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TMA Module for MIRAX Viewer 1.12 User's Guide

1. eeeeennnnnnn nnn 35 6 Exporting Data and IMagES arerin a a aaa 35 oM m doeintucMDB iw 35 6 1 1 Exporting Score Values to 1 Column eeeeerreennenne 3 6 1 2 Exporting Score Values to 2 Columns eeeeeennennn nnne 38 6 1 3 Exporting Score Values for Generating Allred Values 39 6 1 4 Exp rting Clusters Mt AX BS esti asas aeu PEEIREDURDUDENDEEF PAR PR ERU d EOD 41 6 1 5 Exporting Score Values for Cluster Analyzing 42 6 1 6 Exporting Score Values to R Software cceeeeeeenn nnn 42 5 2 EXPOrUNO IMAGO Seer arininn AEE AE EE EEEE EEE 47 7 Analyzing the Data Exported from TMA Module Cluster Analysis 48 June 11 2009 3DHISTECH Ltd 2 49 TMA Module for MIRAX Viewer 1 12 User s Guide Contents Disclaimer Copyright 2001 2008 3DHISTECH Ltd All rights reserved The following patents are pending for 3DHISTECH products WO 2006 0303260 P0700409 and 11 826 752 3DHISTECH Ltd is not liable for damage of whatever nature including but not limited to general or specific damage indirect damage consequential damage or incidental damage including the results of the analysis of the digitized slides for example change of health status related to erroneous diagnosis from the digitalized slide s that stems from or is associated with use of Product digitalized sli
2. Lookup table Output File path D Test_Folder test R Reclassing ranking Visualization Stats Heatmap R Script shading generating Mosaic Plat hel Association Flot Independence Test Pearson s Chi squared Test June 11 2009 3DHISTECH Ltd 45 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring R Script The generated R script is displayed To save it into the formerly defined folder and file click Finish Lookup table Reclassing ranking colName list c 1 48 cf Unique TMA Sample ID TMA Block ID Donor Block ID eee Organ Diagnosis Grade Sender institute Survival Stage Metastasis X Visualization stats B Catenin Cox Z EGFR Ventana p53 SP5 R Scipt tmadata lt aray data 0 dim c 48 15 dimnames colName EUER tmadata 1 2 c 1 Block1 1722 02 liver cimhosis GO Szombathely tmadata 2 c 2 Block1 546 D4 breast DC G2 Bay TOTT OS I 1 11 9 6 tmadata 3 c 3 Block 1 546 04 breast IDC G2 Bay n mom aT py g tmadata cid Block 1 210 04 breast Mucinosus carcinoma G2 Bay oe a REV 3 x4 dU tmadatal5 J c 5 Block 1 210 05 breast IDC G3 Bay TOTT TT nn 1 UPS 713 tmadata b c amp Bock1 16434M breast IDC G3 Bay nn m ae 7 Block 1 14164M breast ILC
3. G2 SE LPatholey 2 0 15 14 Block 1199 02 breast IDC G3 SE LPatholoy O 16 15 9 17 16 Block 657 03 breast connectivdGO Bay p2 a 18 17 Block 1222 03 breast IDC G2 Bay HT 2 19 18 Block 1324 03 breast ILC amp 1 Bay p2 0 20 19 Block 1643 04 breast IDC G2 Bay HT 21 20 Block 1643 04 breast IDC G2 Bay ft 9 22 21 7 23 22 Block 1877 02 breast__ ILC_ G2 NatInstCancer 0 2 24 23 Block 988 03 breast IDC G2 Bay a 25 24 Block1 1023 03 breast IDC GO Bay ft 2 26 25 Block 210 05 breast connectivdG3 Bay 1 0 27 26 Block 1643 04 breast lLC G2 Bay ft 2 28 27 Block2 1416 04 breast IDC G2 SE L Pathdlogy O 29 28 Block 1610 04 breast IDC G3 _ S E I Pathology 0 7 30 29 Block 128 05 breast IDC G3_ _ S E l Pathology 0 31 30 Block 774 02 breast DCIS G2 S E l Pathology 0 0 32 31 2 June 11 2009 3DHISTECH Ltd 39 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring Allred Value Intensity Score Value Rate Score Value For example in the case of 8 4 Rate 2 Intensity 6 Allred Bh T Ex WW ee ee on WENT UN ae m Rr nrimn reme Se ia n Lr Ta mna Stains Scoring Scheme Type Selected Stain Simple Load Scheme Combined Save Scheme Frequ
4. D mc L WIE at s g a E d s L p S E i n T n TA o 3DHISTECH TMA Module for MIRAX Viewer 1 12 User s Guide June 11 2009 TMA Module for MIRAX Viewer 1 12 User s Guide Contents Contents Becan ete ee TTC TTE 3 Font Types and Symbols Keyboard Quick RefErenCe cccccseseeeeeeeeseeeeeeeeteeetenteeas 4 1 About the TMA Module for MIRAX Viewer 1 12 cccccccce cem mnm mnn 5 1 1 Overview of the TMA Module for MIRAX Viewer 1 12 een nm 5 1 2 New Features of the TMA Module for MIRAX Viewer 1 12 eere 6 2 Installing TMA with MIRAX Viewer 1 12 cesse nnnm hne 7 2 PPEFEOQUISICOS eee en eee ee et NDS NU MM E IU MSIE I ee ee 7 2 2 Installing the Application sssesnsssssnnsnsnnnnnnsnrunsnsnnnennnnnnnnenrnnnnrnnnernnnnnnennnn 7 S Serung UP a IMA JPEFOIGCL ees mnia ETER EEA EAA BERETA 8 3 1 Collecting Project Data in an Excel Worksheet and Building a TMA 8 3 2 Creating a New WIMA PEOIBCLicus anm acrtingUrexED dnd crane s dURdUx dua das PDC EE S DP ETS EDS 10 3 1 Berore YOU SE al uesaseebutiiedaxrE PESE UIDES IAEA XR UE RU Dead NE SURE RU 10 3 2 2 Creating a New TMA Project ssseesseeeeem mn mmn 10 3 2 OPENING dd PEOR ossoeciirunadn CH miS E eU RE PEDI DEI renee Pug cM ceed 13 Sm DIGS SClCCUON lt T TT TETTE 14 9 9 ROMOV PTO T wenevccetecsxan staan ecesetnsceeteccaatesssetevencne EESEENEMN UNITS INDEED PESE 15 3 6 Managing MA
5. kA Colec EGFR Vent Erbit B 1 Update Tree 3 Enter a name for the new project in the Project name text box then click OK As it cannot be modified later be sure to incorporate all the necessary information into the name of the new project The TMA Project will be located within the folder of its slides which can be visualized after pressing Update Tree in the bottom left part of the Mirax Viewer screen Project name Test Frapect Cancel June 11 2009 3DHISTECH Ltd 11 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project 4 Di 6 ad b June 11 2009 The TMA Project Wizard dialog appears User Management dialog Since the software offers parallel scoring for several users enter a user name in the User Name text box then click Next The user will score the slides If you need a multi user project you can add further users later on when the project has been created and you first start the project Slide Selection Select the appropriate slides for all the TMA slides e The Path text box displays the path for the TMA slides e To select all slides click ca e To deselect all staining types click al The slides that you leave out are not removed from the file system You can also add them or new ones to the project later HE e To display the thumbnails for the TMA slides click wl To display the TMA slides in list view click ml After selecting the appropria
6. 2 Calcc B Cat Szl B2 fen Calcc Cox2 Erbil B1 sn Calcc Cox2 Erbit B2 fen Calcc Cox2 Szl B1 feen Colec Cox2 S21 B2 e Calcc EGFR Vent Erbit B1 Colec EGFR VentE rbit B2 faras Calcc EGFR Vent Szl B1 sn Colec EGFR Vent SZI B2 E Colec p53 5 P5 E rbit B1 A Colec p53 SP5 Erbit B2 ns Colec p53 SP5 S21 B1 fe Calcc p53 5P5 Sz B2 g Test Froject Remove Project from Tree Colec B Cat Erbit 61 3 6 Managing TMA Projects Having started a new TMA project or opened up an existing project the function buttons available for project management are listed on the top of the panel H Important You can only see all function buttons if it is a one user project or if you are signed in as Consolidate user in a multi user project If you are signed in as another user in a multi user project you cannot access all functions E Galery _ Allows for creating TMA spot galleries according to any filter options represented by each column in the Excel Lookup file June 11 2009 3DHISTECH Ltd 15 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project Cehimport _ Allows importing database from Excel in XLS format La Export _ Allows exporting database to a new Excel file merging original Lookup table data with the newly generated scoring data A Allows opening the selected slides adding or removing slides and creating either simple or combined schemes Look at the details in chapter 4 1 Always put new slides in
7. B Catenin 2 T B Catenin 10 B Catenin 5 1 Function Buttons In the left upper window corner 2 I B Catenin 0 54 B Catenin 8 B Catenin 11 B Catenin 4 Scoring LAM P A 4 ant E ad n 2 iP C a B B Catenin 12 B Catenin Desired spot magnifications can be set up with the Zoom panel to fit on the screen There are 5 function buttons below the Zoom option e To initiate gallery spot collection click Aj Click this button every time after you have changed the conditions to update the gallery Ba e To allow sequential scoring click e To allow saving of the selected image collection click j June 11 2009 3DHISTECH Ltd 32 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring e To display the spots in their original position on the slides click matrix view This function is only active if you have filtered for only one slide or you are using a one slide project e To activate the gallery zoom window click 4 e To position the gallery zoom window click on the window with the left mouse button and drag it to the desired position e o shrink or enlarge it use the mouse scroll wheel e To zoom in or out use the scroll bar 5 2 Labels Below the function buttons Label I Sample ID Iv Stain I Score Slide name TMA Block ID I Dr Brown s Score Value From the La
8. GZ Bay ag m di iot a1 tmadata 8 ceg Black 1 i T610HM breast IDC comedo Gz Bay Er TM ais iae 1m T E ar 1 ar 16 imadata S z cia Block1 128 05 breast ADC metastasis poor Bay pyp cag o Gn 1 g g 10 imadata 10 c 10 Block1 774 02 breast connective tissue GO S E I Pathology uiro Gags ahr a A p um 2 107 tmadata 1 1 J c l t Block 1 559412 breast IC G3 SE Pathology T Hai pb 10 ar 5 imadsta 12 c 1 Block1 322 02 breast IDC G2 S E I Pathology Project for Statistical Computin Getting Started e R is a free software environment for statistical computing and graphics It compiles and runs on a wide variety of UNIX platforms Windows and MacOS To download R select June 11 2009 3DHISTECH Ltd 46 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring your preferred CRAN mirror If you have questions about R like how to download and install the software or what the license terms are read our answers to frequently asked questions before you send an email PCA 5 vars princomp x data cor cor Fertility 1 3 60 Clustering 4 groups Factor 1 41 Factor 3 19 F Examination 0 60 40 20 This server is hosted by the Department of Statistics and Mathematics of the WU Wien 6 2
9. ILC G2 Natl Inst Cancer 25 23 23 Block1 988 03 breast IDC G2 Bay 26 24 24 Block1 1023 03 breast IDC GO Bay 27 25 25 Block2 210 05 breast connective tissue G3 Bay 28 26 26 Block2 1643 04 breast ILC G2 Bay 29 27 27 Block2 1416 04 breast IDC G2 S E I Pathology 6 1 6 Exporting Score Values to R Software June 11 2009 Ol xS En C5V Far Cluster Analysing Eo R Scripk 3DHISTECH Ltd G D 4 Scoring This type of Exporting generates a special txt file for the TreeView software see below It automatically generates a GWEIGHT column and an EWEIGHT row The columns selected by the user are displayed in one column each columns separated with a symbol The points related to different stainings are displayed in separate columns The file can only be imported in this format to the TreeView software for further processing d GWEIGHT B Catenin Cox 2 EGFR Ventana p53 SP5 Hm HP Hm HB H HB HB HB kB iB iR m HR HF HR HI IB HB HB HB I HB HB H HB HH oloimOioimioim ioleiuim iNimioiv eiolweioi ioimivuimeivNimimi Nv aM E F G 1 1 1 i 2 3 11 9 6 ri 9 9 3 7 10 7 2 13 3 1 7 3 2 12 6 1 16 9 0 10 5 2 10 10 7 5 2 1 0 0 3 1 6 1 9 8 8 7 4 2 10 2 0 16 1 1 11 10 9 2 6 7 9 7 2 14 3 1 2 2 8 6 0 11 11 2 10 12 6 R is a statistics producing software that is able to predict an estimated life expectancy
10. PFrOlECUS ss soa cs ieu od Eoo RRERIERROER EPIPRPuPIRETO EEESPORA P IDEE ISRrP XE EE 15 3 7 Managing TMA Slides in a Project sssssssseeeesee nenne nhe enhn nnns 16 3 8 Linking Excel Data to TMA Spots cccc cece ececeee cece eee eeeee eee nennen nnns 18 3 9 Finding TMA SDBOLS is erassesapaxbtun 22 cucscsaaiers racseabandevesteimbesnviestervatarsieieesnenee es 18 3 10 Adjusting Annotations in the MIRAX Viewer 1 12 eee e ee ee nnn 24 A COMM Cie gaamc sane ee Sacee 25 Ai eegjsEeri oe UU 25 4 1 1 Simple Scoring SCHEIN GC iccninntetinaadrataseraedandaensnshehecneieensenexaraseenes 25 4 1 2 Combined Scoring SCNEME cccccceseccecseuctecuctecuaeseuteneunenauress 26 4 2 Scoring UMA DOlS cascencascas sonncecasecescaseeaaasstacoaecacansoanes teensy soeeesvocanseannactess 29 AZ SCONE IMG SZ TDI 29 42 2 Sequential SCONO suse ketrERFREAFESEQENR EQUES PREDA RECS FIN RR EISE 30 4 3 Consensus AUTOS CON Oe cocccccaccuccaszceaaessceatsctonasecoscencasacetotecegscunrseecstcansaseetes 30 5 Managing Spots in the Gall P usssamuvncema 1999 QN PRDSP ERU AUNOSUN I EXO IUE MESURE DEDERE ENG ers 31 SM 8gresreisiereg E ET m E TT 32 DaZ E OPE 33 DO PiLek Pale Pet Ol Siastaacenvetnenecssaunentaneaananeaniumensarnenuiieasembeanarabedenesmannantneet 34 Sd FUCINO FOF Au U CIO masir nirna send E DIPEUEN DUNS Ms MESE QUEN Urat Pe E 34 5 3 2 Filtering Only For Consolidate User
11. analyzes on slides digitized with MIRAX scanner microscopes With this module you can digitally inspect and score stained tissue micro array TMA slides on your computer screen After you scored the slides you can link the image files with pathological and clinical data in a shared database and further submit them to statistical cluster or Survival analyses H Important Slides digitized with other scanner microscopes have not been tested with MIRAX TMA module Therefore the features described in this document are fully supported only for slides that have been digitized with MIRAX SCAN MIRAX SCAN2 MIRAX MIDI or MIRAX DESK 1 1 Overview of the TMA Module for MIRAX Viewer 1 12 The role of MIRAX TMA Module software in the workflow of a TMA project is shown in figure below IHC immunohistochemistry FISH fluorescence in situ hybridization Tissue Microarray TMA Project TMA sections IHC FISH Mirax Scan Virtual slides Mirax TMA Module Ja Nis ri Paraffin Blocks amp H E sections NNI ERO Histological Data Diagnosis v Clinical Data ki Significance WENNS Image Statistics Cluster Export Chi Fischer exact Univanate Kaplan Meier Log rank test Multivariate Cox regression etc June 11 2009 3DHISTECH Ltd 5 49 TMA Module for MIRAX Viewer 1 12 User s Guide 1 About the TMA module for MIRAX Viewer 1
12. may be full name of the detected antigens for example with the given clone used In the block column that is Block1 for TMA Block 1 Block2 Block3 for further blocks in the project one must use the very same labels which had been used for labeling the given slide at scanning In other words a slide in each TMA block column is best defined by the serial number of the slide the applied marker and the TMA block ID that is the slide named 12 E Cadh is the 12th section of Block2 block stained for E Cadherin marker June 11 2009 3DHISTECH Ltd 9 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project A B C 1 Stain Block1 Block 2 B Catenin Calcc B Cat Erbit B1 Colcc B Cat Erbit B2 3 Cox 2 Calcc Cox2 Erbit B1 Colcc Cox2 Erbit B2 4 EGFR Ventana Calcc EGFERE Vaent Erbit B1 Colcc EGFE Vent Erbit B2 5 p53 SP5 Colcc p53 SP5 Erbit B 1 Calcc p53 SP5 Erbit B2 5 T M 4 gt M Lookup Blocki Block2 Slides 2 g For further instructions on slide scanning please refer to the User Guide of MIRAX Scan H Important For identifying the position of consecutive sections in Z dimension depth it is essential to use serial numbers in front of the slide labels when scanning with the MIRAX Scan see in column Block1 It is also useful to select the same order of sections for immuno staining different markers in different blocks for example E Cadherin is followed by EGFR etc in each block However t
13. on the base of the exported to it Score Values using its enormous fact database 42 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring Lookup table Select the points the criteria of the statistical analysis from the panel then mark the needed variables for the statistical calculations When finished click Next Lookup table Outcome Unique TMA MA Block ID LM DENEN ID eet ID Reclassing ranking Diagnosis 1772 02 Visualization Stats _ Block 1 546 04 R Script Variables B Block 1 545 04 Unique TMA Sample IC 4 Bed O nm TMA Block ID 5 Bokt 210 05 eE Bok esm F s mer eos Lom 222 02 131502 wer nes k re nt cea June 11 2009 3DHISTECH Ltd 43 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring Reclassing ranking The software displays an arrangement of the chosen criteria that you can re arrange Make an order of importance for these criteria To enter new values or to modify them double click on the field When finished click Next Lookup table Diagnosis value Reclassing ranking Class Visualization Stats R Script June 11 2009 3DHISTECH Ltd 44 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring Visualization Stats Define the folder and the file to where you want to save the R script If you want to visualize the statistical results mark tick the options Heatmap Mosaic Plot Association Plot When finished click Next
14. Exporting Images Using the Export Images button in the Gallery allows for saving TMA spot image collections either in JPEG BMP TIFF or PNG formats or as an HTML page in JPEG Save ko HTML Save to Picture s June 11 2009 3DHISTECH Ltd 47 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring Images can be saved at arbitrary magnifications and new directories can be defined Save to Image s Dest path Magnification Image format 7 Analyzing the Data Exported from TMA Module Cluster Analysis For Cluster Analysis without using the Deconvoluter program the 4 scale system above 2 negative 0 equivocal 1 weak moderate positive 2 strongly positive has to be used At Saving Data to Excel the program will convert them into a format which can be loaded and analyzed in the Cluster program for example version 2 11 see below The modifier columns intended to appear in TreeView for example version 1 60 see below are also merged into one column and all other columns except those with the scores are deleted The modified Lookup table above is saved as a text file data divided by tabs which can be Loaded into the Gene Cluster program Average Linkage Clustering Correlation uncentered under the option of Hierarchical Clustering will result in relation analyses which can be imported and viewed in heat map and dendrogram representations using the TreeView program June 11 2009 3DHI
15. Values for Cluster Analyzing B 1 UID NAME 2 EWEIGHT 3 1 1 Block1 1722 02 liver cirrhosis GO Szombathely 4 2 2 Block1 546 04 breast IDC G2 Bay 5 3 3 Block1 546 04 breast IDC G2 Bay 6 4 4 Block1 210 04 breast Mucinosus carcinoma G2 Bay T 5 5 Block1 210 05 breast IDC G3 Bay 8 6 6 Block1 1643 04 breast IDC G3 Bay 9 7 7 Block1 1416 04 breast ILC G2 Bay 10 8 8 Block1 1610 04 breast IDC comedo G2 Bay 11 9 9 Block1 128 05 breast ADC metastasis poor Bay 12 10 10 Block1 774 02 breast connective tissue GO S E I Pathology 13 11 11 Block1 659 02 breast ILC G3 S E Il Pathology 14 12 12 Block1 322 02 breast IDC G2 S E I Pathology 15 13 13 Block1 1315 02 breast IDC G2 S E I Pathology 16 14 14 Block1 1199 02 breast IDC G3 S E I Pathology 17 15 15 Block1 4761 02 breast DCIS GO S E 1 Pathology 18 16 16 Block1 657 03 breast connective tissue GO Bay 19 17 17 Block1 1222 03 breast IDC G2 Bay 20 18 18 Block1 1324 03 breast ILC G1 Bay 21 19 19 Block1 1643 04 breast IDC G2 Bay 22 20 20 Block1 1643 04 breast IDC G2 Bay 23 21 21 Block1 1826 02 breast DCIS GO S E I Pathology 24 22 22 Block1 1877 02 breast
16. and clinical data you must first Import data stored in the Excel TMA database file by choosing the corresponding XLS file The software will automatically import all information from the Excel file v Tip TMA Module does not support Excel files newer than 2003 xlsx If you have Excel newer than 2003 you must select in File Save as the following file type Excel 97 2003 worksheet xls 3 9 Finding TMA Spots G ssh Spot Finding on Slide D Slides TMA Histogenex BF_TMA_full_Pike421 C eS e x l E Slide List 3 p m BF TMA full Pike421 4 Q S dr 2 amp ate AN wu Vu Slide Type Q9 Brightfield Fluorescent 77 6006099 5 Slide Rotation 1 8 s 25 ry Pa r g o Cue s Background Threshold 230 a DIOBA G Spot Diameter mm x D 2 00 A BOROS Columns Rows 2 2 T oeo0o0 Save to slide 9060670099 Number Manager E LES 7085 a 600 c 6 3 Finish Cancel An t c 288266 us E June 11 2009 3DHISTECH Ltd 18 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project Having started a TMA project the first task is to identify the individual spots on a TMA Slide by choosing the Spot Finding option which opens up a new window Note that without spot identification no further options of TMA management are accessible The main function of Spot Finding is to link stained tissue spots to relevan
17. profiles June 11 2009 3DHISTECH Ltd 8 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project A B C D E F G H 1 A B C D LELF G X1 X2 X3 X4 2 Y1 3 Y2 4 Y3 5 Y4 6 Y5 7 Y6 Eo gn om F b Ro M 4 H Lookup Block1 Block M 4 H Lookup Blockl Block Either using a manual or automated arrayer a TMA map should be designed for each block to show the location and orientation of tissue cores according to the TMA block ID Block1 Block2 Block3 etc This map will be used later at Spot Finding The serial numbers in the TMA map correspond to the Unique TMA sample ID of the Excel data sheet If there are clusters of cores within a TMA block they should also be numbered on the same basis including all clusters of a block into the same worksheet as they are arranged in the block v Tip The core behind the red label in the corner of each TMA block map is an orientation tissue easily differentiated from the test tissue type for example liver However one can use any other ways for easily recognizing the original orientation of the TMA section Leaving out cores may be an option but please consider that the depth of the cores is different and some may be used up at cutting before others which also result in missing cores The third worksheet in the Excel database file identifies stains used in the particular project In the Stain column one can indicate the official
18. the folder where all the other slides in the project are Consensus Auboscore l This button appears only for Consolidate user to make possible the comparison of the of the ordinary users scores ve ER Hd Matrix view Sorts slides in the following order Each row represents one TMA block displaying consecutive sections Z dimension depth Each column lists sections of consecutive blocks stained for the same marker below one another 3 7 Managing TMA Slides tn a Project Having started a project there are four functions to select from the pop up menu when clicking on a slide with the right mouse button ai TMA Test Project Consolidate ESEEGE W Manage fg Spot Finding ed Gallery OR Import L3 Export A3 Matrix view Bb Consensus Autoscore E Spot Finding Open Slide Marked Remove Slide s Colcc B Cat Colcc Cox2 Erbit Colcc Cox2 Erbit Colcc EGFR Colcc EGFR Erbit B2 B1 B2 Vent Erbit B1 Vent Erbit B2 Block2 Block1 Block2 Block Block2 B Catenin Cox 2 Cox 2 EGFR Ventana EGFR Ventana June 11 2009 3DHISTECH Ltd 16 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project e The Spot Finding option allows for identifying and linking to Excel data of each Stained spots within the given TMA slide block e The Open Slide option allows for opening a slide in the MIRAX Viewer software e By selecting the Marked option you can indicate with a green tick labe
19. xlsx If you have Excel newer than 2003 you must know that although the file extension will be xls but the file structure will be according to XIsx file extension If you have Excel newer than 2003 you must select in File Save as the following file type Excel 97 2003 worksheet xls After finishing scoring the database including all imported data of the patients added with the scores of the TMA spots can be exported into either a new Excel file XLS or a CSV format by choosing the Export option from the project menu Having chosen the Combined option in the Scoring Scheme Type panel the user has got the following possibilities for exportation click Export then select XLS option 3 Export EZ Matrix view L Consensus Autoscore 1 column to CSV 2 colurnn for Cluster Analysing Allred to R Script Clusters June 11 2009 3DHISTECH Ltd 36 49 TMA Module for MIRAX Viewer 1 12 User s Guide 6 1 1 Exporting Score Values to 1 Column It makes possible to save only one Score Value to each spot A B C D E F G H Unique TMA TMA Donor Sample ID Block ID Block ID Organ Diagnosis Grade nder instit Survival Stage 1 Block1 1722 02 liver cirrhosis GO Szombathely 2 Block 546 04 breast IDC G2 Bay S Block 546 04 breast__ IDC G2 Bay 4 Block 210 04 breast MucinosusG2 Bay 5 Block 210 05 breast IDC G3 Bay 6 Bloc
20. 12 1 2 New Features of the TMA Module for MIRAX Viewer 1 12 e Spot finding Batch mode Storing profiles if using several slides the software saves your previous settings Grid retorsion according to rows and columns Grid retorsion according to spots Manual anchoring for spots Activating grid Deleting grids the last grid will be active Reverse spot numbering e Adding new slide to an existing project e Image export without database import June 11 2009 3DHISTECH Ltd 6 49 TMA Module for MIRAX Viewer 1 12 User s Guide 2 Installing TMA Bundled with MIRAX Viewer 1 12 2 Installing TMA with MIRAX Viewer 1 12 TMA is not a standalone application you need MIRAX Viewer to run it Therefore you have to install MIRAX Viewer before installing the application 2 1 Prerequisites Tools to be Used Description of Prerequisites Software MIRAX Viewer version 1 12 Runs on Microsoft Windows XP and Vista 32 Business TMA Module Excel Export Microsoft Office or Open Office Hardware Recommended Pentium C2D DualCore 2 GHz 2GB RAM Minimum Pentium P4 3GHz 1GB RAM Minimum Disk Space 110 MB Mouse with scroll wheel 2 2 Installing the Application 1 Start the MIRAX_Viewer exe file that you received on a DVD or downloaded in a compressed file 2 Follow the instructions in the Installation Wizard 3 Start the 3DHISTECH_Apps exe file that you have purchased Make sure t
21. 3 LL HE F PI Spot numbering To access the Numbering menu click bimbe bagage Automatic Reverse Automatic from DataBase Clear Numbers e Select the Automatic option if the spot map is regular without any rows or column of spots missing and the numbering starts from the upper left corner of a TMA slide June 11 2009 3DHISTECH Ltd 21 49 TMA Module for MIRAX Viewer 1 12 User s Guide Slide List Colcc B Cat Erbit B2 B Slide Type 3 Brightfield Fluorescent Image Zoom Level 5x U Slide Rotation 2 3 p Background Threshold 230 Show B Spot Diameter mm 2 57 i uod Grid Columns Ha Save to slide Frev Slide Next Slide 23 Spot Finding on Slide D tma Colcc B Cat Erbit B2 ae SEDI 7 E T ET HE g L TH cT 3 e eae ds b ec 3 Setting up a TMA Project Fg fon Ex e For reverse order numbering select the Reverse Automatic option e Select the from DataBase option to open a window showing the original spot map from the Excel database file Block1 Block2 and so on Identification of the spots can be done by clicking on one of the corner numbers in the Excel spot map and dragging it over the corresponding stained spot drag and drop Having repeated this by pulling over the number from the uppermost opposite corner and placing it to the opposite corner of the TMA slide will result in the desired numberin
22. ADVISED OF THE POSSIBILITY OF DAMAGE AND ON ANY THEORY OF LIABILITY ARISING OUT OF OR IN CONNECTION WITH THE USE OR PERFORMANCE OF THIS SOFTWARE Further Information For the latest information on 3DHISTECH products and services visit http www 3dhistech com June 11 2009 3DHISTECH Ltd 3 49 TMA Module for MIRAX Viewer 1 12 User s Guide Font Types and Symbols Font Types and Symbols Keyboard Quick Reference Font Types and Symbols Example Words or characters that appear on the screen These include field names screen titles pushbuttons and menu names paths or options Keys on the keyboard For example function keys such as F11 or the Ctri O key combination Example Cross references to other documents A Warning Indicates that you need an additional module to use the feature or that there are prerequisites for the task i Important Contains an important piece of information or a recommendation The application will work if you choose not to follow the recommendation but its performance might be less than optimal w Tip Contains a suggestion about using the application in some other way or to some interesting purpose June 11 2009 3DHISTECH Ltd 4 49 TMA Module for MIRAX Viewer 1 12 User s Guide 1 About the TMA module for MIRAX Viewer 1 12 1 About the TMA Module for MIRAX Viewer 1 12 The TMA Module for MIRAX Viewer allows you to perform molecular morphological
23. O ND 12 Block1 322 02 breast IDC G2 S E I Pathology 13 Block 1315 02 breast IDC G2_ S E I Pathology 14 Block 1199 02 breast IDC G3 S E I Pathology 15 Block1 4761 02 breast DCIS GO0 S E LPathelggy 16 Block 657 03 breast connectiveCO Bay 17 Block 122203 breast_ IDC G2 Bay 18 Block 132403 breast ILC Gl Bay J 19 Block1 1643 04 breest IDC G2 Bay p BERE ah mh mh an amp eet esi fas a fee O ONI 20 Block1 1643 04 breast IDC G2 Bay 21 Block 1826 02 breast DCIS GO SE LPathology 22 Block 1877 02 breast lC G2 jJNalinstCaner 23 Block 988 03 breast IDC G2 Bay 24 Block 1023 03 breast IDC GO Bay J 25 Block2 210 05 breast connectivdG3 Bay J 26 Block 1643 04 breast ILC G2 Bay 27 Block 1416 04 breast_ IDC G2 S E 1 Pathology 28 Block 1610 04 breast IDC G3 jSE LPathlogy 29 Block 128 05 breast IDC G3 SE LPathology 30 Block 77402 breast DCIS G2 S E Pathology 31 Block 1315 02 breast connectivdG2 SE LPathology amp e9esmmss mT SN 3 616 OlS ch ld NS June 11 2009 3DHISTECH Ltd 41 49 TMA Module for MIRAX Viewer 1 12 User s Guide 6 1 5 Exporting Score
24. STECH Ltd 48 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring Gene Cluster Check for update at http rana st BI Tree View CurmreniHode HODET3K Correlation 0 3273 730468 75 Fleloaded iC Donum and Seins T borvisptaf TIR FleFomnat Help BREAST xoc Test D ecesvolute Hob E fed Jot Mame TMAC BREAST cc Tecteccoiote FobndOT Feed Hara Dasa han nm see Fiber Daha Aust Data Horeke Cisterna K Meant Clustering Sel Ceganing Maps PEA Lr ris Juri FW Gerar Anm FP Dnie F Chutes L o Cake Saves me sa tet at ae i 2 231 D ANNE E p z ks c D ae Ch smi t ld 8 2 Ba bi LETIS Ed LH L4 ab n a E Dia aria D haere sy Aveuge Uriage Cheer Complete Linkage Chastang Binge Linkage Lasten Er ur PPP EE CEE EE EER FE errr FRESE ERR REEF Se BF amp am wo ctor om Eee T m mom Recommended software tools used for Hierarchical Clustering Analysis of TMA data may be downloaded freely from the following web pages TMA Deconvoluter 1 11 http genome www stanford edu TMA download shtml by Liu CL Prapong W Natkunam Y Alizadeh A Montgomery K Gilks CB van de Rija M 2002 TMA Deconvoluter 1 11 software by Software Tools for High Throughput Analysis and Archiving of Immunohistochemistry Staining Data Obtained with Tissue Microarrays American Journal of Pathology 61 1557 1565 The conversion was done with the TMA module for MIRAX Viewer 1 12 TMA Cluste
25. ast IDC G3 SE L Pathology 31 30 Block 774 02 breast DCIS G2 SE LPathology 32 31 Block 1315 02 breast connectivdG2 S E l Pathology ceo coc ONI June 11 2009 3DHISTECH Ltd 4 Scoring 37 49 TMA Module for MIRAX Viewer 1 12 User s Guide 6 1 2 Exporting Score Values to 2 Columns 4 Scoring It makes possible to save the Score Values of a given spot recorded on a 2D scale into two separated columns This export type is only valid if the slide was scored with a Combined Scoring Scheme A B C D E F G H Unique TMA TMA Donor Sample ID Block ID Block ID Organ Diagnosis Grade der instit Survival B Catenin Onan ON cm oe 10 Block1 774 02 breast connectivdGO S E I Pathology h en ens pes Ac Nm 14 Block 1199 02 breast IDC G3 SE l Pathology ASRS TAS TAS oe oe oe ok oe WIN OC AN DN 23 Block 988 03 breast IDC G2 NO NO nn 25 Block2 210 05 breast connectivdC3 Bay No NS Oo 27 WO Co CO NM NM M OO amp 32 Block 1199 02 breast IDC poor S E LPathology eo 4v June 11 2009 3DHISTECH Ltd i Block 1722 02 liver cirhosis GO Szombathely 2 2 Block 546 04 breast IDC G2 Bay M S Block 546 04 breast IDC G2 Ba
26. ation of scores by accepting values identical at all assessors of by choosing the median values of all scorers to be June 11 2009 3DHISTECH Ltd 30 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring registered into the score boxes of the consolidator All scoring data are accessible and can be used for filtering in the Gallery The Score label represents the person working under the Consolidate option The order of scores given by different users seen above the gallery spots is the same as their order under the Labels option left hand panel Ep Consensus Autescore Only Identical Lower Median Upper Median 5 Managing Spots in the Gallery In the Gallery stained TMA spots can be sorted into restricted groups irrespective of their physical location for example different glass slides The order of the spots can be defined according to their staining score values slide names spot IDs and parameters which were defined in the database listed under the Filters option lower left panel You can arrange the gallery based on primer and seconder features i Important After changing settings click a to update the gallery June 11 2009 3DHISTECH Ltd 31 49 TMA Module for MIRAX Viewer 1 12 User s Guide E TMA Black ID E Dr Brown s Score Value Order by Primary Disabled 7 Secondary Disabled Slide m Spot C Stain Score of Consoli v J Database ms 14 B Catenin 2 4
27. bels panel one can select what information to be shown above the spots in the gallery There can be as many as the number of patients related feature groups columns in the Excel Lookup table Options in the Filters panel lower left allow for creating selected spot collections based on the listed features alone or combined The default of each parameter is All for selecting all members of the given category Each filter parameter group is defined by one column in the Excel Lookup table and most of them are optional To check whether a spot has been scored or not check the Score check box If the spot has been scored the score number is displayed If not a character is displayed June 11 2009 3DHISTECH Ltd 33 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring 5 3 Filter Parameters 5 3 1 Filtering For All Users Filters Slide Spot Stain Score of Consoli f Database m v Sample ID correspond to the Unique TMA Sample ID in Excel In some cases one sample ID identifies one patient However in most cases particularly at tumor heterogeneity or small core diameter more than one sample 2 5 of a patient may be used which can be managed under different sample IDs and grouped into gallery by sorting together in the Gallery with donor block ID Restricting Sample ID to one sample only and leaving all other parameters at their default All allows for the rapid confirmation of the accuracy of c
28. des quality of staining quality of stained slides quality of used method of staining 3DHISTECH Ltd assumes no responsibility for the functionality and fault free condition of your application programs Workflows VBA macros Commander scripts CAUTION For research and education uses only not for use in diagnostic procedures This product has not been approved or cleared as a medical device by the U S Food and Drug Administration The data and images obtained or viewed using this product are not intended for clinical or diagnostic use Third Party Disclaimer LibTIFF TIFF Library and Utilities 3DHISTECH Ltd is not liable for damage of whatever nature including but not limited to general or specific damage indirect damage consequential damage or incidental damage including damage resulting from lost profits interruption of business loss of business information pecuniary loss or similar damage that stems from or is associated with the incorporated part of this Product that is made by Silicon Graphics Inc THE SOFTWARE IS PROVIDED AS IS AND WITHOUT WARRANTY OF ANY KIND EXPRESSED IMPLIED OR OTHERWISE INCLUDING WITHOUT LIMITATION ANY WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL SAM LEFFLER OR SILICON GRAPHICS BE LIABLE FOR ANY SPECIAL INCIDENTAL INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND OR FOR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS OF USE DATA OR PROFITS WHETHER OR NOT
29. elected the The project handled by one user option you will not be able to add more users to the project later Manage Users P Select a user to open the project Active Dr White m The project handled by one user Do not ask me again To remove obsolete slides from a project click on the annotation of a slide with the right mouse button and select Remove Slide s June 11 2009 3DHISTECH Ltd 13 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project 3 4 Slide selection To select more than one slide for removal keep the Ctrl button pressed and click on the slides to be removed These slides will be removed from the project but will not be deleted from the disk 5 TMA Test Project Consolidate E Manage gl Spot Finding Gallery A Import El Export EZ Matrix view W Consensus Autoscore zi Hi Colcc B Cat Colcc EGFR Colcc EGFR Erbit B2 Vent Erbit B1 Vent Erbit B2 Black2 Block 1 Block B Catenin EGFR Ventana EGFR Ventana H Important To avoid confusion only one project is allowed open at a time June 11 2009 3DHISTECH Ltd 14 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project 3 5 Remove Project Existing TMA projects can also be removed by clicking on the project name with the right mouse button and choosing Remove project from Tree fe Colec B Cat Erbit B1 e Calcc B Cat Erbil B2 fen Colec B Cat SZ1 B1
30. ency Number of Columns 1 10 14g 40 80 4 al oe of Rows la Title of Columns Stron 4 I g l 2 Title of Rows Intensity a EA Intensity Special Scheme Setup Threshold Setup June 11 2009 3DHISTECH Ltd 40 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring 6 1 4 Exporting Clusters into XLS It makes possible to save the Clusters into XLS by the software This export type is only valid if the slide was scored with a Combined Scoring Scheme and you have also set Thresholds in the Scheme A B C D E F G H l J K Unique TMA TMA Donor Sample ID Block ID Block ID Organ Diagnosis Grade Sender in Survival B Catenin i Block 1722 02 liver cirrhosis GO Szombathely 2 Blocki 546 04 breast IDC G2 Bay 3 Block 546 04 breast IDC G2 Bay 4 Block 210 04 breast MucinosugG2 Bay 5 Block 210 05 breest IDC G3 Bay o 6 Block 1643 04 breast IDC G3 Bay 7 Block 1416 04 breast ILC G2 Bay 8 Block 1610 04 breast IDC come G2_ Bay 9 Block 128 05 breast ADC metajpoor Bay 10 Block 774 02 breast connectivdGO S E LPatholopy ii Block 659 02 breast llC X G83 SE LPathdogy G2 Ei O d Loe Ea od EGFR Ventana O N ant WN ot k mh ro e
31. es can be opened in the MIRAX Viewer 1 12 software either by double clicking on their preview slide or by using the right mouse button and choosing Open Slide option from the pop up menu The TMA identifier square linked with the core ID number annotation will appear on each stained tissue core if you have selected the Save to Slide option in the Spot Finding window If there is no such annotation seen the TMA slide needs to be identified by using the Spot Finding function a MIRAX Viewer Local Colcc B Cat Erbit B2 p Ele Settings Window Help E amp ri w EE le 8 rf 8 D Z9 a P p D 3 lm ERO e Lf i LJ 10x E ERI Magnification 27 Progress 00 Iw Fit scanned area A00 fe Preview Viewing history Preview and viewing history Delete Viewing History June 11 2009 3DHISTECH Ltd 24 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring 4 Scoring The latest version of TMA software allows for using different scoring schemes within the Same project for example when markers detected stains in a project cannot be assessed on the same basis The schemes are stored and offered for later use under the Stain option The name and value of empirical scores used in a project can be predefined in the Scoring Scheme menu The first step is to chose the stain in the Stains panel from combo box Selected Stain for defining the score values The second step is to chose from options Simple or Combined i
32. eus cytoplasm membrane into the heads of rows and columns E ES Scoring Scheme Setup Window Stains Scoring Scheme Type Selected Stain Simple Load Scheme Canan G Combined Seve Scheme Frequency Columns om of Co m 10 1040 40 90 Number of Rows Weak 1 3 Title of Columns Frequency Title of Rows Intensity Moderate Intensity Strong Special Scheme Setup Threshold Setup Then enter the score value into each end field text box of rows and columns June 11 2009 3DHISTECH Ltd 26 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring Making Special Schemes You can define V name special schemes giving them a value a number for example 0 Non Evaluable 1 Negative after marking in the Special Scheme Setup option 5 a Scoring Scheme Setup Window mm 5tains Scoring Scheme Type Selected Stain Simple Loo cime M Save Scheme Number of Special Scores Name 2 Non Evaluable Negative OK Cancel To return to the Scoring Scheme menu click Back Threshold Setup After marking in the Threshold Setup option you can define groups of scoring schemes for showing a common visually hidden but known by the researcher biological property of these value groups for example a given DNA section produces the examined protein in various rows and columns You can define the number of Clusters and you can name these Clusters writing a value into the text b
33. g to appear over the cores June 11 2009 3DHISTECH Ltd 22 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project 22 Spot Finding on Slide D tma Colcc B Cat Erbit B2 Les I EG Jie Slide List Colec BCatEbiB2 7 Slide Type Brightfield Fluorescent Image Zoom Level 5 X p Slide Rotation 2 37 E Background Threshold 230 7 Shaw Spot Diameter mm 2 57 Er os Grid Columns Rows i Re Save to slide eae Automatic Reverse Automatic from DataBase Clear Numbers i Important Using an orientation tissue easily differentiated from the test tissue such as liver as suggested above for example when testing breast cancer samples safely assists in the accurate core finding However this is optional and any other system may be used for recognizing TMA slide orientation e To cancel the grid numbering such as the spot numbers select the Clear Numbers option Navigation Prev Slide Next Slide e To move between slides click for the previous slide or for the next slide June 11 2009 3DHISTECH Ltd 23 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project e Click to cancel the spot finding process e After spot identification layout will have been accepted the annotations and Finish numbers attached will be saved by clicking 3 10 Adjusting Annotations in the MIRAX Viewer 1 12 Identified TMA slid
34. hat you also have the dongle or the MView lic licence file 4 Follow the instructions in the Installation Wizard When prompted select the dongle or browse for the licence file Choose the application TMA during installation June 11 2009 3DHISTECH Ltd 7 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project 3 Setting up a TMA Project 3 1 Collecting Project Data in an Excel Worksheet and Building a TMA A TMA project can be started with collecting pathological and clinical data of relevant patients in Excel data sheet format TMA Lookup table and using the individual donor blocks correlated with their H amp E stained sections for punching out representative cores and producing TMA blocks either using an automated or a manual TMA arrayer TMA Master instrument is available to support MIRAX TMA projects Each Excel file supporting a project TMA database file should include at least three worksheets One for the main Lookup table Lookup one or more for linking stained spots with their location in the TMA blocks Block1 Blockn and one for linking the stains to Excel Lookup table data Slides A B C D E L G H J K Unique TMA TMA Block Donor Block 1 Sample ID ID ID Organ Diagnosis Grade Sender institute Survival Stage Metastasis x 2 1 Block1 1722 02 liver cirrhasis G Szombathely 3 2 Block1 546 04 breast IDC G2 Bay 4 3 Block 546 04 breast IDC G2 Bay h 4 Block 210 04 breast Mucinosus carcinoma G2 Ba
35. hese features are optional and slides that are not labeled in this way may also be supported by the software When starting a project all information included in the Excel database file can be imported into the MIRAX TMA software to be exported into a new Excel file added with the scores when scoring will have been finished 3 2 Creating a New TMA Project 3 2 1 Before You Start Make sure that the slides that you want to add to the TMA project are all in one folder at a location that you can access from MIRAX Viewer and that they are not read only 3 2 2 Creating a New TMA Project For creating a new TMA project click on the Slide project folder in the Slide Tree panel June 11 2009 3DHISTECH Ltd 10 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project H k ThA breast demo Clicking on the sign will list all slides found in the given folder while in the right side panel their preview image will be opened with the same labels at the bottom which were used at their scanning 1 Click on the Slide Project folder with the right mouse button 2 Select Create New TMA Project from the menu Then the new project will include all digital slides found in the original Slide project folder File Settings Window Help EE u m Tm d m BI x BH Remove Project from Tree Create Mew 3D Project Upload Slides to Teleconsultation Server Create New TMA Project Create New ImmunaScreener Project i F
36. k 1643 04 breast IDC G3 Bay 7 Block 1416 04 breast C G2 Bay 9 8 Block 1610 04 breast DC comeG2 Bay 10 9 Block 128 05 breast ADCmetappor Bay 1 10 Block1 774 02 breast connectiveCO S E LPathoogy 12 11 Block 659 02 breast l C G3 SELPathelogy 18 12 Block 322 02 breast IDC G2 SELPathelogy 14 13 Block 1315 02 breast IDC G2 SE LPathelogy 15 14 Block 1199 02 breast IDC G3 jSE LPatheogy 16 15 Block 476102 breast DCIS GO0 SE LPathelogy 17 16 Block 657 03 breast connectiveGO Bay 18 17 Block 122203 breast IDC G2 Bay 19 18 Block 1324 03 brest ILC G1 Bay 20 19 Block 1643 04 breast IDC G2 Bay 21 20 Block 1643 04 breast IDC G2 Bay 22 21 Block 1826 02 breast DCIS G0 SE L Pathology 23 22 Block 1877 02 breast ILC G2 Natl Inst Cancer 24 23 Block 988 03 breest IDC G2 Bay 25 24 Block 1023 03 breast IDC GO Bay 26 25 Block 210 05 breast connectivdGC8 Bay 27 26 Block 1643 04 breast l C G2 Bay 28 27 Block 1416 04 breast IDC G2 SE L Pathology 29 28 Block 1610 04 breast IDC G3 SE L Pathology 30 29 Block 128 05 bre
37. l in the grid of a slides annotation that the scoring of that slide is completed Please note that this option will not be activated automatically when scoring has finished Colec Cox Erbit Colec EGFR B2 Vent Erbit B1 Block2 Black1 Cox 2 EGFR V entana e The Remove Slide option is used for eliminating the given slide from a project see above To remove obsolete slides from a project click on the annotation of a slide with the right mouse button and select Remove Slide s These slides will be removed from the project but will not be deleted from the disk H Important You can only remove slides from a project if it is a one user project or if you are signed in as Consolidate user in a multi user project If you are signed in as another user in a multi user project you cannot access this function The slide label including the serial section number Z dimension depth stain label and block number appear in the bottom region of the thumbnail image of each slide A grid label seen in the upper left corner of each slides annotation indicates that the spots within the given slide have been identified by using the Spot Finding The green tick label for Marked appears if activated and may refer to the completed scoring June 11 2009 3DHISTECH Ltd 17 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project 3 8 Linking Excel Data to TMA Spots To identify cores and link to patients pathological
38. lock2 77402 breast DCIS G2 S E I Pathology 0 31 o o EGFR 2 9 9 7 2 2 2 7 2 4 9 2 1 9 7 2 1 2 2 7 1 2 38 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring 6 1 3 Exporting Score Values for Generating Allred Values The software generates an Allred Value from the Score Value given by the user The Allred Scoring Scheme is preferred mainly in the breast cancer diagnosis This export type is only valid if the slide was scored with a Combined Scoring Scheme A B C D s G H J K Unique TMA TMA Donor 1 Sample ID Block ID Block ID Organ Diagnosis Grade fhder instit Survival B Catenin EGFR Ventana 2 i Block 1722 02 liver cirrhosis GO Szombathely 1 2 2 3 2 Block 546 04 breast IDC G amp 2 Bay J 9 4 3 Block 546 04 breast IDC G2 Bay l 9 5 4 Block 210 04 breast MucinosusG2 Bay 2 7 6 5 Block 210 05 breast__ IDC_ 63 Bay H o 2 7 6 Block1 1643 04 breast IDC G3 Bay p2 8 7 Block 1416 04 breest lLC G2 Bay A 2 9 8 Block 1610 04 breast__ IDC come G2_ Bay if 10 9 Block 128 05 breast__ ADCmetajpoor Bay 1 0 11 10 Block 774 02 breast connectivdGCO SE LPathology O 2 12 11 7 13 12 Block 322 02 breast IDC G2_ S E 1 Pathology O 2 14 13 Block 1315 02 breast IDC
39. n activated the centers of the given grid row column becomes red e To align a single grid element drag not the line as above but the dot itself This time only one single dot will become red e If the above steps do not help fitting the grid on the spots for example because it detects specks as specimens you can also align the grids to the spots manually To do this hold the right mouse button on the center dot of one grid element and drag it to the required position e If you delete a grid in the case of multiple grids existing on a slide the grid of the last spot group will be active In spot finding the software stores the last threshold value spot diameter and the grid dimension used If you generate several grids for several spot groups in a slide you can activate those by clicking on the grid inside the grid or on the spot frame When activated the grid color turns blue from gray Select the Save to slide check box if you want to make measurements for individual spots in the different Quant applications These options are only active if you save grids into the slide For the TMA scoring it is indifferent whether you choose to save grids to Slide or not June 11 2009 3DHISTECH Ltd 20 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project Slide List mnm Colcc B Cat Erbit B2 z Slide Type Q Brightfield Fluorescent pm Image Zoom Level 5 m Slide Rotation 2
40. n the Scoring Scheme Type panel 4 1 Scoring Setup 4 1 1 Simple Scoring Scheme Select the Simple option in the Scoring Scheme Type panel Scoring can be defined on a maximum 10 scale system allowing for considering staining intensity or of positive cells To change the number of scores click the Up or Down arrow buttons a 3 Scoring Scheme Setup Window Stains Scoring Scheme Type Selected Stain Simple Lond Scheme B Catenin Combined Save Scheme Number of Scores Name negative equivocal weak to moderate positive strong positive oc Cien June 11 2009 3DHISTECH Ltd 25 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring Though options are flexible and there are many the use of a 4 scale system including 2 negative 0 equivocal 1 weak moderate positive 2 strongly positive scores is practical for later Unsupervised Hierarchical Clustering which may be done without using score conversion Deconvoluter 4 1 2 Combined Scoring Scheme Select the Combined option in the Scoring Scheme Type panel Either enter or select the property you want to evaluate and then define the number of rows and columns max 10 rows and 10 columns Then enter either the ranges of the chosen two properties or the structural functional parts of the cell for Frequency values for example 0 10 9o 1 40 9o 40 80 9o 80 100 for Stain Intensity weak moderate strong for Localisation nucl
41. ore identification Restricting the Score and Stain options into one parameter each will allow comparing spots of a particular staining obtained the same score The inconsistently scored samples can be recored within the Gallery function before final exporting of the results into Excel database for further correlation studies validated scoring This feature of the MIRAX TMA Module allows for using highly reliable score values for statistical survival and cluster analyses In the Gallery the spots which have not yet been scored can be collected with filtering Scored on the base of the Special Scheme in the Scoring Scheme Setup menu spots can be re selected and re scored In equivocal cases doubtfully scored spots can be reseted for non scored allowing their re scoring at the end of a project based on a professional consultation when collected them together The space for gallery images can be extended full screen on the expense of the left hand gallery menu panel by clicking on dark gray bar in the right edge of gallery menu panel To filter based on the Excel database select the Database filter June 11 2009 3DHISTECH Ltd 34 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring 5 3 2 Filtering Only For Consolidate User Only the Consolidate User has a chance to assembling spot galleries on the base of arbitrarily selected scores of all users ls 2 Click the button next to the Score of Consolidate text A menu i
42. out the risk of false conclusions based on inconsistent scoring 4 2 1 Scoring in Gallery The scoring can be done by clicking on the spot with the right mouse button and choosing a score from the pre defined values 1 B Catenin 2 2 B Catenin 0 3 B Catenin 1 4 B Catenin 2 5I B Catenin amp B Catenin Order by Primary Disabled g Secondary Disabled Slide J Spot OMe Stain a Score of Consoli v 7 I B Catenin Database J v weak to moderate positive 1 strong positive 2 10 B Catenin 11 B Catenin 12 B Catenin June 11 2009 3DHISTECH Ltd 29 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring If a score has already been attached it is indicated by a pushed down button in the scoring panel and the score appears in the label above the spot after spot ID and stain 4 2 2 Sequential Scoring 4 Cox 2 zm in i JAW Sequential scoring in the Gallery automatically offers full size image of a spot on the screen which can be further magnified in the magnifying window Scored image will disappear allowing for the next spot to be displayed for scoring 4 3 Consensus Autoscore In case of more than one assessors working in a project the Consolidate option is automatically offered which can be used as a separate scorer user window for creating consensus scores Consensus Autoscore function allows automated consolid
43. oxes of these threshold groups and giving a chosen color by right mouse clicking on color box under the Color option June 11 2009 3DHISTECH Ltd 27 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring En eae 23 Scoring Scheme Setup Window Stains Scoring Scheme Type Selected Stain Simple Save Scheme Number of Thresholds Name Value Color E Weak Moderate 2 Strong E Scoring Scheme Setup Window Stains Scoring Scheme Type Selected Stain omi TUS Combined Save Scheme Frequency M J ibe of Colu ins 10 10 305 40 803 Z e Number of Rows Weak aise 2 Ez 4 l Title of Columns a sono E MANN NNI 2 B Weak 1 Title of Rows 3 i Moderate 2 Intensity B Strong 3 Intensity Special Scheme Setup Threshold Setup June 11 2009 3DHISTECH Ltd 28 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring To return to the Scoring Scheme menu click Back After all these steps click Apply or OK 4 2 Scoring TMA Spots Scoring of stained TMA spots can be done from the Gallery Scoring from Gallery offers much more flexibility in this updated version see later thus it should be preferred for scoring Validated scoring allowing for possible re scoring of spots in Gallery mode when sorted with other spots along with any common feature s is an important function of the MIRAX TMA Module software ensuring reproducible results with
44. pot After releasing the mouse button the distance will appear in millimeters Note that the spot diameter parameter has strong effect on the result of spot finding June 11 2009 3DHISTECH Ltd 19 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project Grid Define the number of spots arranged along the horizontal and vertical axes of the TMA Slide for example 4x6 This can go up to 50x50 but the basis of identification is the same Enter the correct number of spots in a column in the Columns text box and the correct number of spots in the Rows text box e To draw the grid over the spots click E Then click the corner spots in the TMA slide one after the other for example going clockwise A red X sign will appear on the marked corner spots After the fourth corner will have been finished a net of lines will connect the middle of the spots and gray square annotations will surround their borders the size of square annotations was defined in the Spot Diameter option e To remove the grid click zs You can start the procedure again e Sometimes spots do not form straight columns and rows due to barrel or other type of distortion This results in spots being excluded from straight grid lines This problem can be solved by grid retorsion Click H to move grid elements on spots e To align grid elements to whole rows or columns hold the left mouse button on the the row column line and drag it with the mouse Whe
45. r 2 11 and TreeView 1 60 http rana Ibl gov EisenSoftware htm by Eisen MB Spellman PT Brown PO and Botstein D 1998 Cluster Analysis and Display of Genome Wide Expression Patterns Proc Natl Acad Sci U S A 95 14863 14868 June 11 2009 3DHISTECH Ltd 49 49
46. s displayed where you can select the other users After selecting the users you can select the scores After this you can select the appropriate filtering options For example Not Scored 2 at user1 and 1 at user2 In this case the Gallery will only include those spots that satisfy these conditions 6 Exporting Data and Images 6 1 Exporting Data Export column Please specify the column you would like to export Organ Diagnosis Grade Sender institute Survival Stage Metastasis X Userl s Scores User s Scores Select all Clear all Score normalization Lowest Highest When exporting select which Database column s you would like to export June 11 2009 3DHISTECH Ltd 35 49 TMA Module for MIRAX Viewer 1 12 User s Guide 4 Scoring If you are signed in as a Consolidate user you can also export the scores of other users In this case select the names of the users whose scores you would like to export Score normalization There can be slides where more spots are included from the same donor These spots are usually identified with the same Core ID However the Excel sheet has only 1 row for 1 ID therefore it has to be decided which score should the software include in that row In this case the software will calculate the median of all the scores depending on whether the Lowest or the Highest option was selected A Warning TMA Module does not support the newer than 2003 Excel files
47. t patients data in Excel Slide list The batch mode allows you to access spot finding for all slides in a project in one spot finding window You do not have to enter spot finding for the slides separately Select the appropriate slide from the Slide list roll down list To move between slides click Prev Slide Next Slide frame for the previous slide or for the next slide in the Navigation Slide Type Select the appropriate slide type in this frame e To apply Spot Finding for a brightfield slide select Brightfield e To apply Spot Finding for a fluorescent slide select Fluorescent Image Zoom Level To change the zoom level of the slide adjust the Image Zoom Level slider The actual zoom level is displayed above the slider Slide Rotation To rotate the slide adjust the Slide Rotation slider The actual rotation degree is displayed above the slider Background threshold If there is an unwanted background staining or the specific staining is too weak you can adjust the Background threshold level with the slider to make sure that all stained spots will be scanned Check the Show check box to display the scanned areas according to the threshold level Spot Diameter mm To define the diameter of the spots write the correct diameter in millimeters in the text box or set it with the arrows next to the text box To measure the spot diameter click t mum bo E 4 0 and then pull a line over the largest s
48. te slides click Next Project Data To import the Excel database ID specimen diagnosis etc click Find DataBase and browse for the appropriate Excel file When finished click Next Spot Finding To ignore spot finding select Ignore Spot Finding You can access the spot finding dialog later by clicking Spot Finding in the TMA Project window For more information on Spot Finding see section 3 7 Finding TMA Spots For manual spot finding select Manual Spot Finding This will open the Spot Finding dialog instantly after finishing the steps of the wizard Click Finish 3DHISTECH Ltd 12 49 TMA Module for MIRAX Viewer 1 12 User s Guide 3 Setting up a TMA Project 3 3 Opening a Project Existing projects are marked with a TMA icon followed by the name of the project Double clicking on the given project icon will open the project folder The software will ask for the scorer if it is only the same person who created the project go ahead If different Add new user to the list In case of more than one assessors in a project the Consolidate option is automatically added which can either be used as a separate scorer to decide the final consensus scores or this function will offer automated creation of consensus scores based for example on mean values of different scores If the project is a one user project select the The project handled by one user check box to disable the notification i Important If you have s
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50. y M 4 Block 210 04 breast MucinosuSG2 Bay p2 5 Block 210 05 breast IDC G8 Bay M 6 Block 1643 04 breast IDC G8 Bay 2 7 Block 1416 04 breast c G2 Bay M 8 Block 1610 04 breast IDC come G2 Bay J0 9 Block 128 05 breast ADCmetajpoor Bay H 11 Block 659 02 breast IC G3 SE L Pathology 2 12 Block1 322 02 breast IDC G2 SE LPathelogy 13 Block1 1315 02 breast IDC G2 SE L Pathology 2 oF 15 Block1 4761 02 breast DCIS GO_ S E Pathology 1 16 Block 657 03 breast connectivdGO Bay p2 17 Block 122203 breast_ IDC G2 Bay M 18 Block 132403 breast ILC Gl Bay p2 19 Block 1643 04 breast IDC G2 Bay M 20 Block 1643 04 breest IDC G2 Bay 0 21 Block 1826 02 breast DCIS G0 SE LPathoogy 1 22 Block 1877 02 breast lC G2 Natl Inst Cancer 0 Bay 24 Block 1023 03 breast IDC CO Bay 0 o d 26 Block 1643 04 breast lLC G2 Bay 0 oo 28 Block 1610 04 breast IDC G3 SE LPatheogy 0 29 Block 128 05 breast IDC G3 SE LPathoogy 30 B

TMA Module for MIRAX Viewer 1.12 User's Guide

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