control c 9 1 5 Set as Nuclear Marker Set as Nuclear Marker option from the marker manipulation menu will mark another master marker master channel In order to accomplish this action the user has to select the marker to be transformed into a master marker and then access the Set as Nuclear Marker option Changing the ma
78. 7600 16385 win7_rtm 090713 1255 C Windows system32 SHLWAPI dll Version 6 1 7600 16385 win _rtm 090713 1255 C Windows system32 GDI32 dl Version 6 1 7600 16385 win _rtm 090713 1255 C Windows system32 USER 32 dll Version 6 1 7600 16385 win _rtm 0907135 1255 C Windows system32 LPK dll Version 6 1 7600 16385 win _rtm 090713 1255 C Windows system32 USP 10 dll Version 1 0626 7600 16385 win _rtm 090713 1255 C Windows system32 IMM32 BLL Version 6 1 7600 16385 win _rtm 090713 1255 C Windows system32 MSCTF dl Version 6 1 7600 16385 win _rtm 090713 1255 Read Product Improvement Program Terms Figure 7 Opening TissueFAXS project error message The last imported TissueFAXS HistoFAXS project file will be remembered if any The TissueFAXS HistoFAXS project to be imported in HistoQuest must not be opened at the same time in TissueFAXS HistoFAXS management and acquisition software Without license the user won t be able to create a new project from a TissueFAXS HistoFAXS project Also he won t be able to add sample s from a TissueFAXS HistoFAXS project Choose Regions The analysis in HistoQuest is performed on tissue samples Images of your samples virtual samples can be obtained by importing a TissueFAXS HistoFAXS acquisition project Page 20 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com HistoQuest 3 0 User Manual
79. 7638 Hematoxylin Variance of Intensity 331 4565 Hematoxylin STD of Intensity 18 20595 Hematoxylin Perimeter um 27 99555 Hematoxylin Compactness 0 6433494 Hematoxylin Eccentricity 0 750657 Hematoxylin Minimum Width um 6 079618 Hematoxylin Maximum Length um 9 519716 Hematoxylin Feret Ratio 0 6386343 Ki 67 DAB Area um 17 Ki 67 DAB Mean Intensity 40 97059 K 67 DAB Minimum of Intensity 8 K 67 DAB Maximum of Intensity 148 Ki 67 DAB Range of Intensity 140 Ki 67 DAB Sum Intensity 11144 Ki 67DAB Equivalent Diameter um 4 652426 Ki 67 DAB Variance of Intensity 861 9257 Ki 67 DAB STD of Intensity 29 35857 Ki 67 DAB Perimeter um 49 74702 Ki 67 DAB Compactness 0 08632261 Ki 67 DAB Eccentricity 0 7515501 Ki 67 DAB Minimum Width um 6 583135 Ki 67 DAB Maximum Length um 10 40733 al Ki 67 DAB Feret Ratio 0 632548 Figure 401 Event Data panel Internal Page 263 of 351 www tissuegnostics com HistoQuest 3 0 User Manual 13 3 Diagrams A HistoQuest feature of major importance is the ability to display cytometric data of cells in tissue in form of diagrams This permits to visualize cellular subpopulations within a given sample and or compare across ROIs and samples Any of the measurement parameters delivered by HistoQuest may be displayed in a diagram By setting cut off values and or gates positive and ne
80. 768 Recommended Intel Core2Duo Quad Processor 3 GHz or more 4 GB RAM Internet connection USB Port for dongle 1000 GB hard disk space for storing your projects Dual monitor system with a resolution of 1280x1024 or higher 2 2 Software Requirements ff Note Windows 7 x32 Recommended Windows 7 x64 Windows XP Windows Vista x32 Windows Vista x64 Windows 2000 By default HistoQuest needs to be run by a user member of the Administrators group In this case all users will share some settings e g institution data system data users HistoQuest can be also run by a user member of the Users group in which case every user will have its own settings Please contact your Support team in order to get the desired configuration for your workstation Once all the components are successfully installed the application is ready to be used r TissueGnostics provides a set up application that installs HistoQuest and all its ae ote dependencies For more information please contact your support team Page 12 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Notice regarding virtual machines TissueGnostics software is not meant to work on virtual machines Also TissueGnostics does not offer support for these configurations as the software directly uses advanced CPU features and therefore any third layer between the software and the CPU will affect performance and might affect th
81. 96 100 00 5531 097 Positive Ki 67 DAB o uwo ooa aoo O O o ooo aoo 0 000 0 000 D0 000 0 000 0 000 0 000 D oo00 0 000 21 497 13 667 100 00 1251 498 Figure 482 Include Scattergrams Scattergram Statistics e Include Histograms e Include Histogram Statistics Page 316 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Kib5 DAB Mean Intensi ki 7 DAB Mean Intensity 0 10 20 30 ET Ki 57 DAA Mean Intensity Overall Statistics 2 Ki 4 DAE Mean o o6 J o o o f o 6 809 1896 100 00 5531 097 Figure 483 Include Histograms Histogram Statistics e Include Processed Images Page 317 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual colon jpg Shades Marker Hematoxylin colon jpg ShadesOverlay colon jpg Measuremert colon jpg MeasurementOyverlay colon jpg Labeled OF pie T wt ees eee E Ti amp Oe Figure 484 Include Processed Images e Include Overlay Diagrams only for Compare Sets Page 318 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Overlay Diagrams ROI 02 ROI 01 Ki 6 DAB Mean Intensity 40 AKAM nN 4 Ki 67 DAB Mean Intensity Figure 485 Include Overlay Diagrams e Include Observations by pressing the Edit Observations button aE observation page can be edited Page 319 of 351 Internal www tissuegnostics com Hist
82. Area Measurements This method allows you to measure the total area expressed by a marker 11 4 1 Parameters e Automatic Background Threshold this parameter allows you to individually detect or set the threshold value for expressed marker masks Page 229 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e f you check Automatic Background Threshold you will be able to modify the limits between which the threshold will be computed Lower indicates the lower limit of the interval in which the threshold is computed The background threshold will be computed for pixels higher than the Lower value Upper indicates the upper limit of the interval in which the threshold is computed The background threshold will be computed for pixels lower than the Upper value Total Area Measurements Lower Upper Figure 358 Automatic Background Threshold lower and upper limits e Manual Threshold if you choose this then you should provide the threshold value Total Area Measurements Figure 359 Automatic Background Threshold Manual Threshold ote By pressing Reset Parameters button all the parameters settings modified by the user will be restored to their default values 11 4 2 Diagrams Configuration For this segmentation method the Results tab of the detail window will display the following default diagrams set Scattergrams e Master marker Area Non master marker Area Histograms
83. B Mean Intensity 26 61 D 30 80 120 Hematoxilyn Mean Intensity Double cick on a diagram to display it Figure 446 Manage Diagrams dialog rote _ The user should double click on a diagram name to display it on the right e Add Scattergram you can add new scattergram on a region and then propagate them on other regions By pressing the arrow button why the following options will be available Add Scattergram to Current Region Add Scattergram to Current Sample and its Regions Add Scattergram to All Samples and their Regions Add Scattergram to Current Region Add Scattergram to Current Sample and its Regions Add Scattergram to All Samples and their Regions Figure 447 Add Scattergram menu e Add Histogram you can add new histograms on a region and then propagate them on other regions By pressing the arrow button wh the following options will be available Add Histogram to Current Region Add Histogram to Current Sample and its Regions Add Histogram to All Samples and their Regions Page 291 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Add Histogram to Current Region Add Histogram to Current Sample and its Regions Add Histogram to All Samples and their Regions Figure 448 Add Histogram menu e Configure by pressing this button Configure h the following dialog will appear Here you can set the
84. Create ROIs including only those FOVs which belong to the same sample see Chapter 6 1 for details ROIs and or groups of ROIs will represent your individual sample 2 HistoQuest allows you to add more samples by importing images from folders after the project has been created For more details please see Chapter 6 1 1 The images must have three channels and the same size the maximum size of a FOV should not overpass 3200x2400 4 2 2 Step 2 HistoQuest Project This step is identical to the second step of the Import TissueFAXS HistoFAXS project Wizard having a Basic Settings tab and an Advanced Settings tab Since it is used to provide information about the HistoQuest project that will be created project name project description and storage folder any kind of project may be chosen as the functionality will be the same For more information please see Chapter 4 1 2 of this document Page 50 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual HistoQuest Project Please specify a path where the project will be stored and other project settings Choose Images Basic Settings Advanced setings E HistoQuest Project What s the name of the project Choose Markers Project Description Where will the project be stored F Projects Demo_Project Images If the project folder does not exist do you want to create it Create Project Folder Copy images to project folder or leave images in t
85. E A EAA AE E AE E A A EN E 307 1O TS ODON Sarpa E EEE E EAEE E E E E E E SE E E EE E E A E E E 310 NEE NCS PROD ON E EEE A E EA A A A E A E E 310 le oA TOM E i sels EE A del rece ETA wee T E EA E A T E A AE E E seater 314 TAa MIONO D AT aeee aa E E EENE EEE E EE EEEE EEE E E E E E EEEE E 322 UT Ng EE a E E E E A A A E A E A A A E A A 322 MZ OS IVS a cpa eee E 2 sce cin sepa a E Hera cele ots acess E a aeeesdencenseecs 324 Ae WOGIS CUM E EAA AE E aeteuesseedeee qses eset A NE 326 17 3 1 Manage DiagramS 00nn00nnannnannnnenernnnnnrrrsnrrnrennrrsnerrrrsnrrnrrrnrrsnernrrnnernrronrrrrronrrrenarennrronrrerernrennererenne 326 Ge Manage Gal Sussie EE AEAEE E E E EEE E ESES EEEE EEEE 326 E a SCS RODOR ao menene EEEE EEEE EE EEEE EE EEE E EEE EEE OEE EAE 326 EEDS WONG E E E E E A EEIE E A O E E 327 A MMO NU a n E a neg cde ee vet a E E agen dence nese 343 1o MOGU AUO e E A nr eee E E A EE a ee E 345 19 Memory NS SOS os cece ccs ee sec ccs ae aa Sees se sees eat se onde EE nk Se E se ava ieee ct aa OAE 349 ZO EE E E e saps E E AAN E A S T N E Fehler Textmarke nicht definiert 21 Document HISTON scree E A E Fehler Textmarke nicht definiert 22 Document Quality Assurance cccccceeeccccceeceeceeeeeeeceeeceeseaceesseeeeeesaeeeeeas Fehler Textmarke nicht definiert Page 4 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Disclaimers 1 TissueFAXSplus is a microscope based cell analysis system for cells in cryoc
86. Events are hidden only from the display but they are still considered to be part of the result Actions to be performed within a scattergram In each scattergram the user can perform the following actions Cutoff actions e Add cutoff To add a cutoff to a scattergram perform the following steps Click on the Create cutoff button in the scattergram toolbar Move the mouse to the position where to set the cutoff During this operation the mouse is captured inside the diagram area Finally the cutoff is determined by pressing the left mouse button on the desired position in the diagram e Modify cutoff Move the mouse cursor over the cutoff to modify the value of the cutoff It is possible to move both horizontal and vertical cutoffs only the vertical cutoff or only the horizontal cutoff As a result the cursor changes the shape Click on the cutoff using the left mouse button Move the cutoff to the desired new position Finish the cutoff reset operation by pressing again the left mouse button Page 269 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Copy cutoff The Copy action can be performed on one or both axis of the cutoff In order to copy only one of the cutoff axes move the mouse cursor over the desired axis Then press the right mouse button and from the displayed menu choose Copy Cutoff To copy both axis of the cutoff the mouse cursor will be moved over the axis intersect
87. FOV size in the Project Properties the values displayed by the _ caution scale bar will also change The Scale bar feature requires a license WjRequirements 5 5 Show Color RGB Button The Show Color RGB button i shows the color at the current position of the mouse For color images the RGB red green blue is displayed For gray images Shades Measurement etc only one value is displayed because red green and blue indicators are equal To be able to configure RGB Indicator you have two options e Press the Options button on the Region Viewer toolbar e Goto Tools Options General Image Viewer In the dialog that appears you will be able to set the color and the location of the RGB Indicator Page 82 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual RGB Indicator Color Location CUOL Figure 97 RGB Indicator set color RGE Indicator Color Location Top Right Bottom Left Bottom Right Figure 98 RGB Indicator set location Figure 99 Show Color RGB On the processed grayscale images of the regions e g Shades Shades Overlay the indicator will show only the gray level Page 83 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Guay ievel 98 J Figure 100 Show gray level A If the user has pressed the Show Color RGB button HistoQuest will remember it SS fcu next time it runs 5 6 Ex
88. Folder or NanoZoomer File For more details see Chapter 4 from the current manual the steps to be followed are exactly the same as in Chapter 4 excepting the fact you cannot change the storage folder the FOV size and the marker configuration This option works when a project is already opened Save oll Save AS Close Print Print Preview Exit SN wy Figure 494 Add Samples from option The same menu can be accessed by pressing the Add Samples from button a 4dd Samples From from the HistoQuest toolbar Note If the FOV size of the sample to be imported is different from the FOV size of the existing project you will receive a warning message For consistency reasons the images must be of the same type same resolution and same size of pixel Page 323 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual istoQuest WARMING A The selected TissueFAxs HistoF Ass project has a different FOY size 0 6729 0 5034 than the FOY size of the project 0 1 0 1 HistoQuest will use the project s FOY size Do you really want to continue jae Figure 495 Add Samples from option warning message Save this option saves the currently opened project Save as this option saves the currently opened project and you must specify the file name and the file type Close this option will close the currently opened project Print this option allows you printing
89. Here is where the HistoQuest Project File hiqproj extension the processed images and the cache will be saved It is recommended not to choose as location for the storage folder a mapped drive or an external hard drive e The Browse button Browse h helps you finding a previously defined location for your project If the new project folder is different from the initial folder the user can create a folder with another name by checking the Create Project Folder option If the storage folder differs from the TissueFAXS HistoFAXS folder you can decide if the images from your project will be used remaining at their initial location or if they should be copied in the storage folder This is realized by simply checking the Copy images to project folder option in this step of the wizard 9 If you decide to leave images at their initial storage location you will save space rote However if you move delete the original image source files your HistoQuest project might not be usable anymore data will remain though but as images are no more available also the connection of data to original images will fail and you cannot reanalyze the sample s The fully qualified hiqproj file name must be less than 260 characters and the project folder name must be less than 248 characters Please ensure that the selected folder exists Please ensure that you have enough rights on the selected folder Please ensure that you have enough free sp
90. I or group of ROIs Backward Connection The user won t be able to use Backward Connection If the user owns a predefined number of uses the counter will be incremented each time Backward Connection is performed Load profile from an existing project The user won t be able to load a profile from an existing project Load or save marker profile The user won t be able to load or save a marker profile Use Ruler The user won t be able to use the Ruler Use Scale Bar The user won t be able to use the Scale Bar Show Statistics Report The user won t be able to show statistics report If the user owns a predefined number of uses the counter will be incremented after each use Show Raw Data The user won t be able to access raw data If the user owns a predefined number of uses the counter will be incremented after each use Regions of Interest The user won t be able to define the regions of interest Instead he will be able to import a region from TissueFAXS If the user owns a predefined number of uses the counter will be incremented each time a region of interest is created Annotations Page 346 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual The user won t be able to define annotations If the user owns a predefined number of uses the counter will be incremented each time an annotation is created Exclusion Areas The user won t be able to define exclusion areas If the user owns a predef
91. Intensity Cutoff OTSU Ki 67 DAB Mean Intensity Cutoff 5 Quantile Cutoff 5 Quantile Hematoxylin Mean Intensity Cutoff 5 Quantile Ki 67 DAB Mean Intensity Cutoff EYIN ii Figure 415 Autosuggest Cutoff menu Show Percents The Show Percents button will display the following options Results i Show Percents for Count Show Percents for Area of Hematoxylin 9 54 120 ao cc Figure 416 Show Percents options Show Percents for Count for this option the percents are given by the number of events for each quadrant Page 273 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Hematoxylin Mean Intensity Hematoxylin Area p Hematoxylin Area Lum 0 40 a 120 Hematoxylin Mean Intensity Figure 417 Show Percents for Count diagram Show Percents for Area of Hematoxylin for this option the percents are given by the summed area of the events for each quadrant Hematoxylin Mean Intensity Hematoxylin Area p Hematoxylin Area um D 40 a 120 Hematoxylin Mean Intensity Figure 418 Show Percents for Area diagram For Total Area Measurement the percents displayed in the diagrams for each quadrant will be computed based on area of the chosen marker and not based on number of events Gate actions e Add gate HistoQuest provides three types of gates rectangle ellipse and polygon 1 Add rectangular gate C
92. NS ope ceceetcescce os Ss seesaw eee E E EAEE E SEE 72 Ao TNA ascetics serra ce teas serosa cece A sg eataeeseaiceu eee sacesz aie 73 Bs TS IM VION een tiger rte tele tt lea eco E be to tne 15 LRV O Qa tne eo E E E A eee A cee eee ee eer 15 Coe nn eS ne eee eee ee eee cee E ee TT AR GT eae ee E E E A eee ee E E E 80 ICE B a ee E E ee ne nee nee eee eee E E EE 81 gJ SNOW UO ROB BUNGO ercana a a iaa ia a e a e 82 5 6 Excluding and removing FOVSs Flag Button cccccccccscceceeeeceeeeseeeeseeeeaeeeseeeeseeceseseeseeeeseeeseeesaueeseeeaeeetaes 84 5 7 Shape Manipulation toolbar ccccccsscccsscccsecccececeuceceeecaeeecueeseueecsueecueeseeeeueessueessueesaueesseeeeueessueessueeesaeeseas 86 OO SNOW ANOS NAD erica accra i anie EAA R ARE R AEE Aa eenen 86 IN OG BUTO ririn innne A E E A EEA E AEE EAE E EIEEE E EAE EAE 87 SAULO E Aa CONN EO A A A EE E E E E A A E EEE AT E 87 5 11 View Raw Data Button sieraveastadancmotegavatacedancdatendvuncdaaensmetieddeateedandsete adv anciedencantiendvubead jacasutientnenand sedneiedenenatendeuneos 87 M ss AS EEEN E ON E E A ET N EE E ET E EE N E E E 87 SHAD oaea T e a EE E A ASE E N E A O AE E EE A NEA E E 89 Page 2 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 5 14 Project Items Properties Control ccccccccecccssccceeecencecenceceeeecueeceeeceucecaueesaueesaeeceusesaueesaueesueessueessusesseeessas 89 Sea Feral G8 1210 lt 1 COND TE O era e E E
93. Name the Input Gates and the Number of Shown Events for the respective scattergram 2 Scattergram Properties Te Parameter Parameter Name Hematoxylin Area um Ki6 D 46 Mean Intensity Input fates Number of Shown Events eee 100 100 Figure 449 Configure Scattergram Properties Page 292 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Delete you can delete a selected diagram from a region An identical diagram can appear on more regions and can also be deleted from any of those regions By pressing the arrow button hy the following options will be available Delete from Current Region Delete from Current Sample and its Regions Delete from All Samples and their Regions Delete From Current Region Delete From Current Sample and its Regions Delete From All Samples and their Regions Figure 450 Delete menu e Delete All you can delete all diagrams from a region By pressing the arrow button yh the following options will be available Delete All from Current Region Delete All from Current Sample and its Regions Delete All from All Samples and their Regions Delete All 3 Delete All From Current Region Delete All From Current Sample and its Regions Delete All From All Samples and their Regions Figure 451 Delete All menu e Apply Diagram Set you can propagate a set of diagram for one or more regions By pressing the arr
94. New folder E Recent Places 4 Name Date modified Type i Imaqes_MS07 6 3 2011 10 11 AM File folder coy Libraries lt 5 L d Images_MS08 6 3 2011 10 17 AM File folder Documents r re A Miso 6 3 2011 10 11 AM File folder a d M08 6 3 2011 10 12 AM File folder Pictures ae T i d M09 6 3 2011 9 52 AM File folder ideos U MS07 nines 10 14 2008 12 00 MEXS File MS08 mres 10 14 2008 12 00 MRXS File LJ Computer 5 P O MS09 mines 10 14 2008 12 00 MEAS File ch Network ER File name MSO7 mres Mirax files rnrcs Open i Cancel Figure 67 Import Mirax Project browser Page 61 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Choose Mirax file Select the Mirax file from which you want to import images in this HistoQuest project E Choose Mirax file Where is your Mirax file HistoQuest Project F Projects HistoQuest Mirax Projects MSO7 mrxs Choose Mark ne What size should the extracted images have 1024x 1024 v What format should the extracted images have At which zoom level should the images be extracted Description Sample size 74036 px 166152 px Sample size 24 3000 16744737 mm 54 3148736 140258 mm Pixel size 0 32821893058427 um 0 32821893058427 um Phisical magnification 20X Scanned FOVs count 4556 Export information Sample size FOVS 163 73 al FOV size 0 336096 1849 mm 0 3347442738 mm a CoD
95. Select Only Time Regions Slide Slide 1 has 6 regions Comment TMA Grouping Import TissueFAXS HistoFAXS regions V Import TissueFAXS HistoF AXS logical groups Import TissueFAXS HistoFAXS subregions y Create Group of ROIs from TissueFAXS HistoFAXS categories Note Your project will have 2 samples TISSUEGNOSTICS Figure 6 Import TissueFAXS HistoFAXS Project Step 1 If the project to be opened in HistoQuest is simultaneously opened in TissueFAXS or it has been created with an unsupported TissueFAXS version an error message will appear mp Page 19 of 351 Internal www tissuegnostics com F an jv HistoQuest encountered an error G Error description Error detail at a9y a String A_0 Loaded Modules C Program Files TissueGnostics HistoQuest HistoQuest C Windows SYSTEM32 ntdll dll Version 6 1 7600 16385 win _rtm 090713 1255 C Windows SYSTEM32 MSCOREE DLL Version 2 0 50727 4927 NetFxXspW7 050727 4900 C Windows system32 KERNEL32 dl Version 6 1 7600 16385 win _rtm 090715 1255 C Windows system32 KERNELBASE dll Version 6 1 7600 16385 win7_rtm 090713 1255 C Windows system32 ADVAPT32 dll Version 6 1 7600 16385 win _rtm 090713 1255 C Windows system32 msvet dl Version 7 0 7600 16385 win _rtm 090713 1255 C Windows SYSTEM32 sechost dll Version 6 1 7600 16385 win _rtm 0907135 1255 C Windows system32 RPCRT4 dll Version 6 1
96. The user can be interested in statistics computed based on area and count The statistics depend on choosing one of the two options area or count Count is the default option for Various Shapes and area is default for Total Area Measurement Count The statistics will contain the following columns e Count number of detected events Page 231 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Percent calculated based on events number No mm number of detected events per Area Measurement Unit Area The statistics will contain the following columns e Total Area of total area of chosen marker e Percent Area of percent area of chosen marker relative to the total area of the marker e Percent Area of Area Measurement Unit percent area of chosen marker relative to the total area of the Sample ROI Group Page 232 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 12 Image Analysis 12 1 Analysis After having created a project defined the reference shade s for color separation and optionally created some ROls or groups of ROIs on your sample s HistoQuest is ready to perform analysis e Ensure there is at least one marker in the project e Set atleast one nuclear marker e Pick at least one shade for each marker HistoQuest allows the user to perform analysis on an entire sample several samples or regions of interest Because analysis involves many
97. User Manual Hematoxylin Sum Intensity Hematoxylin Sum Intensity Logarithmic Scaling type Linear Scaling wt a08 108 04 QF 0 10000 20000 30000 Hematoxylin Sum Intensity Hematoxylin Sum Intensity Figure 433 Histogram Parameters Scaling Type parameter Logarithmic left and Linear right e Set Cutoff on option will display a menu where it is possible to set new values for the cutoff Also the cutoff can be propagated from this dialog In order to perform the propagation the axes of the different diagrams should be the same AY Set Cutoff Cutoff value For Hematoxylin Mean Intensity Apply to w Current Regiontaroup 2 Current Sample C All Samples Figure 434 Histograms Set Cutoff control box e View Backward Data will display the menu containing all possible options for backward data on a histogram For more details please see Chapter 14 2 e Copy to Clipboard creates a copy of a histogram the user being allowed to paste it into another software application e g MS PowerPoint e Save as will save a histogram as an image file The user will be able to choose the name and the extension of the newly created image file Configure Histogram Page 283 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Marker Parameter Hematoxylin iv Mean Intensity o Mame Hematoxylin Mean Intensity Input Gates a T Da Wa UR Hematosylin Mean Intensity Ki 6
98. a aal PDF File HTML File MHT File RTF File Date ofanalysis 9171 2009 3 31 28 PM PAVER E CSW File i Text File Image File Figure 490 Exporting Print Outs Page 321 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 17 Menu Bar The Menu Bar contains four items e File e Project e Tools e Help 17 1 File Menu File Project Tools Help Figure 491 Menu Bar Project Tools Help Add Samples from T y Save Save Os Close Print Print Preview Exit Figure 492 Menu Bar Open by choosing this option you can browse for the HistoQuest project you wish to open or you can choose your project from the Last Opened Projects list By pressing the Clear button you can 3 empty this list The same menu can be accessed by pressing the Open button PE from the main toolbar this operation will automatically close the current opened project For more details please see Chapter 4 4 Import this option helps you import TissueFAXS Project HistoFAXS Folder or NanoZoomer File For more details see Chapter 4 from the current manual It only works when no project is opened Internal Page 322 of 351 www tissuegnostics com HistoQuest 3 0 User Manual TissueFAsS HistoF AX igs Folder far MHanoz oomer File Figure 493 Import option Add Samples from this option helps you add TissueFAXS HistoFAXS Project
99. ace on the drive where the folder is located If a project with the same name already exists at this location the user is asked if he wishes to overwrite the existing project If the user doesn t agree to overwrite the existing project he must select another name for the new project Page 39 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual rE If the storage folder is the same as the TissueFAXS HistoFAXS project folder then the newly created project will be automatically saved in this folder and all temporary files will also be stored at this location To save the HistoQuest project at another location a different storage folder must be specified Advanced Settings FOV Size Basic Settings Advanced Settings Do you know the size of a Field of View FOV Size predefreds CL FOV Width mm FOVHeight 040200001 Mes Image Width 1600 px Image Height 1200 px Pixel Width 0 3362499925 um Pixel Height 0 3350000083 pmi Do you know the objective that was used to acquire these images Objective Figure 36 Import TissueFAXS HistoFAXS Project Step 3 Advanced Settings The user has more possibilities to enter the size of the FOV For more details please see Chapter 4 6 Objective The Objective value can not be modified it is defined by TissueFAXS HistoFAXS and it just serves for your information Starting from this value the image will proportionally increase or decrease its size w
100. ach kind of shape Samples e Total Scanned Area the initially scanned area of the tissue e Total Excluded Area the excluded area from the analysis when using exclusion areas or flags Regions of interest e Area for analysis displayed on the image viewer and in the project item properties the area of the tissue obtained after removing all exclusion areas or excluded FOVs that intersects with the regions shape Annotations e Area displayed on the image viewer the total area of the tissue delimited by the shape of the annotation disregarding the excluded areas or FOVs Exclusion areas e Area displayed on the image viewer and in the project item properties the total area of the tissue delimited by the shape of the exclusion area disregarding other excluded areas or FOVs Groups e Area displayed in the project item properties the area of the tissue obtained after removing all exclusion areas or excluded FOVs that intersect with the contained regions shapes F Areas are computed based on the FOV size in square millimeters If the project was note created from a TissueFAXS project with stitch then the FOV size used in computations will be the size of the crop stitch rectangle which is smaller than the FOV size displayed in the project properties form Page 171 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 9 Markers and Profiles In HistoQuest the term Marker refers to a certain color which
101. ache Delete Results Add Standard ROL Figure 159 Sample contextual menu Paste ROI j If the copied ROI doesn t fit the coordinates of the destination sample the user will be vf Note informed that this action cannot take place Figure 160 Paste ROI error message e Analyze option will perform the analysis for the selected ROI e Analyze Unprocessed FOVs option if a number of FOVs of a ROI were already analyzed in a previous analysis operation for another ROI these FOVs being common to both ROIs this command will analyze the rest of the unprocessed FOVs e The Delete Results option erases the data obtained by the analysis Image Viewer or by using Image Viewer button from the Region Viewer Q The specific color of a ROI can be set and changed in Tools Options General TIP Page 118 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual General Image Viewer Default Colors Default Locations Region of Interest RGB Indicator Top Left Annotation Bottom Right Group of ROIs mi Top Left Exdusion Area Backward Connection RGE Indicator Scale Bar LI Task Manager Overlay Non selected Conta Non selected Conta La a Selected Contour Co Selected Contour Thickness D La i TY Tl kz r Show non selected events in backward connection mode _ Animate borders of ROIs and Annotations Highlight double cdicked point from Sample Image View
102. ackground Threshold Wo Background Threshold 25 Post Processing Order Remove Labels cytoplasmatic Use Merging Rules Figure 346 Virtual Channel nuclear Non Master Marker Parameters The non master channels are segmented using an especially designed algorithm which starts from the segmentation obtained in the master channel A series of configurable features are available for this process e Use Ring Mask if you select this a ring mask around the objects detected in the master channel is created and included in the segmentation result arious Shapes 2 0 3 Interior Radius i Exterior Radius Use Identified Cell Mask Mo Use Nuclei Mask Mo Automatic Background Th Yes i Threshold Range 5 255 Figure 347 Use Ring Mask Parameter Internal Page 224 of 351 www tissuegnostics com HistoQuest 3 0 User Manual e Use Ring Mask Parameter The Interior radius and Exterior radius parameters allow the user to define in which direction outwards inwards or both and to what extent this ring should be created The value represents the number of pixel layers As this ring construction takes care of neighboring objects and statistical growth process it stops when the growing environment of a neighboring cell is reached e g when cells touch each other The value for exterior values represents the maximum growth The interior radius ring growth process stops when the entire nucleus is covered arious Shape
103. al What is the name of the new group The Group will be propagated only on the samples where it doesn t already exist Figure 171 Create Group of ROls message The name of the group of ROIs to be created must be different of all the other project items e Using the Create Group of ROIs option from the menu that appears after the right mouse button is pressed Before that at least one ROI has to be selected like in the previous case A W oe 4 d a ROILO09 4 iL Add to existing group View Event Data Show in Detail RO 09 Show Interior ROI 09 Remove ROI O09 Copy to Clipboard Figure 172 Create Group of ROIs from context menu Page 127 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual F s RoI Y SA j a S i aa Aa rae 2 er Cte Le aS gt p Figure 173 Group Detail Window e Using Add to Existing Group option from the ROI contextual menu The user will be allowed to choose the group where to add the ROI Page 128 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Create Group of ROIs View Event Data Show in Detail ROI 08 Show Interior ROI OS Remove selected ROIs Copy to Clipboard Save Copy Selected ROIs Figure 174 Add to Existing Group from context menu Figure 175 Add to Existing Group message lf a ROI has to be added to an already existing group select
104. al area of the marker Percent Area of Area Measurement Unit percent area of chosen marker relative to the total area of the Sample ROI Group For Total Area Measurements with count used in computing the percents X Sum i e sum value for the parameter shown on the x axis Y Sum i e sum value for the parameter shown on the y axis For Total Area Measurements with area of a marker used in computing the percents X Sum i e sum value for the parameter shown on the x axis Y Sum i e sum value for the parameter shown on the y axis Percent Area of percent area of chosen marker relative to the total area of the marker Percent Area of Area Measurement Unit percent area of chosen marker relative to the total area of the Sample ROI Group Scattergram An x y diagram showing two measurement parameters for all data units or a selection thereof in linear or logarithmic scale Within the scattergram cutoffs can be set in order to define a threshold for each measurement parameter Profile A set of image analysis and image processing parameters used for proper identification of one or several specific markers that was stored to the hard disk and can be reloaded and reused for another sample at any time Field of View FOV The area of a sample tissue section visible in the microscope and captured at one x y z position on the slide and with a given optical magnification An image of one F
105. ams are a means of visualizing co expressions of different markers two in each scattergram the quadrant regions define the four possible combinations of positive and negative i e above or below cutoff value per marker 1 LL Marker X negative and Marker Y negative 2 UL Marker X negative and Marker Y positive 3 UR Marker X positive and Marker Y positive 4 LR Marker X positive and Marker Y negative Quadrant Statistics Shows the following values according to the segmentation method For Various Shapes 1 0 and Various Shapes 2 0 with count used in computing the percents X Mean i e mean value for the parameter shown on the x axis Y Mean i e mean value for the parameter shown on the y axis Page 8 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Count number of events in the respective unit sample gate ROI quadrant Percentage i e how many events are in the respective quadrant region relative to the total number of events for each of the four quadrant regions No mm number of detected events per Area Measurement Unit For Various Shapes 1 0 and Various Shapes 2 0 with area of a marker used in computing the percents X Mean i e mean value for the parameter shown on the x axis Y Mean i e mean value for the parameter shown on the y axis Total Area of total area of chosen marker Percent Area of percent area of chosen marker relative to the tot
106. an intensity higher than the specified value will be removed from the segmented image Yarious Shapes 1 0 Nuclei Size 5 Discrimination Area 1 Yarious Shapes 1 0 Discrimination Gray i Nuclei Size z Automatic Background Threshold Yes Discrimination Area 1 Virtual Channel no Discrimination ray 1 Post Processing Order Remove Merge Automatic Background Threshold Yes Remove Labels es Virtual Channel no j Smaller Than r0 33 um Post Processing Order Remove Merge T Larger Than 35416 67 pm abels gt Weaker Than 20 Use Merging Rules 3 Stronger Than do not use Use Merging Rules Mo Figure 322 Remove Labels Parameters e Merging Rules Parameters searches for and merges groups of objects that make a round shape when combined This is especially useful in case of undersegmentation where a round nucleus might be detected as 2 separate objects Max combined area specifies the maximum obtainable area by applying merging process Max involved compactness specifies the maximum compactness which an object can have prior to the merging process Group Max specifies the maximum number of neighboring labels involved in the merging process large number of combination possibilities especially when the image contains large Ue Increasing this value more than 3 or 4 might lead to long processing time due to the fi ote clusters of objects Min Resulted Compactness specifies the minimum possible value of an object
107. analysis options do you want to use in your project Analysis Options Segmentation Method Various Shapes 2 0 Strategy for events in ROI Select only events entirely in ROI Color Separation Method Single Reference Shade Figure 55 Import Folder Choose Markers 4dd Samples From A Folder can be added to a project by pressing the Add Samples from button from the main window toolbar then choose the Folder option from the displayed menu TissueFAXS HistoF Aas Figure 56 Add Folder The same wizard will appear but the values from step 2 and 3 cannot be modified 4 3 Import Images from a Hamamatsu NanoZoomer File In order to import a Hamamatsu NanoZoomer file press the Hamamatsu NanoZoomer button af i ER Hamamatsu NWanoZoomer or choose from the menu File Import NanoZoomer File Page 53 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Project Tools Help Fa Open Import Gy Tissueraxs Historaxs Add Samples from Save Save As Close Print Print Preview Exit Figure 57 Import Hamamatsu NanoZoomer file 4 3 1 Step 1 Choose Hamamatsu NanoZoomer file The Import from Hamamatsu NanoZoomer wizard will appear Choose Hamamatsu NanoZoomer file Select the Hamamatsu NanoZoomer file from which you want to import images in this HistoQuest project E Choose Hamamatsu NanoZoomerfile Where is your NanoZoomer f
108. ancbsetaaesdecisacseunpeavecdsenteesdeuiosendccecesusstevecdsce ENEGA A EEEE 185 9 2 2 Load Prolls TGP VO OCI oc ersinnen nisse aAA EEEE AARE EEEE E SA AAEE EOE EEANN EER 185 9 233 Load Marker Prole seriene enee EEEE AEE EA AEEA ENEE aA OEE EEEE INESE EE EEEa 186 9 2 4 Manage Profiles ea seie ences nie denna cae lndeta chia bene scielstranciee dated deielysacid badd d nae cule oeingeets dated cdi dated bale owas dee ioialaatisiednnoneudendenetieds 187 10 Color Separation MethodS jai sciec ce nceneontacer louse hceedensistiiedaisafeuisnncessienniacbileedmasiba ioteie hes Aandanubiouslachiuiedensibadieinesbundeessbeeleantausecs 190 10 1 Multiple Reference Shades ccccccccscccesceceececeeecceeeceucecaueeceneesueessueessueesaueesueeseaeessueeseeesueeseusessueesseeeseas 190 TO ea Fig Pie a2 6 5 ea ene ee ne ea eee ee ee eee 190 10 2 Single Reference Shade ccccccccccsscccsccceececencecaeeeceeeceuceceueesueesseeseueecsueessueesaeessueeceusesueesueessueessueesseeeseas 199 Ti SONIC EI AUIONN NEOUS epee emcee en A A EE E A aside OE E pace E EO 205 11 1 Changing Segmentation Method ccccccccsscccesecceeeceseeceuceceueeceesececeueecaueesaeessaeecsuseseueeseessaeesausesseeesaes 205 11 1 1 Configure Segmentation Method Parameters cccccccccsececececeeeeceeeeseeeeeeeseeeeseueeseeeseeesseeeseeeesaees 207 TEA W ANIONS se oe O acictecsa sich acte ee ediee E palsies sega E E E E E 208 MDZ Mig eA LIN VOUS E EE ton ass
109. anipulation menu Only one marker can be selected at a time If the master marker was previously chosen the Is master option will appear disabled The user can visualize the existing markers by accessing the dropdown list from the Add Marker dialog and select the desired marker then press Ok Figure 251 Add Existing Marker dialog Adding an existing marker will clear the results in the project A warning message will vf note be displayed to the user Page 175 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual HistoQuest Some ROI contain events and adding a new marker will clear them Do you really wank to continue W a Figure 252 Add existing marker warning for a project with existing results 9 1 2 Add Copy of Marker The Add Copy of Marker Name option will create a copy of a selected marker The copied marker will have the same shades and the segmentation parameters like the source marker When using Multiple Reference Shades any copy of a marker will be independent a change to the original marker will not affect its copy If a master marker is copied only the shades will be copied the segmentation Note parameters of the copy are the default ones The name of the copied marker will have the following form Copy_Of_Source Marker Copy OF K 67 DAB Hematoxylin Multiple Reference Shades Figure 253 Copy Marker control Virtual Markers W
110. annot be modified 4 A mrxs file that has different FOV size than the project or different magnification cannot be vf Note added The following message will be displayed WARNING The selected Mirax project has a different FOV size 0 3360961849 0 3347442738 than the FOV size of the project 10 7550779173 10 7550779173 HistoQuest will use the project s FOV size Do you really want to continue Figure 74 Mirax Project Warning message 4 5 Open HistoQuest Project The user can also use the Wizard to open an existing HistoQuest project He will click on the corresponding Open Existing Project button then choose the project from the browser In the Open HistoQuest Project dialog the user can see an overview of the project before effectively opening it He can also see the markers the segmentation and color separation methods the preview of the samples and comments Page 66 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Open HistoQuest Project F Projects HistoQuest Colon 1 Colon 1 hignroj Segmentation Method Various Shapes 2 0 Strategy for events in ROIL Select only eventsentrelyinROl Color Separation Method D i Magnification 20 FOV size is 0 4 x 0 3 Number of ROIs 0 Number of Groups 0 Events count 1705 Figure 75 Open HistoQuest Project dialog e Clear button erases all items from the Last Opened Project list e Show Hide Preview
111. areas will be taken into consideration From the region viewer toolbar choose desired shape rectangular elliptic custom and freedrawn then draw the exclusion area on the sample Figure 213 Exclusion Area The Exclusion Areas have some adjustable settings that can be accessed from Tools Options General Image Viewer e Exclusion Area color choose desired color from the color picker e Exclusion Area transparency move the slider to make the exclusion area more transparent or more opaque e Show name for Exclusion Area mark this checkbox to display the name of the exclusion area on the sample Page 148 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Default Colors Default Locations RGE Indicator Top Left e Scale Bar Bottom Right Item Name Top Left Region of Interest Analysis i Annotation EIE Group of ROIs t i Exclusion Area Exdusion Area Transparency Backward Connection RGB Indicator k Scale Bar E Task Manager Overlay Non selected Contour Color ee Non selected Contour Thickness Skin Selected Contour Color Selected Contour Thickness gt Show non selected events in backward connection mode oa Tt rt _ Animate borders of ROIs and Annotations Highlight double cicked point from Sample Imag 5 j Show grid lines for Samples imported from Folder
112. at shows the entire sample only if the cache is built The part of the sample within the red frame is shown in the Region Viewer window Figure 85 Region Viewer By pressing the Close button E in the upper right corner of the Region Viewer only the displayed image will be closed not the entire project 5 1 Navigator To change the area of the sample visible in the Region Viewer the user has several options Page 75 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e use the map functionality a simple click on the map will make the visible area centered on the chosen point e use the scrollbars of the Image Viewer or the arrow keys on your keyboard to scroll the image on horizontal and vertical axis e zoom to a specified area of the image draw a rectangle around the area you would like to zoom into on the image by keeping the left mouse button pressed this option will automatically zoom centered to that area Right Click F x x pa ick ye b Figure 86 Zoom to a specified area e With a left click you will be able to move the sample on the screen by dragging it The short right click displays the contextual menu of the region while the long right click defines a zoom rectangle The scroll wheel zooms in out Zoom Buttons The Region Viewer also provides Zooming Operations e Zoomin use the Zoom in button 2 of the Region Viewer The zoom in is performed centered
113. ating HistoQuest Project Loading Project Figure 45 Creating HistoQuest Project progress bar By pressing the Cancel button l from the progress bar the following message will appear Figure 46 Creating HistoQuest Project canceling message A TissueFAXS HistoFAXS project can be added to a project by pressing the Add Samples from button a 4dd Samples From from the main window toolbar then choose the TissueFAXS HistoFAXS option from the displayed menu Folder ira Hamamatsu NanoeZoomer Mi I rax bee Figure 47 Add TissueFAXS HistoFAXS project The same wizard will appear but the values from step 3 and 4 cannot be modified Page 46 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 4 2 Import Folder This 3 step wizard will guide the user through the process of creating a new HistoQuest project from an existing folder This option should be used if the samples have not been acquired by TissueFAXS HistoFAXS Th Note The special HistoQuest features for Tissue Microarrays TMAs handling are only available for TissueFAXS projects hence the Import Folder option cannot be used for a TMA project 4 2 1 Step 1 Choose Folder At this point the storage folder for the images to be imported must be selected The newly created project will contain all the images located in this folder T Folder In order to import a Folder press the Folder button Bo o me or
114. button lx 2 5 5x 10x 20n 40x 63x 100 Figure 87 Objective Buttons p Although you can zoom in using the Objective buttons on any source magnification rote the magnification used for acquisition the resolution will NOT CHANGE by this operation The optical resolution is determined by the objective lens used for acquisition If a sample has been acquired using a 10x and the user presses the Magnification Button 63x the magnification will look like 63x magnification but still with 10x resolution pixel will be blown up The only way to obtain a real 63x resolution is to acquire the sample using a 63x objective lens The only way to obtain a higher resolution is to use an objective with higher numerical aperture The resolution is not bound to magnification The value the user already entered as reference for the Objective parameter when the Import Folder option was used will display the image at its original size For any other value the image will proportionally increase or decrease its size The respective Objective button will be displayed in bold letters Figure 88 Magnification Values 5 2 Map A map is provided that shows the entire sample The part of the sample within the red frame is shown in the Region Viewer window Page 77 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual The user can choose to display or hide the map by checking un checking the Map button of
115. button shows hides the preview over the experiment Page 67 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual gt F Projects HistoQuest Colon 1 Colon 1 higproj Figure 76 Open HistoQuest Project dialog hidden preview To be able to see the Preview section of the Open HistoQuest Project panel a license is required Without the license the Show Hide button will be disabled 1 Requirements When a project is corrupted If a project is corrupted HistoQuest will launch a warning message when opening the existing project Here are the most common situations e illumination correction image is missing Events cannot be read ROI cannot be deserialized e Sample cannot be deserialized fn When opening a project the first sample is automatically loaded in the main image MOE 7 viewer If for some reason the images from the project to be opened are missing HistoQuest will display a warning message HistoQuest uses relative paths from the project location to the images In order to use an existing project that was moved to a different location the images should also be moved correspondingly so that the relative path remains the same Page 68 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual HistoQuest generated warning messages A Description Details All images are missing You probably recently moved your project HistoQuest
116. button menu on the empty column header Group By Box E Column Chooser oy Filter Editor Best Fit all columns Figure 395 Filter Editor b Hematoxylin Area uim Is less than 10 Figure 396 Filter Builder 13 2 1 Exporting events The events currently displayed in the grid can be exported to Page 260 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual _ E Region Of Interest B Ee a EE Feld OFView Event Label Hematoxyin Mean 67 AB Mean Intensity ekekeke ere T A Figure 397 Export to Excel format Figure 398 Export to PDF e Export to CSV format Page 261 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual agi Microsoft Excel Colo dEi File Edit View Insert Format Tools Data Window led Bj Arial 210 B 7 DU EP Snagit f Window ie A1 fe Region Of Interest _ Region Of Interest IF eld Of View Event Label Hematoxylin Mean Intensity Kib Colon 65 52631 Colon 79 89726 ff 34454 67 98925 79 11848 58 03286 T3 20446 66 26396 57 19685 55 48815 T0 98343 63 09649 55 55414 1 q 1 2 1 3 1 4 1 5 1 6 1 T L 0 1 g 1 10 1 11 1 12 1 14 Colon lid 4 e bh Co Ready NUM Figure 399 Export to CSV To achieve the raw data export just press the corresponding button and in
117. c and unspecific staining patterns allowing determining positive and negative measure events Event A data unit obtained by HistoQuest algorithms representing a quantum of information about a defined object nucleus cell An event is identified by one or several image processing and pattern recognition algorithms An event contains data for all measurement parameters applied to a single image object Gate A subset of events within a scattergram selected by defining a region in a diagram Gates may have one of the following shapes rectangular elliptical and polygonal and contain any number of cells Gates may overlap each other and be combined or defined as mutually excluding Marker A molecular labeling component chemical or biological reagent e g Hemalaun stain antibody genetic probe that allows the identification of defined cell types tissue types cellular or tissue compartments cellular functions disease status etc In HistoQuest markers have colors picked from the images to be analyzed and also parameters for detection and measurement masks Project A set of tissue samples which are analyzed together with the same parameter settings for the image analysis and pattern recognition algorithms Quadrant Regions Each scattergram is separated into four regions by its x and y cutoff values The four quadrant regions are labeled lower left LL upper left UL upper right UR and lower right LR As scattergr
118. cal dimension is expressed in metrical units The Ruler values are computed using the experiment s FOV size In order to show hide it press the Ruler button faa Depending on the zoom level the Ruler will display different divisions If the user has pressed the Ruler button HistoQuest will remember it next time it runs If the user changes the FOV size in the Project Properties the values displayed by the ruler will also change The Ruler feature requires a license Page 80 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 5 4 Scale Bar Figure 94 Scale bar The Scale bar is a graphical element which shows a sample scale graphically o 10 dalat In order to show hide it press the Scale bar button The color and the position on the sample of the Scale bar can be set in two ways e By clicking the Options button on the Region Viewer Scale Bar Color Scale Location OCOLUL Figure 95 Scale bar color combo box Page 81 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Scale Bar Color Scale Location Top Left Top Right Bottom Left Bottom Right Figure 96 Scale bar location combo box e By accessing the same menu from Tools Options General Image Viewer rea ie If the user has pressed the Scale bar button HistoQuest will remember it next time it runs If the user changes the
119. choose from the menu File Import Folder mmm HES Project Tools Hel p Tr Print e e re Print Preview Exit Figure 48 Import Folder Choose Images step of the wizard has two tabs Basic settings and Advanced Settings Basic Settings Page 47 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Select markers from the existing ones or create new ones to use in this HistoQuest project E Choose Images Basic Settings Advanced Settings HistoQuest Project Choose Markers Where are your images Folder containing your images F Demo Project Images Add Subfolder Images Exclude _hiqtmp folder fe onmanl lawman Tq E Images 9 7 2010 9 7 2010 E GF Slide 1 9 7 2010 9 7 2010 E Region 001 9 7 2010 9 7 2010 Timeregion 4 9 7 2010 9 7 2010 j E Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 w Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Z Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Z Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Z Demo Project Region 001 TimeRe 9 7 2010 9 7 2010 Note Your project will have 6 samples Figure 49 Import Folder Wizard Step 1 Basic Settings e To filter the files from the selected folder the user should use their extensions or a file naming pattern e To select the project s
120. city Hematoxylin Equivalent Diameter Hematoxylin Feret Ratio Hematoxylin Maximum Length Hematoxylin Maximum of Intensity Hematoxylin Minimum of Intensity Hematoxylin Minimum width Hematoxylin Perimeter Hematoxylin Range of Intensity Hematoxylin STD of Intensity Hematoxylin Suro Intensity Hematoxylin Variance of Intensity Kib D46 Area Ki6 D46 Compactness Ki6 DAB Eccentricity Ki67 DAB Equivalent Diameter Ki6b D46 Feret Ratio Ki67 D4B Maximum Length Ki67 DAB Maximum of Intensity Ki67 D4B Minimum of Intensity ki67 DAB Minimum Width Ki6 D4B6 Perimeter Ki67 D4AB Range of Intensity Ki67 D4B STD of Intensity kior DAB Sur Intensity Ki67 DAB Variance of Intensity Figure 393 Customization dialog In order to delete a parameter the user will drag its label from the grid header and drop it on the customization list On the Raw Data window more filters can be added depending on the user s needs The Filter Editor can be accessed in two ways e By selecting Filter Editor from the right mouse button menu on the existing column headers Page 259 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com ail Sort Ascending Sort Descending Clear Sorting ae Group By This Column Group By Box g column Chooser J Best Fit oy Filter Editor Best Fit all columns Figure 394 Filter Editor a By selecting Filter Editor from the right mouse
121. cluding and removing FOVs Flag Button Y The Flag button is used in two cases e Remove it eliminates from the analysis the selected FOVs e Exclude it keeps the selected FOV in the sample and it eliminates from the analysis only the data results Remove Exclude Apply Figure 101 Operations Available for Flag Button In order to accomplish the desired operation select Remove or Exclude then select the desired FOVs and finally press Apply fn i For the Exclude option the eliminated FOVs can be reintroduced by ote selecting Exclude removing the flag from the desired FOVSs by directly clicking on the FOVs surfaces pressing apply The Flag button is only shown for the sample opened in the main window If there is any task running for the opened sample the Flag button will be disabled In order to cancel the operations Remove Exclude uncheck them from the contextual menu that appears when right clicking on the Flag button Excluding or removing a FOV will influence the number of events from a region and also its area Page 84 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com In order to remove or exclude FOVs from an analyzed sample the user should ensure there is enough memory available in order to load all the events ROIs and groups from the sample If the memory is not sufficient the removal operation will not be possible and a warning message will app
122. context menu e The Autoscale on option available for any of the axes will set the maximum value of a chosen axis to be the maximum value of a parameter to the affected axes e The Set Max Value on option will allow the user to set a new maximum value for an axis Set Max Value Maximum value For Hematoxylin Mean Intensity eal Apply to Current Region Group O Current Sample and its Regions D All Samples and their Regions Figure 403 Scattergrams Set Max Value dialog By choosing one of the three options the newly introduced value will be applied to all scattergrams containing this axis If Autoscale on option is checked for an axis Set Max Value on will not be es allowed for it If Set Max Value on is applied with a lower value than the maximum value of the Page 265 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual corresponding parameter not all data points will be shown in the diagram but they are still computed appropriately in the quadrant statistics e Parameters displays a dialog in which the user will be allowed to set max value for the axes make Autoscale and set the scaling type for both axes of the scattergram and propagate the new values to the diagrams from other regions containing similar parameters 2 Auboscale Mo Scaling type Linear Current Sample and its Regions g Hematoxylin Area tum All Samples and their Regions j 160 Current Region
123. d to New Group Create Annotation from ROI Create Exclusion Area from ROI Copy Delete Results Propagate ROI Figure 155 ROI Options context menu e The Show in Detail Window option will display the ROI on a detail window Regions are sorted after their area so that any region can be chosen for an operation e The user will be able to rename a ROI using the Rename option The name can contain maximum 40 characters Qld name Hew name Figure 156 Rename ROI dialog e Pressing the Remove button will erase an existing ROI Page 116 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 157 Remove ROI message e In order to introduce a ROI to a group the Add to Group option should be used Select the group from the displayed list of existing groups in which the ROI should be included For more details please see Chapter 0 Figure 158 Add ROI to Group dialog e The Copy option will create a duplicate of a ROI on the same sample using a different name or on another sample After performing the paste operation of the right mouse button menu on the desired sample the duplicate of the ROI will preserve the coordinates of the original one Page 117 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Show Show in Detail Window Analyze Analyze Unprocessed Fos Snalyze All ROIS Rename Remove Remove All ROIs Build Cache Delete C
124. de in the preview E U La pa Slide 4 Region 037 i L Slide 4 Region 037 Region Os cia sr oups aroup O1 D TET Region 302 pil Slide 4 Region 302 H Region 302 Sal roups EO Slide 4 Region 303 iD slide 4 Region 303 Region 303 l roups CO Slide 4 Region 306 fel Slide 4 Region 306 H Region 306 Groups eo Slide z slide 5 ial TMA Block 00071 401 Figure 480 Print dialog Options Include Scatbergrams Include Scattergrams Statistics Include Histograms Include Histograms Statistics Include Overlay Diagrams Include Processed Images Include Motes page Include Only Visible Diagrams Manually select the regions to be included in the print report or use any of the options displayed in the Options menu Select All ROIs Select Processed ROIs Select Unprocessed ROIs Select All Samples Select Processed Samples Select Unprocessed Samples Select All Clear Selection Figure 481 Print Options menu The following operations are available Include Scattergrams Include Scattergram Statistics Internal Page 315 of 351 www tissuegnostics com HistoQuest 3 0 User Manual Hematoxylin Mean Intensity Ki 67 DAB Mean Int Pasttive Ri 57 DAB Ki 67 DAB Mean Intensity 0 mo 20 2 B Hematoxylin Mean Intensity Overall Statistics LR LR 25 163 4 786 1457 76 85 4250 427 24 339 6 5809 18
125. dialog Page 136 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Import groups of ROIs from HistoQuest Project File Text File F Projects HistoQuest Project 1 higproj Select groups of ROIs you want to import Group 01 Select samples where you want to import the groups of ROIs Coloni Figure 192 Import Export Groups of ROIs dialog You can import groups of ROIs from e A HistoQuest Project File import existing groups from another project e A Text File import groups from a previously saved text file Choose one of these two options then press Browse button in order to load chosen item K J gt Computer gt Data2 F Projects gt HistoQuest Colon1 Search Colon 1 Organize v New folder A Name Date modified Type _ Colon hiqproj 1 7 26 2010 1 16 PM HIQPROJ File Jr Favorites E E Desktop J Downloads i Recent Places T Libraries Ea Documents a Music Pictures E Videos 1 Comniuter File name vy HistoQuest Project hiqproj v Figure 193 Import Groups of ROIs from existing HistoQuest projects Page 137 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Select Groups of ROIs you want to import in this section mark the checkboxes corresponding to the groups of ROls you want to import Select samples where you want to import the groups of ROls in this section mark the checkboxes correspondin
126. dicate the desired filename and location The user can delete one or more events by selecting them and pressing the Delete Events button Gots free The CSV format is designed to be used in statistics For the Export to XLS option the user can export maximum 65 536 rows this is a current limitation of MS Excel 2003 To export a larger number of rows it is recommended to use the Export to CSV option 13 2 2 View Event Data HistoQuest gives the possibility to see data behind any event from an analyzed sample In the detail window choose desired event right click on it and from the context menu that appears choose View Event Data option Page 262 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Create Group of ROI Add to existing group Show Interior ROI 02 Remove ROIL OZ Copy to Clipboard Save Copy Figure 400 View Event Data option Event Data panel will appear it includes the following information e The Id of the FOV the event belongs to e The event label each event has its unique identification number e The parameters values for each marker Event label Hematoxylin Area um 40 125 Hematoxylin Mean Intensity 63 15732 Hematoxylin Minimum of Intensity 28 Hematoxylin Maximum of Intensity 105 Hematoxylin Range of Intensity 77 Hematoxylin Sum Intensity 40547 Hematoxylin Equivalent Diameter um 7 14
127. dius 1 i Exterior Radius Use Nuclei Mask Automatic Background Th Outside i Threshold Range Outside amp Inside Figure 350 Use Identified Cell Mask No e By choosing Outside growing is performed on the identified cell mask but it will not grow inside the nuclei mask even if there is staining in the nucleus arious Shapes 2 0 Use Ring Mask Ves z Interior Radius 1 i Exterior Radius 2 Use Nuclei Mask Automatic Background Th i Threshold Range Outside amp Inside Figure 351 Use Identified Cell Mask Outside e By choosing Outside amp Inside growing is performed on the identified cell mask and might grow inside the nuclei mask if these two masks overlap Yarious Shapes 2 0 Use Ring Mask bo Interior Radius Exterior Radius T Outside amp Inside Max Growing Steps i Skip Steps Use Nuclei Mask Automatic Background Th i Threshold Range 5 255 Figure 352 Use Identified Cell Mask Outside amp lnside e By selecting Outside option or Outside amp Inside option the growing options will appear Maximum Growing Steps The user may limit this growth process on demand by setting this parameter Page 226 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual arious Shapes 7 0 Use Ring Mask Ves a Interior Radius 6 67 um Exterior Radius 13 33 um Use Identified Cell Mask Outside amp Inside H Max Growing Steps t Skip Steps Ma
128. e you can also erase the existing comments all the changes concerning the notes are automatically saved e Delete the annotation by pressing button e Close the edit box by pressing button Manage annotations options e Remove removes the selected annotation Figure 207 Remove annotation message e Remove all removes all existing annotations Page 145 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 208 Remove all annotations message You can delete an annotation by right clicking on it on the sample and choosing from f lt 7828 the contextual men Remove Annotation name option Edit Annotations contextual menu By right clicking on the name of an annotation listed in the Edit Annotation dialog box a contextual menu will appear Rename Annotation Remove Annotation Locate Best Fit Figure 209 Edit Annotation contextual menu e Choose Rename Annotation option to rename the annotation a dialog like the one below will appear where you can write the new name and press Ok to save it ok Figure 210 Edit Annotation dialog Page 146 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual By choosing Remove Annotation option a dialog will appear asking you to confirm the deletion of the selected annotation Figure 211 Delete annotation dialog e Locate by pressing this button the selected annotation will be located on
129. e Chapter 4 6 If there is a predefined template with the same values as the one read from the NDPI file its name will appear in the Predefine combo box Magnification is automatically read from the NDPI file To finish this step press Next 4 3 3 Step 3 Choose Markers Page 58 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Choose Markers Select markers from the existing ones or create new ones to use in this HistoQuest project HM Choose Hamamatsu NanoZoomer file What markers do you want to use in your project E HistoQuest Project E Choose Markers What analysis options do you want to use in your project Analysis Options Segmentation Method Strategy for events in ROI Select only events entirely in ROT Color Separation Method Single Reference Shade Figure 63 Import from Hamamatsu NanoZoomer Step 3 This dialog is the same as for the previously described wizards same functionality The user will be able to choose e Segmentation Method e Strategy for events in ROI e Color Separation Method wf Note a The user should press Finish in order to create the project If the user loads a profile the segmentation method and the color separation method cannot be changed 4dd Samples From A NDPI file can be added to a project by pressing the Add Samples from button from the main window toolbar then choose the Hamamatsu NanoZoomer file option from
130. e Compare Set OF PE aN Pir et ee er re et ee ae Figure 119 Registration toolbar Page 93 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Propagate ROIs e Propagate Regions of Interest copies all ROIs from a sample to one or more different samples By pressing the button a dialog will open where you can choose the ROI s to be propagated and the destination sample s 13187 CD45 Region 002 ROI 15 ROI 17 ROI 18 ROI 20 ROI 21 ROI 26 ROI 27 ROI 28 Figure 120 Propagate Regions of Interest dialog a E ae SOHC e Synchronize samples enables disables the synchronization mode When the button is pressed the synchronize mode is activated and all the items in the viewer share a common scrollbar Page 94 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual iia 13187 CD45 Region 002 Annotaton 08 e a oo Figure 121 Synchronized mode common toolbar Page 95 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 13187 CD3 Region 001 13187 CD45 Region 002 Figure 122 Desynchronized mode individual scrollbars oe e Adjust registration points Ban similar tissue synchronization is realized based on a set of corresponding points from the tissues The option allows effectively editing these points After pressing the button Apply and Cancel options will appear in the toolbar Also three defa
131. e Rename option Page 103 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual lyze lalyze Unprocessed FOVs nalyze All ROIs Remove r Remove All ROIs New name Remove All Groups Remove All Exclusions Paste uild Cache lete Cache elete Results Figure 132 Rename Sample from context menu with dialog e In order to erase a certain sample use the Remove command The sample will be just removed from the project but the files on hard drive will remain Do you really want to remove sample Colon1_1 All data will be lost Figure 133 Remove Sample message e Remove All ROIs for more details please see Chapter 6 1 2 Figure 134 Remove All ROIs message e Remove All Groups will remove all groups from the sample e Remove All Exclusions will remove all exclusion areas from the sample Page 104 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Remove All Annotations will remove all annotations from the sample e The Paste option will introduce on the sample a previously copied ROI shape If the copied ROI overpasses the limits of the destination sample the paste action will not succeed e Build Cache the cache is needed to speed up zooming operations and is built automatically when a sample is opened for the first time in a HistoQuest project It also generates the overview map It is stored on the hard drive to allow fluent wo
132. e Sets manipulation toolbar has to be pressed After pressing this button the same dialogs like in the case of creation of Compare Sets will be displayed and the user will follow the same steps Compare Sets Deletion Deletion of Compare Sets can be done in two ways e Using Delete button Delete deletes a selected Compare Set Figure 238 Delete compare set warning message e Using Delete All button Pelete all deletes all Compare Sets Figure 239 Delete all compare sets warning message _ The creation modification and deletion of Compare sets can be done using the right r TIP mouse button menu on the Compare Sets tree Page 164 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Mer Edit Delete Figure 240 Manage Compare Sets menu a A E T The right mouse button menu on the Compare Set Side by Side tab _ provides the opportunity to change the way the diagrams are displayed un a iri Vertical layout will display the compare set items Samples ROIs Groups on columns Horizontal layout will display the compare set items Samples ROIs Groups on rows Page 165 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Compare Set 04 Hematoxylin Mean intensity Ki b DAB Mean Horizontal Layout Intensity Ki 67 DAB Mean Inte Diagram Options ROI 02 100 Coloni Ki 6 DAB Mean Intensity 0 40o g0 120 H
133. e first sample of the project Measurement Units Page 335 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual General Measurement Units Region area Event Parameters Milimeter mm C Millimeter mm_2 C Micrometer um Micrometer um m sT ee p Figure 513 Options Menu General Measurement Units dialog e Region area the user can choose to display tissue areas in square millimeters or square micrometers The default value is square millimeters e Event Parameters certain event parameters have measurement units i e micrometer or square micrometer The user can choose to display parameter values using both micrometers and millimeters The default value is micrometer The parameter values will be displayed according to the selected measurement unit in diagrams statistics reports raw data Diagram Common this section allows realizing the settings for the layout and the display of all diagrams The Show Grid option will display the grid on all diagrams Page 336 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Common O Fixed horizontal number of diagrams with the size a Fixed vertical number of diagrams with the size Scattergrams Flow Layout with the size il ae Display Small Range Limit Histograms C Show Grid Cutoff Color mz Cutoff Decimals Cutoff Quantile Value s General System Data Figure 514
134. e gate configuration dialog will appear Page 277 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 423 Gate Configuration dialog Name here you can choose the name for the gate lf you want to rename a gate and that name already exists you will receive the following message Figure 424 Rename gate the name already exists message If you want to create a gate without giving it a name you will receive the following message Figure 425 Missing name for the gate message Color here you can choose the color of the events within the gate Show Label by checking or unchecking this checkbox the name of the gate will be displayed or respectively hidden e Delete gate To delete an existing gate from a scattergram Press the right mouse button on the gate contour Choose the Delete option from the menu that appears Page 278 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Propagate Gate In order to propagate a gate press the right mouse button on the gate s contour and from the displayed menu choose the Propagate Gate option Gates can be propagated on the current sample on all samples or the user can choose desired sample Propagate gate to Current Sample and its Regions All Samples and their Regions Select Sample Figure 426 Gate Propagation dialog If a gate with the same name exists in other project items where the gat
135. e must be propagated the user has the option to overwrite existing gates or enter a new name for the gate that does not exist Some regions already contain a gate named sates o what do you wank to do O Rename Gate Figure 427 Gate Propagation name conflict options TE The propagation of gates is only allowed between scattergrams with similar axes Z Propagate gate to desired samples The user can choose to propagate the gate on the diagrams of a desired sample You can choose from the dropdown list where to propagate the gate On the current sample and its regions On all samples and their regions Page 279 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Select sample In this case a new dropdown list will be available including all available samples the user will select the desired one Propagate gate 1 and its Regions es ee Figure 428 Propagate gate to desired sample dropdown list e View Backward Data for Gate for more details please see Chapter 14 3 1 e Copy Gate Paste Gate In order to copy a gate choose the Copy Gate option from the gate contextual menu The gate will be pasted on a scattergram with similar axes when the Paste Gate option is accessed from the contextual menu of a scattergram 13 3 2 Histograms Histograms are another form of diagrams that help to visualize cytometric data obtained by analysis of tissue samples A histogram
136. e of about 200 Figure 307 Intensity control e Mixture Percent Depending on the markers and the staining protocol it may happen that one color is a combination between two shades To be able to set the mixture percent the user has to check the Is Mixture of checkbox If there are two markers in the project the second marker will be automatically selected If there are more than two markers the user will manually choose the second marker The user will be able to select the Mixture Percent whether by moving the trackbar or by typing a value in the edit box The Pure Color indicator will show the resulting color considered the pure one For example in case of the Ki6 7 nuclear marker the brown DAB staining will always be co localized with the blue counterstain of Hematoxylin making picking pure brown impossible In such cases the brown shade picked from the original image contains a mixture of pure Hematoxylin and pure brown of DAB The Page 203 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual parameter described in this section allows the user to specify the amount of blue present in the mixture picked from the original image A value of O means no blue is present in the mixture and it shows a pure brown color A value of 99 is the maximum allowed and means that the mixture contains 99 of blue and only 1 of brown Mixture Percent D H Figure 308 Mixture Percent control A marker that has already
137. e results of the analysis in an unpredictable way Page 13 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 3 Starting HistoQuest 3 1 Login Dialog After starting the application a login dialog will appear In order to login to HistoQuest you must enter a username into the User Name field and type the proper password into the Password field Press Login in order to be authentified If the username and password are correct the login dialog will close and you can start using the application Otherwise the application will prompt again for a valid username and password combination If you press Cancel the login dialog and the application will close The login dialog is used to restrict access to the application There is one Administrator and any number of users who may be managed when the Administrator is logged in for more details please see Chapter 17 3 4 TISSUE A9 GNOSTIC HEDI CG AL amp Bio Te amp amp H SOtrluUTIOWN S TISS UE ANALYSIS SOFTWARE CE A ATTENTION CONSULT OPERATING INSTRUCTIONS FOR USE Disclaimer TissueFAXSplus is 2a mcoscopehased cel anales system for cells in oyot parai A ections and THAS It consist of the softwere modules TissvelAxNS TesveQvest and WistoQuest and is used for acquisition of images in ihe Muerescence and brightield mode for counting fe number of positive and negative ale and for quanificaton of staining
138. e the sample s rote If you decide to leave images at their initial storage location you will save space ce The fully qualified hiqproj file name must be less than 260 characters and the project Page 63 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual folder name must be less than 248 characters Basic Settings Advanced Settings Do you know the size of a Field of View FOV Size ine ____ 0 3547442 758 FOV Width 04360361843 liam FOV Height Image Width 1024 px Image Height 1024 Pixel Width 0 3282189306 um Pixel Height 0 3268987049 pm Do you know the objective that was used to acquire these images Figure 70 Import Mirax Project Step 2 Advanced Settings FOV Size is automatically read from the mrxs file and put in Width and Height edits the FOV size can also be modified manually please see Chapter 4 6 If there is a predefined template with the same values as the one read from the mrxs file its name will appear in the Predefine combo box Magnification is automatically read from the mrxs file To finish this step press Next 4 4 3 Step 3 Choose Markers Page 64 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Choose Markers Select markers from the existing ones or create new ones to use in this HistoQuest project E Choose Mirax file T 7 a E HistoQuest Project What markers do you want to use in your project eate
139. ear Excluding a FOV from the sample will generate the following message Figure 102 Excluding FOV message Removing a FOV from the sample will generate the following message Figure 103 Removing FOV message Reintroducing a FOV into the sample after it was excluded will generate the following message Figure 104 Reintroducing excluded FOV message y Only the excluded FOVs can be reintroduced in the sample the removed ones being vf note completely deleted Page 85 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 5 7 Shape Manipulation toolbar HistoQuest helps the user better interact with the virtual sample by using a set of regions to be drawn e Regions of Interest ROI may be used in a HistoQuest project to either restrict analysis to certain relevant regions and or compare these regions with each other e The Annotations tool can be used in order to add relevant information directly to the digital sample The user may also add clinically relevant information for documentation purposes or Pa opinion experts consultations e The Exclusion Areas help you eliminate from the analysis the non relevant areas The Shape Manipulation Toolbar includes four buttons corresponding to four kinds of shapes rectangular elliptic custom and freedrawn Each button will display a menu that allows adding a ROI an Annotation or either Exclusion For more details please see Chapter 6
140. ed before you install the HistoQuest product Install them in any order but make sure HistoQuest is the last one to be installed HistoQuest software will not run and operate properly if any of the components below VF Note is not installed on your HistoQuest workstation Installing software programs and Windows system components will require Administrator privileges on your computer You might need to ask for support at your IT department if you do not owe the rights to install software or you don t have sufficient IT knowledge 2 2 Windows 3 1 Installer for Windows XP SP2 The Microsoft Windows Installer is an application installation and configuration service 2 3 NET Framework 2 0 Runtime The HistoQuest product is built over the version 2 0 of NET Framework which can be also found at the Microsoft official website The Microsoft NET Framework version 2 0 redistributable package installs the NET Framework runtime and associated files required to run applications developed to target the NET Framework v2 0 It is recommended to install the latest service pack available for the Microsoft NET Framework version 2 0 For further information about this package please visit the Microsoft homepage 2 4 Microsoft Visual C 2005 Redistributables The Microsoft Visual C 2005 Redistributable Package x86 installs runtime components of Visual C Libraries required to run applications developed with Visual C on a computer
141. eeeeeee cece ceseeeeseeeeseeesseeeseeeseueesaeeeseeesaeeeseeeseees 11 2 5 Intel Peformance Primitives 9 3 vecesscussendnneniawtendeonadueedsunsnsdaidusunewnenevedondudneeesedseadebunduasubeariedandsbuneieusdvachendenden 12 2 6 MARX Dongle 8 i 1 gt eee meee ne eee ee N aE EEN an eee ee a meee ene N 12 2f OyStem PROCS NOTING sisinio toned cee adaceeipsceta EAEE A ESEA EEEE 12 ZF Nee Hardware ReguiremenlS oe te toe acdc teietad is u ERE A EEEE 12 2 1 2 Software IRC QUINCIMICING viene caedsacscinices chose tnetedndddaas donids wigcedsaatedsadeaagcuedhededentnecedsideaneddedawnodsedhadedeadiesedaenscedsdehana 12 nea UNC TANS OS SU sts cars Sots gece iota ce ae ees asc aces sac ess ea eee et een opened eee mw tind EA 14 rts MBO GIM DIAO Jere aer EEE scueietecavencenss EE E ue aieh ease ace enectsuaetaceseemecteuse 14 OZ A INO OY a Gating nose cictan E anete pease eee E E 15 EONS sa es a es tics oe ae tama ce de esate aici nels epee E E E E 17 4 1 Import TissueFAXS HIStoFAXS Project cccccseccceececeseccseeceueeceeeseueeceeseaeeceueesaueeseessueecsueeseeeseeessseeseueess 17 4 1 1 Step 1 Choose the TissueFAXS HIStOFAXS Project cccccceccceceececceeeecaeeeeseeeeeseueeeseesesseeeesseeeseeeeees 18 WT POS es ee etn nected ames sce eed S A ae neu ted tad epedendecdeatereucap dein meneametegenas 31 4 1 2 Step 2 Hist oQuest PIO CCl srini snie i n ea EEEa aE EEE TEERADA NE AEEA 37 7 eve Pa 1 8 ero OS o E E E en es AET A eee ee ree
142. ematoxylin Mean Intensity eA ve ov Hematoxylin Mean Intensity Ki 67 DAB Mean Inte z ROT 04 Coloni wn E a i C EC E a 2 1 oO ill oO ma wo A oy 40 a0 120 Hematoxylin Mean Intensity a_l Figure 241 Compare Sets Side By Side display option 1 Page 166 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Compare Set 04 uw k i l a D i Coloni ROI 02 Coloni ROI 04 atensi Ei Pj TE Al in Hean Intensi c6 Hean LAL Diagram Options tensity Ki 67 DAB Mean Inte Hematoxylin Mean Intensity Ki 67 DAB Mean Inte 100 H Ta C LB 4 L E a EJ i oo Ci m wD a2 ne Ki 6 DAB Mean Intensity 4 BO 0 40 a 120 Hematoxylin Mean Intensity Hematoxylin Mean Intensity Figure 242 Compare Sets Side By Side display option 2 Hematoxylin Mean Intensity Ki 67 DAB Mean I The user can show hide row columns in the compare set side by side tab using and buttons Page 167 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Compare Set 04 Coloni ROI 02 Side by Side G5 Hog Hematoxylin Mean Intensity Ki 67 DAB Mean Inte Ki 6 DAB Mean Intensity 40 80 120 Hematoxylin Mean Intensity Ee Figure 243 Compare Set Show Hide buttons The Diagram Options menu is displayed if the opt
143. en A E E 92 o PrO 1 ON bee ren EEE E E ee e EE cee ee ee 101 Delea MOS Aa R gt a E ee ee ene eee eee ee ee 101 0 Js APIE eet on re oo ee ee ee 101 sample O70 00 00 gt 8 eee ene eee ere eee ee ee eee eee 105 MON LOAN OMG CU OI ota ssc ce ceases asec E A E E A ad oeasaueat ene aseesestoeeee 106 EAR ret geese eects E E E ssunacoseestecteeevosqneserorceeds 108 BOM NS cero ce tenn ss eae E E 120 AUTSI RIG oases teeta tenes pseeeeeaeeracae condos E EEE E A 121 6 eG OUP OTR oe E E EE E A AE eee ee eee ee 126 AO AO e a S E oe ee eee eee ee es eee 138 6 2 1 Annotation Manipulation Toolbar cccccccccecccseceeeeeeceeeeceeeeseeeeaeeeeae cess eeseeeesaeeesecesseeseneesaueesaeeeseeeeaes 138 02 2 Grealng FUMIO VOI So ssictsraese sic ans csetvdese scene cited EE iE OSE EE E EEE 139 6 2 3 Managing ANNOTATIONS irncnerscdonsest enina aa aai aE aaa o a aai i 141 OZA ELANNON S ponire s i i a ce ee eee ee ee E 143 OE K USTO I NOA a e eo eee E E A E O E E 148 6 4 Transiorm SINAC S soccer er tee serena tee deat atte goes aiciee decider ge runctia iE E ain a aiea R a eiaa enis Eoia 151 CONDA eee eee ee ee eee ee eee ee ee eee 154 yd Wes Create Compa S615 arisane iiaa ee eS nee eee ee eee eee 154 2 Side by Side Diagrams site cernes ase javemedeaanencedaanedadeasuosidenacGnetedakedenddanasete advance de aai aa iaaii aaea 155 D9 OVA TAN AUS sessin a en a A sete od oncte deacon ede adeu bead sacesutieavesete an e iaeei 159 7 4 Managing Com
144. er Show grid lines for Samples imported from Folder _ Show grid lines for Samples imported from TissueFAXS HistoFAXS or Hamamatsu NanoZoomer L Show name only for selected ROI eee _ Show name for Exclusion Area _ Indude user name in annotations contents E Show annotations in browser Measurement Units System Data Figure 161 Set ROI Color dialog Show ROI in detail window Show in Detail ROI name option is available in the contextual menu of the ROIs on the sample or from the Project Browser by accessing the contextual menu that appears when right clicking on an item s name and choosing Show in detail window option This option will open the selected ROI in the detail window Page 119 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Y Roos a 0 0004 mm 2 am Remove ROI 0S Copy to Clipboard Figure 162 Show ROI in detail window option Option on Open ROI Also open its sample in the left If choosing Show corresponding sample for regions opened in detail from Tools Q TIP Options General Automatization the corresponding sample will open in the left ROI Comments You can add your own comments for ROIs by using the Comments tool from the Project Items Properties control TissueFAXS comments are also available rote The comments cannot be edited while the analysis is running Page 120 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Man
145. er in other HistoQuest projects As mentioned above all information available for a marker is saved in a profile The following items will be saved in a profile The markers The parameters of color separation method The segmentation method and the parameters of that method The strategy for events in ROI By choosing a certain profile the user will implicitly set the markers the color separation method and the corresponding parameters the segmentation method and the strategy for events in ROI for the project as well Profiles To access the profile menu the user must press the Profiles button nit Save Marker Profile Load Profile From Existing Project Load Marker Profile Figure 270 Profiles menu Page 184 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 9 2 1 Save Marker Profile In order to save a profile the project must contain at least one marker empty profiles cannot be saved To open the Save Profile dialog press the Save Marker Profile button The user will have to set a name for the profile mandatory and a description optional Identification Name Hematoxylin Ki 6 DAB Description Figure 271 Save Profiles dialog To overwrite an existing profile you must firstly choose it from the drop down list A message box will appear notifying the user that the new profile will overwrite the existing one wf Note To
146. er manipulation menu removes a selected marker Internal Page 181 of 351 www tissuegnostics com HistoQuest 3 0 User Manual Figure 265 Remove marker warning message e Remove All Markers option from the marker manipulation menu removes all markers Figure 266 Remove all markers warning message Removing markers will invalidate the results Before the removal you will receive a warning message l CAUTION 9 1 7 Manage Markers Accessing the Manage Markers from the marker manipulation menu will open the Manage Markers dialog where the following operations are available Add Rename Remove Remove all Page 182 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual System Data Markers Templates Figure 267 Manage Markers dialog To visualize the Description field for a marker click on the corresponding cell in the dialog Hematoxylin stain is one of the most commonly used stains in histology Cancel Figure 268 Marker description field A marker belonging to a profile cannot be removed An error message will be displayed Page 183 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 269 Remove marker error message The same option is available by accessing Tools Options System Data Q TP Markers 9 2 Profiles Any set of markers used by a HistoQuest project can be saved to be used lat
147. er to continue the process the user has to press am the Resume button h Page 244 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual io Stop io Resume Clear ETR 05 46 20 Figure 377 Task Manager buttons The Clear button S removes from the task manager list all finished tasks Task Manager B 4 i Analyze ROI Slide OF ROI 06 PT Finished 9 10 2009 2 54 39 PM ETR 00 00 40 Analyze ROI Slide O7 ROI O05 Doo Finished 9 10 2009 2 53 55 PM Analyze ROI Slide O7 ROI 04 Doo fk Finished 9 10 2009 2 54 16 PM Analyze ROI Slide O7 ROI 03 PT Finished 9 10 2009 2 53 22 PM Analyze ROI Slide OF7 ROI 02 PT Finished 9 10 2009 2 52 35 PM Figure 378 Clear button before applying a Task Manager ETR 00 00 00 Figure 379 Clear button after applying b Page 245 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 13 Analysis Results 13 1 Image Results After analyzing the samples and or regions the user will obtain for each marker a set of image results Shades Measurement only for Multiple Reference Shades Color Separation Method Labeled Shades Overlay Measurement Overlay only for Multiple Reference Shades Color Separation Method Color Overlay The images for markers will be displayed if the user presses the marker s name and the name of the processed image The S
148. es 1 0 ja Interior Radius Exterior Radius Use Identified Cell Mask Max Growing Steps 26 67 um 3 Skip Steps 0 um Use Nuclei Mask Mo Automatic Background Th Yes Page 213 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 325 Use Ring Mask Parameter e Use Ring Mask Parameters The Interior radius and Exterior radius parameters allow the user to define in which direction outwards inwards or both and to what extent this ring should be created The value represents the number of pixel layers As this ring construction takes care of neighboring objects and statistical growth process it stops when the growing environment of a neighboring cell is reached e g when cells touch each other The value for exterior values represents the maximum growth The interior radius ring growth process stops after the specified distance is reached or if the entire nucleus is covered Yarious Shapes 1 0 Use Ring Mask Interior Radius Exterior Radius Interior Radius Use Identified Cell Mask bo ar Growing Steps QIRAAACOCORCHACOCORCNACAOCCORCRIACCCRARANCCCRARAACCCOCRAACOCOCRARIOCOORANENACOCCAORACHOCCORCNUCOCORCNUCOCORCNACAOCCORACAOCCORCCACCOCRCRENRCOCRCRCACOCOCRCRIOCCCRCRTOANOCRININ Skip Steps Use Nuclei Mask Aa ee et Hm 666 G M Automatic Background Th Default Apply Figure 326 Use Ring Mask Parameter Interior Radius Yarious Shapes 1 0 Use Ring Mask Yes 6 67 um 1
149. ess it measures how much the segmented object shape resembles a disk The more likely it resembles a disk the expressed compactness is closer to 1 If not its value will be closer to 0 Eccentricity it measures how much the object shape resembles a circle or a line segment When it is closer to 0 the object resembles a circle when it is closer to 1 the object is more elongated Minimum Width minimum value a micrometer would express taken into account all possible measurements positions orientations Maximum Length maximum value a micrometer would express taken into account all possible measurements positions orientations Feret Ratio the ratio between minimum width and maximum width For identifying an event the following columns appear in the Raw data grid e Region of Interest e Field of View e Event Label There are several options to display Raw Data for a region oy e Use the View Raw Data button from the toolbar of the results tab on the detail window Page 252 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 40 20 Hematoxilyn Mean Intensity Hematoxilyn Mean Intensity 40 a Hematoxilyn Mean Intensity Figure 385 View Raw Data button Page 253 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Ei Di 000001 44 693430 q o00000 ooo0o1 1O4 128600 0 000000 ooo001 31 075890 0 000000 000001 28 904520 0 000000 000001 35 569770 0
150. ess ors Svcd A weg ite EE E A E E E E 208 122 Dagan Conmigo ssa ne t ne ener me ne ener ee eee eee eee 217 1123 Raw Data Columns COMMOUNAUOM aricessncecececvesdus donnvereimeweacsdnnsdace cavenduecoteuededmedeoestanedavecavesduecanseecaearecess 217 A 232 AU SUG COMMON ANIONS aaescaeasncenaccuaracgsenn nance aE Eaa a EAE N so unsaunca dew EER 217 Woe WV AIOUS NO S126 a EE E E A A E se ase nes eam E 218 Ie Mee FAV AMI VSO oy n E agescauecaaasner ence taun season neceangatsaacaanee se ysacc eqns camueneeaasedangaisansess 219 Tio Dagan COMMOUNAWON senrega oacsges cate EE EEEE EEA E EEE EEEE awsean eae 229 11 93 33 Raw Data Columns COMmOUFAUOMN acsdesescececs ca neangan anaE aai a aT Aaa 229 113 4 Slatstics ConNguraliO aossen E E a E E EEEE 229 11 4 Total Area Measurements ccccccecccsecceececencecaeecceeceueecsueeseueeseeseueecaueesaueesaeessaeecsusesaueeseessaeessueesseessgas 229 TEEL PIT O o E ee E ET E ee 229 T42 Diagrams ConhgurallON siiict sete cscescecencsaecd E NEE E E E 230 Page 3 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 11 4 3 Raw Data Columns COntiguration cccccccccseccceececeeeeceeeaeeeeee cece eeseeeeseeeesaeesseesaueeseesseeesaneeseueesaees 230 TAA eo iol iol C OO 11 6 g aera Ene rae eee E enc ae eee ee ee eee 231 Tea oa y fe fe 8 oem F Koad t ieee nent en oe nee re eee ne ee ee eee 231 SZ SIGN AS PVA S16 ee sh pct E as caren bansonc E E E E E AE 233 TEAR a E ee e
151. ete Cache Delete Results Figure 361 Analyze Sample from context menu e By pressing the Analyze button nal ze from the sample viewer You will have to select the desired samples in the list Page 234 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Select Input Please select the project items you wish to analyze Colon F ROT 08 _ Analyze Only Unprocessed FOV Figure 362 Analysis menu from the Sample Viewer for samples e lf the sample is loaded in the main window or in the detail window by pressing the Analyze button ig from the toolbar fate For the Analyze Unprocessed FOVs option please see Chapter 6 1 Analyze for the sample will delete all previously obtained data for any region defined on it 12 1 2 ROI Analysis In order to analyze a ROI there are four possibilities e Analyze from the right mouse button menu on the ROIs name from Project Items Page 235 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Analyze Unprocessed FOVs Rename Remove Add to Existing Group Add to New Group Create Annotation from ROI Create Exclusion Area from ROI Copy Delete Results Propagate ROI Figure 363 Analyze for a ROI from context menu e Analyze button on the toolbar of the ROI detail window if the ROI was loaded e Analyze All ROIs option from the right mouse button menu on the sample s name from
152. ethod e Strategy for events in ROI e Color Separation Method vf Note When loading a profile the following buttons are disabled pelea Remove Marker However if the markers from the loaded profile are not suitable you can press button in order to remove them and then add a new set of markers manually Load Profile A profile is a set of markers defined at an earlier occasion A profile also contains information about the segmentation method the color separation method the description of the markers and the segmentation method parameters A profile created and saved in a HistoQuest project can be used for another project This operation can be realized when creating a project in the Choose markers step of the wizard A list of the available profiles is presented to the user The information available for each profile contains the segmentation method the markers and their type master or non master To load a profile simply select it from the list and then press the Ok button re Marker Segmentation Method Color Separation Method Ts Master Strategy For events n ROI Description DE D be Hematoxylin Ki 67 DAB Figure 38 Select Profile Dialog Page 42 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual This method of choosing markers for a HistoQuest project can be combined with the other two available methods If the project to be created already contains chosen markers
153. ew value in a specified range Master Marker Parameters The Default button Default of the configuration window allows the user to reset the value of the parameter to default To keep the last changes and close the window you can e click the Apply button Apel e press the Enter key after typing a value in the edit box To skip the changes and close the window click anywhere outside the window The Master Marker parameters are e Nuclei Size specifies the size of the nuclei This is an arbitrary value and it should be increased if your nuclei are oversegmented Page 209 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual arious Shapes 1 0 Discrimination Area Nuclei Size Discrimination Gray Automatic Background Threshold E a a a a a a a a a a a a a a virtual Channel Post Processing Order a Remove Labels DePaul Apply Use Merging Rules Figure 316 Nuclei Size Parameter e Discrimination Area by increasing this parameter smaller events are removed from the result of the segmentation Yarious Shapes 1 0 Nuclei Size Discrimination Area Discrimination Gras Automatic Background Threshold Virtual Channel Post Processing Order Remove Labels Use Merging Rules s eae _ Default Apply Figure 317 Discrimination Area Parameter e Discrimination Gray by increasing this parameter weaker intensity events are removed from
154. existing project If the user doesn t agree to overwrite the existing project he must select another name for the new project If the computed size of the pixel is not square the following message will be displayed CAUTION within the dialog Pixel is not square Vatning Pixel is not square The user can recheck the input data or continue with the given values 4 7 Finalize Analysis A finalized project is a project in which the user is unable to make any further modifications in order to influence the already obtained data By checking the Finalize Analysis option from the Project menu the user will obtain a finalized project Internal Page 72 of 351 www tissuegnostics com HistoQuest 3 0 User Manual Figure 82 Finalize Analysis Message box a The user can also continue the analysis of a finalized project by unchecking the Finalize Analysis option from the Project menu Figure 83 Finalize Analysis Message box b Finalizing a project will double the size of the higproj file on the hard disk so u consecutive finalizations of the same project can lead to a very large file 4 8 TMA If you are working with Tissue Microarrays TMA HistoQuest offers you special support for dealing with big sample numbers which TMA analysis is usually associated with The following aspects should be taken into account e The special HistoQuest features for TMA handling are only available for
155. ffect only the selected sample Project Illumination Correction this option will affect all samples in the project items list for which the option was selected The user will be displayed a dialog in which he can choose yes or no for the image illumination correction he can also verify the existence of correction image The Delete Correction Image button will allow the user to erase these correction images If for a sample illumination correction is computed and Delete Correction Image is used the status of the project items properties control will display the word No and any previously results will be deleted Illumination correction has effects on the analysis uneven illumination gives uneven intensity information Illumination correction can improve the results of the analysis Internal Page 106 of 351 www tissuegnostics com HistoQuest 3 0 User Manual Please choose the samples you wish to use Illumination Correction from below m Sample 01 Sample O2 Mo Samp io K EC AN EAA e E Delete Correction Image Figure 137 Illumination Correction Properties e Pressing the Select button will display two options All Samples selects all the samples from the list Clear Selection unchecks all previously selected samples All Samples Clear Selection Figure 138 Select button options use it In this case in the Illumination Correction Properties dialog the Has Correction Image section will di
156. fferent regions starting from three predefined values Area Events Count and Density Statistics _ ea Project Items SO sa pe e e eo Sample 01 Editcoumn p Sample 01 a m ROI Og eM Ror10 7 Groups eo Sample 02 g Sample 02 eM Rorot Sa Groups gu Sample 03 g Sample 03 LP faroups eu Sample 04 p Sample 04 a ll Groups eo Sample 05 HO Sampie 05 Es Drag a column header here to group by that column ao Sample 06 E Sample 06 i P Groups B 0 Sample 07 ga Sample OF LP Groups eo Sample 08 _ Sample 08 i if Groups eo Sample 09 Sample 09 Export to Excel to Excel Export TOPDF To POF Export to csv to CSW cose Figure 473 Statistics Report dialog The regions can be selected whether by checking them in the Project Items list or from the menu that appears by pressing the Select button Page 310 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual All ROIs Processed ROIs Unprocessed ROIs All Samples Processed Samples Unprocessed Samples All Samples and ROIs Clear Selection Figure 474 Statistics Report Dialog Select menu Predefined From Histogram C From Scattergram Column Mame Events Count Predefined Value Greg Density Figure 475 New Column dialog Predefined The Predefined selection will display two items Column Name and Predefined Value Page 311 of 351 In
157. for cell identification is the segmentation of nuclei i e object recognition and background rejection The application uses three methods of segmentation used separately Various Shapes 1 0 Various Shapes 2 0 and Total Area Measurements The user is able to choose which method to use The segmentation algorithms use the colors extracted from the images markers Each marker contains a set of parameters for the shades to be used in the color separation analysis and a set of parameters for the shape detection method All these parameters are configurable 11 1 Changing Segmentation Method The change of color separation methods can be done in two ways e By accessing Project Project Properties Page 205 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Description Location CiHistoQuest ProjectsColonl Browse Segmentation Method Various Shapes 2 0 Strategy For events in ROI arious Shapes 2 0 Various Shapes 1 0 Color Separation Method Total 4rea Measurements Foy Size Predefined Objective 20 CMount 0 5 Top Pork PixeLINkK 160 POY Width 0 676056338 mm FOW Height O 507042254 mm Image Width 1600 px Image Height 1200 px Pixel Width 0 42255521153 Lm Pixel Height 04225352117 Lm ok corel Figure 310 Changing Segmentation Method dialog a e By accessing Tools Options General Analysis Various Shapes and one for Total Area Measurements Inter
158. g to the samples where the groups of ROIs will be imported Now press Import button to effectively accomplish the import action Save Groups to Text File Press this button to save selected groups to text file format so you can import them later You must choose a location for the text file give it a name and press Save button Save Groups of ROIs to text Files F Computer Data2 F Projects HistoQuest Colon 1 Organize New folder Sr Favorites Bs Name Date modified Type BD Desktop 2 Demo 1 bt 3 3 2011 12 06 PM Text Document m Downloads SEN Recent Places oy Libraries E Documents al Music lea Pictures F Videos ae Filename Dero zi Save as type Text Files txt Hide Folders Figure 194 Save Groups of ROls to text files 6 2 Annotations An annotation is a shape you can create by drawing it on a sample which can contain a set of information written by yourself In order to add relevant information directly to the virtual sample the Annotations tool can be used The user may also add clinically relevant information for documentation purposes or 2nd opinion experts consultations 6 2 1 Annotation Manipulation Toolbar The annotations share the same controls with the regions of interest and with the exclusion areas The annotations can be easily created by choosing the desired option shape from the region viewer toolbar Page 138 of 351 Internal www tissuegnostics co
159. gative cell populations with respect to the markers stained can be determined The values of the event parameters can be used to build diagrams There are two types of diagrams e Scattergrams e Histograms 13 3 1 Scattergrams Definitions 1 A scattergram is an x y diagram showing two measurement parameters for all data units or a selection thereof on a linear or logarithmic scale Within the scattergram cutoffs can be set in order to define a threshold for each measurement parameter 2 A gate is a subset of events within a scattergram that is selected by defining a region of interest ROI Gates may have any polygonal shape and contain any number of cells Gates may overlap each other and be combined or defined as mutually excluding 3 A cutoff is a numerical parameter used as a threshold value in a diagram in order to determine positive and negative measure events 4 Quadrant statistics are scattergram related statistics that show values depending on the Segmentation Method used In a scattergram there are two parameters displayed against each other Each parameter may be associated with a cut off value Whenever these two cut off values one on the x axis and the second on the y axis are different from zero which is the normal case they define four quadrants within a scattergram These four quadrants are e upper left UL quadrant represents cells that are positive for the y axis parameter but nega tive for the x a
160. group is loaded in the detail window the status will be Not available for overlapping regions In this case the events count will always be 0 Annotation 06 0 001305 mm Demo comment Figure 115 Project Items Properties Control Annotation fn For the Line Annotation the ength will be displayed instead of area ote Collapse Expand all option Below Project Items panel there are two buttons that help managing the display of the experiment items e Collapse all ROIs Groups Exclusion Areas Annotations if pressed collapses all the items in the Project ltems panel e Expand all ROIs Groups Exclusion Areas Annotations if pressed expands all the items in the Project ltems panel If you want to collapse expand only certain type s of items press and select desired action in the dropdown list Collapse all ROIs Collapse all Groups Collapse all Exclusion Areas Collapse all Annotations Excduded FOVs Count 0 Total Scanned Area 0 120000 mm Total Exduded Area 0 004283 mm Page 91 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 116 Collapse all Expand all buttons 5 15 Images Compare Sets Images Compare Sets is a visualization mode that is used to see two or more similar tissues at the same time It has a synchronization feature so that operations like zoom and scroll are performed in parallel It allows visual side by side comparison of multiple
161. hades image obtained at the moment when the user presses the Shades button 78985 displays the image used for segmentation Page 246 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual fin Ki 67 DAB Shades Labeled Shades Overlay Color Overlay Original Hematax Figure 380 Shades Image By pressing the Labeled button 44 4 a pseudo coloring of the events will be displayed The colors are chosen randomly and do not indicate different staining intensities They simply help showing the segmentation results Page 247 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Original Hematoxsdlin Ki 67 DAB Shades Labeled Shades Overlay Color Overlay Figure 381 Labeled Image The Shades Overlay image displays the contour of the events on the Shades image The Shades Overlay button Shades Overlay will display this image Page 248 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual jin Ki 67 DAE Shades Labeled Shades Overla Original Hervakos Color Overlay Figure 382 Shades Overlay Image The contours of the events will be visible on the Color Overlay image displayed when the Color Overlay button Color Overlay is pressed Page 249 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Original Hematoxylin Ki 6 DAB Shades Labeled Shades Overlay Color Overlay 4 l a or t d ai apr Fig
162. he ROI will be taken into account for evaluation Figure 367 Select only events entirely in ROI e Select events entirely or partially in ROI an event placed on the edge of the ROI will be taken into account for evaluation even if it overpasses this edge Page 238 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 368 Select events entirely or partially in ROI e Crop events to ROIs shape for an event placed on the border of the ROI it will take into account only the part inside the ROI belonging to the group are not overlapping If regions are overlapping events roe When Crop events option is used the events will be computed if the regions computation is not yet available Page 239 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 2 Sa amp Figure 369 Crop events to ROI s shape Changing this strategy will only show effect on the data level after the algorithm has lt jf Note been run again This strategy is recommended for Total Area Measurement where total area within the selected region is required In a single cell analysis this strategy cuts the cells on the border and measures the cell fragments creating thus measurements which might not be relevant or desired If a ROI is modified and it also contains results the user will be asked whether he wants or not to reanalyze it again Page 240 of 351 Internal www tissuegnostics co
163. he analysis process the user will have to pick shades for each of them To pick shades for a marker the user has to work directly on the images using the Color Picker by pressing the Color Picker button Adding shades to marker Steps to perform in order to add a shade to a marker Page 191 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com e Choose the desired region from the Project Browser double click on it e Choose the area of the image from which you want to pick the actual The user is also allowed to zoom into a small region Separated e Next the number of colors in the image can be reduced by clicking on the Separated button the following menu will appear Options Figure 284 Separated Button and its menu e From the menu chose Separated e A square will appear on the image and you will move it to the desired region Figure 286 Initial Image Figure 287 Separated Image Use Toggle between Original image and Separated image control TIP 2d image to alternatively visualize the initial and the Page 192 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual segmented image e Now the user is ready to add colors to a marker The desired marker must be selected in the Project Browser e Click on the Color Picker button before starting to pick colors from the separated image e Pick the shades by directly choosing them from the image
164. he width of the desired ROI then press Add in order to include the new ROI in the list Page 114 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Name 0 3xX0 4 Height 0 4 mm Use default naming sve J cna Figure 153 Add Standard ROI dialog c 7 For the elliptic ROIs the values from the Width and Height fields refer to the Note VS diameters of the ellipse radius value x 2 The last used standard ROI is always remembered It is selected automatically when opening the Add Standard ROI dialog Note Choose Add to Sample to add Standard ROI 0 3 X 0 4 to Sample Slide 1 Region 001 Rectangular Elliptic Figure 154 Add Standard ROI dialog d Add to Sample button will include the ROI to the sample If the size of the ROI overpasses the sample the user will be informed by a note above the Add to Sample button lis All ROIs created using this method will be rectangular Edit button will modify the properties of a ROI from the list Delete button deletes a selected ROI from the list Page 115 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Delete All button deletes all ROIs from the list Options To see the Options contextual menu the user has to press the right button of the mouse on the ROI name Show in Detail Window Analyze Analyze Unprocessed FOVs Rename Remove Add to Existing Group Ad
165. heir folder Figure 53 Import Folder Wizard Step 2 Basic Settings To choose a new location for your project use the Browse button You can optionally add a description of the project If the new project folder is different from the import folder the user can create a folder with another name by checking the Create Project Folder option If the storage folder differs from the import folder you can decide if the images from your project will be used remaining at their initial location or if they should be copied in the storage folder This is realized by simply checking the Copy images to project folder option in this step of the wizard If you decide to leave images at their initial storage location you will save space However if you move delete the original image source files your HistoQuest project might not be usable anymore data will remain though but as images are no more available also the connection of data to original images will fail and you cannot reanalyze the sample s jf Note The fully qualified hiqproj file name must be less than 260 characters and the project folder name must be less than 248 characters Page 51 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual HistoQuest Project Please specify a path where the project will be stored and other project settings Choose Images E HistoQuest Project Choose Markers Predefined FOV Width 0 4 dam FOV Height o
166. hen the other objective values are used 4 1 3 Step 3 Choose Markers Page 40 of 351 Internal www tissuegnostics com Markers like Hematoxylin stain antibody stain are used for the identification of defined cell types tissue types cellular or tissue compartments cellular functions disease status etc In HistoQuest markers are represented by a name and a set of parameters depending on the Color Separation method and the Segmentation Method In this step the user can choose the markers to be used in the current HistoQuest project To analyze the images provided the HistoQuest product needs to have markers parameters set A set of completely defined markers can be saved for further use obtaining what we call a profile Choose Markers Select markers from the existing ones or create new ones to use in this HistoQuest project E Choose TissueFAXS HistoFAXS Project What markers do you want to use in your project E HistoQuest Project E Choose Markers What analysis options do you want to use in your project Analysis Options Segmentation Method Strategy for events in ROI Select only events entirely in ROI Color Separation Method Single Reference Shade Figure 37 Import TissueFAXS HistoFAXS project wizard Step 4 When choosing the markers for the current project the user has more options Existing markers can also be added one by one by clicking the Add Marker button Thi
167. hen using Single Reference Shades color separation method the Copy Marker option will create a virtual copy This virtual marker will inherit the color separation parameters shades and parameters from the original marker The advantage of virtual markers is that you can use the same color but measured with different parameters color separation segmentation If the marker that the virtual copy was created from changes color separation parameters the virtual copy will automatically update too For the virtual copy of a marker color separation will not apply but all the information will be inherited from the original marker Copying a virtual marker will actually copy the original marker Page 176 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual bar tual Copy OF KROZ DAS Hematoxylin K 67 DAB Single Reference Shade View Gay image _vew colr image r IN Coster pacts C is Mixture of Figure 254 Example of virtual copy of a marker The name of the copied marker will have the following form Virtual Copy_Of_Source Marker ro Copying a marker will delete the existing results A warning message will be displayed For Measure in different compartments a license is required UJ Requirements 9 1 3 Rename Marker The user will be allowed to rename a marker by accessing the Rename Marker option from the marker manipulation menu The name of the marker will be changed for
168. hese parameters have default values and controls that describe the effect on the result level Although a high level of image analysis expertise and many different sophisticated mathematical operations are running beneath the graphical user interface of HistoQuest the user does not have to deal with these image analysis functionalities and mathematical operations as such Markers can be saved in sets called profiles which can be then reused in other projects Working with markers involves the following steps e When creating a new HistoQuest project choose markers to be used from the existing ones or add new ones e Since only the markers that were added from a profile can contain a list of shades and a marker that does not have shades is not usable the next step after adding new markers is to choose at least one shade for each marker e Finally the set of markers used in the current project can be saved creating a new profile to be used later 9 1 Markers Markers To access the markers menu the user must press the Markers button Page 172 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Markers Autodetect Color Picker Single Reference Shade View Gray Image View Color Image color IN color Palette Intensity 200 ts Mixture of Various Shapes 2 0 Nudei Size 4 Discrimination Area 1 Discrimination Gray 1 Automatic Background Threshold Yes i Threshold Range 20 255 V
169. hoosing the Side by Side Diagrams option the user will be able to select the diagrams to be introduced in the new Compare Set Page 155 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Compare Set Name Compare Set 01 m Hematoxylin Mean Intensity Ki 67 Mean Intensity Scattergram Hematoxylin Mean Intensity Histogram Ki 67 Mean Intensity Histogram Figure 226 Side by Side Diagrams dialog Add Histogram Add Histogram this button opens the Histogram Properties dialog where you can select the parameter to be compared in histogram form Page 156 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Marker Parameter Hematoxylin Mean Intensity Mame Hematoxylin Mean Intensity Input Gates a Figure 227 Histogram Properties dialog The user will be able to select the desired markers and parameters The Input Gates will be modified only on already created diagrams The name of a diagram can be modified starting from the creation of the diagram Add Scattergram this button opens the Scattergram Properties dialog where you can select the parameters to be compared in scattergram form Page 157 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Hematoxylin ki6 DAB sa Parameter Parameter Mean Intensity se Mame Hematoxylin Mean Intensity Ki67 DAB Mean Intensity Input Gates per M
170. ield of View may contain several markers 1 4 Conventions In this section you can find listed and explained the conventions used throughout this manual Note s describes important features or instructions WY TIP Y Tip s highlights features or hints that can help you save time or avoid difficulties Page 9 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual CAUTION Caution s warns readers about possible damage to equipment or to data or about potential problems in the outcome of what they are doing ij Requirements Requirement s informs the user about the demands that must be met in order to have a fully functional system Italics are used in order to highlight a title and also new or unfamiliar terms usually at their first appearance in the text Bold are used in order to highlight important terms or to mark the names of the features GUI elements throughout the text of this manual the arrow indicates a menu choice For example choose File Save as template means you have to choose Save as template from the File menu Page 10 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 2 Installing HistoQuest 2 1 Application Dependencies There are some special requirements concerning the installation of HistoQuest You may check below for the components required to be installed on your computer It is recommended to have all these components install
171. ile teats A A Choose Markers Image Map Ce Figure 58 Import from Hamamatsu NanoZoomer Step 1 a Page 54 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Then choose Browse Browse h and select a NDPI file then press Open m amp Projects HistoQuest Organize by Favorites gt So Libraries gt i Computer D th Network NanoZoomer instrument Mew folder File name pl SeorhHistoouet o Name 1 2008 08 01_14 01 49 ndpi 2008 08 01 _16 11 25 ndpi _ 2008 08 06_13 19 50 ndpi Figure 59 Import from Hamamatsu NanoZoomer browser sro The user must choose a brightfield experiment acquired by a Hamamatsu Q Page 55 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual E Choose Hamamatsu NanaZoomerfile where is your Nano Zoomer file Choose Markers Image Size 1600 x 1200 Image Format jpg Z position Image Map Width 13 2mm Height 10 5mm Next Cancel Figure 60 Import from Hamamatsu NanoZoomer Step 1 b At this point the following data are available e Image Map the preview of the region as it is stored in NDPI file e Width Height the width and the height of the sample in millimeters with two decimals as displayed by Hamamatsu NanoZoomer e Image Size the desired FOV size in pixels the recommended value is 1600x1200 e Image Format the recommended format is JPG
172. import folder the user can simply type its path or press Browse button Browse for the desired folder The user should ensure that the selected folder exists and that he has enough rights frote on it otherwise he won t be able to use it in this case an error message should appear If a problem occurs while reading the folder HistoQuest will display a message box like the one below Figure 50 Message Box Jote The last imported folder should be remembered if any Page 48 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e From the listed images check the ones you wish to import e On the right side of the dialog you have a preview of the currently selected image e Add Subfolder Images if you mark this checkbox HistoQuest will import images from multiple folders From the listed subfolder images check the ones you wish to import For each subfolder containing images and belonging to a folder a sample will be created This option is available only for projects from folders e Exclude HistoQuest generated folder Exclude _hiqtmp folder mark this checkbox in order to exclude HistoQuest generated folder s from your new project Select markers from the existing ones or create new ones to use in this HistoQuest project E Choose Images Basic Settings Advanced Settings HistoQuest Project Choose Markers Where are your images Folder containing your images F
173. ined number of uses the counter will be incremented each time an exclusion area is created Tissue Detection The user won t be able to use tissue detection To use tissue detection a license is needed to define ROIs To create a group of ROIs from detected ROls a license to use groups of ROIs is needed lf the user owns a predefined number of uses the counter will be incremented after each tissue detection The counter of ROIs and groups of ROIs will increment after each newly added ROI or group of ROIs Virtual Channel The user won t be able to use virtual channels A license to use Various Shapes is also required If the user owns a predefined number of uses the counter will be incremented only if the analysis for each FOV succeeds Scattergrams The user won t be able to use scattergrams Basic HistoQuest contain only histograms scattergrams require license Import From Multi Folders The user won t to import images from multi folders Also to import images from multi folders a license for Import Add Folder is required ForwardConnection The user won t be able to use Forward Connection If the user owns a predefined number of uses the counter will be incremented after each successful Forward Connection Group Of Rois The user won t be able to create a group of ROIs If the user owns a predefined number of uses the counter will be incremented each time a group of ROls is created Shape Measurement The user won
174. ion Then from the right mouse button menu choose Copy Cutoff 4utoscale on Hematoxylin Mean Intensity 4utoscale on Hematoxylin Area Set max value on Hematoxylin Mean Intensity Set max value on Hematoxylin Area Parameters Set Cutoff on Hematoxylin Mean Intensity Set Cutoff on Hematoxylin Area Paste Cutoff Delete Cutoff View Backward Data Copy To Clipboard Save a5 Paste Figure 410 Scattergrams Copy cutoff context menu e Paste cutoff In order to paste cutoff press the right mouse button on the diagram area and access the Paste Cutoff action The Paste Cutoff action is available only on diagrams with similar axes jf Note Page 270 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 4utoscale on Hematoxilyn Mean Intensity 4utoscale on Hermatoxilyn Area Set max value on Hematoxilyn Mean Intensity Set max value on Hematoxilyn Area Parameters Set Cutoff on Hematoxilyn Mean Intensity Set Cutoff on Hematoxilyn Area Copy Cutoff Delete Cutoff View Backward Data Copy To Clipboard Save das Figure 411 Scattergrams Paste cutoff context menu Delete cutoff The cutoff will be deleted by pressing the right mouse button on the diagra the displayed menu the Delete Cutoff option New defined regions should automatically take cutoff values from the parent sample 4utoscale on Hematoxylin Mean Intensity 4utosca
175. ion with the same name is accessed from the Display Compare Sets menu The same menu can be accessed from Tools Options Diagrams for more details please see Chapter 17 3 4 Vertical Layout Horizontal Layout Figure 244 Diagram Options menu Page 168 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Scatkergrams Histograms Figure 245 Diagram Options dialog 7 5 Compare Sets Print Out Reports The user can preview and print out a compare set report To print the report of a compare set simply select it in the Print dialog Page 169 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Please select the items from the project rou wish to include in the print Options Indude Scattergrams Indude Scattergrams Statistics Indude Histograms Indude Histograms Statistics Indude Overlay Diagrams _ Indude Processed Images Indude Observations Indude Notes page Indude Only Visible Diagrams print Cancel Figure 246 Compare Set print out report Ar z Only one compare set can be included in a report 0 If a compare set is added to a report no other item Sample ROI Group can be added For more details about print out reports see Chapter 16 2 Page 170 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 8 Shapes Measurements Below there is a list that describes the area that will be measured for e
176. ions Delete Event in order to delete events press the Delete Event button Then select a group of events creating a shape which surrounds them or delete separate events pressing the Ctrl key and selecting them In order to delete more groups of events keep the Ctrl key pressed while creating the shapes Then press the Apply Manual Correction button Figure 467 Delete Events e Create Event Q in order to create one or more events press the Create Event button define their shapes and press the Apply Manual Correction button Figure 468 Create Events i A newly created shape is not allowed to cover entirely an existing event the system Y i rejects such user input The desired event will not be created Page 307 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 469 Create Events Exception e Merge Events x the Merge Events operation can be applied only on connected events First press the Merge Events button then create the desired shape and press the right mouse button in order to finish it After that press the Apply Manual Correction button Figure 470 Merge Events e Split Events two or more events can be created from an existing event First press the Split Events button and then define a cut which splits the original event Then press the Apply Manual Correction button Figure 471 Split Events e Add disc with certain radius for master cha
177. ions The user won t be able to access auto cutoff suggestion If the user owns a predefined number of uses the counter will be incremented each time a cutoff is automatically suggested Page 345 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Images Compare Sets The user won t be able to create images compare sets If the user owns a predefined number of uses the counter will be incremented each time an images compare set is created Compare Sets The user won t be able to create compare sets If the user owns a predefined number of uses the counter will be incremented each time a compare set is created Various Shapes The user wont be able to use Various Shapes 1 0 and Various Shapes 2 0 If the user owns a predefined number of uses the counter will be incremented only if the analysis for each FOV succeeds Total Area Measurements The user wont be able to use Total Area Measurements If the user owns a predefined number of uses the counter will be incremented only if the analysis for each FOV succeeds Propagation The user won t be able to propagate cutoffs gates diagrams ROIs group of ROIs This license also includes deleting a diagram from all samples and their regions and gates To propagate a ROI a license for ROIs is required Also a license is required in order to use groups of ROIs If the user owns a predefined number of uses the counter will be incremented after each newly added RO
178. ir areas the user can select the desired number Default this button restores the last default detection parameters saved by the user It also has two options Save s Tissue Detection Default Parameters Restore Tissue Detection Default Parameters Figure 165 Autodetect ROIs Default Parameters Save as Tissue Detection Default Parameters allows you to store this set of parameters values for future detection Restore Tissue Detection Default Parameters restores all the default settings established by the manufacturer of the application Advanced Settings Thresholds These thresholds help to discriminate specific staining against background and artifacts thereby facilitating proper sample detection Automatic The RGB red green blue channels values are automatically determined Apply same value for all channels by checking this option all channels will have identical values you must set the value for the red parameter this value will appear automatically for the other two channels Red set the threshold for the amount of red in the picture Green set the threshold for the amount of green in the picture Blue set the threshold for the amount of blue in the picture Page 123 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Tissue Advanced Settings Thresholds Point Generation Automatic 4oply same value For all channels Figure 166 Advanced Settings Thresholds tab
179. irtual Channel nudear Post Processing Order Remove Merge Remove Labels No Use Merging Rules No Profiles Figure 247 Markers manipulation menu Page 173 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Add Marker Add Copy of Hematoxylin Rename Hematoxylin Copy Hematoxylin parameters Set Hematoxylin as master marker Remove Hematoxylin Remove All Markers Manage Markers Figure 248 Marker Manipulation menu 9 1 1 Add Marker The Add Marker option from the marker manipulation menu adds in the opened project a new marker that has never been used before The user can provide a name and a description for the newly created marker If the project does not contain a master marker the user can check the Is master check box If the project already contains a master marker the check box will appear disabled Description Figure 249 Add New Marker dialog e Adding a new marker will clear the existing results in the project A warning message will be displayed to the user Page 174 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 250 Add marker warning for a project with existing results Add Existing Marker An existing marker is a marker name which has already been created before and HistoQuest introduced it in the list of existing markers In order to add an existing marker the user has to choose the Add Marker option from the marker m
180. issueFAXS software in T ion as shown istoFAXS Reg issueFAXS H T 14 igure F Figure 15 HistoQuest Region of Interest Page 26 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Import TissueFAXS HistoFAXS logical groups When Import TissueF AXS HistoF AXS logical groups option is checked the HistoQuest project will import them The imported logical groups from the acquisition module will keep the original name and will be treated as groups of ROIs j This option is available only for TMA projects The Import TissueFAXS HistoFAXS vf Note logical groups option is available only if previously checking Import TissueFAXS HistoFAXS regions option Different TMA spots belonging to separate TMA blocks could be gathered in the same group Figure 16 Import TissueFAXS HistoFAXS logical groups When Import TissueFAXS HistoF AXS subregions option is checked the HistoQuest project will open them The imported subregions from the acquisition module will keep the original name and will be treated as ROIs Page 27 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual SS es Yj Fa 7 AV N vA hs Wy Figure 17 TissueFAXS HistoFAXS Categories Page 28 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Cate oo ea ae eat jory A i Hi i a i we Fe ci ay ie re ed m be T Ta rel Se Dal ig e i a4 allego AZ wer ea _ pam
181. issuegnostics com HistoQuest 3 0 User Manual Hematoxylin Mean Intensity Hematoxylin Area Areg as rag ba Hematoxylin Hematoxylin Mean Intensity Figure 420 Elliptic Gate 3 Add custom polygonal gate Click the Custom button v inside the scattergram toolbar The starting point first corner of the region of interest is defined by pressing the left mouse button inside the diagram area Thus the first point of the polygon is defined and a line to the current mouse position appears This line connects the starting point with the mouse cursor To define the next corner of the polygon the left mouse button must be pressed somewhere in the diagram area Repeat the previous step until all desired points are defined The final step of setting a gate is to close the polygon This means that the previously defined point is connected with the first point of the polygon To perform that action the right button of the mouse will be pressed inside the diagram area All events inside the gate change their color to a predefined hue Additionally the gate becomes labeled with a default text e g Gate 1 The color the label and the visibility of each gate can be modified later Hematoxylin Mean Intensity Hematoxylin Area 2100 4rea onli Hematox lin Hematoxylin Mean Intensity Page 276 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 421 Cu
182. isting Templates Objective 1x CMount 0 5x Top Port PixeLINk 1600x1200 06 June 2006 12 10 43 m Objective 10x CMount 0 5 Top Port PixeLINK 1600x1200 06 June 2008 12 10 43 m Objective 20x CMount 0 5x Top Port PixeLINk 1600x1200 06 June 2008 12 10 43 m Objective 40x Oil CMount 0 5 Top Port PixeLINk 1600x1200 06 June 2008 12 10 43 m Objective 63x Oil CMlount 0 5 Top Port PixeLINK 1600x1200 06 June 2008 12 10 43 m Hamamatsu NanoZoomer Objective 20x 1600x1200 11 August 2009 14 11 27 Template Info Mame lt lt New Template gt gt 06 June 2008 12 10 43 06 June 2006 12 10 43 06 June 2006 12 10 43 11 August 2009 14 11 27 06 June 2006 12 10 43 06 June 2006 12 10 43 Figure 81 FOV Size user predefined values Optionally a short description of the template can be given in the Description field Enter the values in the Width size of the x axis of your images and in the Height size of the y axis of your images field Press the Update button to permanently store the new settings in your HistoQuest configuration and then press the Close button to exit this dialog Please ensure that the selected folder exists Please ensure that you have enough rights on the selected folder Please ensure that there is enough free space on the drive where the folder is located If a project with the same name already exists at this location the user is asked if he wishes to overwrite the
183. isting marker Please ensure that the name provided for the marker is valid it does not contain any unaccepted characters lt gt To add existing markers press Add Marker button then choose desired marker from the list available in the dropdown box and press Enter key Page 44 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com Name Figure 42 Select Existing Markers Decide which marker should be used for cell identification Master Marker The wizard asks which marker should be selected as master marker nuclear marker It is always the master marker that has to be used for primary cell identification in all current algorithms The appropriate box must be checked Figure 43 Select Master Marker dialog If there is no selected master marker the user will receive a message as in the dialog below Figure 44 Select Master Marker Message box Page 45 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual You may leave this field Currently chosen markers empty and select the markers vf Note later in the Markers manipulation menu of the detail window While the project is loaded a progress bar will appear Add existing or new marker means that the name of a marker is used only To obtain a marker that includes parameters choose Load Profile After choosing the markers the wizard is ready to create the HistoQuest project Cre
184. k Hemat CD3F Count Percent No mm2 oa fess am efo isao k sesi nase teo 3 40 8750000 u ese me rejis toes e fse e snr oveos ovea onae 27 879 aas 100 00 107675 eee i0717 oss slios soe ETATS ja forser fiero olua i5000 ue fason iras 16 25 72 260 000 e fees fons aloe lion ik flow sas zs 27rleoa7e 6525000 ovaa 107176 0355 3 i00 00 i085 Figure 441 Example of Diagram Detail Window Statistics tab An Each Segmentation Method has its own Statistics configuration For more details Vf Nol please see Chapters 11 2 4 11 3 4 11 4 4 e Copy Copy Diagram to Clipboard copies the image of the diagram to clipboard Copy All to Clipboard copies the entire Diagram Detail Window to clipboard Saver fooml foome Zoom 3 Copy Diagram to Clipboard Copy All to Clipboard Figure 442 Diagram Detail Window Copy menu e Save Save Diagram As you can save the diagram under different image extensions and to a desired location Save All As you can save the entire Diagram Detail Window under different image extensions and to a desired location zoom 1 Zoom 2 Zoom 3 Save Diagram As Save All As Page 288 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 443 Diagram Detail Window Save menu zoom i Zoom Zoom 3 f e Zoom _ ____ offers three predefined window sizes for the Diagram Detail Wi
185. l display its properties Also if selecting an item in the Project Items it will appear highlighted on the sample Page 125 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Fished 2 9 2011 3 09 39 PH Figure 170 Displaying properties for selected ROI The same option is available for TMA spots Select a spot directly on the sample to Q TIP see its properties 6 1 3 Group of ROIs If several ROIs shall be analyzed together then groups of ROIs can be created A group of ROIs will create one group result where the data cells and measurement values are all put together Several groups of ROIs may be created and used for comparison e g tumor area versus normal adjacent tissue A Group of ROIs is an assembly of at least one ROI created by the user according to his needs The ROIs maintain their individual shapes but they are displayed within the same detailed widow sharing the same specific color The data obtained through the analysis for individual ROIs are merged in diagrams and marker images Create Group of ROIs A group of ROIs can be created in four ways e By pressing Create Group of ROIs button from the ROI manipulation toolbar after having previously selected at least one ROI by pressing the left mouse button inside the ROI bounds For a multiple selection the CTRL key should be used for every selection Page 126 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manu
186. le on Hematoxylin Area Set max value on Hematoxylin Mean Intensity Set max value on Hematoxylin rea Parameters Set Cutoff on Hematoxylin Mean Intensity Set Cutoff on Hematoxylin Area Copy Cutoff Paste Cutoff View Backward Data Copy To Clipboard Save a5 Paste Figure 412 Scattergrams Delete Cutoff context menu Inherit cutoff values Internal m area and choosing from Page 271 of 351 www tissuegnostics com HistoQuest 3 0 User Manual If on the sample there is a cutoff all the regions groups to be created will inherit the sample diagrams cutoff e Autosuggest cutoff In HistoQuest there are two methods that can be used in order to suggest a cutoff value Otsu Method based on images processing sets the cutoff on one or both axes based on automatic detection OTSU Cutoff OTSU Hematoxylin Mean Intensity Cutoff OTSU Hematoxylin Area um Cutoff Figure 413 Otsu method options Quantile Method splits the events population according to a percent given by the user Write the desired percent in the edit box Cy Then click on the desired option from the dropdown list for one or both axes 40 Quantile Cutoff 40 Quantile Hematoxylin Mean Intensity Cutoff 40 Quantile Hematoxylin Area um Cutoff Figure 414 Quantile method options Page 272 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual OTSU Hematoxylin Mean
187. lick the Rectangle button E inside the scattergram toolbar Page 274 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual The starting point of the region of interest is defined by pressing the left mouse button inside the diagram area The left mouse button must be pressed to resize the rectangular region in order to obtain the desired size The left mouse button must be released to finally determine the gate All events inside the gate change their color to a predefined hue Additionally the gate becomes labeled with a default text e g Gate 1 The color the label and the visibility of each gate can be modified later Hematoxylin Mean Intensity f Hematoxylin Area 4rea omnis r34 Ed Hematoxylin Hematoxylin Mean Intensity Figure 419 Rectangular Gate 2 Add elliptic gate Click the Ellipse button O inside the scattergram toolbar The starting point of the region of interest is defined by pressing the left mouse button inside the diagram area The left mouse button must be pressed to resize the elliptical region in order to obtain the desired size The left mouse button must be released to finally determine the gate All events inside the gate change their color to a predefined hue Additionally the gate becomes labeled with a default text e g Gate 1 The color the label and the visibility of each gate can be modified later Page 275 of 351 Internal www t
188. listoQuest and is used for aoquisiton of images in ihe Tuorescence and brughtied mode for counting the number of postive and negative cells and for quantiicaton of saining intensities TissuefAxsous amp used for the standardization of tissue analysis in combination wth immunensiaqenical and inmunefiverescence staining HistoQuest Version 3 0 Copyright 2006 2011 TissueGnostics GmbH All rights reserved WWW Dssuegnostcs com Figure 529 About HistoQuest splash screen Page 344 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 18 Modularization HistoQuest application requires license for some of its features that you can find listed below rote Some licenses have a limited number of uses that will be incremented as described below Import Add Hamamatsu NanoZoomer The user won t be able to create a new project from Hamamatsu NanoZoomer file Also he won t be able to add sample from Hamamatsu NanoZoomer file Import Add Mirax The user won t be able to create a new project from Mirax file he won t be able to add sample from Mirax file Import Add Folder The user won t be able to create a new project from an existing folder Also he won t be able to add sample s from an existing folder Import Add from TFAXS HistoFAXS The user won t be able to create a new project from a TissueFAXS HistoFAXS project Also he won t be able to add sample s from a TissueFAXS HistoFAXS
189. lly open the application and the corresponding project when double clicking on the hiqproj file r ONLY data associated with HistoQuest will be transferred to the TissueGnostics support department Image Viewer Page 333 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual General Image Viewer Default Colors Default Locations Region of Interest eee top Lett Aani Scale Bar Bottom Right Group of ROIs Item Name Top Left Exclusion Area Exdusion Area Transparency RGB Indicator A a P Scale Bar 0 100 Overlay Non selected Contour Color Non selected Contour Thickness Selected Contour Color Selected Contour Thickness Show non selected events in backward connection mode _ Animate borders of ROIs and Annotations Highlight double cicked point from Sample Show grid lines for Samples imported from Folder _ Show grid lines for Samples imported from TissueFAXS HistoFAXS or Hamamatsu NanoZoomer _ Show name only for selected ROI _ Show name for Exclusion Area _ Indude user name in annotations contents Show annotations in browser DoS see a ae ee A nna Figure 511 Options Menu General Image Viewer dialog Default Colors it allows you to set the colors for ROI Annotations Group of ROIs Exclusion Areas RGB Indicator and scale bar Default Locations it allows you to set the location for the RGB Indicator the Scale Bar and the Item Name Exclusion Area Transpare
190. ly for two markers Figure 303 Singe Reference Shade Autodetect choosing colors Page 200 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual The assignment of colors to the defined markers can be blue to Hematoxylin and brown to Ki6 DAB The order and color of displayed shades can be changed by checking the respective empty checkboxes Also the user will be able to visualize the color and gray images of the corresponding shades By pressing the Advanced button Advanced the following dialog will appear gt o Advanced Parameters Low Outlier Per million Range is 0 100000 High utlier 999990 Per million Range is 900000 1000000 k Range is 0 100 Figure 304 Advanced Parameters dialog The Default button is used to reset the values in the dialog to the default ones By modifying the settings from this dialog the shades of your color separation method can be adjusted and improved depending on your needs The Advanced Parameters are e Low Outlier by increasing this value non interest shades but close to value of the interest shade are not considered for the detection process If the results are not as expected it might be possible that the Low Outlier was increased too much e High Outlier by decreasing this value non interest shades but close to the other interest shade are not considered for the detection process If the results are not as expected
191. ly press the button During the definition of a new ROI an approximation of its area is displayed to the user i 0 0029 mm 2 Figure 144 Drawing new ROI area approximation Add standard ROI with predefined size e Standard ROIs option available next to other ROIs options Figure 145 Add Standard ROI button Page 110 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Once clicked the user has the rectangle of the ROI following the mouse so that he can center the ROI on the desired area Ba EER A tt ay i Le ae a F ROIO7 0 2 X0 2 W 0 0299 mm2 Ep aN a L t A pyi i ae j n P i 4 m i Ei s Y i i ii g i i z gt A rh i t T i s n a k E 7 i a j F i Figure 146 How to add Standard ROI The name of the standard ROI will include its size Add standard ROIs to all samples By choosing Propagate ROI option from the contextual menu of a standard ROI the standard ROI will keep its name on the samples where it will be propagated Only the samples suitable for propagation will appear in the dropdown list Propagate Region Of Interest ROI 02 0 2xX0 23 13187 CD45 Region 002 Figure 147 Propagate ROI dialog When propagating one or more ROIs or when performing copy paste action two _ situations may occur Zh Note if the ROI s is are pasted on the image viewer or on the detail window the propagated ROI s
192. m HistoQuest 3 0 User Manual a a 15 De 25 Se 10x 20 40x 63x 100x H 4 4 F 22 el O Figure 195 Annotations Manipulation Toolbar By using the buttons from the Shapes Manipulation Toolbar the following operations can be performed see also Chapter 5 7 T e Add rectangular annotation C e Add elliptical annotation Q e Add freedrawn annotation es e Add custom annotation Ea e Show hide annotations choose Show Hide Annotations option from the Show Shapes context menu see also Chapter 5 8 Show Hide ROIs Show Hide Exclusion Regions Figure 196 Show Hide Annotations option 1 e Edit annotations t 6 2 2 Creating Annotations In order to create an annotation the following options are available e Add rectangular annotation Page 139 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Annotation 01 0 0006 mm e Add elliptical annotation imi a O lt 0002 mime e Add freedrawn annotation L at Annotation 03 0 0004 rare e Add custom annotation Internal Page 140 of 351 www tissuegnostics com HistoQuest 3 0 User Manual Annotation O04 4 4 0 0003 rime 7 Annotation 05 0 0479 mm Figure 201 Line annotation The line annotation does not show the area but the distance between two points on rore the sample The area of an annotation depends on the FOV size 6 2 3 Managing
193. m HistoQuest 3 0 User Manual ROI modified Do you wank to reanalyze the ROT IF not the result will be cleared p Figure 370 Reanalyzing modified ROI message As previously explained the toolbar Analyze button 4 4lyz will display a more comprehensive menu where the user will be able to set different regions for which the analysis will be done 12 1 3 Toolbar Analyze Button Select In put Please select the project items you wish to analyze All ROI Processed ROIs Unprocessed ROIs All Samples Processed Samples Unprocessed Samples All Sarnples and ROIs Clear Selection _ Analyze Only Unprocessed FOV Figure 371 Analyze Select Input dialog elect a The Options menu opened by pressing the arrow button i will display a set of selection options Page 241 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Select All ROIs Select Processed ROIs Select Unprocessed ROIs Select All Samples Select Unprocessed Samples Select All Clear Selection By marking the Analyze Only Unprocessed FOVs check box H Analyze Only Unprocessed FOV only the unprocessed FOVs of a region will be analyzed After the Analyze button is pressed a dialog will appear displaying a list of the regions which are about to be analyzed and the number of FOVs You are about to start the analysis Please read the following summary before proceeding You have 2 regions to be
194. m2 Zoom3 Hematoxilyn Mean Intensity Ki 67 DAB Mean Intensity al a 33 5 Plowdont__ Nemoftenatny MeaneFtt67000 cont Peet Nok gt UL 344 8 10 644596 498 uR 9 35 743909 330 ju 53 94 4292937 722 LR 28 61 2276699 839 5 Overal 100 00 7958143 389 Gate 2 0 54 43098 022 E G Gate 2 om 0 0 00 0 000 a UR 100 00 43098 022 7 Ju 0 00 0 000 LR 0 00 0 000 Overall 100 00 43098 022 40 80 Hematoxilyn Mean Intensity 0 20 Figure 440 Diagram Detail Window In a Diagram Detail Window you can perform the same actions available for a diagram in the detail window of the region see Chapters 13 3 1 and 13 3 2 e Configure a He e Define cutoff a e Show Percents e Define gates O O v The specific actions available in the Diagram Detail Window are Sh tatisti e Show statistics nana by pressing this button the Statistics section will appear The statistics offer summary data concerning the diagram axes depending on the used Segmentation Method The same data are offered for the quadrants formed by the axes of the cutoff and for the gates defined by the user For the gates defined by the user the statistics will appear separately the same data as the overall statistics will be included but they refer only to the gate Page 287 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual _ Statistics gt G PT Quac
195. might behave erratically or even stop working if the drive completely runs out of space Usually systems delivered by TissueGnostics have a dedicated storage medium for projects it is advisable to use it in order to increase TissueGnostics products performance and avoid issues with low disk space Also in case the system needs to be reinstalled virus infection system files being deleted you might lose data located on the system drive 4 1 Import TissueFAXS HistoFAXS Project The TissueFAXS and HistoQuest management and acquisition software applications are powerful tools They are the core of TissueGnostics microscope based cell analysis systems for cells in tissue sections culture monolayers cytospin preparations and cell smears An existing TissueFAXS brightfield or a HistoFAXS project file may be used to create a HistoQuest project This operation is realized by importing a project from TissueFAXS HistoFAXS into HistoQuest To guide the user through this process we provide the Import TissueFAXS Project Wizard In order to import a TissueFAXS HistoFAXS project press the TissueFAXS HistoFAXS button i TissueF Aes HistoF Ass or choose from the menu File Import TissueFAXS HistoFAXS Page 17 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Folder Mano oomer File Print Print Preview Exit Figure 5 Import TissueFAXS HistoFAXS project A _ The TissueFAXS
196. mplicit Gate Figure 478 New Column dialog From Scattergram Propagated Gate y A Column Name for both Cutoff and Propagated Gate has to be set ote In order to effectively see the data press the Fill Report Data button The Clear Report Data button can be used to delete the displayed columns The existing columns can be modified and the user can also perform changes for the parameters using the Edit Column button Edt ol The Remove Column button Remove Column will remove the selected column from the list The Statistics Report option also provides three possibilities of export e Export to XLS Export to Excel e Export to PDF L EXEot a POR e Export to CSV Export to csv 7 For the Export to XLS option the user can export maximum 65 536 rows this is a rote current restriction of MS Excel 2003 To export a larger number of rows it is recommended to use the Export to CSV option Page 313 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 16 2 Print Out The Print option is available by accessing e File Print from the main window menu of HistoQuest Im save Save AS Close a Print Preview Figure 479 Print Option 5 e The Print button Frint on the main window toolbar Internal Page 314 of 351 www tissuegnostics com HistoQuest 3 0 User Manual Please select the items from the project you wish inclu
197. n the histogram System Data System Data Instituton Data Institution Data Instituton Name Department Laboratory Figure 523 Options Menu System Data Common settings box Page 340 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Institution Data in this section the user may define the name of his institution the department and the laboratory he belongs to These data will be used in Print Reports System Data i Institution Daka Institution Data Figure 524 Options menu System Data Institution Data Users this section is helpful in order to manage the users of the application New HistoQuest users can be added by pressing the Add user button The information about the already existing users can be modified from the Modify user button Any user can be deleted by selecting it and pressing the Delete user button The password can be changed by pressing the Change Password button Modify user and Delete user options are available only for the Administrator The vf note Standard user is allowed only to change his password System Data i Users Administrator Real name Administrator Description Figure 525 Options menu System Data Users dialog Page 341 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Markers All the existing markers are displayed in a list The following operations are available
198. nal In order to change the Segmentation Method a license is required one for Page 206 of 351 www tissuegnostics com HistoQuest 3 0 User Manual General Analysis New Project Settings Segmentation Method Various Shapes 2 0 Various Shapes 1 0 Total Area Measurements Single Reference Shade Note Changing these settings will not affect opened project Analyze Project Beep after finish L Shut down after finish La ft 5 i Upport System Data Figure 311 Changing Segmentation Method dialog b method unless a profile with a different segmentation method is loaded This pre selection becomes relevant when separate markers not profiles are chosen for analysis during the New Project Wizard Q This action means that the projects that will be created later will use this segmentation TIP If a new segmentation method is added to a project which has obtained results using another profile with certain segmentation and separation methods the previously obtained data will have to be updated i e the analysis has to be performed again If a project is opened which uses a different segmentation method the parameters of the markers available in HistoQuest will change even if a different selection was made in the Options menu for the segmentation method before opening that project 11 1 1 Configure Segmentation Method Parameters Each segmentation method contains a set of parameters that are
199. ncy using a slider set the desired transparency value for the Exclusion Areas Overlay it allows you to set the color and the thickness of the selected and non selected events in backward connection Checking Show non selected events in backward connection mode will show the non selected events on the marker images in backward connection mode Animate borders of ROIs and Annotations option will animate the borders in order to make them visible on any background Enabling this may decrease the overall performance as it requires relevant computing resources Highlight double clicked point from Sample it highlights on the detail window the point where a double click was performed on the main image viewer in order to open the sample Page 334 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Show grid lines for Samples imported from Folder all the FOVs on the image will be delimitated by grid lines Show grid lines for Samples imported from TissueFAXS HistoF AXS all the FOVs on the image will be delimitated by grid lines Show name only for selected ROI the label of the ROI will be shown only when the respective ROI will be selected Show name for Exclusion Area the label of the Exclusion Area will be shown only when this checkbox is marked Include user name in annotations contents if marking this checkbox the user name will be included in the annotation edit box Show annotations in br
200. ndow 700x400 800x500 900x700 7 In the Diagram Detail Window can be performed the same diagrams operations as in vf note the diagrams from the Results tab of the region detail window Export histograms and statistics as image e You can copy to clipboard only the diagram the entire Diagram Detail window e You can save as image bmp png gif tiff jpg only the diagram the entire Diagram Detail window Saver Zoom 1 Zoom 2 Zoom 3 Copy Diagram to Clipboard fooml Zoomsz Zooms3 Save Diagram As Copy All to Clipboard Save All As Figure 444 Export histograms and statistics as image Manage Diagrams In order to access the Manage Diagrams menu press The Manage Diagrams button ERRER from the toolbar of the results tab on the detail window The right mouse button on the results section of the region viewer in order to access the diagrams contextual menu and the corresponding option Page 289 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Manage Gates View Raw Data 0 40 ao Hematoxilyn Mean Intensity Figure 445 Diagrams Context menu Manage diagrams Manage Diagrams option will display a dialog where it is possible to create configure delete and propagate diagrams Page 290 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual z Hematoxilyn Mean Intensity Z Ki 67 DAB Mean Intensity draic Ki 6 DA
201. ng Rules Figure 336 Discrimination Area Parameter e Discrimination Gray by increasing this parameter weaker intensity nuclei are removed from the result of the segmentation Page 219 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual arious Shapes 7 0 Nuclei Size Discrimination Area Discrimination Gray Automatic Background Threshold i Threshold Range Virtual Channel Post Processing Order Remove Labels Use Merging Rules Figure 337 Discrimination Gray Parameter e Automatic Background Threshold the background threshold level can be computed automatically selecting Yes or can be set manually by selecting No and providing the desired value You can estimate the value of the background by using the mouse and enabling the RGB tool You can see the value of the gray level by holding the mouse above an area with the background This is a good starting point in estimating the best value for the background threshold e Automatic Background Threshold Range you can specify the interval in which the background threshold is searched The gray level values outside the interval are disregarded This is when you want to exclude the areas of the holes in the tissue which might have a very low value of gray or the saturated areas high level of gray Lower lower limit of the search interval should be increased when you have high background intensity above the level of the areas with no tissue i e h
202. nnel this is a feature in manual correction that helps create events more easily you can create events by using circles of certain radius Set the desired value for the radius in the available field then draw circles over desired events After you finish drawing press Apply to create events Page 308 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 472 Add disc with certain radius a e Apply Manual Correction by pressing this button any of the previously mentioned operations will be applied If this button is not pressed none of the changes indicated by the user is made e Exit Manual Correction E by pressing this button any current manual correction operation will be canceled and the system will not interpret further manual correction commands returning to the normal visualization mode Th Note al Manual Correction can be performed only on master marker images from Labeled to Color Overlay Manual Correction cannot be applied for samples that have results not updated Without a license the user will not be able to apply manual correction Also in 1 Requirements the raw data dialog the user will not be able to delete events Page 309 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 16 Reports 16 1 Statistics Report The Statistics Report is a HistoQuest tool that allows the user to access various statistic data for di
203. ns dialog Default Colors Default Locations teen nese RGB Indicator Top Left Image Viewer Amatai Scale Bar Bottom Right Group of ROIs Item Name Top Left Exclusion Area Exdusion Area Transparency O Scale Bar l E 100 Overlay RGE Indicator Non selected Contour Color Non selected Contour Thickness Selected Contour Color Selected Contour Thickness Show non selected events in backward connection mode _ Animate borders of ROIs and Annotations Highlight double dicked point from Sample Show grid lines for Samples imported from Folder L Show grid lines for Samples imported from TissueFAXS HistoFAXS or Hamamatsu NanoZoomer L Show name only for selected ROT L Show name for Excusion Area L Indude user name in annotations contents Show annotations in browser Figure 110 Region Viewer Options dialog For a detailed description please see Chapter 17 3 4 5 14 Project Items Properties Control For each project item selected in the project browser from the Samples tab or the Compare Sets tab HistoQuest will display a list of properties for example project item type name rows count columns count FOVs count area events count cache built status and illumination correction setting The properties displayed in this list depend of the selected project item Page 89 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Name Rows Count Columns Count FOVs Count Exduded FOVs Count 0
204. nu General Support settings dialog Page 330 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Use Submit Command check this in order to access an external executable file that will send the files to a server e Auto Save Report check this option in order to save the log files on the hard drive no matter if the report is sent or not e Test Configuration this option helps you verify whether you introduced the valid data in order to send your message e Generate Exception this option simulates generating an error message in order to check once again the validity of the data introduced in the fields e Open Reports Folder if a report is not sent it will be automatically stored in a local folder press Open Reports Folder to open that folder e Send Saved Reports press this button to send all unsent reports if any After effectively sending saved reports they will be automatically erased from the local folder where they were stored e Clear Reports Folder will erase all reports from the folder HistoQuest encountered an error ad Error description Generated test exception Error detail Application HistoQuest Version Stack Trace Loaded Modules C Program Files TissueGnostics HistoQuest C Windows SYSTEM32 ntall di Version 6 1 7600 16385 win _rtm 090713 1255 Cc Windows SYSTEM32 MSCOREE DLL Version 2 0 50727 4927 NetFXspW7 050727 4900 C Windows sy
205. nual Hematoxylin ki6 DAB sa Parameter Parameter Mean Intensity Mean Intensity se Mame Hematoxylin Mean Intensity Ki67 DAB Mean Intensity Input Gates per Mumber of Shown Exents Oo Figure 234 Scattergram Properties dialog In the case of overlay diagrams the user can modify only the name the markers and vf note the parameters Page 162 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Compare Set 01 gt 2 n p a 0 a co 1 T a 2 Ba gt E Ww p At _ pa a i gt x 3 dat p pi Ki67 DAB Mean Intensity 40 Hematoxylin Mean Intensity Figure 235 Overlay Diagrams Scattergram the default diagrams will be set depending on the segmentation method this button removes the previously checked diagrams Figure 236 Remove diagram warning message this button removes all diagrams Figure 237 Remove all diagrams warning message Page 163 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual After all steps for the Compare Sets creation were accomplished the user has to press the Create button create rote Gates and cutoffs cannot be defined on overlay diagrams Y 7 4 Managing Compare Sets Compare Sets Modification In order to modify a Compare Set the user has to select the desired Compare Set Then the Edit button Edit from the Compar
206. nuclear expression from all channels This is indicated for cases of nuclear staining i e estrogen receptor ER progesterone receptor PR proliferation markers i e Ki67 etc Yarious Shapes 2 0 Nuclei Size 5 Discrimination Area 1 Discrimination ray l Automatic Background Threshold Mo 5 Background Threshold 25 virtual Channel Post Processing Order Remove Labels nuclear cytoplasmatic Use Merging Rules Figure 340 Virtual Channel Parameter e Post Processing Order Parameter specifies the order of execution for the following operations Remove Labels and Merge Labels arious Shapes 2 0 Nuclei Size S Discrimination Area 1 Discrimination Gray 1 Automatic Background Threshold Mo 2 Background Threshold 25 Virtual Channel Post Processing Order Remove Merge Ferge Remove Labels Use Merging Rules Merge Remove Page 221 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 341 Post Processing Order Parameter e Remove Labels Parameters Smaller Than all objects with the area smaller than the specified value will be removed from the segmented image Larger Than all objects with the area larger than the specified value will be removed from the segmented image Weaker Than all objects with the mean intensity lower than the specified value will be removed from the segmented image Stronger Than all objects with the mean intensity higher than the
207. o draw the rectangle e click on Run Selection lf you want to erase the detected regions press the Clear Selection button Remove Existing ROls if this box is checked any previously created region or group on the sample will be deleted and the newly detected regions will be added to the project Create Group of ROIs from Detected Regions if this box is checked all the detected regions will be included in a group Priore i The numbering of the regions will be realized on the size of the regions criteria starting with the largest one Page 122 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Detection Parameters In order to refine the results of the automatic detection HistoQuest provides a set of parameters you can adjust according to your needs in order to obtain the desired results Minimum Tissue Area the smallest area in square micrometers a tissue region can have All the detected regions with a smaller area will not be taken into account Tissue Closing Radius try to increase this parameter if your probe is split into multiple small regions The result will be a single region containing smaller subregions Exclude border connected regions the regions touching the border of the detection area will be ignored The detection area can be the entire overview image or a crop you can define by using the Run Selection button Regions from the number of detected regions ranked by the
208. oQuest 3 0 User Manual Insert below the observations bo be included in the report oe cnet Figure 486 Add observations dialog Date of analysis 02 02 2009 15 2452 TISSUE W GNostics SB FEegatiks amp FRESE BARE Ga Fon F Observations Figure 487 Include Observations e Include Notes page the Notes page will be included in the report Date ofanalysis 30 03 2009 16 20 31 TISSUE GNOSTICS Hw FCO ec amp A L FEGeEA FRC KE G E O U fF Notes Figure 488 Include Notes page The Notes page is a white page on which the user can add handwritten notes signatures stamps second opinions etc Print Preview The Print Preview option is available by accessing Page 320 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e File Print Preview from the main window menu of HistoQuest Add Samples from Save Save Os Close Print Figure 489 Print Preview option 4 e The Print Preview button Print Preview on the main window toolbar Exporting Print Outs The information in the Print Preview is exactly the same as in the Print Report except for the fact it is in electronic format Exporting Print Outs can be done from the Print Preview Dialog There you can choose the format type for the file to be exported by pressing the Export Document button e 7 I File View Background a e mig oy Ala 1S foe Wali i gt 1 P A
209. of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Add to existing group View Event Data Show in Detail ROI 04 Show Interior ROI 04 Remove selected ROIs Copy to Clipboard Save Copy Selected ROIs Figure 149 Create Group of ROIs contextual menu Any type of ROI can be obtained from the corresponding annotation This operation can be done whether Figure 150 Create ROI context menu b Create ROIs on a ROI detail window You can create ROIs on the detail window of an already existing ROI In this case a message box will appear asking whether you want to switch or not to the detail window of the newly created region By pressing Yes button the detail window of the new region will be opened By pressing No the old window will remain opened but your new region will still be created Page 113 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 151 Create ROIs message box Add Standard ROI option 4x5 Another way to create ROIs is by pressing the Add Standard ROI button from the viewer s toolbar The Add Standard ROI dialog will open Note There are no standard ROIs defined Please use the buttons in the right side to create new ones Rectangular C Elliptic Figure 152 Add Standard ROI dialog First you should choose the desired type of shape rectangular or elliptic Now press the New button to set the height and t
210. oles The gray levels can be measured directly on the image using the Show Color RGB tool Use 0 if you want to disable this option and include all low gray levels Upper upper limit of the search interval You can set it to 255 to include all high values In case you want to exclude saturated areas from searching the background threshold you can decrease this value i e to 250 Yarious Shapes 2 0 Nuclei Size 5 Discrimination Area 1 Discrimination Gray l Automatic Background Threshold Yes z Threshold Range Virtual Channel Threshold Range Post Processing Order Remove Labels Lower Use Merging Rules PETES Ta EE Ga EE Apply Figure 338 Automatic Background Threshold Threshold Range Page 220 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Yarious Shapes 2 0 Nuclei Size 5 Discrimination Area 1 Discrimination Gray 1 Automatic Background Threshold No e Backgror nd Threshold virtual Channel Background Threshold Post Processing Order Remove Labels DUPED DED C OECD Use Merging Rules Default Figure 339 Automatic Background Threshold Background Threshold e Virtual channel you can choose to run the segmentation on the original nuclei image or on a virtual channel The nuclear virtual channel is build by mixing the nuclei channel with the rest of the channels This mixing is computed in the following in a way that takes into account high
211. on 9 2 4 Manage Profiles Accessing the Manage Profiles option from the profiles menu will display a dialog in which the user will be able to see the constituents and the properties of the profiles Profle Warker Segmentation Color Separat ls Waster Svategy for Desapton DE al Various Shape Single Referen Figure 276 Manage Profiles dialog In the same dialog three operations are available e Rename the user can change the name of the selected profile The new name of the profile cannot be empty Page 187 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 277 Empty profile name warning message The new name cannot contain any of the following characters lt gt Figure 278 Profile name not allowed characters warning message The new name cannot be the same with one of an already existing profile Figure 279 Profile name already existing name warning message e Remove deletes a selected profile Figure 280 Remove profile warning message e Remove All deletes all profiles Page 188 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 281 Remove all profiles warning message The same dialog can be accessed from Tools Options System Data Q re Profiles Page 189 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 10 Color Separation Methods Color se
212. on methods and color separation operations cannot be applied to any region of the current project if there are markers with no shades Without a license the user won t be able to use Multiple Reference Shade method ij Requirements Page 198 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 10 2 Single Reference Shade Hematoxylin Ki 67 DAB Single Reference Shade View Gray Image View Color Image Color sl Color Palette Intensity 200 Is Mixture of Ki 67 DAB Mixture Percent 0 99 Figure 300 Single Reference Shade choosing colors dialog In order to choose the shades for the markers one per marker there are three possibilities e The Color Picker for more details please see Chapter 10 1 1 e The Color Palette by pressing the Color Palette button S20 Tate the desired color from the dialog that appears a 0 TLL Tee ll SEER Sat 240 Green 119 Define Custom Colors gt gt ColoriSolid Lum 176 Ae 255 Add to Custom Colors 77 SemEEn E EEE nan Eng EEE CE A A A A E EE 1d Figure 301 Color Palette Page 199 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Autodetect button i etl Sein by pressing this button after selecting a certain region a dialog will appear displaying the two main colors of the region The Autodetect method is available on
213. on of the mouse on the name of the group and it will display the group in the detail window Analyze Analyze Unprocessed FOVs Rename Remove Add to Existing Group Add to New Group Create Annotation from ROI Create Exclusion Area from ROI Copy Delete Results Propagate ROI Figure 180 Show option from context menu A group can be renamed using the Rename option from the mouse right button menu on the name of the group Page 131 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Show in Detail Window Analyze Analyze Unprocessed FOVs Remove from Group 01 Add to Existing Group Add to New Group Create Annotation from ROI Create Exclusion Area from ROI Copy Delete Results Propagate ROI Figure 181 Rename option from context menu A dialog will be displayed where the user can modify the name of the ROI me oon New name Group 02 _ Rename in all samples Figure 182 Rename ROI dialog A ROI will be removed from a group if the right mouse button menu is accessed by clicking on the name of the ROI from the group Then the Remove from Group name option will be chosen This option erases only from the group the individual ROIs still remain Page 132 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Show in Detail Window Analyze Analyze Unprocessed FOVs Rename Add to Existing Group Add to New Group Create Anno
214. one scattergram or histogram The data of different data sets are displayed in different colors Page 159 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Compare Set Name Compare Set 01 m Hematoxylin Mean Intensity Ki 67 Mean Intensity Scattergram m Hematoxylin Mean Intensity Histogram Ki 67 Mean Intensity Histogram Figure 231 Overlay Diagrams dialog By choosing the Overlay Diagrams option the user will be able to select the diagrams to be introduced in the new Compare Set this button opens the Histogram Properties dialog where you can select the parameter for all samples to be compared in histogram form Page 160 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Marker Parameter Mame Hematoxylin Mean Intensity Input Gates lS Figure 232 Histogram Properties dialog Sez The user will be able to select the desired markers and parameters The Name and the J Note Input Gates will be modified only on already created diagrams Compare Set 01 Hematoxylin Mean Intensity Hematoxylin Mean Intensity 40 Hematoxylin Mean Intensity a Figure 233 Overlay Diagrams Histogram Add Scattergram l this button opens the Scattergram Properties dialog where you can select the parameter for all samples to be compared in scattergram form Page 161 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Ma
215. or e View Backward Data for Left Right Quadrants shows the backward data of the events placed on any of the two quadrants created by the vertical axis of the cutoff e View Backward Data for Upper Lower Quadrants shows the backward data of the events placed on any of the two quadrants created by the horizontal axis of the cutoff e View Backward Data for UL UR LL LR Quadrant shows the backward data of the events placed on any of the four quadrants created by the intersection of the cutoff axes Backward Data can be accessed from the contextual menu of gates Page 302 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Configure X Delete Propagate sate 1 View Backward Data For Gate View Backward Data Around This Point Copy Figure 463 Gate Backward Data context menu View Backward Data for Gate shows the backward data for the events inside a gate This option will open the raw data window for all events within a gate and highlight those events on the images in a special color or with a labeled binary mask overlay depending on the user s selection Page 303 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Raw Tints for gt gt Colon ch S070 events of POS oa PERERA i 4 pple a oar keer Sais Tao inter oer Sia Da am B aoa Caer fi ary SOR GRO TORE Be eee we Figure 464
216. ough memory is available to load events for the sample e Modify existing exclusion area this operation is allowed only if events can be loaded e Analysis if there is no more memory available to continue the analysis the user is asked to restart HistoQuest and try analyzing unprocessed FOVs Page 349 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual i Not enough memory available to continue the analysis Please restart HistoQuest and analyze unprocessed fovs in order to complete the analysis Figure 531 Message example Not enough memory available Page 350 of 351 Internal
217. oups will appear as having no results For the Analyze Unprocessed FOVs option from the right mouse button menu on the group s name on the Project Items please see Chapter 0 If a ROI belonging to a group is modified and it also contains results the user will be asked whether he wants or not to reanalyze it again Page 243 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual HistoQuest e ROI modified Do you want to reanalyze the ROT IF not the result will be cleared Figure 375 Reanalyzing modified ROI from group message 12 2 Task Manager The Task Manager will display e The progression of the cache building process and the analysis process e The estimated time for each task e The estimated time remaining for all tasks Task Manager O Stop 0 Pause 2 Clear ETR 01 09 42 Analyze ROI Slide 15 Slide 15 Ol tila O Analyze ROI Slide 14 Slide 14 OC mm O Analyze ROI Slide 13 Slide 13 OL meom Y O Analyze ROI Slide 12 5lide 12 Ol tile O Analyze ROI Slide 11 Slide 11 OL O mm O Analyze ROI Slide 10 Slide 10 Ol tila O Figure 376 Task Manager If the Stop button OQ is pressed any operation in the task manager will be canceled The same operation can be done for each separate task by pressing the Stop button displayed near each progress bar The Pause button a will temporarily interrupt all tasks In ord
218. ow button wh the following options will be available Apply Set to Current Sample and its Regions Apply Set to All Samples and their Regions Apply Set to Current Sample and its Regions Apply Set to All Samples and their Regions Figure 452 Apply Diagram Set menu e Select by pressing this button Select you will be able to Select Unselect all Scattergrams Histograms Diagrams depending on your needs Page 293 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Select all Scatkergrams Unselect all Scattergrams Select all Histograrns Unselect all Histograms Select all Diagrams Unselect all Diagrarns Figure 453 Select menu Diagram Options In order to access the Diagram Options menu press the Diagram Options button af from the toolbar of the results tab on the detail window the right mouse button on the results section of the region viewer in order to access the diagrams contextual menu and the corresponding option Page 294 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Manage Gates View Raw Data 40 a0 Hematoxilyn Mean Intensity Figure 454 Diagrams Context menu Diagram options Diagram Options will display a dialog where it is possible to configure diagrams e The Common option allows setting the Layout and the Display of diagrams Page 295 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual
219. owser by checking this option annotations will be displayed in the Project Items panel Automatization In this section mark corresponding checkbox in order to enable Automatization for the below listed actions General Automatizaton Build cache for all samples in a new created project Analyze all samples in a new created project Show scoring backward connection when opening item in detail Start analysis and load in the right newly added ROI Group Items opened in the right should display color overlay hot ie h wt Automatically Autodetect ROIs for the first sample oe E gimme i in heen F an a ae a Figure 512 Options Menu General Automatization dialog e Build cache for all samples in a newly created project when creating a new project you can choose to have the cache automatically built for all samples e Analyze all samples in a newly created project if loading a marker profile while creating a project it is possible to automatically start analysis for all samples in the project e Start analysis and load in the right newly added ROI Group the analysis can be automatically started for a newly added ROI group e Items opened in the right should display color overlay default setting for items opened in the detail window will be displayed in color overlay during backward gating e Automatically Autodetect ROls for the first sample HistoQuest will automatically autodetect ROIs belonging to th
220. paration methods classify the pixels of each image based on colorimetric properties The resulting channel images show only one color each one marker The advanced algorithms of HistoQuest permit also separation of mixtures of markers Multiple Reference Shades realizes likelihood images for each marker taking into account the shades indicated by the user The user can select any combination of shades and adjust the shade parameters for subsequent color separation This method is suitable for counting cells and for differentiation between marker positive and marker negative cells Single Reference Shade computes the amount of marker color and generates images similar to those in fluorescence staining The grayscale images resulted after the color separation are further used for both segmenting the cells and for measuring the reactivity of each marker 10 1 Multiple Reference Shades 10 1 1 Shades Each marker used in a HistoQuest analysis should contain a defined set of shades By default each shade is also considered a measurement shade If you want to use a shade only for the detection and not for the measurement you can indicate this by just unchecking the corresponding check box This method for color separation performs a probability transformation for each pixel as to which probability a certain pixel has to belong to a given reference shade The closer in color space the actual shade of a given pixel is to the reference shade
221. pare Sets ccccccccsccccseecscesaseeeseeceseesaueecaueecaeeceueesaueesaueesaeessseeceeesaueetaeeesaeeseseeesaeeseaeeseeeens 164 7 5 Compare Sets Print Out Reports cccccccsccccseeceececneeceeecseeceueeceueesaueeceeesueeseueesaueesaeeesaeeseueesseeessensnaeeeas 169 8 Shapes Measurements ccccccccecccceeccescececececccsecceuceceucecneecueeseueecsucesaueesseeeeaeessusecsueesaeeesseeessaeesseeesseeseueessaeess 171 eNO S 2G PrONGS arsana eE E EE E N EEE EEEN ESE SEA ETE E EE 172 SME E E AA EE E EA E AEA A EA O N AE AE EA O OON A ET 172 SME E alo I WE E T A PE E A A E E A A A A E E A E E 174 Oy le Add Copy of IMAI ICOM serseri norne EE EEEE EEEE SEE TEENE EE EEE E E EEEE EENEN EEN 176 La Ranae MaK serek E N EREE S EEA NE EEEE ENE E EEEN EEEE ENN 177 9 1 4 Copy Marker Parameters cccccsecccssccceeecceeeceuceceueecueeeaeeceucecsueesausesaeessaeecsueetaeesueesueessueessueesseeensas 178 9 1 5 Setas Nuclear Marker ccdecncnavnssisncvepenstostbodaedeasaceansesereaudenenstnstbadandcepaccansenoredeseundasestbedealuenessseuveddaestversades 180 9 1 6 Remove Markers wes dae orccteie tna cal nde acta ance sees shrgnciea deca deka dane bale enue cutiotsianies dance ceieiialecbes pemas cee icdtedistnneenbendenesiods 181 91 4 Manage BVA CUS a crt ssee ice aoe cieets vreccects EEEE RNE EEE A accu EENE EE EEE EEE AEE NES EREE 182 De P OMO e E eee e ee ee ee eee ee 184 9 2 1 Save Marker Profil s geccedconcsnscouensiecdvced
222. project If the user owns a predefined number of uses the counter will be incremented only if the project creation Succeeds Multiple Reference Shades The user wont be able to use Multiple Reference Shade method If the user owns a predefined number of uses the counter will be incremented only if the analysis for each FOV succeeds Single Reference Shade The user won t be able to use Single Reference Shade method If the user owns a predefined number of uses the counter will be incremented only if the analysis for each FOV succeeds Event Parameters Count The user won t be able to use more than two parameters for each marker The number of parameters is chosen by the user when buying the license and it goes up to 15 Illumination Correction Images The user wont be able to compute Illumination Correction Images Instead he will be able to use correction images from TissueFAXS If the user owns a predefined number of uses the counter will be incremented only if the illumination correction Succeeds Show preview The user won t be able to see the preview of an existing project If the user owns a predefined number of uses the counter will be incremented each time the preview of an existing project is shown Print Report The user wont be able to print the report or show the print preview If the user owns a predefined number of uses the counter will be incremented each time print report or show print preview Auto cutoff suggest
223. r ooo dr E Choose Markers F TTE What analysis options do you want to use in your project Remove Marker Remove All Markers Analysis Options Segmentation Method Strategy for events in ROI Select only events entirely in ROI Color Separation Method Single Reference Shade Figure 71 Import Mirax Project Step 3 This dialog is the same as for the previously described wizards same functionality The user will be able to choose e Segmentation Method e Strategy for events in ROI e Color Separation Method be If the user loads a profile the segmentation method and the color separation method r cannot be changed The user should press Finish in order to create the project When creating the HistoQuest project the images are exported from the Mirax project vf note and a progress bar like the one below will be displayed Creating HistoQuest Project Exporting Mirax images step 3026 of 11899 Page 65 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 72 Import Mirax Project progress bar 4dd Samples From A mrxs file can be added to a project by pressing the Add Samples from button from the main window toolbar then choose the Mirax option from the displayed menu sea TissueFAXS HistoFAXS Eg Folder far Hamamatsu NanoZoomer Figure 73 Mirax Option The same wizard will appear but the values from step 2 and 3 c
224. re possible Cutoff actions e Add cutoff To add a cutoff on a histogram perform the following steps Click on the Create cutoff button in the histogram toolbar Move the mouse to the position where to set the cutoff During this operation the mouse is captured inside the diagram area Finally the cutoff is determined by pressing the left mouse button on the position in the diagram e Modify cutoff Move the mouse cursor over the cutoff to modify As a result the cursor changes the shape Click on the cutoff using the left mouse button Move the cutoff to the desired new position Finish the cutoff reset operation by pressing again the left mouse button e Copy cutoff To Copy the cutoff press the right mouse button and from the displayed menu choose Copy Cutoff 4utoscale on Hematoxylin Mean Intensity Auboscale On Y Axis Set Max Value For Hematoxylin Mean Intensity Set Max Value For 4xis Parameters Sek Cutoff Delete Cutott Paste Cutoff View Backward Data Copy To Clipboard Save AS Figure 437 Histograms Copy Cutoff context menu Page 285 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e Paste cutoff In order to paste cutoff press the right mouse button on the diagram area and access the Paste Cutoff action Note The Paste Cutoff action is available only on diagrams with similar axes Autoscale on Hematoxylin Mean Intensity Su
225. represents one defined structure in a tissue sample For example a Hematoxylin stain that labels all nuclei in blue would be one marker An antibody stain directed against the molecule Ki 67 proliferation marker visualized by the chromogen diaminobenzidine DAB which gives a brown colored precipitate would be another marker In order to measure the amount of Ki 67 positive cells with respect to all cells in the analyzed tissue section HistoQuest needs to identify these two markers in this example the blue and the brown To do so HistoQuest offers several methods For each marker the user is able to set a name and depending on the color separation method used one or several color shades used as reference for color separation There are custom methods where the reference shades are configurable while for automated detection the definition of reference shades is done by the HistoQuest software In order to analyze your samples all markers of the project must have assigned an appropriate reference shade In every HistoQuest project there should be a main marker master marker or master channel which contains a nuclear stain HistoQuest needs a nuclear staining in order to identify individual cells This process is explained in Chapter 9 1 1 of this document All markers have a set of parameters that depend on the used segmentation method and on the type of the marker In order to increase the ease of use all t
226. rkers a positive negative decision does not represent a qualified approach Neither does a S estimation Rather the question has to be addressed how much of a certain marker is present where in cells and tissues Moreover measurements of simultaneously determined markers patterns and functions are required In order to optimally fit therapies to individual patient s needs specimens have to be analyzed for an entire set of markers and or marker combinations in an observer independent manner In order to meet these requirements computer aided processing of histological samples is required 1 1 Purpose The purpose of this document is to help the user understand how HistoQuest works It describes HistoQuest software s capabilities and it guides the user through the features of the application in order to master all the information required for an optimal usage The document covers installation of the application system requirements main features In the next section you will read more about the goals of the application and its place in a larger system You will also understand the purpose and the advantages of this application 1 2 Goals of the application The HistoQuest software has been designed for cytometric analysis of tissue sections including TMAs and biopsy material as well as cytological preparations The initial point of the process chain in tissue analysis using the HistoQuest software is impo
227. rking with your samples If the cache was deleted or the user wants to build it for multiple samples at once the Build Cache option should be used e f the user wants to erase the data of the already built cache he must use the Delete Cache option Deleting the cache makes sense for archiving a HistoQuest project The cache needs hard drive space which is not needed for archiving a project If the cache is needed at a later point in time it can be easily re built from original image data so there is no need to archive the cache e The Delete Results command erases the data obtained through the analysis Sample Colon 1 contains results Do you really want to delete them Figure 135 Delete Results message Sample Comments You can add your own comments for samples by using the Comments tool from the Project Items Properties control TissueFAXS comments are also available Page 105 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Columns Count FOVs Count Exduded FOVs Count Total Scanned Area Total Exduded Area Events Count Use Illumination Correction Cache Built 0 120000 mm 0 000357 mm Task Manager Stop Pause 9 Clear Figure 136 Add comments to a sample Illumination Correction This option is available for all samples It can be accessed in two ways note From the project items properties control of the sample in this case this option will a
228. rs from the import folder you can decide if the images from your project will be used remaining at their initial location or if they should be copied in the storage folder This is realized by simply checking the Copy images to project folder option in this step of the wizard 4 If you decide to leave images at their initial storage location you will save space rote _ However if you move delete the original image source files your HistoQuest project might not be usable anymore data will remain though but as images are no more available also the connection of data to original images will fail and you cannot reanalyze the sample s The fully qualified hiqproj file name must be less than 260 characters and the project folder name must be less than 248 characters Page 57 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com Basic Settings Advanced Settings Do you know the size of a Field of View FOV Size Predefined Hamamatsu NanoZoomer Objective 20x 1600x1200 aaa badas mm FOV Height hacias mim Image Width 1600 px Image Height 1200 px FOV Width Pixel Width 0 459579375 pm Pixel Height 0 4550625 Do you know the objective that was used to acquire these images Figure 62 Import from Hamamatsu NanoZoomer Step 2 Advanced Settings FOV Size is automatically read from the NDPI file and put in Width and Height edits the FOV size can also be modified manually please se
229. rting of images to a new project Analysis parameters for sophisticated image processing and pattern recognition algorithms have to be optimized for your specific needs After optimization of analysis parameters segmentation is done for all images in the project which results in a set of numerical data These data are visualized in scattergrams and or histograms and are subject to further processing and finally statistical analysis 1 3 Definitions Acronyms and Abbreviations Algorithm A sequence of mathematically realized processing steps applied to an image or part of an image with the aim to improve image quality and to extract specific information or segment identify image objects What an algorithm might do 1 Separate color images Page 7 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Identify colored regions Discriminate background from specific cell objects Oo NN Recognize individual cells where does one cell end and neighboring cell begin 5 Identify cytoplasmatic areas Analysis An analysis is the process of identifying individual nuclei cells cellular compartments and other structures of histological pathological relevance respectively This operation is accomplished by applying the algorithms of the HistoQuest software to the images of tissue sections Cutoff Value A numerical parameter used as a threshold value in a scattergram in order to distinguish between specifi
230. s Custom Select Select Only Standard Regions C Select Only Time Regions Figure 11 Select acquired regions section e Custom Select by checking this option the user will be able to manually select the desired items in the Select Region section e Select Only Standard Regions by checking this option only standard regions will be selected e Select Only Time Regions by checking this option only regions acquired with time lapse will be selected 4 If the check mark is set on Select Only Standard Regions or Select Only Time rote Regions and the user manually modifies the selection in Select Regions section the check mark will automatically move on Custom Select Depending on the number of time lapse regions present in the Select Regions section you can choose the way they are displayed by using the two buttons placed below the Select acquired regions section e Expand Time Regions will expand all time lapse regions in the Select Regions section e Collapse Time Regions will collapse all time lapse regions in the Select Regions section Page 23 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Select Regions Demo_Project pu Slide 1 3 v ogi w Timelapse Regions Z Region 001 Timelapse 1 of 3 4 Region 001 Timelapse 2 of 3 W Region 001 Timelapse 3 of 3 F Region 002 Ho Region 006 HE ee at Select Regions M Demo_Project 5 Slide 1 g z pemo 001
231. s 2 0 Use Ring Mask Yes 2 Interior Radius Exterior Radius Interior Radius Use Identified Cell Mask Lise Muclei Mask PUEDE DEE E EDDC Automatic Background Th Threshold Range aaa al bO6 66HM Default Appl Figure 348 Use Ring Mask Parameter Interior Radius arious Shapes 2 0 Use Ring Mask Yes 6 67 um j Interior Radius i Exterior Radius Use Identified Cell Mask Exterior Radius Use Nuclei Mask Automatic Background Th Ag QIRAAHCACORCHACOCORORACAOCCORANRNACCORCNANCOCRANCACOCRCRANRNOCNCRANRTACOCRANENACOCCAORACHACCORANUCOCORCNACOCORONACOCCRORACHOCRUROCHCCCCRANCNCOCRCRCCCOCCCRACEOCCCRCRIONOLEENIN i Threshold Range 66 0 uM 13 33 Hm B66 BGLM Default Apply Figure 349 Use Ring Mask Parameter Exterior Radius e Use Identified Cell Mask By selecting Identified cell mask as measurement strategy the nuclei are used as seeds e g starting points of image analysis and cell recognition This algorithm searches for specific staining patterns in the other channels of one field of view and causes binary mask objects to grow along those specific structures To obtain this result the existence of at least one other channel than the nucleus channel is required By choosing No no growing is performed on the identified cell mask Page 225 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Yarious Shapes 2 0 Use Ring Mask Yes j Interior Ra
232. s com HistoQuest 3 0 User Manual Figure 459 Forward and Backward connection Marker Image backward connected data cells within the source gate are displayed with red labels cells outside the gate with a green label The cell marked with the square blinking indicator is also blinking in the scattergram so that the user can determine the position of each cell in the scattergram Hematoxylin Mean Intensity Ki 67 DAB Mean Inte Ki 67 DAB Mean Intensity Hematoxylin Mean Intensity Figure 460 Forward and Backward connection Diagram Page 300 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Raw Data for gt gt Coloni showing 280 events of 1668 3 g Colon Coloni Backward connection for gate Positive Ki 67 DAB from scattergram Hematoxylin Mean Intensity m Region OF Interest Field OF View Event Label Hematoxylin Mean Intensity Ki 6 DAB Mean Intensity e Colon oo0001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo00 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 ooo001 a Sire Peal i a4 995250 31 317460 35 862070 44 153590 40 652650 33 562920 54 195550 a4 02600 47 054670 36 231 960 44 090660 50 775160 39 659090 a4 144650 34 191560 a4 172000 36 9 5720 35 563530 43 4797 90 35 839290 So 921290
233. s com HistoQuest 3 0 User Manual Basic Settings Advanced Settings What s the name of the project Demo Project Project Description Where will the project be stored F Projects TissueFAxS Demo Project If the project folder does not exist do you want to create it Create Project Folder Copy images to project folder or leave images in their folder _ Copy images to project folder Figure 34 Import TissueFAXS HistoFAXS Project Step 3 Basic Settings Project Name mandatory Name of the project it must contain minimum 1 character maximum 50 characters It must also not contain any forbidden characters lt gt The corresponding HistoQuest project file will have this name and it will be stored in the folder selected in the first step of the wizard The default name of the project is the name of the import folder Page 38 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Basic Settings i Advanced Settings j l m What s the name of the project p Project Description Where will the project be stored Browse F Projects TissueFAxs Demo Project oe k a s or ee ai ka F ai ka E ia uf d x ki pre i iaie a a oa Hate hah i Pro A Figure 35 HistoQuest Project project name Project Description optional e Short description of the experiment max 255 characters Project Folder mandatory e The storage folder of the project
234. s may overlap with the excluded area Excluded FOVs count You can exclude FOVs from the analysis in two ways by using the flags or the exclusion areas e Flags see Chapter 5 6 Coloni Figure 219 Flags option e Exclusion areas that include entire FOV s if the exclusion area completely covers a FOV the respective FOV will be excluded from the analysis 6 4 Transform Shapes Any ROI annotation or exclusion area can be converted into another type of shape You have three options e Using Transform Shapes button from the Region viewer toolbar e Using item s contextual menu in the Project Browser e Using an annotation s contextual menu directly on the sample Transform Shapes button Press Transform Shapes button from the Region viewer toolbar in order to access the Transform Shapes panel Coloni Bil Figure 220 Transform Shapes option In the Transform Shapes panel select desired item s by marking corresponding checkbox es you can also perform multiple selection by using Ctrl control from the keyboard or press Select button for selecting all ROls all exclusion areas or all annotations Page 151 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Select all ROMs Select all Exclusion Areas Select all Annotations HE Exdusion Areas Exclusion Area 03 Clear Selection a Exdusion Area 04 g Annotations _ Annotation 07 Select the t
235. s way duplicate names are avoided since regions with the same name can exist on different slides in TissueF AXS HistoFAXS Sample Options By pressing the right button of the mouse on the sample s name a menu containing a set of options will be displayed Page 102 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Show Show in Detail Window Analyze Analyze Unprocessed FOVs Analyze All ROIs Rename Remove Remove All ROIs Remove All Groups Remove All Exclusions Remove All Annotations Build Cache Delete Cache Delete Results Figure 131 Sample context menu An Duplicate names are not allowed f NOTE The new name cannot contain any forbidden characters lt gt the characters allowed in a file directory name are also the ones allowed in the name of a sample The name of a region can have maximum 40 characters e Show option displays the sample in the main window e Show in Detail Window option will open the sample in the detail window e lf the user wants to analyze the sample he can use the Analyze command for more details please see Chapter 12 1 1 e The Analyze Unprocessed FOVs option will analyze only the rest of FOVs which were not already analyzed before within a region in order to complete the computed data for a sample e Analyze All ROIs for more details please see Chapter 12 1 2 To give a different name to a sample choose th
236. s way only the name of the marker is loaded the parameters have to be defined later A new marker can be added to the project also by clicking the Add Marker button Addmarker This marker will only have a name and you will have to choose its parameters later in order to use it An existing profile set of markers can be loaded in order to be used in this project This operation can be done by clicking the Load Profile button Lead Profle You will be able to see the markers from the selected profile in the list of currently chosen markers the segmentation method the color separation method and the strategy for events in ROIs fr e You can also use a profile from an existing project Note Add Marker Page 41 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com HistoQuest 3 0 User Manual The markers already added as described above can also be removed one by one by pressing Remove Marker Se or all at the same time by pressing Remove All Markers These two options are available only if at least one marker is available in the list After specifying the markers that will be used in the current project the user has to pick a main marker master marker The wizard cannot move to the next step until one of the available markers is chosen as master marker This is realized by simply checking the Is Master option corresponding to a marker The user will be able to choose e Segmentation M
237. scale representation of the shade Just go on the desired shade and choose the View Shade Image option from the contextual menu b pe Lap e kas e gt IS eae Figure 293 View Shade Image left original image right shade image View Gray Image if you want to see the grayscale representation of all shades belonging to one marker choose the View Gray Image option from the contextual menu Page 195 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 294 View Gray Image left original image right gray image View Measurement Image this option displays the gray image only for the shades that have the Measure option checked Figure 295 View Measurement Image left original image right measurement image Check All this option checks the Measure option for all the shades of a marker Uncheck All this option will uncheck the Measure option for all the shades of a marker Remove Selected Shade this option removes a shade you have selected Remove All Shades this option removes all shades of a marker Advanced Mode this option makes available the advanced parameters Gain Sigma Gauss Color Space Toolbar by choosing this option all the items from this menu will be included as a toolbar for the markers Page 196 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Hematoxylin Multiple Reference Shades SAY x lt X _ 0 LAB 0
238. se fessor alare lowes ovaa foose 27 573 15100 2002 tors esei 107 176 50 85 sfioro i0025 aar remat ce count Percent romz ja ferae fieno efua i5000 Je fais iras 16l33 72 aes 000 e fesso foins aloe 100000 ik flow ans zsa area 6525000 oveen 107 176 0 355 s22 sono ioe Figure 334 Example of Various Shapes Statistics section 11 3 Various Shapes 2 0 HistoQuest provides you a method created especially to detect structures with various shapes and sizes For tuning this method a parameter set is available Page 218 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 11 3 1 Parameters Master Marker Parameters e Nuclei Size specifies the size of the nuclei You should increase this parameter if you have large nuclei in your sample arious Shapes 2 0 Discrimination Area Nuclei Size Discrimination ray Automatic Background Threshold LEELEE i Threshold Range Virtual Channel Post Processing Order Default Remove Labels Use Merging Rules Figure 335 Nuclei Size Parameter e Discrimination Area by increasing this parameter smaller nuclei are removed from the result of the segmentation Yarious Shapes 2 0 Nuclei Size scrimnaCoOn Area Discrimination Gray Discrimination 4rea Automatic Background Threshold e Threshold Range Virtual Channel Post Processing Order Remove Labels Use Mergi
239. sing the Configure button on each scattergram a a dialog will be displayed where these parameters can be set e Anew Name for a scattergram The name of a scattergram should be unique In case you want to introduce an already JS existing name an error message will appear Internal Page 268 of 351 www tissuegnostics com HistoQuest 3 0 User Manual HistoQuest x A diagram with this name already exists Figure 409 Scattergram name error message e The Input Gates created by the cutoffs and gates from the other diagrams e The Number of Shown Events on a scattergram Choosing Input Gates for diagram Choosing some Input Gates for a diagram will show in the diagram only those events from the Input Gates If more input gates are selected the union of the events from the input gates is considered as input from the diagram Not all gates can be selected as input gate for a diagram Gates defined on that diagram and gates that could create interdependencies between diagrams are not allowed to be selected they are marked by this red sign G in the list instead of checkbox Choosing Number of shown events for diagram For diagrams with a large number of events that are overlapped setting the Number of shown events could improve their display Setting this value will have the effect to select and display only a certain percentage of events Events to be displayed are chosen following a uniform selection pattern
240. software provides stitch support and HistoQuest can open and f Note i i properly display samples with stitch applied In the case of a project with stitch in project properties are displayed the sizes of the original FOVs not the crop size 4 1 1 Step 1 Choose the TissueFAXS HistoFAXS Project Start by selecting a TissueFAXS HistoF AXS project file to be imported to HistoQuest This operation can be done by typing the path to the project file or by browsing for it The user should ensure that the selected file exists at that location and that he has of Note 5 i j enough rights on it otherwise he won t be able to use it an error message should appear in this case It is recommended choosing a local drive both for ensuring performance and avoiding unpredictable errors For TissueFAXS versions compatible with your HistoQuest version please contact your TissueGnostics support team Page 18 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Choose TissueFAXS HistoFAXS Project Select the TissueFAXS HistoFAXS project file from which you want to import regions in this HistoQuest project E Choose TissueFAXSHistoFAXS Project Where is your TissueFAXS HistoFAXS project HistoQuest Project F Projects TissueFAXS Demo_Project Demo_Project agproj Browse Choose Markers GE Demo_Project HM Side 1 HQ Side 2 Select acquired regions Custom Select O Select Only Standard Regions
241. specified value will be removed from the segmented image arious Shapes 2 0 Nuclei Size 5 Discrimination Area 1 Yarious Shapes 2 0 Discrimination Gray 1 Nuclei Size Automatic Background Threshold Yes Discrimination Area 1 i Threshold Range 5 255 Discrimination Gray 1 Virtual Channel na Automatic Background Threshold Mo Post Processing Order Remove Merge Background Threshold 25 Remove Labels Virtual Channel no H Smaller Than 2708 33 ym Fost Processing Order Remove Merge 1 Larger Than 135416 67 um Remove Labels Weaker Than z0 Use Merging Rules i Stronger Than do not use Use Merging Rules Mo Figure 342 Remove Labels Parameters e Merging Rules Parameters Max combined area specifies the maximum obtainable area by applying merging process Max involved compactness specifies the maximum compactness which an object can have prior to the merging process Group Max specifies the maximum number of neighboring labels involved in the merging process Min Resulted Compactness specifies the minimum possible value of an object after the merging process Page 222 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Yarious Shapes 2 0 Nuclei Size 5 Yarious Shapes 2 0 Discrimination Area 1 Nuclei Size z Discrimination Gray 1 Discrimination Area l Automatic Background Threshold Yes Discrimination Gray l L Threshold Range 5 255 Automatic Background Threshold Wo ETE ne
242. splay the word Yes If there is no correction image available the word No will be displayed An If a TissueFAXS HistoFAXS project already has correction image HistoQuest can ote If the settings for illumination correction change the results of the processing if any will be invalidated Illumination correction settings affect the detection and the measurements so the user has to do the analysis again Changing correction mode When changing the correction mode Use illumination correction Don t Use illumination correction the results will be invalidated but not erased Page 107 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Project Browser g Group 01 Exclusion Areas Rows Count Columns Count FOVs Count Exduded FOVs Count 0 Total Scanned Area 0 120000 mm Total Exduded Area 0 002128 mm Events Count Figure 139 Changing the correction mode 6 1 2 ROIs A ROI Region of Interest is an area within a sample which represents a certain interest for the user s analysis Types of ROIs e Rectangular Gl For the creation of rectangular ROls the user has to select its shape from the shapes manipulation toolbar then the left mouse button should be kept pressed on the sample the pointer is dragged over the interest area and then the mouse button is released Figure 140 Rectangular ROI Page 108 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual
243. ssage is shown e Manage diagrams the events of the project item should be loaded when the user double clicks on a diagram If there is not enough memory a warning message Is displayed e Manage gates the events of the project item should be loaded when the user double clicks on a gate If there is not enough memory a warning message is displayed e Print preview a short report is presented to the user if some or all events could not be loaded before actually showing the report e Statistics reports a short report is presented to the user in case some or all events could not be loaded before actually showing the report e Delete exclude FOVs delete exclude FOVs is allowed as for delete events from raw data below For adding previously excluded FOVs HistoQuest will ask if analysis should be runned for those events only if enough memory is available to load events for the sample and perform the analysis e Delete events from raw data deleting events from a sample requires enough memory available to load events for all ROls groups belonging to that sample If not the operation is not possible and the user will be guided to try again later e Manual correction requires that HistoQuest has enough memory available to load events for all ROls groups belonging to that sample If not the operation will not be possible and the user will be guided to try again later e Remove all exclusion areas this operation is allowed only if en
244. stem32 KERNEL32 dll Version 6 1 7600 16385 win _rtm 090715 1255 C Windows system32 KERNELBASE dil Version 6 1 7600 16385 win _rtm 090713 1255 C Windows system32 ADVAPT32 dll Version 6 1 7600 16385 win _rtm 090713 1255 C Windows system32 msvet dl Version 7 0 7600 16385 win _rtm 090713 1255 C Windows SYSTEM32 sechost dll Version 6 1 7600 16385 win _rtm 0907135 1255 C Windows system32 RPCRT4 dll Version 6 1 7600 16385 win7_rtm 090713 1255 C Windows system32 SHLWAPLdll Version 6 1 7600 16385 win _rtm 090713 1255 Ci a ea dil Version 6 1 7600 16385 win7 _rtm 090713 1255 2 dll Version 6 1 7600 16385 win _rtm 090713 1255 I agree Read Product Improvement Program Terms Figure 507 HistoQuest error dialog Error description here you can see a short description of the error Error detail here you can read detailed data related to the error Product Improvement Program checkbox In the Product Improvement Program participating systems send information to TissueGnostics about how they use certain products Received data is combined to help TissueGnostics solve problems and to improve Page 331 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual the products and their features Click the link in the lower left corner of the dialog shown above to read about this program and check I agree checkbox if you want to participate If you don
245. steps of sophisticated image processing and high level mathematical operations that make possible the analysis depending on number and size of samples ROIs may be time consuming and because analyzing a sample actually means analyzing all contained images the user has the option to choose whether analysis should be run for entire samples or just relevant parts thereof This way the best configuration can be chosen for analysis of samples Er Before starting analysis for any region the user has to ensure that the desired color note separation and segmentation methods as well as the parameters for these methods had been set While the analysis is performed for any region some of the operations cannot be done and they appear disabled in the menus operations with flags finalize analysis marker shades and segmentation parameter modification load profile add copy rename remove marker s set as nuclear marker copy marker parameters remove sample and ROIs 12 1 1 Sample Analysis The analysis for the sample can be performed in three ways e By pressing the Analysis option that appears when pressing the right mouse button on the sample name from the Project Items Page 233 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Show Show in Detail Window Analyze Unprocessed FOVs Analyze All ROIs Rename Remove Remove All ROIs Remove All Groups Remove All Exclusions Paste Build Cache Del
246. ster marker will determine the loss of the existing results in this cauTION situation a warning message will appear Set as Nuclear Marker option will appear disabled in the menu if the selected marker is already master marker Page 180 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 262 Set as Nuclear Marker warning message This operation will preserve the color separation method settings but will set the default parameters values for the current segmentation method for both previous and new master marker The previous master marker will become non master changing its parameters accordingly Hematoxylin Multiple Reference Shades BE Beeeee RE EPRURERRURRREREEE arious Shapes Discrimination Area 1 aaa dare i Discrimination ray Automatic Background Threshold Yes Virtual Channel na Post Processing Order Remove Merge Remove Labels No a Use Merging Rules i Figure 263 Ki 67 DAB Non Master Marker tab Hematoxylin Ki 67 DAB Multiple Reference Shades Ree ee ee ROR ER ERR RRR R ER ORR ERROR ERR ER ERR ERE EERE RRR ERR REPRE EERE arious Shapes Use Merging Rules hd Mo Figure 264 Ki 67 DAB Master Marker tab Q You may recognize the master marker by the fact its name appears in bold letters on TIP its tab 9 1 6 Remove Markers There are two possibilities for marker removal e Remove Marker option from the mark
247. stom Gate e Modify gate Any gate can be resized and moved To move a gate follow the steps below Begin the movement of the region by pressing the left mouse button on the contour of the shape Hold the left mouse button down while dragging the shape inside the diagram area Release the left mouse button to finally determine the gate s new position To resize a gate a set of resizing points are defined For a rectangle these points are the corners for an ellipse there are eight resize points and for the polygon these points are the actual points of the shape To resize a gate follow the steps below Begin the resizing of the region by pressing the left mouse button on a resize point Hold the left mouse button down while dragging that point inside the diagram area Release the left mouse button to finally determine the gate s new size Gate Contextual Menu To access the Gate Contextual Menu press the right mouse button on the contour of the gate Configure X Delete Propagate Gate 1 View Backward Data For Gate View Backward Data Around This Point Copy Figure 422 Manage Gate menu e Configure Gate For any gate there is a set of management operations that can be performed like changing the color renaming the gate and choosing whether the label will be visible or not To configure a gate press the right mouse button on the contour of the shape and choose the Configure option from the menu Th
248. tation from ROI Create Exclusion Area from ROI Copy Delete Results Propagate ROI Figure 183 Remove from Group option from context menu Create annotation and exclusion area from ROI By choosing Create Annotation from ROI or Create Exclusion Area from ROI option the selected ROI will automatically transform into an annotation respectively into an exclusion area it will also change its name and move to the annotations or exclusion areas section Show in Detail Window Analyze Analyze Unprocessed FOVs Rename Remove Add to Existing Group Add to New Group Create Annotation from ROI Create Exclusion Area from ROI Copy Delete Results Propagate ROI Figure 184 Create Annotation Exclusion Area from ROI Analyze In order to analyze a group choose the Analyze option from the right mouse button menu on the name of the group This option will run the algorithm for all the regions of the group Page 133 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Analyze Unprocessed FOVs Rename Remove Add to Existing Group Add to New Group Copy Delete Results Propagate ROI Figure 185 Analyze option from context menu Task Manager GO Stop 0 Pause Clear ETR 00 02 07 Analyze ROI Region 001 ROI 01 MZ co Step 7 22 31 Analyze ROI Region 001 ROI 02 CME oi Step 7 19 36 Figure 186 Task manager Analyzing ROIs in a Gro
249. te Sample For each TMA block Figure 29 Create Sample for each TMA slide Figure 30 TMA slide in TissueFAXS Page 35 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Bertie Ais Count THN Scarred Aree Pita Erie dare Geert Count Cache Bult Figure 31 TMA slide in HistoQuest If the acquisition was realized with different objectives for different regions the user will be asked if he wants to open the regions acquired with the objective value of the first region in the project In this situation the user has the possibility to manually choose the regions acquired with the same objective The selected regions are acquired with different objectives Do you want bo select only those acquired with 20x Figure 32 Message Box for Different Objectives When the analysis is started identical image processing and object segmentation note steps are applied to all samples You cannot analyze samples acquired at different magnification in a single analysis You can change analysis parameters for individual samples and perform the analysis only for those but this will cause inconsistency between analysis parameters and obtained data results Hence it is HIGHLY RECOMMENDED to analyze samples acquired at different magnification in different HistoQuest analysis projects Page 36 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual For creating a sample from
250. te eon eee ee ee ere 233 lat T Sample ANalySIS oeiras eee eee 233 TES ROAT E E E A AE ee ee eee ee 235 12 1 3 Toolbar Analyze B tONe isiisnsisiirnaisiinninrninenniiinina raana ad AEE EEA EEEE 241 12 1 4 Group f ROIs AnalySiS sisssirirrasisiinniriinnniia inian ETEA aAA EA EANA E 243 12 2 Task MaMa gO oniran ne EERE A EE EEE E 244 TANN S RO 6 era E E EE E E 246 Tet m ge RS INS ei a E EE E E E EE EO E S E 246 TZ RW O ae E E E EEE 251 13A Ms BON GIN OV CUMS siraspa OE EEEE OE E E ESEE EEE 260 gs eam ial E VOD eaa eee eee eee eee E eee 262 Toa DAO 9 ee oe neice cee ne nee ee se 264 e a In e fo 19 oe ee ee eee el ee N E eee eee eee ee 264 Mss WON EEE A EIT EI A A sie jected ges S EOE socteoetie gost 280 133 3 Diagram Detail WINGOW iussisse eniani enina aa a a a a aa a E i 287 Manage Na Me AN YS eect cette aes Ee a aa E aE a E aa aa a 289 14 Forward and Backward Connection ccccccccccseccceececeeeeee cece eeseeeseeeeseeeeseeeeseeeseeeeseeeeseeeeseeesseeseueeseeeeseeeesaeeess 299 14 1 F rward COnnectoN sisinta nee iaiia Aniane i eita iiaii aneia ad a iaa eiei 301 14 2 Backward OIC COM ses cece nse etiaiesnd oer teob eseete etc nalotostacieia eeteb ea Aa aeiiaaie Aa aAa aE A aaa EEEa 302 143 Backward Dala caresses dete pastes cnr nee sins eea eA RA EAR Aae Aaa aa ANERER 302 a Se PES COLONY INS eE A P A E E E EE E EE E A S E casas 302 Toes PISO I eE ENE E E E EEE EEE EEE NEEE E E E EE 305 1D BULAN eE e E a A
251. ternal www tissuegnostics com HistoQuest 3 0 User Manual O From Scattergram Column Mame Marker Parameter Mean Intensity Event Type Positive Unit Percent Cutoff Figure 476 New Column dialog From Histogram From Histogram selection will provide data in conformity to the following criteria Marker Parameter Event Type Unit Cutoff The user will also set a Column Name From Scattergram selection offers two possibilities for data computing e Cutoff the data will be computed according to the following issues Unit Markers Cutoff and Implicit Gate C Predefined From Histogram From Scattergram Cutoff Propagated Gate Column Name Unit Propagated Gate X Axis Marker Y Axis Marker X Axis Parameter Y Axis Parameter Mean Intensity Implicit Gate Upper Figure 477 New Column dialog From Scattergram Cutoff Page 312 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual rE From Scattergram option contains an extra predefined value Unit Total rote Area If choosing Total Area you must also select the marker in the dropdown box on the right please see image above e Propagated Gate the data will be computed according to the following issues Unit Propagated Gate Predefined From Histogram From Scatbergrarn C Cutoff Column Mame Unit Propagated Gate Axis Marker Y Axis Marker 4 Axis Parameter s Cutoff I
252. the Add to Existing Group option of the right mouse button menu on the name of the ROI If the group already contains the ROI the user wants to introduce he will receive a warning message Page 129 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 176 Add to Existing Group warning message e Using Add to New Group option from the Project Items ROI contextual menu Show in Detail Window Analyze Analyze Unprocessed FOVs Rename Remove Add to Existing Group Create Annotation from ROI Create Exclusion Area from ROI Copy Delete Results Propagate ROI Figure 177 Add to New Group from context menu What is the name of the new group Propagate Group to all Samples The Group will be propagated only on the samples where it doesn t already exist Figure 178 Create Group of ROls message Create group with propagation to all samples When a group is created the user has the possibility to propagate it to all samples i e have group Tumor Non tumor in all samples Internal Page 130 of 351 www tissuegnostics com HistoQuest 3 0 User Manual What is the name of the new group Group gil Propagate Group to all Samples The Group will be propagated only on the samples where it doesn t already exist Figure 179 Propagate Group to all Samples option Show in Detail Window option is accessed from the right butt
253. the displayed menu Page 59 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual P TissueFAXS HistoFAXS Eg Folder Figure 64 Hamamatsu NanoZoomer file Option The same wizard will appear but the values from step 2 and 3 cannot be modified A NDPI file that has different FOV size than the project or different magnification cannot be added 4 4 Import images from a Mirax file In order to import a Mirax file press the Mirax button 1 or choose from the menu File Import Mirax File Project Tools Help rr ManoZoomer File Save As Close Print Print Preview Exit Figure 65 Import Mirax file 4 4 1 Step 1 Choose Mirax file The Import Mirax Project wizard will appear Page 60 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Choose Mirax file Select the Mirax file from which you want to import images in this HistoQuest project E Choose Mirax file Where is your Mirax file ganas Doo Choose Markers What size should the extracted images have 1024 x 1024 What format should the extracted images have K ao g E gt zoom level should the images be extracted Figure 66 Import Mirax Project Step 1 a Then choose Browse Browse h and select a mrxs file then press Open a ps aa e L e Data2 F Projects HistoQuest Mirax Projects Search Mirax Projects pl Organize
254. the entire experiment For instance if a vf note _ diagram had a name set by the user the name will be reset in order to show the new name of the marker Old name Hematoxylin Hew name Figure 255 Rename Marker dialog The new name of the marker cannot be empty Page 177 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Figure 256 Empty marker name error message The new name of the marker cannot contain any of the following characters lt gt Figure 257 Marker name not allowed characters The new name cannot be the same with one of another marker in the project Figure 258 Identical marker name error message 9 1 4 Copy Marker Parameters The Copy Marker Parameters from the marker manipulation menu will copy the shades and the segmentation parameters from one marker to one or more markers The color separation parameters can be copied between master and non master markers l CAUTION When copying parameters the old shades are erased at Multiple Reference Shades Any modification on parameters will invalidate the existing results if any You cannot undo Copy Marker Parameters Page 178 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual For Measure in different compartments a license is required Ki67 DAB Copy_OF_Ki67 DAB Multiple Reference Shades arious Shapes 1 0 a Interior Radius
255. the sample and displayed at the current size of the sample e Best Fit by pressing this button the selected annotation will be located on the sample and displayed at the current size of the region viewer e Close To display the user name for the annotations go to Tools Options General Q TIP then check Include user name in annotations The specific color of an annotation can be set and changed in Tools Options General Region Viewer General Image Viewer Default Colors Regon free i Annotation Group of ROIs Item Name Exdusion Area RGB Indicator j a Q 1 B a iO mM uw C 5 oO po _ Animate borders of ROIs and Annotations Highlight double dicked point from Sample Show grid lines for Samples imported from Folder 0O Show grid lines for Samples imported from TissueFAXS HistoFAXS or Hamamatsu NanoZoomer C Show name only for selected ROI C Show name for Exclusion Area _ Indude user name in annotations contents Show annotations in browser Figure 212 Set Annotation Color dialog Page 147 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 6 3 Exclusion Areas This feature allows excluding from the analysis some events by selecting on the sample the tissue region to be excluded from the analysis From the total area of the scanned tissue the exclusion area will be subtracted no events detected in these
256. the value of an axis has is higher than the Small Range Limit Value the number of decimals will be the one from Cutoff Decimals If the value of an axis has is lower than the Small Range Limit Value the number of decimals will be 2 Scattergrams the point style and the point size can be set and adjusted Diagram ii Scattergrams Point Style Point Size Point O ACrOSS O Circle Figure 518 Options Menu Diagram Scattergrams settings box Page 338 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual XCross Figure 519 Options Menu Diagram Point Style Histograms the user can set here the bins count the style and the color for the histograms Diagram i Histograms Bins count Line Style with Fill Figure 520 Options Menu Diagram Histograms settings box Bins count adjusts the number of bins using the trackbar Style three styles are available in order to graphically represent a histogram Bin Style Line Style and Line Style with Fill Line Style with Fill Line Style with Fill Figure 521 Options Menu Diagram Histograms Style dropdown menu Page 339 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual 10000 20000 30000 10000 20000 30000 10000 20000 0000 Line Style ai ayie with Fill Line Style Figure 522 Options Menu Diagram Histograms Styles Color here you can adjust the color the events i
257. threshold is searched The gray level values outside the interval are disregarded This is when you want to exclude the areas of the holes in the tissue which might have a very low value of gray or the saturated areas high level of gray Lower lower limit of the search interval should be increased when you have high background intensity above the level of the areas with no tissue i e holes The gray levels can be measured directly on the image using the Show Color RGB tool Use 0 if you want to disable this option and include all low gray levels Page 227 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Upper upper limit of the search interval You can set it to 255 to include all high values In case you want to exclude saturated areas from searching the background threshold you can decrease this value i e to 250 arious Shapes 2 0 Use Ring Mask j Interior Radius _ Exterior Radius Use Identified Cell Mask zc Max Growing Steps Skip Steps Use Nuclei Mask Automatic Background Th z Threshold Range Ves al 2 Outside amp Inside 4 T Mo Threshold Range Lower e m Figure 355 Automatic Background Threshold Threshold Range arious Shapes 2 0 Use Ring Mask Yes be Interior Radius 1 h Exterior Radius zZ Use Identified Cell Mask Outside amp Inside Max Growing Steps 4 Skip Steps 0 Use Nuclei Mask Mo Automatic Background Th Mo 25 Backgro
258. tissuegnostics com HistoQuest 3 0 User Manual Rename Annotation Remove Annotation Locate Best Fit Transform into ROIs Please insert annotation content below Figure 203 Edit Annotations dialog In the Edit Annotations dialog you can perform the following actions e Inthe Visibility column mark the checkboxes corresponding to the annotations you want to visible on the sample e Locate press this button and you will see the location of the selected annotation on the map of the sample Best Fit press this button and HistoQuest will give an optimum display of the annotation in the viewer Remove press this button to delete the selected annotation s Remove all press this button to remove all the annotations from the sample Transform into ROls this button will transform all the selected annotations into ROIs Insert some comment regarding the annotation in the lower left corner of the Edit Annotations dialog and press Save to keep it 7 You can also delete all annotations from a sample by right clicking on sample s name rote in the Project Browser and choosing from the contextual menu Remove All Annotations option 6 2 4 Edit Annotations The annotations can be managed using the Edit Annotation menu This menu can be accessed in two ways e By pressing the Edit Annotation button L from the annotation manipulation toolbar e By pressing the right button of the mouse on the surface of an
259. tistics for Various Shapes 1 0 and Various Shapes 2 0 will display the following columns e For Various Shapes 1 0 and Various Shapes 2 0 with count used in computing the percents X Mean i e mean value for the parameter shown on the x axis Y Mean i e mean value for the parameter shown on the y axis Count number of events in the respective unit sample gate ROI quadrant Percentage i e how many events are in the respective quadrant region relative to the number of total events for each of the four quadrant regions No mm number of detected events per Area Measurement Unit Page 217 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual e For Various Shapes 1 0 and Various Shapes 2 0 with area of a marker used in computing the percents X Mean i e mean value for the parameter shown on the x axis Y Mean i e mean value for the parameter shown on the y axis Total Area of total area of chosen marker Percent Area of percent area of chosen marker relative to the total area of the marker Percent Area of Area Measurement Unit percent area of chosen marker relative to the total area of the Sample ROI Group Show Percents for Count Show Percents for Area of Hematoxylin Show Percents for Area of Ki F Toad Hemat COS count Percent No nm Fla forser fiero ofo fiso Je nsee i322 i60 3 e aren 000 e fesa ie valisa ise e f
260. to group by that column a Edit Column Remove Column Figure 500 Statistics Report main window Manually select the different regions to be included in the report or use the Options menu for different selection criteria For more details please see Chapter 16 1 from the current manual 17 3 4 Options The Options menu can be accessed from Tools Options This menu has three sections General Diagram and System Data General Page 327 of 351 www tissuegnostics com HistoQuest 3 0 User Manual Seals hoa New Project Settings Segmentation Method Various Shapes 2 0 Strategy for events in ROI Select only events entirely in ROI Color Separation Method Single Reference Shade a gt 5 i in i Note Changing these settings will not affect opened project sy Ww ou y O m 4 T Analyze Project 13 ia Beep after finish C Shut down after finish Skin Support G gt m D a O O Ig N O oY o gt fi ct m ov Ww be m 3 m oF C 5 o vi Figure 501 Options Menu General dialog In the Analysis section the user is allowed to change the color separation and segmentation methods and the options for the Analyze Project General Analysis Mew Project Settings Segmentation Method Various Shapes 2 0 Strategy For events in ROT Select only events entirely in ROT Color Separation Method Single Reference Shade Note
261. toQuest provides you a method created especially to detect structures with various shapes and sizes For tuning this method a parameter set is available 11 2 1 Parameters In the figure below a comparison is shown of the default parameters for a master marker left side and a non master marker right side for the Various Shapes method Page 208 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Yarious Shapes 1 0 arious Shapes 1 0 Nuclei Size 5 Use Ring Mask Yes Discrimination area i i Interior Radius 6 67 um Discrimination Gray i oe Exterior Radius 13 33 um Automatic Background Threshold es ai Identified Cell Mask Yes virtual Channel no i Max Growing Steps 6 6 um Post Processing Order Remove Merge Skip Steps 0 urm Remove Labels Mo Use Nuclei Mask No Use Merging Rules Mo Automatic Background Th Yes Figure 314 Various Shapes 1 0 Default parameters for a master marker left and a non master marker right All these parameters are configurable To change the default value of a parameter simply press the left button of the mouse on the value to be configured arious Shapes 1 0 Nuclei Size Discrimination Area 1 Discrimination ray l Automatic Background Threshold Yes Virtual Channel na Post Processing Order Remove Merge Remove Labels Heo Use Merging Rules Mo Figure 315 Changing the Default Value of the Parameter This will display an editor where the user can provide a n
262. toscale On Y Axis Set Max Value For Hematoxylin Mean Intensity Set Max Value For 4xis Parameters Sek Cutoff Delete Cutoff Copy Cutoff View Backward Data Copy To Clipboard Save AS Figure 438 Histograms Paste Cutoff context menu e Delete cutoff The cutoff will be deleted by pressing the right mouse button on the diagram area and choosing from the displayed menu the Delete Cutoff option Autoscale on Hematoxylin Mean Intensity Auboscale On Y Axis Set Max Value For Hematoxylin Mean Intensity Set Max Value For 4xis Parameters Set Cutoff Copy Cutoff Paste Cutoff View Backward Data Copy To Clipboard Save AS Figure 439 Histograms Delete Cutoff context menu Page 286 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual i e Show Percents 7 see Chapter 13 3 1 13 3 3 Diagram Detail Window Both scattergrams and histograms can be opened on a separate window called Scattergram Histogram Detail Window It contains only the data related to the respective diagram To access this Detail Window simply double click on the desired diagram The advantage of the Diagram Detail Window is the wider perspective offered on the analysis At Sca etail gt eo attergram Detail gt gt Sample 01 ROI 09 gt gt Hematoxilyn Mean Intensity Ki 67 DAB Mean Intensity x X BGS 00 7 Show statistics Copy Save Zoom1 Zoo
263. ts and comments if any e The slide number of regions and comments if any For TMA blocks the number of spots will be mentioned e The region acquisition status FOV matrix size number of images objective used for acquisition FOV size and comments if any On the right side of the panel there is a preview image of the slide If a TissueFAXS project was previewed with illumination correction when importing the respective project in HistoQuest the preview image of the slide in the wizard will be shown with illumination correction If a TissueFAXS project was acquired with illumination correction when importing the respective project in HistoQuest the samples will already have applied illumination correction Select Regions section The TissueFAXS HistoFAXS project file is not enough to run analysis in HistoQuest the user must also provide the acquired images TissueFAXS HistoFAXS regions cannot be imported without the stored image files Page 21 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Moreover rather than analyzing all samples in a project you may select which samples should be included from the items that are valid and available The images of a TissueFAXS project are structured in slides and for each slide there can be several regions of interest Select Regions M Demo_Project 5 e Slide 1 B oe n cela 001 ome aa 006 GID Region 036 HD Region 039 28 Side
264. ts of 41966 Sample be Sample 01 eB Sample 02 loses D oa LL Sample 03 OOOOO1 E Sample 04 m Sample 01 o00001 o Sample 04 000001 E Sample 05 o00001 i Sample 05 oo0001 G Sample 06 ooooo1 m i Sample 06 mme i G Sample oF aes m be Sample OF B Sample 08 Sample 08 goodi Sample 09 000001 p Sample 09 o00001 m Sample 10 Oooo o be Sample 10 oomoo m E Sample 11 o00001 E i Sample 11 memm E G Sample 1 mem B be Sample 12 and 5 ae 13 T m o be Sample 13 E Sample 14 OOOOO1 m 3 i Sample 14 o00001 Sample 16 oo0004 m i Sample 16 D A amp M Y e o00001 o00001 41966 Figure 388 Raw Data window a By default the Raw Data window will display a grid with the following columns For Various Shapes 1 0 and Various Shapes 2 0 segmentation methods Region of Interest Field of View Event Label Mean Intensity parameter value for each marker For Total Area Measurements segmentation method Region of Interest Field of View Event Label Area parameter value for each marker e Delete Events deletes selected events e View Backward Data displays backward data for filtered events Exit Backward Connection closes backward connection Summary Row Sies Eal 1 4 125600 31 075890 20 904520 35 5097 70 35 195490 31 150300 32 964570 36 518420 31 375000 32 9666 70 39 084670 26 500000 a2 207500 29 002 4050 30 397 730 62 251500 270797
265. ttings Default values are filled for Project Name and Project Folder If the Project Folder does not exist the user will be asked if he wants to create it Basic Settings Advanced Settings What s the name of the project MSO Project Description Where will the project be stored F Projects HistoQuest Mirax Projects MS07 If the project folder does not exist do you want to create it Create Project Folder Copy images to project folder or leave images in their folder Figure 69 Import Mirax Project Step 2 Basic Settings If you do not want to use the default project folder provided by HistoQuest you can add the project name you desire and HistoQuest will create this project folder if you check Create Project Folder 7 The fully qualified hiqproj file name must be less than 260 characters and the project rote folder name must be less than 248 characters If the storage folder differs from the import folder you can decide if the images from your project will be used remaining at their initial location or if they should be copied in the storage folder This is realized by simply checking the Copy images to project folder option in this step of the wizard However if you move delete the original image source files your HistoQuest project might not be usable anymore data will remain though but as images are no more available also the connection of data to original images will fail and you cannot reanalyz
266. ual Type egio Name ROI 05 Sample Coloni Rows Count 1 Columns Count 1 FOVs Count 1 Exduded FOVs Count 0 Area 0 000715 mm Events Count 0 Figure 163 Add comments to ROIs Autodetect ROls This is a tool provided by HistoQuest which can help you save time and improve the accuracy of defining the area to be processed e g the section contour In order to detect the ROIs press Autodetect ROIs button from the ROI manipulation toolbar To improve the results of Autodetect ROIs option apply Illumination Correction on the sample before performing the detection wf Note The following dialog will appear The sample must already have the cache built Page 121 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Slide Preview Detection Parameters p ia aee Tissue Advanced Settings Minimum Tissue Area m 180 22 ume Exclude border connected regions Regions a E E ka 2 Detected Regions v Region Ol 11 Region ooz Options C Remove Existing ROIs L Create Group of ROIs from Detected Regions Run Selection Clear Selection OK Figure 164 Autodetect ROIs panel By default the detection is run on the entire preview image If you want to refine the results you can run the detection on a smaller area e select the desired area by drawing a rectangle on the displayed image keep the left mouse button pressed t
267. ult registration points will appear on the samples Page 96 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Paa r 5 JIR l Figure 123 Add registration point Operations available for registration points Add registration point adds a new point to previously mentioned registration points Remove double click on any registration point and from the contextual menu that appears choose Remove Point option View Event Data Copy to Clipboard Save b Figure 124 Remove registration point Page 97 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Move you can move a registration point by dragging it on the sample to the desired location You can also move all the registration points simultaneously see image below by holding Ctrl key pressed select the points with the mouse 1 the color of the points will turn from red to green and they will be surrounded by a black frame that can be rotated or moved 2 After moving or rotating the registration points 3 press Apply button in order to effectively synchronize the sample 4 1318 7 CD45 Region 002 lt a 13187 CD45 Region 002 AEE egion U egion 13 1453 m 13 1453 1 13187 CD45 Region 002 ie egion i 13 1453 m Figure 125 Moving registration points simultaneously Page 98 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual When modifying more
268. umber of Shown Exents Oo 100s Figure 228 Scattergram Properties dialog The user is allowed to modify the markers and parameters The Name the Input Gates and the Number of Shown Events options can be set for the newly created scattergrams and also modified at any time after creation Ar For any scattergram to be created the markers have to be different MOTE Different behavior of diagrams in Compare Sets e Propagation of axes properties can be done for Current Region Group or for the entire Compare Set e View Backward data from a Compare Set will not show the Results tab from the Region Detail Window Also in the region viewer the user will not be able to create or modify project items and make manual correction e Except this the behavior of axes is similar to the one described in Chapter 13 3 and Chapter 14 3 Page 158 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual the default diagrams will be set depending on the segmentation method erases a selected diagram Figure 229 Remove diagram warning message emove All erases all diagrams Figure 230 Remove all diagrams warning message changes the position of a diagram to an upper one changes the position of a diagram to a lower one 7 3 Overlay Diagrams In this form of diagrams different data sets e g different ROIs or different samples can be compared within
269. und Threshold Background Threshold Figure 356 Automatic Background Threshold Background Threshold e Use Nuclei Mask By selecting Nuclei mask the area of the binary objects in the nucleus mask is also used as measurement mask for all other channels Page 228 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Yarious Shapes 2 0 Lise Ring Mask Yes Interior Radius i i Exterior Radius Use Identified Cell Mask Outside amp Inside Max Growing Steps 4 i Skip Steps Automatic Background Th Background Threshold Figure 357 Use Nuclei Mask vf Note By pressing Reset Parameters button all the parameters settings modified by the user will be restored to their default values 11 3 2 Diagrams Configuration For this segmentation method the Results tab of the detail window will display the following default diagrams set Scattergrams Master marker Mean Intensity Master marker Area Master marker Mean Intensity Non master marker Mean Intensity Histograms Master marker Mean Intensity Non master marker Mean Intensity 11 3 3 Raw Data Columns Configuration For this segmentation method the Raw Data window will display the following default columns Region of Interest Field of View Event Label Master marker Mean Intensity Non master marker Mean Intensity 11 3 4 Statistics Configuration Please see Chapter 11 2 4 11 4 Total
270. up e Analyze Unprocessed FOVs option if a number of FOVs of a ROI belonging to a group were already analyzed in a previous analysis operation for another ROI these FOVs being common to both ROIs this command will analyze the rest of the unprocessed FOVs Show in Detail Window Analyze Rename Remove Add to Existing Group Add to New Group Copy Delete Results Propagate ROI Figure 187 Analyze FOVs option from context menu Page 134 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Region Viewer The same option is available from the Change Color Option that Q The specific color of a group can be set and changed in Tools Options General TIP appears on the right mouse button menu on the name of the group Default Locations RGE Indicator Top Left A Scale Bar Bottom Right Group of ROIs Item Name Top Left oe Exclusion Area Backward Connection RGE Indicator Scale Bar al La A Selected Contour Th E E E E E a E E Show non selectce ua a rrr E i oa i t _ Animate borders of ROIs and Annotations Highlight double dicked point from Sample i i Show grid lines for Samples imported from Folder C Show grid lines for Samples imported from TissueFANS HistoFAXS or Hamamatsu NanoZoomer P L Show name only for selected ROI Automatization a L Show name for Exclusion Area _ Indude user name in annotations contents E
271. ure 383 Color Overlay Image When the image is not processed a grid appears on the region detail window when note the marker images are accessed Page 250 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Original Hematoxylin Ki67 DAB ades Labeled Shades Overlay Color Overlay Figure 384 Grid on unprocessed image 13 2 Raw Data Raw Data for a project item Sample Region of Interest or Group of ROIs consist of a set of events and their corresponding numerical values For each event the following parameters are computed Area the area of the segmented object Minimum of Intensity minimum gray level expressed by the pixels of the object Maximum of Intensity maximum gray level expressed by the pixels of the object Range of Intensity the gray level interval max min Sum Intensity sum of all gray level intensities expressed by the pixels of the object Mean Intensity the average gray level expressed by the pixels of the object Equivalent Diameter the diameter of the disk which has the same area as the object Page 251 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Variance of Intensity the measure of the amount of variation of all the gray level intensities expressed by the pixels of the object STD of Intensity the average difference of the gray values from the mean of intensity Perimeter the perimeter of the segmented object Compactn
272. uts paraffin sections and TMAs It consists of the software modules TissueFAXS TissueQuest and HistoQuest and is used for acquisition of images in the fluorescence and brightfield mode for counting the number of positive and negative cells and for quantification of staining intensities TissueFAXSplus is used for the standardization of tissue analysis in combination with immunohistochemical and immunofluorescence staining The system does not give any direct diagnosis and there is the possibility that the samples do not contain enough information to give a clear diagnosis The results of the analysis are purely statistical values Users must reevaluate images and likelihood of the Statistical data Pure interpretation of statistical data is a high risk Observations TissueFAXS is similar to TissueFAXSplus but is for fluorescence samples only and must not be used with brightfield immunohistochemical samples TissueFAXS consists of the software modules TissueFAXS and TissueQuest HistoFAXS is similar to TissueFAXSplus but is for brightfield immunohistochemical samples only and must not be used with fluorescence samples HistoFAXS consists of the software modules TissueFAXS and HistoQuest 2 Each and any product should be used only after training performed by TissueGnostics or authorized distributors of TissueGnostics A list of authorized distributors is available here http www tissuegnostics com inde
273. ward data the corresponding events contained in the Raw Data window and in the marker images will be marked with a different color than the unselected events In order to exit backward connection press the Exit Backward Connection button Exit Backward Connection or simply close the detail window Page 305 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Forward and backward connection can also be accessed from any raw data window opened by View Raw Data option please see Chapter 13 2 Any of the operations for backward data can be accessed from detail window of Jf Note diagrams Backward data cannot be accessed from the overlay diagrams of compare sets From Raw Data you can perform backward connection only for filtered events Configure backward connection Displaying mode for selected and unselected events this can be configured from Tools Option General Backward Connection For more details please see Chapter 17 3 4 Controlling how many FOVs are considered for backward connection this can be configured from Tools Option General Backward Connection For more details please see Chapter 17 3 4 Page 306 of 351 Internal HistoQuest 3 0 User Manual www tissuegnostics com 15 Manual Correction Manual Correction menu can be accessed from the Manual Correction button of the detail window toolbar Manual correction contains the following opt
274. www tissuegnostics com HistoQuest 3 0 User Manual HistoQuest 3 0 User Manual Page 1 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Table of Contents TapE Or COON eee ene E en ee ee ee ee ee eee ee eee 2 BES CI NGS AE E EENE caste ore atl E A A A A ga cepa shi E E A 5 Notices about symbols and labels ON product ccc cceccceccceeeceeece cece eeseeeseeeseeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeseeeseeeseeegeeegas 6 Me MMP OG GTN O eaen es pe panes nae inte EE EAE EE EREA EA esd tec asia ee melee a su a E EAEE EAEE ER T eE sa 8 910 Lo E A EE E ee EAA E A E E A EE EEES T t2 CO Alor Ofthe NO ING AU ODN sainia uaa aa AN E AEREA EE EE a wate SE A Aa E aS E E ETE ER S aaa Ea aaea T 1 3 Definitions Acronyms and ADDreviatiONS cccccceccscceeeeeseeseeesecsueeseeeseeeseeeseeesaeesaeetseeseeeseeeseeteeeeeeteeeeeeees T Ee E E g e EENEI AP A neta seein S OE eee EA EIE IE E EA A A A YE 9 2A ANO TIS QS ena eE E AEA AEA E E EAA RAE E AEE A AEE EAE A 11 2 1 Application Dependencies cccccccseecceeeccseeceuceceucecuecsueeceucessueessueeseeeeeecsusesaueeseessaeeseueecaueessueessueessaeenaags 11 2 2 Windows 3 1 Installer for Windows XP SP2 cccccccceeccceeeeceeeeaeeeeeeeeseeeseeeeseeeeseeesseeeseeeeseueeseeeeseeesaneesneesaees 11 2 3 NET Framework 2 0 RUNUME oped cceisacdecueshsdecctinecitnctcindscasencendis AE A Eaa aa 11 2 4 Microsoft Visual C 2005 Redistributables 0 0 00 cece cccccceece
275. x Growing Steps Use Nuclei Mask Automatic Background Th Pir rrrernrrnrraerrrrrrrrrerrrrarrrrerrutrttrrrrttrrraerirrraigvady ie Threshold Range Oum 26 67 um 333 33H Default Apply Figure 353 Use Identified Cell Mask Max Growing Steps Skip Steps in order to reach the marker staining some additional growing steps might be required In this case the growing process will not start immediately at the border of the nucleus but outside at a distance specified by this parameter Yarious Shapes 2 0 Use Ring Mask Yes Interior Radius 6 67 Hm i Exterior Radius 13 33 pm Use Identified Cell Mask Outside amp Inside Max Growing Steps 26 67 um a Skip Steps Use Nuclei Mask kd Automatic Background Th n Threshold Range E UU UU UU UU UU a a OT OU UU a a a Uy ou om 599 334 Default Apply Figure 354 Use Identified Cell Mask Skip Steps Automatic Background Threshold Automatic Background Threshold the background threshold level can be computed automatically selecting Yes or can be set manually by selecting No and providing the desired value You can estimate the value of the background by using the mouse and enabling the RGB tool You can see the value of the gray level by holding the mouse above an area with the background This is a good starting point in estimating the best value for the background threshold Automatic Background Threshold Range you can specify the interval in which the background
276. x php load xml en home company distributors xml amp lang en 3 The names of actual companies and products mentioned herein may be the trademarks of their respective owners 4 In order to use HistoQuest application it is essential for the users to have sufficient knowledge of PC and Microsoft Windows operating system usage 5 The information from this document is subject to changes without notice 6 The information contained in this document is the proprietary and exclusive property of TissueGnostics GmbH except as otherwise indicated No part of this document in whole or in part may be reproduced stored transmitted or used for design purposes without the prior written permission of TissueGnostics GmbH 7 The information in this document is provided for informational purposes only 8 TissueGnostics GmbH specifically disclaims all warranties express or limited including but not limited to the implied warranties of merchantability and fitness for a particular purpose except as provided for in a separate software license agreement 9 Copyright 2006 2011 TissueGnostics GmbH All rights reserved 10 Release date of this document 77 June 2011 11 Document version 4 3 Page 5 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Notices about symbols and labels on product The following symbols appear on the labels of the product HistoQuest Workstation HistoQuest is a trademark of SN o ee Ag
277. xis parameter e upper right UR quadrant represents cells that are double positive for the parameters on both axes e lower left LL quadrant represents cells that are double negative for the parameters on both axes e lower right LR quadrant represents cells that are positive for the x axis parameter but negative for the y axis parameter eg The precondition for the calculation of the quadrant statistics consists in already frote defined cutoff values for the axes of the scattergram The rows of the quadrant contain information about the four quadrants and the sum overall A separate statistic is provided for the scattergrams without gates as well as for each gate that is defined Scattergram Options The scattergram toolbar a 0 eRT x is placed on each scattergram above the diagram area Page 264 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual In addition by pressing the right mouse button on a scattergram a contextual menu like the one below will appear 4utoscale on Hematoxylin Mean Intensity 4utoscale on Ki 6 DAB Mean Intensity Set max value on Hematoxylin Mean Intensity Set max value on Ki 67 DAB Mean Intensity Parameters Set Cutoff on Hematoxylin Mean Intensity Set Cutoff on Ki 6 DAB Mean Intensity Copy Cutoff Paste Cutoff Delete Cutoff View Backward Data Copy To Clipboard Save a5 Paste Gate 2 Figure 402 Scattergram
278. y this parameter specifies the number of desired color groups present in the separated image e Method specifies what distance measure is used between colors during clustering process Euclidian it is the length of the straight line between two points Manhattan the distance between two points is measured along axes at right angles gt Clustering parameters Color Space LAE Afinity Method Maximum Iterations Minimum Percent Figure 291 Clustering parameters Method parameter e Maximum Iterations the number of separation steps after the clustering is stopped Increasing this parameter yields better results but requires more time e Minimum Percent represents the percent of pixels which switch between groups of colors from the previous step to the next one If this is achieved separation stops regardless the iteration number Advanced mode for shades In order to improve the quality of the analysis HistoQuest provides a series of advanced parameters that you can access in the contextual menu that appears when right clicking on any shade Page 194 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual View Shade Image View Cray Image View Measurement Image Check All Uncheck All Remove Selected Shade Remove All Shades Advanced Mode Toolbar Figure 292 Shades contextual menu View Shade Image if you want to spot a certain shade on your image this option will display the gray
279. your project or just a certain part of it For more details please see Chapter 16 2 from the current manual Print Preview this option gives you a preview of the information you are about to print For more details please see Chapter 16 2 from the current manual Exit choose this option if you want to quit HistoQuest 17 2 Project Menu In the main toolbar press Project to display the main menu Help Project Properties Project Summary Analyze Build Cache View Raw Data Delete Cache Delete Image Results Delete Project s Results Finalize Analysis Illumination Correction Figure 496 Main menu The main menu includes the following items Project Properties displays the Project Properties dialog for more details please see Chapter 4 6 from the current manual Page 324 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual Project Summary is a feature that displays in a single dialog a series of data for all the regions in the project status number of FOVs number of events number and type of regions The main utility of the feature is that it offers overview of the data concerning the project items Project Summary Project Ki 67 Project 1644 Fws 55161 Events 3 ROIs Sample Sample 01 Partially processed 13 out of 143 Fons 145 Fos 31166 Events 2 ROIs Groups Region OF Interest ROI 09 Processed 9 out of 9 FOys 9 FOYS 12466
280. ype of the shape in which you want to transform re Figure 221 Transform Shapes panel Now select the output type of shape in the lower part of the panel then press Transform selected shapes button in order to effectively accomplish the operation Item s contextual menu in the Project Browser In the Project Browser right click on the desired item in order to access the contextual menu Show in Detail Window Analyze Analyze Unprocessed FOVs Rename Remove Add to Existing Group Add to New Group Create Annotation from ROI Create Exclusion Area from ROI Copy Delete Results Propagate ROI ROI contextual menu Create ROI from Exclusion Area Create ROI from Annotation Create Annotation from Exclusion Area Create Exclusion Area from Annotation Exclusion Area contextual menu Annotation contextual menu Figure 222 Contextual menus for ROI Annotation and Exclusion Area in Project Browser Page 152 of 351 Internal www tissuegnostics com HistoQuest 3 0 User Manual The following options will be available For ROls e Create Annotation from ROI e Create Exclusion Area from ROI For Exclusion Areas e Create ROI from Exclusion Area e Create Annotation from Exclusion Area For Annotations e Create ROI from Annotation e Create Exclusion Area from Annotation Once you will select one of the options above the chosen shape will automatically transform Annotation

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