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1. Parameter s CD25 PE A PE A CD11C APC A APC A HARKAK M C RT Simulated time Keep file number separation 117886 gt Mixed in one file Save in independent files The user can export only the parameters which are necessary for the analysis avoiding extra memory requirements resulting from unnecessary information Initially all the parameters are selected for export If the user wants to eliminate some of these parameters unclick the parameter to eliminate it from the export Simulated time Last parameter to export is always simulated time If you activate this checkbox your export file will contain the chronology simulated time of the acquisition of the events tubes as a new parameter and it will be possible to represent this simulated time against any other parameter Notice that in a merged file you already have the parameter File Number as the chronology of the tubes then is not necessary to add the simulated time parameter CY XX 090507 Rev 002 Issue date April 29 2010 Page 119 of 127 Events This box allows the user to decrease the number of events selected to export If the user is trying to export a merged file Infinicyt will give you the option to Keep the file number separation will maintain the separation in the original files or mix all the events in a file without separation between original files Exported events will be
2. ccccccccssseseeeceeeeceeeaeeseeeeeeees 40 4 7 1 3 Virtual Parameter Configuration cccccccccssseeseeeeceeeeeeaeeseeeeeees 40 4 7 1 4 COMPE I ON e E ecueielnsestenmendied eds 4 CY XX 090507 Rev 002 Issue date April 29 2010 Page 3 of 127 4 7 1 5 EXP SC Low Conistra sien orenian un a at 4 4 7 1 6 Font Legend Scale Configuration ccccccccsssseecceeeeceeeseeseeeeeeeeeeaas 4 4 7 2 Conti sire Visualization Dala erreren cot sh sie Cuclee Deas tai naiactennactite 42 4 7 3 MiG aL 97 SAA Yc fs acs sacs oh cece sad saa cagu ves Secchi eceewieeweeoteteans 44 4 1 4 Number Or Events Displayedrcrrann e 45 4 7 5 Window Miami pulaiOn eerie a TE T 46 4 7 5 1 Max nize and MINIMI Esee E 46 4 7 5 2 D phedite Dias fason O 46 4 7 5 3 COPY DAUTIT aiia aiwsestonnsnetidacnse shemebeendak 46 4 7 0 ASSON POPULO rese on dean tatsneshom heater decane dean aaa wienoms baa 46 Al Bido Redo ppemememerertrre Bieri Ri reer ne ere Ree eee 46 4 7 8 ET S bane eae eee EEE ene eee A AET Mr ne pen ne reel EE EE E E 47 4 7 8 1 TAVE ACS e EEEN 47 4 7 8 2 Invert Diagram Color Black White Background Color 47 4 7 8 3 Invert Gate Sele Ct On x yessdaraccttareiceucenhen dates a ddtequidetedd 47 4 7 9 Diasto Mactuas tere daelaatbesdem Maneaeuicatiationts 47 BO PVOUeS aenn E OT 48 A9 Rerronce IMa apes oes Seat a E N A TE N E E teeeeey ee 49 4 9 1 Save dR Clerence Imas iak a a A 49 4 9 2 Load Reference MIM a
3. Gate 0 events Total 0 00 Visibility 0 00 j23 of 1013 MB used Save Analysis Strategy as Analysis Strategy name Strategy n 1 Name the strategy This new Strategy will be stored in the profile 4 13 5 7 Load Analysis Strategy To Apply Strategies Open a new file and Go to Profile Load Analysis Strategy in the Manual Analysis Window Menu Bar and select any of the saved strategies in the profile Load Analysis Strategy Analysis Strategy name Strategy n 1 The analysis has been applied on the new file click on the lens of each population to review them CY XX 090507 Rev 002 Issue date April 29 2010 Page 74 of 127 4 13 5 8 Unload Analysis Strategy The same as with the Reference Image it 1s recommended to upload the Strategy when finishing our process in the Manual Analysis Window Menu Bar in the option Profile Unload Analysis Strategy Otherwise the software will try to apply it and the parameters won t match obtaining the warning message of Parameters Concordance 4 13 5 9 Analysis Info In this option the user can see all the steps of the analysis strategy Analysis Info Current Analysis Strategy Populations to review fj Leucocytes Gate 1 Events gt p 7 2D Events APC 4 CD45 Logic S5C A Exp 55C Low Gating steps or j T Cells Gate 2 Leucocytes 2D Leucocytes FSC 4 Lin SSC 4 Exp SSC Low 2D Leucocytes FITC 4 CD3 Logic S5SC 4 Exp 55C Lo
4. F CD19 PerCP Cy5 5 PerCP Cy5 5 A F CDS APC A APC A Use previous groups Manual Analysis I Configure Save As gJ Main Menu The previous groups already done can always be checked in this tab of the window beiei SS bo w Fie meyra Gap prior PEHA oes Dh LES po oe i ihe ae oh 1G fo td Gr i Dr F TE E oe iD Di G fi o i o i x The previous groups information can also be checked in the Manual Analysis Window Menu Bar option Profile Configure Tools Previous Groups In this case the previous groups stored will be saved in the application Infinciyt instead of be stored on the profiles It means that it will be always available to use independently of the Profile it is open each time The Dictionary contains Drag and Drop tools to move the labels and the groups The group s configuration can be exported as a file dic dictionary and imported from this window CY XX 090507 Rev 002 Issue date April 29 2010 Page 98 of 127 Configure LQ Profile Configuration 3 Preferences 4 Analysis Diagrams Controls Statistics Virtual Parameters Stored Compensations Gates and Analysis Strategies Lo Reference Image J FCS file be Configure APS Parameters i Properties BY Previous groups FSCH wll FSC Height FSC H lll FSC Height MO FSC H oll FSC Height FSC H hall FSC Height FSC H CD19 PerCP Cy5 5 A Lai CD19 PerCP
5. INFINIcyt USER S MANUAL 1 4 Version Qiine E SUMMARY I INTRODUCTION gustine a onreunensigeutiat vadeemesteted ereien scotia 8 2 INSTALLATIONJIAND SE FUP seccsccsus weecuce rent vavensctaneart E E 9 Dilys Licence IDC ails asks cers e a a a ad vete ta taeteaces 9 2 2 JVimimUnasSystem requirements eia E E ES 9 25 dpstallaton Procedur Siea a neoeedanieaseeeeedaeas 10 2 PORTOT yO aara ee diawiuakednmotietucathentuaies 11 2 5 Language and regional configurations sseseeessssssssseeersssssssseeeressssssseeeeeee 11 ZO UII S CA A OM e a retail ieesenaadeea heed neeas 11 Dds SUPPO SCLVICES aiT T 12 ZS UPd S rE RS Oe RE Ree ee METER ged Le Tan RTE eC rere 12 Zs SPCCIAl Consideration S z fo ai uetertioha di ioa densi E TTN 13 De MAINSCREEN ernn e a a a reer eo eect 14 4 MANUAL ANALY SIS iemssecsesuseedenndenedsoncveadvandensanovdeedenndueedseneveddvendeeeasoudeedeoadentes 16 Abe UOC UC OI sacs le se eite dete a cee dse specious EA 16 4 2 Getting Started on Manual AnalySsis ccccccccsssssssseccccceeeeeeeeeeeceeeeeeeaeeeeeees 16 4 3 FCS files with more than one Dataset Coulter Files cece eeeeeee 20 4 4 How to analyze and select populations c cc ceecessscccecceceeeeeeeeeceeeeeeeaeeeeees 21 4 4 1 Assign label to populations eccna a 21 4 4 2 Ree ATC G1 OM asc c ecinsinsaadiciie host etn A eaten canst 22 4 4 3 REGIA WW Ae Oa anhauehsarda iy saanasienseedeetelamtauehsaneeesynanacwi
6. NEW DIAGRAM Histogram 2D DotPlot Parameter Band APS diagram 3D Density Multidimensional O Stat diagram Number of parameters 2D Dot plot It is a Graphical representation of events cells or particles in two dimensions The value on each of the two dimensions parameters defines the location of the event in the plot E l lt w T oO w n I g oO w w FSC H FSC Height Lin To change the parameters or the scale click on the label in the axis and you will have available all the possible parameters to show or the different scales As exposed in chapter 4 7 each diagram contains an internal menu which can be obtained right clicking in the middle of the diagram Parameter Band DotPlot CY XX 090507 Rev 002 Issue date April 29 2010 Page 32 of 127 As described in the Parameter Band Histogram the user can select as many different bands as parameters desired to represent the events in a dot plot Each band will represent the expression of the parameter X in those cells with the Time parameter of each band in the Y axes The events will appear in the Time scale of each parameter in the order they were acquired Pi a Called FSC LIMITAR S50 A Exp 350 Low FTE A TOJE LOGICAL Axes Configuration n i PE APC OSE LOGICAL Number of Parameters 6 v cypeeaccots Looe Band 1 FSC A LINEAR x a Fie Band 2 SSC 4 vlExp
7. Infinicyt 1 4 includes a new tool to solve the problem of merging parameters with different labels which improves the Goup tool of versionl 3 When there is a label discrepancy on parameters that should be common just click Group a File preview Add 1 A Group Y Parameters File E m E E Ge jssc a iM e icb22 FITC A FITC 4 fv C cb23 PE A PE A W C CD19 PerCP Cy5 5 A PerCP Cy5 5 A lel a Ure AAM A 1 2 314 It appears a dialog box in which the user may click on the labels that should be grouped CY XX 090507 Rev 002 Issue date April 29 2010 Page 96 of 127 7 7 f 7 7 d d 4 4 f 7 7 The first label selected will give the name to the grouped parameter but it can be changed in the box Name as Add Group Mame as FSC r a FSC ba E a FSC Height E a 55C Height bait F CD103 FITC A FITC 4 ba F CD25 PE A FE A eet F a CD19 PerlP Cy5 5 4 PerCp Cy oo E a CO1 1C APC ALAPC A m E a CD34 APC A APC A jar F a LAMBDA FITC A FITC 4 ian E a KAPPA FE A FE A fv F a CD19 PerlP Cy5 5 PerCP Cy5 5 4 i E a CDS APC A APC A E Use previous groups Done As soon as a first label is selected some parameters disappear from the list These are the ones that belong to the same file therefore can not be grouped with the same name Add Group Name as FSC A a Parameter Groups liv a F5C A FSC 4 a FSC a
8. The calculate data process finished withthe folowing results Ss Population Non calculated events 201 amp The calculate data process has successtully finished amp The calculate data process Tailed Click OK to continue the process STEP 4 VERIFY AND SAVE RESULTS In this step the user directly goes to a pre manual analysis with certain limitations as described in step 2 and will be able to check the not calculated events because the will be painted in highlight within a label under the original population In the following figure the violet events have been calculated and can be visualized in all the parameters On the other hand black events have been rejected by the controls and the can be visualized only on the real markers for each one Then it is easy to visualize only those events and paint them in a different color as debris or as part of the population again If you click next without painting black events in a different color then those events will be included as part of the original population Fie Edt Cisqrams Statistics Profile Steps A H xirs Aad Pogedofar Firs Y 1 Setect thes n g tiens P f z b l 2 GateACiassity everts Popasten 3 k 3 f i V5 a a ki i pi 3 Choose posadations bo caleudate J a read events E E R aja 4 Verify and seve results Norn cekuistod evert ott ute o4att NA NA NA FEY gt e Ea o Pi
9. SSC A Exp SSC Low CD19 PercP cys 6 2 R j oe ee ee 2 ey SSNS _ ae i I SATTA ra ee 2 e tin file Se 3 r ees ib rea po vent in file one CD19 PercP Cy Si pare pce aed fe finding the equivalent l Gaam nanana T in the rest of the files CD19 PerCP CySHy wes 105 COOP eve PCS SA Even more when the common markers have some differences in the expression the software is able to find the equivalent cell in the other file In this part is where the controls apply depending how homogeneous are the populations the area to find the nearest equivalent neighbour can be bigger or smaller In the Calculate Data wizard you will find the Configure button If you click this button you will see the controls dialogue box of the profile with the Calculation data controls where you can manually configure the following values CY XX 090507 Rev 002 Issue date April 29 2010 Page 115 of 127 nan dn ds dE dE 6 Profile Configuration Preferences Analysis Diagrams Controls Statistics Virtual Parameters Stored Compensations Gates Reference Image Threshold parameters for coherence controls AN Merge Control Minimum difference of global aspect required for warning 20 A Population Control Show message when differences are detected in visible populations Minimum difference for each parameter required for warning 5 Calculation Data Controls Advanced Controls Calculation Da
10. 1 88 lt 10 35 D CD56 PE Cy7 A 2 92 S CD3 APC A g CD20 APC Cy7 A 3 6 CD45 Pacific Blue A 2 09 z a v 10000 1000 CDZAPC A LOGICAL CY XX 090507 Rev 002 Issue date April 29 2010 Page 86 of 127 CONFIGURE APS PARAMETERS This option allows eliminating from the profile one or more parameters for the APS calculation Remember that this action will apply globally while it is always possible to do it locally from each APS graph in the right click menu Diagram Configuration APS parameter Configuration The software will take into account only the parameters selected Configure D Profile Configuration Configure APS Parameters Preferences H Analysis H Diagrams Controls H Statistics H Virtual Parameters Stored Compensations H Gates and Analysis Strategies i Reference Image 5 FCS file Current Compensation Negative Visibility Configure APS Parameters Properties I Tools Previous Groups K LINEAR OGICAL OGICAL FITC A cMPO PE A cCD79a OGICAL B3 A CD34 PerCPcy55 OGICAL PE Cy7 A CD19 OGICAL APC A CD7 OGICAL R2 A cd3 OGICAL Pacific Blue A cCD3 OGICAL V2 A CD45 PO LOGICAL lt lt SOOO0O008 lt PROPERTIES This window corresponds to the one previously described on section 4 13 1 8 about the characteristic of the parameters on a given
11. 5 z Non cakuiated everts 44 SSC A Exp SSC Lew SSC A Exp SSC Low e amp F 2 w w Non cakusated everts 17699 amp x 2 s l CD19 PerCP Cy5 5 A PerCP C Non Caksated everts au z x amp a i lOO Non calculated everts 2741 a Gate O eventa Tawm 000 Viseaty 0 00 Save As g Man Meru _ Previous File Number 2 4 CD19 PercP Cy5 5 A PorcP C APS 1 Once the non calculated events are verified it is time to save this file as a cyt if we want to maintain the population analysis or as a fcs file if we want to save only the calculated events CY XX 090507 Rev 002 Issue date April 29 2010 Page 113 of 127 If you don t want to save the file you can go to the Manual Analysis to analyze this sample without saving to disk Both if you go to Manual Analysis or Main Menu you will find the following warning before closing the Calculate Data wizard ves No The diagrams you used for Calculate Data will be kept as part of the profile as a second list of diagrams that will always be open when you open again the Calculate Data wizard If you go to Profile Configure Diagrams you will find a second option called Diagrams list In this box you will see that apart of the diagrams of the profile you have saved a calculation desktop with the diagrams you need anytime you perform a Calculate Data process Profile Configuration Glob
12. At some point the Infinicyt installation process will ask the user to install the USB key drivers necessary for the correct operation of the program The user should carry on this procedure and the Infinicyt installation will resume and conclude If these drivers are not installed Infinicyt will not be able to recognize the USB key and the program will not work The only case in which the user can safely cancel this secondary installation process is when he or she knows for sure that the drivers have already been installed during a previous installation of Infinicyt or from any other software using the same licensing devices CY XX 090507 Rev 002 Issue date April 29 2010 Page 10 of 127 2 4 How to run Infinicyt When the installation process is concluded you can start to use Infinicyt First of all you should connect the license key to any available USB port of your computer After that double click on the shortcut created on the Desktop or on Start Menu gt Programs gt Cytognos gt Infinicyt gt Infinicyt You can also execute Infinicyt by double clicking on the file named InfinicytBW exe placed in your Infinicyt installation folder usually c Program Files Cytognos Infinicyt 2 5 Language and regional configurations The current version of the software is available in English and Spanish At the beginning of the installation it is possible to choose the language used during the installation p
13. Consecutive gates AND If the user considers that the made selection could be improved in a different graph it is possible to draw consecutive regions in other DotPlots In this case only the events included in all the consecutive steps as the intersection will be considered in the selection 4 4 5 Population Tree description As previously described the program visualizes the results in two different ways a table with populations and numerical results Analysis Window and a set of graphical representations configured by the user in independent windows The former window shows a Population Tree with one line per population identified by the user in general with color labels where the color coincides with that of the dots or lines used to represent this population on the diagrams For each population the program provides the following information as shown in the next figure A population is defined when we establish a group of events as an element in the hierarchy on the Population Tree Assign label to populations J 0000 001 Manual ANALYSIS oe File Edit Diagrams Statistics Profile Help Population C unclassified events 4 ae Other unclassified events 0 42 0 42 0 00 a 140 a 14 SF a9 3 82 4 02 42 61 a4 OB ot 06 0 00 0 13 0 25 0 00 ri 37 Yo 45 250 0 005 30 56 56 53 0 00 2 roo EE i 0 00 33 FB 33 FO 0 00 Events d d d d d Gate 0 events Total 0 00 Visibilit
14. Once this is done you can click Next STEP 4 BCR ABL REPORT The software will show you the result of the analysis on a report The software shows you a report by default but it can be customized by the user Advanced Analysis Sic S Steps File Edit Sheet 1 Select an analysis 2 Select controls and samples a Infinicyt 4 BCR ABL report BCR ABL REPORT File 1 name 19796_CONTROL NEGATIVO fes File 2 name 149796_CONTROL POSITIVO fes 150000 File 3 name 2 09 08_20086 fes File 4 name 19 08 08_82776 fes ir SSC A Exp SSC Low A EA File 5 name 19796_19796 fes 0 File 6 name 20168_20168 fes FSC A LINEAR Population 19796 CONTROL NEGATIVO fcs Mean Population 19796 CONTROLPOSITIVOfcs Mean 914 4 Population File 2 09 08_20086 fes 19 08 08_82776 fcs 19796_19796 fcs 20168_20168 fes Results 4 05 10 10 12 Signed by Print Restore Report g Main Menu Yext CY XX 090507 Rev 002 Issue date April 29 2010 Page 126 of 127 CONTROL COMMANDS Ctrl A All Graphics to Front Ctrl B Close All Windows Ctrl D New Diagram Ctrl E Export Ctrl G Configure Profile Ctrl I File Properties Ctrl L Duplicate Graph Ctrl N Open Next File Ctrl O Open Ctrl P Open Previous File Ctrl R Reset Analysis Ctrl Y Redo Ctrl Z Undo Ctrl 0 Delete all Selections Ctrl T Tile of diagrams Ctrl Maximize Window Ctrl Minimize Window Ctrl 1 2 3 Se
15. Properties I Tools Previous Groups Format Decimal separator Character separator in CSY files Semicolon Define decimal places 22 ABSOLUTE COUNTS Absolute Counts Events ul of the reference population beads 490 tad BEADS v If the user is working with beads for absolute counts there is no need to calculate a formula for each sample and each population With this tool the user can choose which population will be taken as reference for the calculation and with the number of events per microlitre the software will automatically show a new column in the Analysis Window with the events microlitre data amp ANALYSIS x000x 001 BAX File Statistics Selection Profile Windows Help Kj i 158575 156481 0 0 0 0 0 1099 995 O0 K K K K a a Gate 0 events Total 0 00 Visibility 0 00 AVAILABLE FUNCTIONS It is possible to show the Mean Standard Deviation Median Mode or even a New Function which can be defined by the user All the statistics available can be shown or not in the population tree and in the report CY XX 090507 Rev 002 Issue date April 29 2010 Page 80 of 127 The user can choose the functions of interest to show just moving to the right with the button from Available Functions chart to Show Parameter Statistics chart In the Available functions chart the user can Add a New Function Then i
16. Select type of Ai Gagra ta Front Cime All Diagram Apps Defiealt Valkea ter Al Diarani SEED ERR EF i EEEER EERE E EF SEEEEERRERE w g g a g g g g are oat venir bi CY XX 090507 Rev 002 Issue date April 29 2010 Page 17 of 127 E SAMPLE ANALYSIS Draw a region with the mouse around the cells of interest The region is automatically closed as soon as the user releases the mouse The selected events will be shown in black in all plots Go to the Population Tree and click on the label of the selected population Then the events will change the black color to the color selected 886 8 Eap 38 Law hed Ce ee er ae i im a ry ELLI BPC COAG LOGICAL 5 g f k d eee EEE EEE SEELEREREEEE EESES SE ES Dast cols Pathologcal clonal cel Sins id ee ee eg e wees s w w 4 ee 4 Gate O events Tawi G00 APC WCD45 LOGICAL F OPEN A NEW FILE If you want to open another file just click on File Open then go to the directory and select another file d Laa dep pe T Tes as ae Antistes Probie Help tiga A Fils p Open Preadud Fie CtriepP i Open Analyned File Tive Arahyted Fda LOL oe ba ee a z a a a Li a PEERS ER EEE EEEEEEEEEEEE 3 eak 0 00 ey es O00 a d G REPORT Go to File and click on Report A page report opens with different components with standard information about the analyzed populations wit
17. c6 B49 Port P Eyt P A LOGARITHMIC i iiH a mii PE ch APPA PE LOGARITHMIC CY XX 090507 Rev 002 Issue date April 29 2010 Page 28 of 127 4 6 Types of graphic representations The representation and manual analysis of cytometric files is performed by several graphical representation windows which can be in the current version of the following types described in this chapter Each one of these diagrams is included in its own individual window which can be moved throughout the screen or amplified reduced or hidden according to the user s needs Since they can be amplified to full screen size the diagrams can provide a very detailed view of the different event sets When the program is closed it remembers the characteristics of all the diagrams and when it is restarted it regenerates automatically the diagrams with the same characteristics that they had at the closing of the last session To eliminate a diagram just close its window All diagrams contain an internal configuration menu with a right click in the middle of the graph The complete menu in the right click is explained in detail in chapter 4 7 To create a new graphic Click Diagram in the menu of the Analysis Window Menu Bar and choose New diagram or Ctrl D see 4 13 3 1 Diagrams menu Then the user may choose between Histograms DotPlots APS views MultiDimensional graphs or Statistical graphs and choose the number of dimensions to be represented 4 6 1 H
18. diagrams you won t be able to select events in all the files at the same time Therefore it is recommended to create some new diagrams only with common parameters If the panel is correctly design the common markers will be enough to clearly identify a homogeneous population that will be later calculated for all the rest of the parameters The diagrams you create will be saved as a new desktop in the profile that will be showed only when you are performing this step of the Calculate Data CY XX 090507 Rev 002 Issue date April 29 2010 Page 111 of 127 r 7 y men g y o O Caleutate Data Llata 7 Pie Cdt D yau Raitis Profile Shepit tie AA Pomuaton Mes i I Eee Tike E 2 CaieSClassity eremia E f vi J Chons one jo cauaa H H 4 Werty and cave resuia i r Fs E gj E ml i i Ea a ae a rey q q J ra a aaa auol n j K Poo e a y me aoo oo oo hee a ii TF aia iba ee a a a a a ae ee ee 4 Hal OF pian hirri Jj hrinn Hid hee FSC A LINEAR CD19 PerCP Cy3 5 A PercP Cy3 5 A LOG FE etic Piece ae iq Ja 4 A bie Ea 4 a a p H ea an cn wl on celle eles H eT gj T t q pi J X 1 SS5C A Exp S5C Low 1 SS A Exp S5SC Low Fil Muimber T J 4 1 4 Oe ee fe oe a a SH DH HEP HE HE HS A ane 10 w id TE CD20 APC AAPC A LOGARITHMIC APS 1 045 PercP cy5 5 A PercP cy5 5 4 LOG FS A LINEAR Once the population s of interest to cal
19. 55C FMC FITC A FITC A a CD24 PE A PE 4 a CD19 PerCPCy5 5 4 PerCP Cy5 CD34 APC 4 4PC 4 FSC Height 55C Height a LAMBDA FITC A FITC amp KAPPA PE 4 PE 4 CD19 PerCP Cy5 5 PerCP Cy5 5 4 E CDS APC A APC A QO oc Ss I Add Group Name as FSC A JEEE aar a Parameter Groups J a FSC 4 a FSC 4 a FSC a FSC Height 55C Height LAMBDA FITC A FITC A a KAPPA PE A PE 4 a CD19 PerCP Cy5 5 PerCP Cy5 5 4 CDS APC 4 4PC 4 Ss L Use previous groups Use previous groups CY XX 090507 Rev 002 Issue date April 29 2010 Add Group Name as FSC A a Parameter Groups 7 8 FSC A 7 a FSC A g FSC J FSC Height Page 97 of 127 Once all the desired parameters are selected click Done Automatically the matrix will change to show the grouped parameters as Common At the same time the software will store these actions in Previous Groups and will be possible to remember them later It means that at the same time the user creates groups of labels the software creates a dictionary The second time we load these files to make a merge the software will make the groups automatically if we click on Use previous groups W SRRBIKAPPAPESAIPEA OOOO F 55C Height _ Ges pD 9 PerCP Cy5 5iPerCP CyS S A F LAMBDA FITC A FITC A v C ICDS APC A APC A F a KAPPA PE A PE A
20. Ctrl and Minimize or Ctrl the user can increase or decrease the size of the dot plot 4 7 5 2 Duplicate Diagram The option Ctrl L creates a duplicate of the same graph with exactly the same configuration preferences and analysis that the copied one 4 7 5 3 Copy Diagram The option Ctrl C allows copying the diagram for a later paste in different software such as PowerPoint 4 7 6 Assign population This option allows the user to classify a population without clicking in the population tree The same populations and colors defined in the tree are also present in the assign population list Therefore after drawing the gate the user can right click on the diagram and go to Assign Population 4 7 7 Undo Redo This option gives the user the possibility of cancelling or doing again the last modification in the analysis procedure These functions undo and redo respectively the last operation performed or undone in any of the currently open diagrams There is unlimited number of actions of undo or redo CY XX 090507 Rev 002 Issue date April 29 2010 Page 46 of 127 4 7 8 Invert 4 7 8 1 Invert Axes This command acts by inverting immediately the axes represented in a 2D dot plot 4 7 8 2 Invert Diagram Color Black White Background Color This option allows the user to configure the background of the graphics initially shown in white Click on black or white if you prefer respectively a black or whi
21. Export Ctrl E Report Documentos recientes File name Sample 1025 cyt CY XX 090507 Rev 002 Issue date April 29 2010 Files of type Infinicyt analyzed files cyt iv 2 E ISG GES CCCEECCCCEEGGOGG Page 123 of 127 10 ADVANCED ANALYSIS This option is not available in the Basic Version of the software In this Module different Advanced Analysis will be included on each update In this version Automatic analysis for BCR ABL fusion protein kit is available 10 1 BCR ABL Analysis Introduction Philadelphia translocation is a specific chromosomal abnormality It is the result of a reciprocal translocation between chromosome 9 and 22 and is specifically designated t 9 22 q34 q11 The result is a fusion gene created with a part of the BCR breakpoint cluster region gene from chromosome 22 region qll to the ABL gene on chromosome 9 region q34 Within EUROFLOW PROJECT it has been developed an automatic system to study the presence of BCR ABL fusion proteins with BCR ABL Protein Kit This Module is available on the Advanced Module and it can be analyzed trough a wizard in the Advance Analysis from the Main Screen 10 2 BCR ABL Wizard STEP 1 SELECT AN ANALYSIS ee EEE 4 Steps Select an Analysis Select an analysis Select controls and samples Merge info BCR ABL report l Help g main Menu Previous In this screen t
22. Issue date April 29 2010 Page 95 of 127 W Select Files select Files if Look inc Sample 1345 E EES 2 Sample 1245 kzotypic Corirol ia 5 Sample 1245 Merged cyt Doci E Hi r c es LEIN File name Sampe 1245 Merged cyt Escriorio Fies oftype lau Files Group with this action you will be able to correct typing mistakes of the labels by writing a new name for them and which parameters should all have exactly the same label Detailed information of this tool can be found on next chapter 6 4 Manual Analysis with this button you can go directly to Manual analysis without saving to disk the new file resulting of this process The new merged file will be open and ready to analyze but without saving This can be done to avoid memory space if you prefer to check and analyze your file before saving Configure File merge options allow manipulating the controls that will warn you in case your common parameters are not similar enough to perform a merge process See chapter 6 4 for more information on this Save as when the selection of the data files 1s done and the parameters to be merged are the correct ones the user can save this information The software will ask you for a new name and directory for the new FCS file Help document with the User s Manual chapter of this Module will be displayed To go back to the main screen of the program click the Main Menu command 6 4 Group tool Dictionary of labels
23. M 6 Cells Gate S Leucocytes NE Cells Gate 4 Leucocytes 4 11 Save Analyzed files Infinicyt allows saving the files already analyzed keeping the classification of the populations the statistics To save the analyzed sample when the analysis has finished go to File Save Analyzed File on the Manual Analysis Window Menu Bar and Infinicyt will immediately ask you where you want to save that file It will be saved with the extension cyt WARNING In the cyt files it is also included the profile in which it was created Then a way of calling a profile can also be opening a cyt file that was analyzed with the profile of interest Notice that whenever we perform this action there is the opportunity of CY XX 090507 Rev 002 Issue date April 29 2010 Page 57 of 127 Include the FCS data in the Analyzed file as a default the new cyt file will include also the raw data If the user unclicks this option only the data analyzed will be saved The advantage is that this file is much lighter in memory but if you want to open this cyt file the software will ask you to open also the raw FCS file Anonimize the file This can be done selecting the option anonymous in the saving window The fields not matched will be excluded from the file to save o Save As Save in MergeFiles 2 og Documentos recientes Escritorio e Filename FCS2_0 fcs cyt Gai documentos oOo Eo ta type In
24. Preferences H Analysis Diagrams Controls H Statistics H Virtual Parameters H Stored Compensations Gates and Analysis Strategies i Reference Image 5 FCS file H Current Compensation Negative Visibility Configure APS Parameters i Properties Stored Reference Images normal B cells normal maturation blast patient xxx Diagnosis Blast Patient yyy Diagnosis I Tools Previous Groups Default Visualization Dots Highlight Lines _ Highlight Contour Density isolines As shown in 4 9 it is possible to store a group of events as a Reference Image in the graphs All the groups of events we Save as will appear listed in the profile Through this window we can delete them or change the appearance of the image CURRENT COMPENSATION This option shows the information of the compensation values which is stored in the data file These values can be changed manually and be Saved as It is also possible to Choose a stored compensation and change the values to modify it Profile Configuration Preferences Analysis Graphs Controls Statistics Stored Compensations Gates FCS file Current Compensation i Negative Visibility Configure APS Parameters H Properties Compensation data in Column Row A B C D 1 10 84 3 49 0 32 35 3 2 64 CY XX 090507 Rev 002 Iss
25. Properties lt gt If you click on Add Parameter the following window appears and you can give a name to the new parameter and choose which calculation you want to represent e g dividing a parameter by a constant number rest one parameter from the other PE kd Parametar EJ waa APE Once the new parameter is created it will appear in the list of parameters to represent in the diagrams and will contain all the statistics as usual STORED COMPENSATIONS In this part of the profile the user may have stored different compensation with different values See also Current Compensation Option this same section to learn how to store them The user can change the current compensation for one of the compensations already stored When clicking OK then the selected compensation will apply to the file but whenever you open a new file it will be always opened with its internal compensation CY XX 090507 Rev 002 Issue date April 29 2010 Page 83 of 127 D Profile Configuration 4 Stored Compensations B Preferences H Analysis H Diagrams H Controls H Statistics H Virtual Parameters Stored Compensations H Gates and Analysis Strategies ie Reference Image FCS file Current Compensation Negative Visibility H Configure APS Parameters Properties I Tools Previous Groups Compensation 2 Compensation 3 0 00 1 34 0 13
26. Remember that for a faster analysis it is recommended to have the statistic view option deactivated in the view menu until you need it All these statistics can be shown on the report You can build a report that always shows the statistics of interest or you can export them to csv files as described in next chapter 4 13 4 2 Export Statistics The export option from the Manual Analysis Window Menu Bar Statistics Export Statistics is a tool to export to other platforms the data of the analysis When the export option is opened this window will appear where and user can configure which population which parameters and which value of each to export It is possible to Save different configurations of statistics to avoid repeating the selection of fields every time the user opens this tool Configurations Selected Configuration Select only populations with events Default mera Show Files Default Events All files stats on lymphocytes absolute counts Other Events NEUTROPHILS Columms Events otal Partial Visibility B CELLS NK CELLS DEBRIS aose 0 K aO OOOO KKOO K oO O K K FE EEEE Median Then clicking on Export the software will ask where to save this new file with the statistic data The proposed extension for this statistic file will be csv coma separated CY XX 090507 Rev 002 Issue date April 29 2010 Page 70 of 127 values which can be r
27. SSCLow iv PE Cy A LOOTA Band 3 FITC 4 CD38 v Band 4 PE A CD56 v Band 5 PerCP Cy5 5 A CD19 Band 6 PE Cy 4 LOGICAL v Scale These parameter band representations are especially useful to visualize the whole phenotype of a population in a merged data file containing all the parameters in the same file The scale of each band can be represented in the X axes If the user prefers to hide the scale right click on the diagram choose Diagram Configuration Legend and Scale Configuration and choose or not Show Scale Diagram Configuration b Axes Configuration Chrl Configure Visualization Data Negative visibility Configuration Local Visibility virtual Parameter Configuration Number of Events Displayed Compensation window Manipulation bo Exp S5C Low Configuration Assign Population f Legend Scale Configuration Undo Redo e Invert b Diagram Info o S Legend Scale Configuration ta i i z oe ci Medeia S3C A Erpse Low e aes Coole v Show Legend 15 o pore asoiy CAPMCT LOGARITHMIC L stow sea Cia i ACR LODARITHINIE Parameter Bard Legend Postion Righi Cy SACOS LOGARITHMIC The complete menu in the right click is explained in detail in chapter 4 7 CY XX 090507 Rev 002 Issue date April 29 2010 Page 33 of 127 3D Graph Graphical representation of events cells or particles in three dimensions The value on each of the three dimensions param
28. Sarir n Soki Pigula alawated eens ow Cther File Selected Summation This option is useful when the user needs to manually calculate the variables and needs the summation of a column Whenever the two parts of the formula have been chosen the user can choose which type of calculation has to be performed Then clicking on the signal the software will display the different mathematical operations available Then click OK 9 Add Function Meme RATO CDAM TS l e Al ie Apply Cancel FORMAT Decimal separator For the values showed in the statistics report and txt exportations Character separator in CSV files The user may choose the appropriate value separator Define decimal places It is possible to define it for the values showed in the statistics CY XX 090507 Rev 002 Issue date April 29 2010 Page 82 of 127 VIRTUAL PARAMETERS This version of the software allows creating new parameter as a combination of others In this sense the way to create them is similar to create new statistics with the difference that the new created parameter can be chosen in the diagrams to be represented X Profile Configuration Preferences Available Parameters Analysis Diagrams Controls Statistics eae ee StorecGompensations Gates mi Reference Image L FCS file Current Compensation Negative Visibility Configure APS Parameters aa
29. in the option Files of Type in the save window so you will have cyt files available to calculate W Select Files Look inc Sample 1245 H SEG Select Files Sample 1245 lsotypac Control kia Z Sample 1245 Merged cyt Domenie i reciaigies LS Fiename Sampe 1245 Merged cyl ancel Escritorio ries of type lag Files Remove with this button you can select a file to remove from the list of files to be calculated If no file is selected and you click this button all of them will be erased You can select and move the files on this list with te and V cursors in order to obtain the best sequence to represent the tubes in the graphics The first one will be the closest to the axis in the graph representations Once you select the data files the matrix parameters file appears in the lower part of the dialogue box In this matrix the user can Add or not each parameter in order to avoid parameters that are not interesting because there is no marker on it With this we will optimize the memory of the new data file reducing the number of parameters to be merged For more information about Merge process go to chapter 4 2 CY XX 090507 Rev 002 Issue date April 29 2010 Page 110 of 127 Calculate Data Jol Steps File list 1 Select file s 1 C 008 LLC K L 19 5 2 Gate amp Classify events r 2 C 012 LLC 22 23 19 20 Select Files 3 Choose populations to calculate 3 C 013 LLC 10
30. new upgrades can be downloaded as well as many other features and advantages are available CY XX 090507 Rev 002 Issue date April 29 2010 Page 12 of 127 2 9 Special considerations Coulter Cytomation users Infinicyt does not apply compensation to Log acquired values To see the compensation in Area and Lin parameters you need to check log acquisition to get the compensation matrix Sentinel Drivers If you are installing Infinicyt in a computer with DIVA software which includes Sentinel drivers as well please make sure that the version of Infinicyt Sentinel Drivers is higher only in that case install the drivers CY XX 090507 Rev 002 Issue date April 29 2010 Page 13 of 127 3 MAIN SCREEN File Merge File Editor Manual Anslysis Calculste Deta File Export Adwanced Analysis When starting INFINICYT the first window to appear is the Main Menu Window with the basic functions of the software File Merge to allow the user to merge flow cytometric data from different files obtained from different immunostained tubes into a single file representing a complete immunostaining protocol of one sample Calculate Data only included in advanced version The user may calculate several characteristics of the cells which have not been directly measured based on the knowledge of those characteristics in phenotypically identical cells which have already been measured File Editor to modify certain p
31. of 127 When a logical transformation is represented the user has the choice of configuring the negative part of the scale for a better visualization with the Negative Visibility Configuration See detailed information on section 4 7 1 2 and also in the Manual Analysis Window Menu Bar in Profile Configure Negative Visibility on chapter 4 13 5 10 Side Scatter transformation Side Scatter may also be represented with a mathematic transformation to expand the values of the lower part of the scale for a better visualization of the populations Exp SSC Low Exp SSC Low transformation can be used with a default transformation or it can even be configured by the user See section 4 7 1 5 max D boen 2 25000 N 25000 N 100000 100000 FITC A CD22 PE 4 CD23 Percp cy5 5 4 CD19 PE Cy 4 APC A CD20 APC Cy 7 A Pacific Blue 4 AmCyan 4 FITC 4 CD22 File Number PE 4 CD23 Exp 55C Low IE Percp cy5 5 4 CD19 Logic PE Cy 4 APC 4 CD20 APC Cy 7 4 Pacific Blue AmCyan A SSC A Exn SSC Lo SSC A Exp SSC Low Lin Log Default Exp 55C Low Configurable Exp S5C Low File Number Logic Lin Log Negative Visibility Configuration 4 13 3 2 Tile The diagrams will resize and the program will rearrange them to cover the whole screen of the computer In the menu Diagrams there is a short command for this option Ctrl T If there is no diagram open Tile function will op
32. process For example if you merge 4 files containing 50 000 events each the result will be a file containing 200 000 events All the parameters measured will be merged into a single data file with n parameters but each parameter is saved separately As you can see in the following figure after the parameter based merging process the user may study the expression of all the markers of the panel in the same data file Phenotypic description of cell population P8 0P FSC Height FSC H LiNeaR SSC Height SSC H Exp SS Low CD 103 FITCIFL 1 H Locanrrnme er yy we as Poe Ou CO25PE FL2 H LOGARITHMIC 2 gt CD19 PERCPCYS SILSH LOGARITHMIC CD11 APC FL4 4 LOGARITHMIC S Aarets ie C022 FITCFLT H LOGARITHMIC CD23 PE FL2 H LOGARITHMIC ig We RES Bo CO20APC FL4 H LOGARITHMIC FMC7 FITC FL 1 H Locanrrumic aoe 1 Merged File CD24 PEFL2 H LOGARITHMIC oe Bere CDI4APC FL4 H LOGARITHMIC base 7 2 r o v a 9 bol oa a a v n w w w wa Deea r re CD19 PERCPCYS SEFLIH LOGAN COS APC FL4 H LOGARITHMIC xe 5 KERN LELE 6 2 Merge conditions for global analysis Infinicyt will merge all the data files required but if the user wants to perform a global analysis on all the tubes at once be aware of the following Acquisition settings The parameters and files must be similar regarding acquisition settings PnE PnR and Data Type If
33. see chapter 5 1 2 4 i he n 9 v g E E E z g g c Fie 1E1 i qE1 1E2 1 1 TCRgd FITC FL1 H Log TCRgd FITC FL1 H Log TCRgd FITC FL1 H Log Selection Select File File 1 Selection Select File File 3 Selection Select File File 5 CY XX 090507 Rev 002 Issue date April 29 2010 Page 106 of 127 7 CALCULATE DATA This option is only available in the Advanced Version of the software 7 1 Description The user can calculate several characteristics of cells in which a parameter has not been directly measured based on the knowledge of those characteristics in phenotypically similar cells which have already been measured in a different aliquot Then once the same cells have been measured in different aliquots the software will find for each cell the equivalent one on the other files and will apply the values for the other markers not directly measured on them This process is valid to study phenotypically identical cells in which is not possible to measure all the characteristics in a same aliquot due to parameters limitations In order to merge and calculate the parameters the original files must have as many as necessary common parameters that allow the identification of the population in which we want to calculate different parameters The necessary number of common parameters as well as the appropriate marker panel design is fundamental in order to obtain satisfactory results with the calcul
34. see how to configure new statistical functions In the Statistics option of the menu you can visualize the statistics by having this option Show Statistics activated V The new columns will appear in the right part of the population window Normal peripheral blood CD3 CDS6 CD19 001 RAF My Profle wirdons Help came Total 2141 LISA 12 4 Berle Bl he rae 718 LHR LHR aver Lh he Tie 0 OD DU ir Dire ITFA u Qh TTA HEPA m Gate O eis Tote 00 Vey he 000 Sa ANALYSIS Normal peripheral blood CD3 CO56 C19 001 CY XX 090507 Rev 002 Issue date April 29 2010 Page 69 of 127 The information columns can be moved by click and dragging the column right or left ANALYSIS Normal peripheral blood CD3 C056 CD19 001 fie Statistics feedin Profle Windows Heb a 2 z 2 Pupdietion 2 40 i vis Tota i a D s 4 at i Mean CV Mean D a Everts j 2000 1372 MOO M23 69 A 77843 Other Everts 24 4 28 4 28 v 8 0 0 00 om 3 oo fnno 0 dw OwA Das 14 7 BTN B7IN LATA Srey ISS 11 57 11 57 0 0 07 oom ors 0 00 0 0 00 O00 040 ver N 2 a a 0w O00 0 165 442 121 49 50D W 4l 7 18 32 655 meS 4 13 4 23 G40 aa 2 37 SH 7P Rw mA 0 00 oo OM 0 00 0 00 0 00 0 00 oao aw 0 00 0 00 0 00 00 OM 0 0 0 00 aC pics o 5 aA a Gaei O evens tad D viy i ODD WARNING
35. stores together with the analyzed file the Diagrams Population Tree Report and settings used in that previous analysis which it means a profile Warning A This action will upload a previously saved profile The current profile will be lost iF unsaved 4 13 1 5 Save Analyzed File This option is used whenever the user wants to store the analysis of a file The software will ask the directory where to save it and the file will be saved as cyt The original data file will remain the same in the corresponding folder because the cyt file does not affect to the original one A cyt file will include the analysis of populations with the corresponding Population Tree the Diagrams the Statistics the Report and all the information contained on the profile used to analyze it 4 13 1 6 Export When this button is clicked Ctrl E the program goes to the File Export Option and in this case the file to export will be the one currently opened with the analyzer For that reason an additional option is included in this window and it is used to export only visible populations With this tool it is possible to export gated events or some specific population File Export xX o File Basic Advanced Parameter FITC amp CD22 FITC A PE amp CD23 PE A PerCP Cy5 5 4 CD19 PerCP Cy5 5 APC 4 CD20 APC A Simulated Time Y OK Events See Export chapter 9 for mor
36. 0 00 0 02 1 54 1 94 0 01 0 00 0 00 0 00 0 28 0 25 0 00 0 00 GATES AND ANALYSIS STRATEGIES This window as explained in chapter 4 10 shows the different Strategies saved and gives the option of checking or delete them In the lower part of the box it possible to change the color of the events and the contour of the regions D Profile Configuration Stored Analysis Strategies 5 Preferences Analysis Strategy List pe Analysis screening Diagrams HT Cells Gate 1 Events invalid 3 amp Controls E CD4 T lymph Gate 2 T Cells invalid i Statistics E CD8 T lymph Gate 3 T Cells invalid H Virtual Parameters Stored Compensations Gates and Analysis Strategies i Reference Image FCS file Current Compensation Negative Visibility Configure APS Parameters W pathological 8 cells Gate 4 Events invalid Kappa Gate 5 pathological B cells invalid fj Lambda Gate 6 pathological B cells invalid J NK Cells Gate 7 Events invalid ER Rl Rb AR Re ij CD34 Gate 1 Events JJ BEADS Gate 2 Events E E Gate Color ERATA Selected Events Color In this window it is also possible to change the color of the contour of the selection region and the selected events before an assignation CY XX 090507 Rev 002 Issue date April 29 2010 Page 84 of 127 REFERENCE IMAGE Profile Configuration 5
37. 002 Issue date April 29 2010 Page 99 of 127 In this sense the Merge Control allows to compare the main populations represented in the file to detect for example that a common MoAb has not been added while the Populations control allows comparing populations less represented which can go unnoticed in the global coherence control In both controls there are some threshold values to define the strictness of the software controls to show a warning of no coherence between files or populations The threshold values explained in the next chapter can be obtained in the Manual Analysis Window Menu Bar option Profile Configure Preferences Controls menu 6 5 1 Merge Control WARNING OF NO COHERENCE BETWEEN FILES This validation process evaluates the global image differences between the respective distributions of events of the different tubes in the n dimensional space defined by the n common parameters In the current version this image difference is represented by a number from 0 to 100 as follows value Q no significant difference the distributions are identical value 100 the distributions are completely different The values between these limits represent different degrees of similarity Although in general values close to 0 will indicate very similar distributions and those close to 100 very different ones To activate this control check the Merge Control option The parameter indicated as Minimum difference of
38. 1 Manual ANALYSIS RAX File Edit Diagrams Statistics Profile Help 9 2 a CJ E 5 gt T Population 5 5 E EA VIS fi 2 fa 2 a W unclassified events Other unclassified events 0 42 0 42 0 00 5 14 5 14 57 39 3 82 3 82 42 61 54 06 54 06 0 00 0 13 0 25 0 00 23 37 43 23 0 00 30 56 56 53 0 00 2 78 2 78 0 00 33 78 33 78 33 78 100 00 0 NA NA OOOO ECO K Gate 0 events Total 0 00 Visibility 0 00 Whether you Add a population or Edit a population you will be able to write the name of the label you want in the box name and also choose its color by using the standard or advanced options for colors CY XX 090507 Rev 002 Issue date April 29 2010 Page 24 of 127 Add Population Name E Basic mestras HSB RGB visualization O Normal Highlight If you want to visualize your population with Highlight you may enable this option in the Edit Population Dialogue Box If you want to visualize populations with automatic highlight whenever they represent less than a given percentage then choose Profile Configure Graphs from the menu and define the maximum percentage as described in 4 13 5 10 4 4 7 Drag and drop labels It is possible to move a label to be included as a subpopulation of a pre existing one just clicking and moving the label without releasing the mouse as a drag and drop tool With Up Population and Down
39. 2010 Page 20 of 127 4 4 How to analyze and select populations 4 4 1 Assign label to populations Once the user has opened a data file and the diagrams to perform the analysis the selection of a population of interest is made by drawing a line with the mouse around the events cells This line is automatically closed as soon as the user releases the mouse Once the selection is closed go to the Population Tree and click on the label of the population you are interested in The events included in the region delimited by the line you drew will take the same color as the label GATE 2D Joe MM_001 fcs Manual ANALYSIS BAX Fie Edit Diagrams Statistics Profile Help Population 300 400 600 900 50000 Ee 50000 100 00 al 0 NA 200 100 00 NA 100 00 NA Monocytes 0 NA NA NA 0 NA NA NA gag Eosinophils 0 NA NA NA SSC H SSC Height Exp SSC Low Gate 18060 events Total 36 12 Visibility 36 12 FSC H FSC Height Lin MM_001 fcs Manual ANALYSIS File Edit Diagrams Statistics Profile Help Population 300 400 600 3900 50000 31940 63 88 63 88 63 88 0 NA NA a Monocytes 0 NA NA 18060 36 12 36 12 ii Eosinophils 0 NA NA SSC H SSC Height Exp SSC Low 200 FSC H FSC Height Lin Gate 0 events Total 0 00 Visibility 0 00 To help the user during the analysis if a gate has been drawn in any of the diag
40. 3 25 19 11C 4 C0014 LLC FMC 24 19 34 4 Verify and save results av File previews nfo Parameters File FSC A SSC A LAMBDA FITC A FITC A KAPPA PE 4 PE 4 CD19 PerCP Cy5 5 A PerCP Cy5 5 amp CDS APC A APC A ga 0022 FITC A FITC A CD23 PE A PE A CD19 PerCP Cy5 5 PerCP Cy5 5 4 CD20 APC A APC A CD103 FITC A FITC A CD25 PE A PE A CD19 PerCP Cyaninas 5 PerCP Cy5 5 4 CD11C APC A APC A FMC FITC A FITC A CD24 PE amp PE A EQ CD19 PerCP PerCP Cy5 5 4 D J CD34 APC A APC A_ al M M v If some of the labels of the common parameters are different and they are not classified as common then you can click on Group to change the labels See chapter 6 4 Once you are sure that these are the files and parameters you want to calculate click Next on the wizard to follow with the next step Gate and classify events STEP 2 GATE AND CLASSIFY EVENTS In this second step the software shows a desktop set of Diagrams similar to manual analysis but with certain limitations The goal of this step is to select manually which is the population to obtain information based only on common parameters You can gate and classify as many populations as you want to calculate but only in diagrams with common parameters represented You will find the comment gates disable in some diagrams if you are showing no common parameters because in these
41. 6 54 7 NEGATIVE ut LAMBDA FITC A FITC A 7 0 58 1 NEGATIVE KAPPA PE A PE A 14 2 17 3 POSITIVE CDS APC A APC A 108 2 66 5 POSITIVE Paste Remove Sheet CY XX 090507 Rev 002 Issue date April 29 2010 Page 88 of 127 You can complete this standard report deleting and adding some elements by right clicking in the middle of the report A menu will appear with the different elements you can include population trees data image diagrams or text All of them can be moved and resized in the report page Add Component Choose a Component Each of these elements will also have a menu for example if you choose Population you will obtain a list of your populations If you double click on it you will obtain a complete menu where you can choose the populations and parameters to be shown on the report amp Population Options Advanced Select only populations v VIS Population Neutrophils Display Options _ Indent Show background Data font rim NK Cells Header font Cancel If you choose Data a box with some sample data will appear as a default Clicking twice on it you can configure this element and choose the data of interest All the fields will have to be filled any time you want to do a report of a new sample If you want this data to be automatically updated then select all the fields from the FCS info and will be directly read from the data file CY X
42. 7 Rev 002 Issue date April 29 2010 Page 30 of 127 The second one is a Histogram of two dimensions The scale in this case will be shown only if both parameters include the same scale If they are not the same no scale will be shown Both diagrams can be obtained from New Diagram Histogram on the Manual Analysis Window Menu Bar and then the user selects as many parameters as desired to be represented in the same graphic NEW DIAGRAM Histogram C DotPlot APS diagram O Multidimensional gt Stat diagram Number of parameters The user can choose the number of dimensions with the only limitation of the number of parameters measured F Peng Jot Pe aliia O KAPPA PE A Log wr LAMBDA FITC A Log lene CD24 PE A Log AON CD5 APC A Log a ee FMC7 FITC A Log tN CD19 PercP Cy5 5 A Log a N Ea CD20 APC A Log CD22 FITC A Log Fa M SSC A Exp SSC Low Ps j FSC A Lin A To increase the number of bands decrease or change the order once it is done it is possible to do it by right clicking in the diagram and the choose Diagram Configuration Axes configuration 4 6 2 2D DotPlot A DotPlot is a statistical chart consisting of a group of data dots plotted on a simple scale DotPlots are one of the simplest plots available They are useful for highlighting clusters and gaps as well as outliers CY XX 090507 Rev 002 Issue date April 29 2010 Page 31 of 127 We can choose different types of DotPlots
43. As an example clonal pathological cells from B lymphoproliferative disorder fit the conditions of being identical clonal therefore if they are appropriately selected with common markers the information from the different tubes can be calculated in the others 7 3 How to use the Calculate Data wizard Calculate Data has to be used following different steps Whenever you click on the Calculate Data in the Main Menu you will obtain a wizard with the different steps you will have to follow E Calculate Data o Steps File list 1 Select file s J Select Files 2 Gate amp Classify events av File preview 3 Choose populations to calculate 4 Verify and save results CY XX 090507 Rev 002 Issue date April 29 2010 Page 109 of 127 STEP 1 SELECT FILE S In this first step the user has to select the files in which the data has to be calculated You can select either independent files or you can select an already merged file In both cases the software will consider them as a merged file and the window will show a matrix as it is shown in the merge screen Select files this command opens the directory where the user can select the files to be merged and calculated Click Shift to select consecutive files in the folder or Ctrl to select alternative files Once the selection is done click Select If you want to calculate and already merged and analyzed file you will have to choose all Files
44. C A FMC7 FITC AcFITC amp CD24 PE A PE A CD19 CD34 APC 4 APC A The option Info in the second tab of this window will show us any comment written on the original data file during the acquisition Even in a merged data file you can visualize also the names of the files Sample 1025 Manual ANALYSIS aax Statistics Profile Help Fie Edit Diagrams Controls A Population Files Open Ctrl 0 Open Next File Ctrl N Open Previous File Ctrl P Events Open Analyzed File Save Analyzed File Export Ctrl E Total Partial Visibility amp Profile Configuration Report Preferences Diagrams Controls Statistics Bids Gates K K K lt EJ FCS file Properties virtual Parameters Stored Compensations Reference Image Current Compensation Negative Visibility Configure APS Parameters Properties File Parameters Comments Comments CY XX 090507 Rev 002 Issue date April 29 2010 Page 62 of 127 4 13 1 9 Exit When choosing this option the program will exit the Manual Analysis mode closing all the windows associated with this process including the Manual Analysis Window and all the Diagrams The Manual Analysis session can also be finished by closing its Analysis Window with the same result 4 13 2 Edit In this section we described all the o
45. C HiGSC Height Exp 6 Low FSC HFSC Height Lin Ihana Ye 31 Lite L tence Default EXP SSC Low If you click this option in the DotPlot menu a default mathematical transformation will be applied to the SSC parameter of the file to improve the separation between populations in the lower part of the SSC scale 4 7 1 6 Font Legend Scale configuration This option allows controlling the appearance of the parameter label The user may change the appearance of the parameter labels by changing the font size bold Italics If you want to have no legend in the parameter label disable Show Legend or Scale in this window Legend Scale Configuration Show Legend Show Scale Font option allows controlling the size and appearance of numbers shown in the scale If you want to show no scales and legend just unclick both CY XX 090507 Rev 002 Issue date April 29 2010 Page 41 of 127 4 7 2 Configure Visualization Data Configure Visualization Data Visualization Data Diagram Configuration gt Dots Configure Visualization Data Means of files F a Local Visibility gt Medians of fies Number of Events Displayed gt Density Configuration Window Manipulation gt OCEN Total Density Undo Redo gt Population Invert b Density Representation d Contour Lines Diagram Info Density increment Min i Max Visualization data In the dialogue box the user can select dots mean value or
46. Configuration Undo Fedo b Invert p Diagram Info 4 7 1 1 Axes Configuration Through this command the user may configure the parameters on each of the axes of the graphic see figure Axes Configuration Number of Parameters 6 v Band 1 FS5C v Axes Configuration Number of Parameters Band 2 55C A w Exp 55C Low Band 3 FITC A CD38 w I LOGICAL v Band 4 PE 4 CD56 LOGICAL v Band 5 PerCP cy5 5 4 CD19 LOGICAL v x Axis APC 4 CD45 w LOGICAL v PE Cy 4 LOGICAL v Y Axis SSC A wI Exp 55C Low CY XX 090507 Rev 002 Issue date April 29 2010 Page 39 of 127 4 7 1 2 Negative Visibility Configuration Whenever the FCS file has been obtained from an instrument with electronic compensation negative values that are usually close to the axes may be generated This option allows visualizing the negative part of the scale to study the events shown on that region When you click this option the window with the negative visibility percentage is displayed and you can modify the values This is the same window that appears in profile configuration and this information will be saved as part of the profile lees 100000 Diagram Configuration p Axes Configuration Ctri G Configure Visualization Data Negative Visibility Configuration Local visibility gt Number of Events Displayed p Compensation Window Manip
47. Cy5 5 4 mE CD19 PerCP Cy5 5 A 1 il CD19 PerCP Cy5 5 FL3 H il CD19 PERCPCY5 5 FL3 H lll CD19 FLIH ll CD19 PecyS FL3 H Import S E CD8 LAMBDA FITC H ll CDB LAMBDA FL 1 H kl CD8 LAMBDA FITC FLIH S CD440D19 PerCP Cy5 5 A a CD4 CD19 PerCP Cy5 5 A m CD4 CD 19 PerCP Cy5 5 A il CD19 CD4 PerCP Cy5 5 A lll CD4 CD19 PerCP Cy5 5 A 6 5 Merger process Controls Infinicyt includes a process of Coherence control to determine if the user is performing the merge process with the optimal files analyzing in different aspects the similarity of expression in the common parameters The control contains different threshold values so the user can modify them in the corresponding option Nevertheless the users are strongly advised not to change the threshold values predefined in the program when working with real data unless they understand perfectly these processes Failure to comply with this warning may produce unreliable results There are two major types of coherence controls Global coherence control Merge Control 1 the software analyzes the similarity of the total events from each file between all files merged regarding the common parameters Populations coherence control Population Control renever the user analyzes a merged file and tries to define a group of events from different tubes as a unique population the software analyzes the similarity between these populations from each file CY XX 090507 Rev
48. E Fsc A E ssc A FITC A CD22 PE A CD23 PercP Cy5 5 A CD19 PE Cy7 A APC 4 CD20 APC Cy7 A Pacific Blue 4 AmCyan 4 100 0 1E2 1E3 APC A CD20 Logic File Number Logic Lin Log Negative Visibility Configuration Type of scale representation Log and Logical Scales Negative visibility The selection of the parameters scale can show the information in linear scale Lin in Log scale Log or with logical transformation Logic Logical scale shows negative values if they exist as a result of compensation The part of the negative scale that we want to visualize can be configured in the right click menu of the diagram in the option Diagram Configuration Negative visibility Configuration a 5 236000 ALLI 100000 SSCA Cip Si30 Low 163 v 163 Sw Diagram Configurstion Axes Confgureton Cote Neyotrre Vinbity Configuration Confgze Vausiseoon Data Local visb ty Number of Events Dapiayed amp Window Marga daton gt DOS Low Cons b Unde Sede Imet b P inks aPc 4 0034 LOGICAL nme on a i Negatwe Visability Percentage fe Automasc 1 10000 negeavity 100 2 Py ameter FOC A 0o soc A 0 01 t FITC A 0015 001 a PE A CO16 0 01 J PerCPCy5 5 A CO45 0 01 a PE Cy A 0 ac ADs o t APC y7 A 0 53 Pack hea 0 42 d AMCE A 0 9 gt Apply to alt 0 01 a Lar cance CY XX 090507 Rev 002 Issue date April 29 2010 Page 66
49. Ge nren isan siadeendbseeuatneiermnbiaievaseneeoines 49 4 10 Save Apply and Review Analysis Strate gies ccccccccccccccsssseseeeeeeeeeeeaes 51 AVON Review a SUALCEY tuoi ie eieruta cl eaaest E esc 51 AA SAVES FAMALY SIS SAIE IESni a a a aa 53 ANOS APP SE E S ea E E seas 54 CY XX 090507 Rev 002 Issue date April 29 2010 Page 4 of 127 AOA Matemu OF Pardime leis scsi siieass te ieb ah a a E 55 4 11 Save Analyzed IES yae T T een 57 4 12 Export statistics from Manual Anal ySsis cccccccccccececessseseeeeeeeeeeeaeeeeees 58 4 13 General description of the analysis MENU ccccccccececeeseeseeeeeeeeeeaeeeeees 59 Te Pe n dears tid aretha Us aoe lates N aN ara 59 Abhe OPEM ty POE RNR TI A PO nO 59 AN Dele 2 MCN CT EO erenn a a 60 ANS Noe Open Previous FiCS EA 60 Alak Open Analyzed Filer a 60 ALS Save ANIV ed File enine Ta 6l PELO EO aa T T Pecmce mtn Teer earner ter 6l PEET RDO E T E aS Tre rer ann 62 ES PrOD S a ane Uae sam cotati cau tn cus 62 GMO EX a D 63 A Pieras aa el arsapieadedason theasionoearadadeeuba seein ated 63 AMS MUNGO RedOne e suena aceetantteceeeconeten 63 AND 22 Reserandl ysi icion 64 kla Delete All SClCCHONS oa a E E E T Sauces 64 Mlb SCLCCE FING wisiesd ccasiaiciwes Sotetia thioteiaalocadanhiaes isan teapucinelaceneeadiann ee 64 Alo Drar te toocus cen ieeuies terete usu eaineeau tcuies eae tinoeais ue wmeaiaautacilesomainiwes ces 65 Alle Ney ddorau E r etanstabedn
50. PE Cy7 4 APC 4 APC Cy 7 4 Pacific Blue 4 AmCyan A Time eee siecle o am Grew e D It is very important to select the correct Beads Parameter this beads parameter comes from the fluorescence where the beads were measured In this case 1s PE Select parameters oo Low 55C 55C 4 l Beads Parameter FE A CY XX 090507 Rev 002 Issue date April 29 2010 Page 125 of 127 The software validates if the parameters FSC SSC and the Beads Parameter are common to all files If there is something wrong it will show an error message i 4 Sepa Bo Select panareters T Cais an aly ih Fat FEA l as ao Awl of ie Cea ee iriak fleas Parameter PSH 3 Merge reien File pr a oe bili aa el hakt fete Paras Fie Advanced Analysis Joey 5 BEF AEL Rope Ea aA N E DETS r i E h pSr al FITC Error fal PEA Select ye r i an haa ocurred while isa on process f I Pent PaA 2 Setect corbis orci sargees There ate sising information in parasrtese Fat fl i Pier A fel APEA 2 Marge ras Pi ibe Lape Cyt 4 Walidation a Pwt fiA a I lanl jane 5 BOR ABL Repot Petar l r me man menu C Previous t Hate Of Man Meru _ PReveus Mezi gt Sed eM a E Then in case there are discrepancies between common parameters we can use the dictionary to solve the discrepancies See Dictionary tool explained on section 6 4
51. Population in the right click of the label you may change the order of the populations of the tree and the population will move one place up or down depending on the command you select 4 4 8 How to add a Function in the Population Tree With Add Function you will go directly to a window similar as the one shown on chapter 4 13 5 10 Profile Configure Statistics where you can create your own function to relate populations The new function added will not be a new column with values for each population but it will be a unique value shown as a new population 0000 001 Manual ANALYSIS File Edit Diagrams Statistics Profile Help o l CI Population Partial _ 0 42 D giso 0 00s aore Di4 514 a aio 1910 3 82 oo 42 61 efQ23 54 06 a4 6s 0 00 1359 2 70 eae be 0 00 16892 33 70 33 70 0 00s NOOB Gate 0 events Total 0 00 Visibility 0 00 CY XX 090507 Rev 002 Issue date April 29 2010 Page 25 of 127 4 4 9 How to remove population labels You can delete a population from the Population Tree by selecting the option Remove population in the right click menu of the label or even delete all of them from the tree with Remove All Populations 4 4 10 How to select show or hide a population VIS Column The column VIS located in the left part of the window contains checkboxes indicating whether the corresponding population must or must not be visible in the graphical panels If the box is d
52. Process File List O M gC 19 PercP Cyanina5 5 PerCP Cy5 5 A M fe icb11c APC A APC A O M FMC FITC A FITC A E r fe ee Manual Analysis Save As g Main Menu Select files this command opens the directory where the user can select the files to be merged Click Shift to select consecutive files in the folder or Ctrl to select alternative files Once the selection is done click Select Remove with this button you can select a file to remove from the list of files to be merged If no file is selected and you click this button all of them will be erased You can select and move the files on this list with theA and V cursors in order to obtain the best sequence to represent the tubes in the graphics The first one will be the closest to the axis in the graph representations Once you select the data files the matrix parameters file appears in the lower part of the dialogue box In this matrix the user can Add or not each parameter in order to avoid parameters that are not interesting because there is no marker on it With this we will optimize the memory of the new data file reducing the number of parameters to be merged You can merge a FCS file with an already analyzed file cyt In this case of merging files in the step you are selecting them you will have to choose all Files in the option Files of Type in the save window so you will have both cyt and fcs files available to merge CY XX 090507 Rev 002
53. SSC Low SSC Exp SSC Low E Current Ansiyss Srategy D By Leucocytes Gate Everts B T Cell Gate DiLeuxocytes Wk Gre Miecccytes Bak Gate Leucccytes Gate 0 events Total 5 0 00 Vidalty a 0 00 PS of 1013 MB used SSC Exp SSC Low PerCP CyS 5 A CD19 LOGICAL w Ss EuroFlow CY XX 090507 Rev 002 Issue date April 29 2010 Page 56 of 127 Looking in detail all information regarding the populations to review T 7Eyenrs i Other Events 72 Wf L eucocybes 90 20 Other Leucocytes 90 26 Modify the gate v M N Check where Y 3 2 the Gate has a chirds lt font colorsreds REVERONENGrafcc a iam j E 4 z Fj Nt been made J ae Me Pi rer F al Current Analysis Strategy EJ Leucocytes Gate 1fEvents 2D Events 4PC 4 CD45 Logic 55C Exp 55C Low T Cells Gate 2fLeucocytes E E Cell Gate 3 Leucocybes H E NK Cells Gate 4fLeucocytes S3 Eap 35C Low Lt T j mi LLE APC A CD45 LOGICAL The and the means if the gates are positive or negative In this example the gate for Leucocytes was positive therefore some events were designate as Leucocytes The negative gates indicate that some events called Leucocytes were moved to different populations T Cells B Cells and NK Cells Current Analysis Strategy o Leucocytes Gate 1fEyvents 2DfEventsAPC 4 CD45 Logic 55C Exp S5C Law T Cells Gate 2 Leucocytes
54. X 090507 Rev 002 Issue date April 29 2010 Page 89 of 127 C Value font F Add FCS Info If you choose Image you will be able to insert a picture or a logo stored on your hard disk Image m Keep spect You can also choose a Diagram and a dialogue box will open to choose the graphic of your interest see figure As you can see the diagram can be configured on parameters visibility density etc c r IT A FITC A C25 Plata 1 GDi i FITCAFITOA i CD05 PEAJEA COLIC APTAAEA PROT FTC AFTCA ED3 PR sie io ODI AA AA j LAMEDA FITC A FDTC4 ODS APC AAR A Show Parameters Defeit Show fie Afer m Representaton by hie invert fies C Diagram Pont Conmfiguintion Ec ae A Showy scale Font Tiyper nd wihi Gobel sibel C Local visis a _ Contour Relerence Image Configuration E Show reference image in tae diagram Name af reference mage kapoa Debil visb iy C Local visbity Contigure _ Humber ol Events Displayed Tba events vieusizalion m E Loca percentage 0 01 Jus goy LAS number of events Vive 0 shoves al aventi Visualization Data Mansal fies O Medians offer CJ Total density 2 Population Apart from choosing the type of diagram you can configure the diagram in the report with the following options CY XX 090507 Rev 002 Issue date April 29 2010 Page 90 of 127 Show Parameters Population in case of Parameter Band
55. a representative sample of the original data file A merged file can also be exported in independent files as they were originally Help Document with the User s Manual chapter of this Module will be displayed Select file Allows changing the file you want to export and the name of the file will appear on the top of the screen Save as Once the user has selected the appropriate export options if you click this button data currently opened will be saved to a disk file with the characteristics indicated by the selected options Main menu At any moment the user can return to the Main Menu of the program by using this option ADVANCED FUNCTIONS O File Export cles Byteord Datatype wt Restore Values PrE formatSource w rJ 102 SC A AMBDA FITC A FITC A 102 APPA PE A PE A 102 DS APC A APC A 102 Exp S8C Low Bip E a m D22 FITC ASFITC 4 D23 FE A PE A D20 APC 4 APC A D103 FITC A FITC 4 D25 PE 4 PE 24 D110 APC A APC A ME FIC ASFITC 4 SS UG 16 o Select File CY XX 090507 Rev 002 Issue date April 29 2010 102 102 102 102 102 102 Bibl R S e E Page 120 of 127 Byteord Datatype and PnE Format These parameters are available for the user to obtain compatibility with other formats of Flow Cytometry Standards In principle there is no need to modify them unless you want to change to a different format Restore Values This button gives the oppor
56. age 51 of 127 001 Tutorial cyt Manual ANALYSIS X File Edit Diagrams Statistics Profile Help Population to review Lens Symbol pressed Population 50000 A 4707 9 57 0 00 Se Leucocytes 45213 90 43 a 100 00 Other Leucocytes p 71 82 71 82 J 25 48 28 18 28 18 NA NA NA 25 48 100 00 28 18 NA NA NA NA NA NA Current Analysis Strategy Polulation to review M T cells Gate 2 Leucocytes 2D Leucocytes F5C A Lin SSC 4 Exp 55 M FSC 4 LINEAR ill 5C A Exp 55C Low Steps of the gating 2D Leucocytes FITC A CD3 Logic 5SC 4 Exp strategy W FITC 4 CD3 LOGICAL Gated Parameters m 5SSC 4 Exp 55C Low Looking at the diagrams Infinicyt shows in which diagram and with which visibility we have made the gates to identify a population Gates can be positive to include envents n this population or negative when the aim has been excluding events from that population ow Total 0 00 Gate 0 events For T Cells Infinicyt shows us the following diagrams lt html gt lt font color red gt R snl y lt html gt lt font color red gt Ra E l l oO wo wi A i i lt q oO w wh er wa AES O TE 1E3 1E4 FITC A CD3 LOGICAL D 50000 100000 150000 200000 6250000 FSC A LINEAR CY XX 090507 Rev 002 Issue date April 29 2010 Page 52 of 127 By clicking on Profile An
57. al Configure Diagrama List Preferences Analysis Diagrams List Diagrams Controls Defaut desktop ctatistics 2D FSC H FSC Height LINEAR File Number valid virtual Parameters 20 CDS 4PC 4 4PC 4 LOGICAL File Number inwalid Stored Compensations 20 0019 LOGICAL SSC H S5C Height Exp 550 Loy s invalid Gates 20 FL1 H TCRgd FITC LOGARITHMIC SSC H SSC Height Exp SSC Low valiq Reference Image 2D FL3 H FL3 Height LOGARITHMIC FL2 H CONTROL LOGARITHMIC valic tile 2D FL4 H FL4 Height LOGARITHMIC SSC H SSC Height Exp SSC Lovw valiq Current Compensation Calculation desktop Negative Visibility 2b FSC 4 File Number o e ee 8 oe Configure APS Parameters 2D lt 0D5 4PC 4 4PC 4 File Number 20 lt 4PC 4 CD3 LOGARITHMIC SSC A ra Properties Disease Categories Configuratio nF Diagram Info CY XX 090507 Rev 002 Issue date April 29 2010 Page 114 of 127 7 4 Calculate Data Controls In the wizard you can see the bottom Configure which can be used to set the merge and calculate controls During Calculate Data process in a given population the software will try to find for each event the equivalent event in the other files based in the position for the common markers and then apply the same values of the rest of the parameters The way to find the equivalent event is by choosing the nearest neighbour in the same position a LLLI 3
58. alization in one or more of the diagrams The special visualization can apply to the population tree and to the reference picture Diagram Configuration H Configure Visualization Data Local Visibility Population Number of Events Displayed o FETErence Window Manipulation Undo R edo Invert b Diagram Info In both cases if you need a specific visualization in a concrete diagram locally just disable Global Visualization option and clicking on Local Visibility you can mark the populations you want to visualize This will apply only on that diagram Configure Global visibility Local visibility Population Configure Show reference image in this diagram Global visibility Local visibility 1 Visibility When the local visibility is active a warning VIS will appear in the frame of the graphic to remind you that you are working on special visualization The rest of the dot plots will follow the operations you make in the Manual Analysis Window in the checkboxes VIS of the population tree but the dot plots with special visualization will work independently The warning message VIS will appear in the left part of the upper side bar the same place as GATE REV CY XX 090507 Rev 002 Issue date April 29 2010 Page 44 of 127 4 7 4 Number of Events Displayed Diagram Configuration I Configure Visualization Data Local Visibility i Humber of Events Displaye
59. alysis Info on the Manual Analysis Window Menu Bar we can see the whole strategy made to get the results W001 Tutor cyt Manual ANAT eee E Edit Chagrams Stabsties Proite Help Lead Profit Siwe Profile Pagal Sane Hebererce inte Save Cokes Srategy F HAUIA F E Current Analysis Strategy Anaha kie q E W Leucocytes Gate 1 Events 2D Events 4PC 4 CD45 Logic SSC 4 Exp 55C Low T Cells Gate 2 Leucocytes 2D Leucocytes FSC A Lin SSC A Exp 55C Low 2D Leucocytes FITC A CD3 Logic 55C 4 Exp 55C Low B Cells Gate 3 Leucocytes 2D Leucocytes FSC A Lin SSC A Exp 55C Low Te Gate Laaseestybeet 2D Leucocytes PerCP Cy5 5 4 CD19 Logic SSC 4 Exp 59 i a a e cad HJ NK Cells Gate 4 Leucocytes IGE Ansy rateg 0 oom Gates D evenk Total Yei DDG Viakey Tei C00 A oF Lod HE ed 4 10 2 Save Analysis Strategies After performing the analysis of the populations is possible to save the gate strategy Go to Profile Save Analysis Strategy on the Manual Analysis Window menu bar File Edit Diagrams Statistics Profile Help g 001 Tutorial cyt Manual ANALYSIS Load Profile z 2 ra Save Profile hs 5 Population VIS 4 Save Reference Image gt w E Load Reference Image ya Unload Reference Image ee a Fi eUCOC vE es 3 100 00 z Other Leur ave A aLegy B 67 18 E Load Analysis Strategy 32 32 82 z Unload Analysis St
60. ameters Show a message when there is a discrepancy in the dassificatio a E O Popas Show a message when you don t save the current analysis IL Tools Previous Groups Co Bcll merged Manual ANALYSIS HUO File Edit Diagrams Statistics Profile Help Controls AA All file v o i a Population Review Events Total Partial K 8624 7 32 7 32 552 0 47 0 47 13557 11 50 11 50 2718 2 31 2 31 92435 78 41 78 41 92435 78 41 100 00 0 NA NA Gate 0 events Total 0 00 Visibility 0 00 j 19 of 1055 MB used K K x fs x Number of comments fields As a default the analyzer will show only 1 column for comments about the different parameters of the sample In case you need to have more that one column for comments you can choose the number of them and their names through this option Show a message during the analysis each time the user selects events associated to two or more populations and assigns them to a new one Show a message during the analysis each time the user selects events whose populations do not derive in the Population Tree from a previously selected population Show a message if you want that Infinicyt warnings you when you change to another sample without saving analysis CY XX 090507 Rev 002 Issue date April 29 2010 Page 77 of 127 DIAGRAMS Infinicyt graphs can be customized by the user as part of the profile regarding bac
61. arameters on FCS files such as parameter s label or typing corrections File Export to export data in other formats readable by external analysis software or database software CY XX 090507 Rev 002 Issue date April 29 2010 Page 14 of 127 Manual Analysis with multiple options and possibilities for easy handling of files Advanced Analysis not included in basic version with different applications of automatic analysis that will be included in the following updates In this version Advanced Analysis includes an automatic recognition for BCR ABL fusion protein kit In addition the following extra options are available in the Menu bar File With all the main functions of the software File Exit Exits from Infinicyt program Alternatively you can close the main window of the application or Ctrl Q Profile Load Before starting analyzing the user may load a previously defined profile A profile consists on a default configuration with different options regarding data statistics visualization graphs configuration and other advanced options Profile Save To save a profile configured or modified by the user Profile Configure To modify the values of the data statistics visualization graphs and other advanced options of the profile See section 4 13 5 10 Help Users Manual Displays the User s Manual Help About Infinicyt Displays a single window with relevant information regarding the current version and in
62. ase the whiskers extend to a maximum of 1 5 times the inter quartile range The points outside the ends of the whiskers are outliers or suspected outliers With a right click on the diagram you can go to Diagram Configuration Box Plot configuration and find several options to display the graph specially useful when you are working with merged files If you choose to show extreme values you will visualize the outliers 100 Box Plot Configuration L Show extrem values Global box representation Mean files representation Box representation by File Cell Width CY XX 090507 Rev 002 Issue date April 29 2010 Page 38 of 127 4 7 Diagrams right click menu By right clicking in the middle of the diagrams we will obtain the following menu All these options will allow us to configure LOCALLY the display of the graphs Diagram Configuration a Configure visualization Data Local Visibility b Number of Events Displayed P window Manipulation Assign Population Undo Fedo b Invert a Diagram Info To apply a change globally to all the graphs then go to the Manual Analysis Window Menu Bar and choose configure Diagrams 4 7 1 Diagram Configuration Diagram Configuration b Axes Configuration Chrl 6 Configure visualization Data Local Visibility b Virtual Parameter Contiguratior Number of Events Displayed P Compensation Window Manipulation b Exp 550 Low Configuration sign Populatic Legend Scale
63. ashed the population appears otherwise it is not shown 4 5 Statistic Information kll merged Manual ANALYSIS File Edit Diagrams Statistics Profile Help im A E Comments K NA NA 0 33 0 33 Neutrophils 1 1 11 89 11 89 2204 Lee 1 877 i 94563 80 22 80 22 i NK Cells 2460 2 09 2 09 lt K E e Gate 0 events Total Di 0 00 Visibility 2 0 00 42 of 1055 MB used 4 5 1 Events column This column indicates the total number of events counted by the program for the population identified in the GROUP column 4 5 2 Total column This column will indicate the percentage that the events of this population represent with respect to the total number of events included in the data file or file chosen in the pop up list if we are analyzing a merged file The decimal separator and the number of decimal places can be changed at Profile Configure Statistics and it will apply to all the values shown in all the columns of the control Analysis Window and Report CY XX 090507 Rev 002 Issue date April 29 2010 Page 26 of 127 4 5 3 Partial column This column will indicate the percentage of events attributed to the considered population relative to the total number of significant events of the population that can be considered the hierarchical superior of the population under consideration 4 5 4 Visibility column This column will indicate the relati
64. ate data FILE No PARAMETER 1 2 3 4 5 6 7 9 FSC HEIGHT PARAMETER 1 FSC HEITGHT 5C HIGHT 5 C HIGHT FITC CD13 PE CD45 Derce CD45 Perce CD34 APC CD34 APC CD2 FITC CD2 FITC CD56 PE gt CD56 PE HLADR FITC HLADR FITC CD13 PE CD0117 PE CD117 PE CD123 PE C A LCU LATE CD123 PE CD15 FITC CD15 FITC CD16 PE D AT A CD16 PE CO22 FITC CD22 FITC CD25 PE cCD65 FITC CD25 PE CD65 FITC 7 1 PE CD61 FITC CD61 FITC CDO33 PE CD71 FITt CD71 FITC Glyn onhoria PE cesar Cora 1 SAMPLE 9 TUBES 9 FILES 1 SAMPLE 21 PARAMETERS 1 FILE CY XX 090507 Rev 002 Issue date April 29 2010 Page 107 of 127 The tables show an example of 9 tubes that make up a panel with the analyzed parameters of each tube Characteristics that have been measured repeatedly in all tubes common parameters are marked with a and will be used by the program to base its calculation of similar cells Parameters actually measured in a tube real parameters are shown with an ts and the new calculated data of these parameters with an D Infinicyt multidimensionally reconstructs the new data files containing both the real and calculated data for those parameters not directly measured The overall number of parameters in the new file can be as large as the total number of parameters measured in the whole group of tubes This allows a more powerful identification and characterization of the different pop
65. ce image can be applied in that parameter If it is white it means that there is a quite similar parameter but not exact maybe typing differences and the user should check in the pop up list that this is the right one The user can look in the pop up list but some parameters will appear in red light for being not possible to establish a correspondence If there is not parameter to correspond CY XX 090507 Rev 002 Issue date April 29 2010 Page 49 of 127 choose None and the process will continue although the reference image wont be shown in a parameter with none correspondence Check parameters Reference Image normail B cells Concordances Previous Groups Parameters concordance aires cenrance ACS a S COOMO Com o uU A FITC A Lambda FITC A Help Ok Can i amp PE A Kappa PE A PerCP Cy5 5 A CD 19 Per Once this step is performed the Reference Image will be shown in all the possible diagrams In the population tree a new label under each color will appear because the reference image can also be shown or hidden The statistics can also be compared because the Statistics of the reference image event are the ones that the sample had in the moment was saved CY XX 090507 Rev 002 Issue date April 29 2010 Page 50 of 127 4 10 Save Apply and Review Analysis Strategies In version 1 4 the user can review the strategy of gating for each population the user can also save a Strategy of gating and apply it ov
66. culate are selected and classified in a color click Next on the wizard to follow with the next step choose populations to classify STEP 3 CHOOSE POPULATION TO CALCULATE In this third step all the populations with events classified in the previous step will be listed in this window The user has the opportunity to choose which population to calculate is and avoid unnecessary calculation with debris and other cells At this moment all the events are non calculated until you go to the next step Calculate Data Joa tep F Calculate Data Population Mon calculated events 1 Select filets 0 unclassified events 14060 14060 UOU RIE 6306 16306 2 Galee Classity events 49099 449099 M 2595 REE A 6140 18140 3 Choose populations to calculate 4 Verify and save results Once the populations are selected click next to start the calculation process and go to the next step In this action is when the software performs the calculation process It consists on finding for each of these cells the equivalent one in the other aliquots tubes and then applies the values of the parameters not directly measured As soon as it ends the process it will appear a summary with the number of events none calculated by the controls on each population In the next step the user can see in detail those events Controls are detailed in chapter 4 3 3 CY XX 090507 Rev 002 Issue date April 29 2010 Page 112 of 127 Calculate Data Info
67. d amp e Global Configure Window Manipulation t Local Configure Assign Population b Undo Redo b Invert b Diagram Info This option allows the user to reduce or increase the number of dots displayed in the dot plot This action can be performed locally on the chosen dot plot Local or globally applied to all the diagrams Global When one of these options is selected a dialogue box appears where the user can select the percentage of dots by moving the cursor with the mouse or choosing an exact number of events to show 50 01 Global Dots 25005 Events Global Percentage O Global Number of Events Value 0 shows all events Once you have selected the appropriate percentage click OK and the graph will contain the new percentage of events and a warning in the title of the diagram will warn you about this change lolx 300 400 600 900 300 400 600 90 SSC Height SSC H Exp SSC Low 200 SSC Height SSC H Exp SSC Low 200 1E1 1E2 1E3 1E3 1E4 CD3 APC FL4 H LOGARITHMIC CD3 APC FL4 H LOGARITHMIC CY XX 090507 Rev 002 Issue date April 29 2010 Page 45 of 127 4 7 5 Window Manipulation Diagram Configuration b Configure Visualization Data Local Visibility b Number of Events Displayed P Window Manipulation Maximize Chril Mas Minimize Ctrl Menos Undo Reda Duplicate Ctrl L Invert b Copy Diagram Cre Diagram Info 4 7 5 1 Maximize and Minimize With these commands Maximize or
68. d to you 2 1 Licence Details The standard licensed version of Infinicyt is delivered in a pack containing the installation disc as well as one USB key per individual license The program will work only if there is a USB key connected to one of your computer s USB ports It is possible to install Infinicyt in several different computers but it will work only in those to which a USB key is connected at that moment In case of malfunction of a USB key we will replace it at no cost in exchange of the malfunctioning one If the malfunction is the responsibility of the client the key will be replaced at the cost of the key itself In case of loss of the key the client will have to purchase a new license so we strongly recommend taking care of these keys If the use of USB keys represents an inconvenience for you you can consult us about the possibility of using alternative licensing techniques without hardware components Please feel free to contact supportinfinicyt cytognos com for any questions regarding network compatibility or any further aspects of the above points 2 2 Minimum system requirements e Operating System Windows 2000 XP VISTA and 7 32 or 64 bits version e CPU Intel Pentium AMD or equivalent 32 or 64 bits version e RAM IGB 2GB recommended e Atleast 150 MB of hard drive free space to install the program e VGA compatible graphics card CY XX 090507 Rev 002 Issue date April 29 2010 Page 9 o
69. data file TOOLS PREVIOUS GROUPS This part shows all the Group actions done 6 4 and stored in the computer As can be seen in the figure it is possible to import or export dictionaries of labels from different users or computers Configure D Profile Configuration 4 2 Previous groups 3 Preferences Say FSc H i Analysis oll FSC Height FSC H Diagrams HM FSC Height MO FSC H Controls M FSC Height FSC H H Statistics Hm FSC Height FSC H H Virtual Parameters CD19 PerCP Cy5 5 FL3 H Stored Compensations Ww CD19 PerCP Cy5 5 A Gates and Analysis Strategies gt i CD19 PerCP Cy5 5 A i Reference Image ilf CD19 PerCP Cy5 5 A 1 z B FCS file CD19 PERCPCY5 5 FL3 H H Current Compensation gt i CD19 FL3 H H Negative Visibility lf CD19 Pecy5 FL3 H Configure APS Parameters 3 CD4 CD 19 PercP Cy5 5 A i Properties H E CD4 CD 19 PerCP Cy5 5 A I Tools llt CD4 CD 19 PerCP Cy5 5 A Previous Groups E CD19 CD4 PerCP Cy5 5 A lll CD4 CD19 PerCP Cy5 5 A CY XX 090507 Rev 002 Issue date April 29 2010 Page 87 of 127 5 PRINTED REPORTS The printed report is a key element of the analysis and is part of the profile Once the user completes the analysis it is possible to define a customized summary of all the information which can be printed and stored The analysis report will be updated each time a new file is loaded Infinicyt gives the user the possibility of edit
70. dotPlot or Parameter Band Histogram you can directly choose to show as many bands as Populations and parameters with comments Populations with events or default to configure one by one In this case of parameter band graphs you can ask for the representation by file the band will be at the same time divided in files and even invert the order that the parameters will appear in the graph Show File in case of merged files it gives you the choice of show All files population files or each file independently Diagram Font Configuration To choose legend and scale to be shown and the font size Reference Image Configuration From version 1 4 it is possible to show a Reference Image on the diagrams of the report In this part the user can choose which image to show and even with the visibility choose the order of populations included in the Reference Image to send to front the one of interest It is also possible to configure it if we prefer the Reference Image with lines dots hightlighted Number of events displayed Same as in the right click of the diagram here you can configure the number of events to show Visualization Data It is possible to choose Means Median Dots or a combination of them Density configuration Here are included the same options as in the diagrams menu All the diagrams here configured will be kept in the profile and will be shown updated any time you run a sample with the same parameters a
71. e Ctr z b all Files Reset Analysis Ctrl F Comment Select File Ctrl 1 Chrl 2 3 Ctrl 3 Ctrl 4 Cbrl 5 Gate 0 events Total 0 00 Visibility 0 00 1 Control buttons this buttons will appear on red color if the software finds important differences related to the coherence controls among the files M Merged Control P Population Control See controls information in next chapter 6 5 2 All Files Whenever the user is working with a merged file the statistics and percentages Total and Partial can be given as data from the total merged file data from each original file or data from the files containing the chosen population For this purpose a pop up menu is present in the right upper side of the Analysis Window where the user may select the information regarding all files only from files in which population is present or only from each of the tubes CY XX 090507 Rev 002 Issue date April 29 2010 Page 104 of 127 sample 4 Manual ANALYSIS ix File Edit Diagrams Statistics Profile Help All Files Seanlettan Population Files Comments s unclassified events 78296 amp Other unclassified events 1444 0 63 leucocytes 225419 98 74 Other leucocytes 95 0 04 176139 77 15 4864 2 13 lymphocytes 44521 19 41 aE Perfect Count BEALS 1433 0 63 lt k lt lt fs Gate 0 events Total 0 00 Visibility o 0 00 If you want that the files a
72. e APS Parameters 2D lt FSC A LINEAR SSC A Exp SSC Low gt Properties 2D lt CD19 PerCP Cy5 5 A PerCP Cy5 5 A LOGARITHMIC SSC A Exp SSC Low gt IL Tools Previous Groups A Global Configuration Diagrams List IEN Diagrams List av Diagram Info DotPlot Number of parameters X Axis FSC A LINEAR Y Axis SSC A Exp SSC Low In case you have used the Calculate Data option the software will keep in the profile the diagrams used for the calculate data wizard Then if you use different diagrams to select the population to calculate which are different from the diagrams you use in your manual analysis the software will keep both lists of diagrams and will use them CONTROLS This window corresponds to the different values and commands of the Merger Process Controls are explained in detail in chapter 6 5and the controls for Calculate Data process explained in chapter 7 4 CY XX 090507 Rev 002 Issue date April 29 2010 Page 79 of 127 STATISTICS This window is used for adding as many new functions as needed Configure D Profile Configuration Absolute Counts guns Events ul of the reference population beads nalysis i Diagrams Available Functions Show Parameter Statistics Controls Statistics H Virtual Parameters Stored Compensations Gates and Analysis Strategies 2 E i Reference Image 5 FCS file lt Configure APS Parameters i
73. e information CY XX 090507 Rev 002 Issue date April 29 2010 Page 61 of 127 4 13 1 7 Report Through this option the user may do printable reports Initially a blank page appears and the user includes different elements such as graphics statistics and comments of interest See chapter 5 Printed Reports for more detailed information about this issue The report will be saved as part of the profile and will be automatically uploaded when you open new data files 4 13 1 8 Properties With this command Ctrl D we open the Profile Configuration Properties window of the data file to check all the parameters measured This is especially useful when working with merged data files chapter 6 because in this window we will always have available the matrix with the information for all the parameters and files Configure T Profile Configuration C Preferences Analysis Diagrams Controls Statistics Virtual Parameters Stored Compensations Gates Reference Image FCS file Current Compensation Negative Visibility Configure APS Parameters Properties Properties File Parameters Info Parameters File a 2 3 4 FSC A SSC A LAMBDA FITC A FITC 4 KAPPA PE amp PE A CDS amp APC 4 amp 4PC 4 CD22 FITC A FITC A CD23 PE A PE A CD20 APC 4 APC A CD103 FITC amp FITC A CD25 PE A PE A CD11C APC A AP
74. e list of parameters in the order of contribution It means that the first parameter in the list is the most important one to get a good separation from one group to the others APS x Joes Diagram Info APS Number of parameters APS Parameter APS 1 Global View Parameters Listed by Significance PE Cy7 A CD19 LOGICAL R2 A sCD3Apch7 LOGICAL FSC A LINEAR B3 A CD34 PerCPcy55 LOGICAL SSC A Exp SSC Low Diagram Configuration gt PE A cCD79a LOGICAL Configure Visualization Data FITC A cMPO LOGICAL V2 A CD45 PO LOGICAL Pacific Blue A cCD3 LOGICAL Number of Events Displayed gt APC A CD7 LOGICAL gt Undo Redo gt Invert gt Diagram Info Local visibility b 4 6 4 MultiDimensional Graph Multidimensional Graph Graphical representation of the events cells or particles in n dimensions The user can choose the number of dimensions with the only limitation of the number of parameters measured The value on each of the dimensions parameters defines the location of the event in the diagram The main advantage of this representation is that the user may get a better separation of populations than with any other diagram except APS which will do the separation automatically FSC H FSC H CY XX 090507 Rev 002 Issue date April 29 2010 Page 36 of 127 Density 3D Multidimensional Graph In the same way of the 2D Dot Plot there is an additi
75. e the Manual Analysis although all this actions are detailed later in different chapters A OPEN INFINICYT Launch Infinicyt by double clicking on the icon that appears in the desktop once it is already installed B GO TO MANUAL ANALYSIS OPTION When you click on Yente pala a file selection window will be opened in order to search for a sample file into the directory Once you open a file you get an example of a template with some diagrams and a Population Tree CY XX 090507 Rev 002 Issue date April 29 2010 Page 16 of 127 ra i een eee a i Fee a epes Gites Profle Het De Hoima ii i is a ii iis tik al tik kik ia i i iii th iii tah ih tah isi iii iii ii isk th aae eret ramiame vey This Population Tree is an example and can be removed and built with different colours and labels C EDIT A POPULATION TREE Right click on any of the colored labels to obtain the population menu In this menu you will find different options Add Population Add Function Edit Population and Remove All Populations in order to customize your Population Tree t Tetors Manual ANALYSIS File Ede Dingrerme Statisties Profile Help FTES ET ETT TEE EEEEEEEEEEEE STCTTT TTT TTT en 8S 8 0 Gate O ewes tate 0 00 weky ws 0 00 D CREATE DIAGRAMS To create a new diagram go to the Diagrams New Diagram or Ctrl D On the Manual Analysis Window Menu Bar
76. e with all data base Infinicyt gives the option to save the results of the report in this format and this can be done in both of the export option File Dedit Sheet Print Ctrl F recientes ay Escritoria Files of type XML files t xmi D 9 5 Export files n TXT format In this version it is possible to export the results in TXT format as well just choosing this option in Files of Type when you save as the results of the Export amp File Export oles gt File g Only visible populations Se T hgt sca SSCA Events 200000 ca Keep file number separation ka W Pt Bile Documentos recientes 3 File name Escrioria Files of type Cancel TXT files FCS files CY XX 090507 Rev 002 Issue date April 29 2010 Page 122 of 127 9 6 Export files in Anonymous format When you export both in FCS or TXT format you will have the option to activate the checkbox Anonymous You will have to click Configure to choose the information to delete name of the patient code number date The parameters to include exclude will be saved in the program configuration for next times so you don t have to configure it any time you repeat it The anonymous option is also available when you save an analyzed file amp anua C 5 dit Diagrams Statistics Open Ctrl O Open Next File Ctrl N Open Previous File Ctrl P Open Analyzed File
77. ead for any worksheet database or word processing third party tool To choose the character separator in the CSV files or the number of decimal places go to Profile Configure Statistics Format See chapter 4 13 5 10 4 13 4 3 Configure This option opens the Profile Configuration Statistics window In this window the user may choose which statistical data will be shown on the analysis window and may create new functions or absolute counts See on chapter 4 13 5 10 Profile Configure Statistics the tools included on this window 4 13 5 Profile In this section we described all the options that can be found on the Profile Menu in the Manual Analysis Window Menu Bar Load Profile Save Profile Save Reference Image Load Reference Image Unload Reference Image Save Analysis Strategy Load Analysis Strategy Unload Analysis Strategy Analysis Info Configure Ctrl ts A profile is all the information about the software configuration as explained in detail on section 4 8 It is possible to create different profiles for different studies so the user can always work with the same Graphs Population Tree Controls Strategies Statistics Report etc with the same type of study 4 13 5 1 Load Profile Through this option the user may open a previously saved profile with Diagrams Population Tree Configure options and Report Depending on the type of study it can be useful to work with specific diagrams and configuration T
78. eeaeeees 93 6 2 Merge conditions for global analysiS ccccccsssseeseececceeeeeeeeeeeeceeeeeeaeeeeeees 93 O3 INICP SCO CESS carat ie dlr teuitateeaaaie a 95 6 4 Group tool Dichondry Of labels seroren a ea E 96 CY XX 090507 Rev 002 Issue date April 29 2010 Page 6 of 127 O52 Morser process Conto agrenn Abas lard attache 99 6 5 1 Merre Contool se esa are ate Serer sedis ca ere ena neat aeons 100 6 5 2 POPUTAMOM COME ON Dcacet aiid aed A Deaceien tie sesaaetneds 102 60 Andos Or The Mer eed MICS a senciucuisasiacedieelcaawissnadeeedtieuenrauretneetuaestausinaex 104 T CAELECULCATE DA A cna a 107 Tale De SCR O en T TN 107 Fa aO S er a tea aeaeeslieteele 109 7 3 Howto use the Calculate Data Wizard errorean eeens e T EE Ores 109 Tae Cak lite Data COMO isene a A E 115 S FILE EDITOR csr O 117 Sbe Odu O E E 117 8 2 Available commands 1n the edito ascua i a A 117 Oe AEREA PORT oeni E E E E E 119 Oke Tarodd HoN essea E EE 119 9 2 Available commands in the File Export window cccceseseeeeeeeseeeseeeeeees 119 9 3 Export a population from the Manual Analysis cccccccsssseeeeeeeeeeeeeees 121 04 Export ies 1 XME TORI AG sis scot said E 122 99 Export Tiles sn IAT TOM aes R 122 9 6 Export files in Anonymous format essesssseeeerssssssssecrerssssssseceressssssseeeeeos 123 10 ADVANCEDANALY SIS ceee a 124 10 1 BCR ABL Analysis Introduction cccccccccssseesseeeeeeeeeeeeseeecee
79. eeeeeaeeeeees 124 10 2 BCR ABEW 1a sa saa E eae ieee 124 CY XX 090507 Rev 002 Issue date April 29 2010 Page 7 of 127 1 INTRODUCTION In our commitment to the innovation and development of flow cytometry Cytognos presents Infinicyt state of the art software for data integration and multidimensional analysis of flow cytometry files This software opens a wide range of possibilities as it can generate files with a great number of parameters from a single sample From our web site www infinicyt com it is possible to download trial versions of the product or order the licensed version Other materials are also available in this site such us Tutorials Demo Files and a Users Area for Upgrades and on line Seminars This User s Manual will explain in detail all the capabilities of this software and how to handle the great amount of information provided with the different tools Please contact us for any further information at supportinfinicyt cytognos com CY XX 090507 Rev 002 Issue date April 29 2010 Page 8 of 127 2 INSTALLATION AND SETUP Installation instructions including data about the minimum hardware and software requirements can be found in this section However if possible we recommend referring instead to the Readme file of the installation disc which should contain similar information but possibly more complete up to date and or adapted to the specific version of Infinicyt that has been delivere
80. en several plots displaying all the different combinations of parameters CY XX 090507 Rev 002 Issue date April 29 2010 Page 67 of 127 Y f C 4 A N EEEREN muse n emamaa me ic ee 4 ai m m 4 13 3 3 All Diagrams to Front In case the plots are hidden behind the Manual Analysis Window with this command or Ctrl A they will be brought to the front In case the user wants to maximize the Analysis Window when it has been minimized just double click in the middle of any diagram 4 13 3 4 Close All Diagrams Choose this option or Ctrl B to remove all the windows shown in the screen if you want to create new different graphics The diagrams which are part of the profile are listed in the following box Profile Configure Diagrams Diagrams List from the Manual Analysis Window Menu Bar If any diagram is closed in the analysis will remain in the list and will be automatically opened when their parameters appear in a new file opened The only way to delete a diagram from a profile is from this window Diagram List select the diagram and click trash or with Ctrl B close all diagrams which will delete all the current diagrams in the profile T Configure gt E i m r oF er Daagiasos aet o Dot 4 LOLILA Ate ete z 4 Pa wta hras o Iho ssatta ai lt ad 4 13 3 5 Apply Default Values to All diagrams Whenever we resize or change the appearance of the graphs we can alway
81. eneettunlceakennts 65 A es Gm Wt lt P CR N 67 AAD eds Al DVAST amis to TOM seee a 68 AA Soe AClOSe ANDI Sais e ected wckedeccntoushcid tee 68 CY XX 090507 Rev 002 Issue date April 29 2010 Page 5 of 127 4 13 3 5 Apply Default Values to All diagrams cc ceeeeeeseeeeeeeeeees 68 2 Bo cs A O err Pee So Pen eee ee 69 ANAT SOW SEAL SUC S coset ca sece N seen ieee 69 AA EXPO SEAUIS ICS a a Ceoalas aa caratal ined oaadtaneaiviecet edd vigaunsceantoand tgaaendiace 70 ANDAs COMM OUT Gi deee sues a elbacsate ved ala nate ses eowed de memo ble anes 71 Mee EEO MC i e N 71 EN DM Load Prole rican are eastside E een aree 71 kI SAVE Proe eea Eaa T2 41 3 Save Rerrecnice Mate ameipa a e 12 ATSA Loit Re rerenCe Tma Cca T T2 ATS Unload Reference Mate aearrasionnr iaa entire iee 73 LNO SIC ANAYSSI OEE e E E iS eaeanau 73 ANS 1s Lda AMAlySIS Stale OY a taucontenestileeanonin edu taien 74 41338 Unlodd Amal ysis Strate ey se siccdicancesiuaiiaeiacenasseeseiiadad oategseuladiadeaass 75 AVS S90 AVAL SIS MIO sonansa a 75 AM Seb COn SUr enen a 76 53 PRINTEDREPOR TS cariosna nn eicuanteeeseeemicenieeeanieds 88 Sule Howto Create a Report ecas N tactile 88 S25 EXPOR Report 10 AM Dorrie a wie loscadeaidioes mii gastaad nie oueedeaadeen ahs 92 6 PLEMERGE corrie e na etennsuceee i eedusennih utes mae seawteerersmceaeenntee 93 6 1 Introduction and merge conditions cccccccccccceesseeeseececeeeeeaeeeseeeceeeeee
82. entaness 22 4 4 4 Conseculive Gales AND Jeary e noneeneers 23 4 4 5 Population ree GescnpiOM esns T 23 4 4 6 Gor DE Le eee ame rear ne nye reer nr ere eRe eee 24 CY XX 090507 Rev 002 Issue date April 29 2010 Page 2 of 127 4 4 7 Drag and drop labe S wcsconneine tence cece a ae ah hare eea ne 293 4 4 8 How to add a Function in the Population Tree seeeeneesssseeenesssssssee 25 4 4 9 How to remove population labels vi ccdeacesnnsiioditeinieuiinnienindctracen 26 4 4 10 How to select show or hide a population VIS Column eee 26 As OSC I LOTIMALLON aio ceseva sore iieadtetaee E 26 4 5 1 Ee CO e a T ete eies 26 492 Total o COUM ie E r 26 4 5 3 Partial o COUM aes TRO 2 4 5 4 SN ISIDI Zo CoN e ees veatemsen dea useneunsaebesacuccseeemcentts 27 4 5 5 COMMEN COM es RN 27 4 5 6 Saase COUM S anena E ES 28 4 5 7 ReView C OMA ROW haian eh eons A heaton 28 AO TYpes Ol Skapnic representations ces E E eteenontesn 29 4 6 1 FItSCO STAM eene E N 29 4 6 2 2D DOFO a a aa 31 4 6 3 Automatic Population Separator Diagram cccccccsssseeeseceeeeeeeeeeeeees 35 4 6 4 Mul Dimensional Gtapn sscehsninsiosni venteidioanincelaasdiorviniadeusionnimcadias 36 4 6 5 Statistical Diagrams BOXPlots rererere concise 37 Ade Diasramstiehtehek menise a a imental 39 4 7 1 Diagram Coni CUratOM ae a 39 4 7 1 1 AXES COI OUPATIOM ie a tenebuhtwinc nea liadewkeleass 39 A EAA Negative Visibility Configuration
83. er other files 4 10 1 Review a Strategy Every time you need you can review the selection of the populations Go to the label of T Cells and by right clicking select the option Load Gates Review At this time a new column will be created with the symbol in all populations with events It will appear also some diagrams showing the strategy of gating you made for characterize the populations T Cells in this case lt himi gt lt tontcoloreredsR_ Bk 2 alki amp Sl EE a 001 Tutoriel cyt N Manual ANALYSIS Pore a a E y Fil bot Disgrome Sather Proble Ae Akika Pt a heey SC A Exp SSC Low SC A Exp SSC Low 1 A bia it 1i id wia PercP Cy5 5 4 C019 LOGICAL APC A CD45 LOGICAL E RA LLE curent ite raba 6B Ces Gate dilaut Dt bewtotybes HFEA Lin SRA Boe SS Low a PS LIAR a SSA Pepe SSL Lin leois HETE ASD Loge SA Ene Low a PTA Sco LA 25508 Bape Sc Low ake Pe ee Eimear oly tiled ae owe PereP cy5 5 A CD413 LOGICAL SSC A Exp SSC Low SSC A Exp SSC Low AEF g iis ia il fi I HEF hit 3 7 a FITC A CDI LOGICAL FITC A CD7 LOGICAL Gae events Total 000 verbit 9 0 00 fio oF IOUS used _SSSsS aT IF F Mid cytognos EuroFlow ir _ ___ _ ____ ___ lt lt lt o Looking in detail the Manual Analysis Window CY XX 090507 Rev 002 Issue date April 29 2010 P
84. eters defines the location of the event in the diagram 4 Ae FL H c098 Fite Powe Mia z FL4 H CD45 AFG Log 3D Diagrams can be resized clicking and moving the scroll of the mouse It can also be rotated in all directions and also with the numeric keyboard can be rotated with the numbers 2 4 6 or 8 Density 3D DotPlot This diagram represents a 2D DotPlot with a third axe where the density of events in the 2D plot is represented 3D Density BHX Fse y H FSC Hatght Uin Page 34 of 127 CY XX 090507 Rev 002 Issue date April 29 2010 4 6 3 Automatic Population Separator Diagram This graphical representation is an automatic separation of the events where the software is able to distinguish as many cell clusters as possible analyzing all the different choices of parameter combinations based on Principal Component Analysis The parameters represented in these APS graphics are not a real measured parameter They are defined by calculations of different percentages of each parameter for their contribution to the most optimized separation of clusters In the dialogue box of Diagram New Diagram from the Manual Analysis Window Menu Bar you will find the APS Graph APS graph The aim of this representation is allow the user to be able to distinguish in these graphics as many different populations as possible especially useful when working with a high number of parameters for a further character
85. f 127 e Recommended graphics card compatible with OpenGl 1 3 or later or with DirectX 9 0 or later to visualize 3D diagrams e Preferably large screen over 20 21 with at least 1280 x 1024 pixel resolution 24 recommended or a double monitor Apart from the above system requirements an extra limitation will be given by the characteristics of the data files the more events and parameters you want to study the higher computer requirements RAM memory and processor you will need These requirements should be considered approximate and an orientation The exact behaviour of Infinicyt in a specific computer may depend on specific configuration factors which would be complex to document and on the type and size of the files to be treated For this reason to determine for sure if Infinicyt will run in a specific computer with full user satisfaction we recommend to install a demo version and test it with files as real as possible 2 3 Installation procedures Your installation CD ROM is prepared to automatically start the installation as soon as it is inserted into your CD drive In that case please follow the instructions of the program installer If it doesn t start automatically the CD ROM contains a file named setup exe please run it and follow the instructions to install the program The user who is carrying out the installation process should have write permissions on the selected folder to install Infinicyt
86. find a equivalent in the new file to load the Reference Image If you click OK no Reference Image will be loaded Check parameters Reference Image kappa Xx Concordances Previous Groups Parameters concordance FSC A FITC A LAMBDA FITC A PE A KAPPA PE A PerCP Cy5 5 A CD19 PerCP Cy5 5 A APC A CD5 APC A WARNING It is important to unload the Reference Image or Strategies when you finish the analysis Then go to Profile Unload Reference Image in the Manual Analysis Window Menu Bar to continue the analysis of the different sample with new parameters 4 13 5 6 Save Analysis Strategy After performing the analysis of the populations is possible to save the gate strategy For detailed information on this procedure go to chapter 4 10 How to save Analysis Strategies Go to Profile Save Analysis Strategy on the Manual Analysis Window menu bar CY XX 090507 Rev 002 Issue date April 29 2010 Page 73 of 127 001 Tutorial cyt Manual ANALYSIS File Edit Diagrams Statistics Help Load Profile m Save Profile Population Visibility Comments Save Reference Image Load Reference Image ex Unload Reference Image ie 0 00 3 100 00 Other euco Save Analysis Strategy l8 67 18 M es 32 32 82 a Unload Analysis Strategy NA NA Wi p Analysis Info 5 28 18 v TA 19 3 90 iv L Configure Ctrl G 16 0 74
87. finicyt analyzed files cyt Cancel 4 12 Export statistics from Manual Analysis The export option from the Manual Analysis Window Menu Bar Statistics Export Statistics is a tool to export to other platforms the data of the analysis When the export option is open this window will appear where and user can configure which population which parameters and which value of each to export It is possible to Save different configurations of statistics to avoid repeating the selection of fields every time Configurations Selected Configuration r Select only popiatiaons with events Defaut sve a yee P pisis ioiei Sog0gJggu0gdg a Then clicking on Export the software will ask where to save this new file with the statistic data The proposed extension for this statistic file will be csv coma separated values which can be read for any worksheet database or word processing third party tool CY XX 090507 Rev 002 Issue date April 29 2010 Page 58 of 127 4 13 General description of the analysis menu 4 13 1 File In this section we described all the options that can be found on the File Menu in the Manual Analysis Window Menu Bar Open Ctrl oO Open Next File Ctrl N Open Previous File Ctrl P Open Analyzed File Save Analyzed File Export Ctrl E Report Properties Ctrl I Exit 4 13 1 1 Open When choosing this option Ctrl 0 a File selection Window will appear where t
88. global aspect required for warning contains a threshold value configurable by the user which determines the minimum degree of difference in the scale from O to 100 explained above between any couple of files over which the program will issue a warning indicating a significant lack of coincidence between the distributions of the events of the different files Current predefined value 20 CY XX 090507 Rev 002 Issue date April 29 2010 Page 100 of 127 4 Configure Profile Configuration Threshold parameters for coherence controls I Preferences Analysis Ma Merge Control Diagrams Minimum difference of global aspect required for warning Contrals statistics Virtual Parameters iP Population Contral Stored Compenzations ates Reference Image Minimum difference for each parameter required for warning Show message when differences are detected in visible populations L Calculation Data Controla Disease Categories Configuratio im lide When the program detects significant differences between the distributions of the events of the different files in the common parameters space as determined by the aforementioned threshold values it shows a warning message The warning will indicate the of difference and will also list by significance the parameters and files which make the differences he Controls Maximum difference between tiles 12 difference between some files Files and parameters with differences li
89. gram When clicking this option Ctrl D the following dialogue box appears NEW DIAGRAM Histogram C DotPlot APS diagram C Multidimensional Parameter Band O Stat diagram Number of parameters The user may select among different kind of diagrams see details in chapter 4 6 Histogram DotPlot Automatic Population Separator Diagram APS MultiDimensional Graphics where the user may select the number of dimensions Statistical Diagram which includes Box Plots Once the user selects the diagram the appropriate parameters can be chosen by clicking on the axes of the figures only in case of DotPlots The configuration of the axes is also modifiable through the right click diagram menu Diagram Configuration Axes Configuration see 4 7 1 Diagrams Configuration CY XX 090507 Rev 002 Issue date April 29 2010 Page 65 of 127 When you click the axes to select the appropriate parameter you can select both the parameter in the upper part of the pop up list and the scale lower part of the pop up list When you are analyzing a merged file the common parameters will appear with a C in the list of parameters N 25000 i To 100000 ENN S ae ZiGiFsc a Sa ssc a amp FITC A CD22 APC A CD20 APC Cy 4 Pacific Blue 4 D Bay L AmCyan 4 File Number Exp 55C Low Logic Lin Log Default Exp SSC Low Configurable Exp SSC Low SSC A Exp SSC Low T
90. gure Preferences Controls window we will see the values taken as threshold same window as Configure in the Merge process and we will be able to correct these values 4 Configure Profile Configuration Threshold parameters for coherence controls Preferences Analysis Ma Merge Control Diagrams Minimum ditterence of global aspect required for warning Contrals statistics Virtual Parameters JP Population Control Stored Compensations ates Reference Image Minimum difference for each parameter required for warning 5 ee Show message when differences are detected in visible populations E Calculation Data Controls Disease Categories Configuratio Mm ode CY XX 090507 Rev 002 Issue date April 29 2010 Page 103 of 127 As previously described the commands for fixing the warning thresholds shown above can be obtained in 2 different ways when the user is working with a merged file 1 Profile Configure Preferences Controls from the Analysis Window menu The commands Controls dP of the analysis window change to red when an incoherence is detected 2 From the File Merge window in the option Configure 6 6 Analysis of the merged files Once you go to the Manual Analysis option note that if the user is working with a merged file the Manual Analysis Window includes some functions useful in this analysis J PATIENT SAMPLE 3 Manual ANALYSIS ota File Edit Diagrams Statistics Profile Help i Undo Change Fil
91. h the data included in the analyzed file CY XX 090507 Rev 002 Issue date April 29 2010 Page 18 of 127 _001 Tutorial Manual ANALYSIS pe x File Edit Diagrams Statistics Profile Help Open Ctrl 0 Open Next File Ctrl N Open Previous File Ctrl P Partial Open Analyzed File Save Analyzed File Export Report Properties Exit D Or St a ey 1 Blast cells Pathological clonal cel The main components you can add to the report are the following l Image pictures or logos 2 Text 3 Data of the sample or from the patient 4 Population tree with statistics 5 Diagrams Right click on the report sheet and choose the component to use Then you can right click on the component to configure it Name Last name Sarnple Type Doctor Hospital Akquisition date g Infinicyt ANALYSIS REPORT z 6 ao a w q o n wre 2 w we 25 FITC A CD3 LOGICAL FSCA LINEAR SSCA Cap SSC Low FITSArSO LOOISAL PEA COEE LOCICAL E Core O 6 peA Leorcar 7 APCACB4E LOGICAL p Population Events To Partial Vicwilty 25552 5310 43 90 70 3403 oo 600 773 413317 38 63 s o a LELI 1805 201 11 04 400 1 1909 5 46 PM E gred by CY XX 090507 Rev 002 Issue date April 29 2010 Page 19 of 127 4 3 FCS files with more than one Dataset Coulter Files Infinicyt supports all kind of FCS files that follows the Data File Standards for flow cytometry www isac net o
92. he user can select the FCS file to analyze Once this is made the user must click the Select button of this screen to load the file and see the results By clicking on the Cancel button the selection screen disappears The FCS file may be in any folder of the computer where the user has reading permission If the user had already opened a file the software will ask 1f he wants to save the manual analysis performed in the previous file before continuing opening a new file Info This warning can always be activated reminding to save the analyzed files when finishing This message can be activated in Profile Configure Analysis of the In this section we described all the options that can be found on the File Menu in the Manual Analysis Window Menu Bar If this option is deactivated the files analyzed won t be saved unless you do it through the option File Save Analyzed File in the menu CY XX 090507 Rev 002 Issue date April 29 2010 Page 59 of 127 Configure Profile Configuration A 4 Visibility Options Preferences cn Define maximum population percentage for highlight 1 00 pe CEN Make population visible when you assign events to it amp Controls n Statistics Show in analyzer Virtual Parameters Number of comments fields 3S H Stored Compensations Gates and Analysis Strate Warnings Reference Image Show a message when you assign events associated to two or more popu
93. he profiles are saved in inp format Infinicyt Profile in the directory chosen by the user CY XX 090507 Rev 002 Issue date April 29 2010 Page 71 of 127 4 13 5 2 Save Profile If the user wants to save the profile of an analysis with the Diagrams Population Tree Configuration and Report options click this option and the software will open a dialogue box that asks you to choose a directory to save it It will be saved with a inp extension 4 13 5 3 Save Reference Image It consist on a tool to save pictures of the diagrams which can be used as a reference in similar data files to analyze This reference pictures can appear in the same dot plot you are analyzing so the user can always have the Reference Image to compare 4 9 To create a Reference Image you have to consider that the data file of reference and the data file you will compare later need to have exactly the same parameters acquired Typing mistakes can be corrected if the labels don t match when applying the Image Once this is checked the user must analyze the file which will be the Reference one and show in the population tree only the populations of interest Then in the Profile you only have to choose Save Reference Image and give a name to that group of events It is possible to save as many different Reference Images as desired 4 13 5 4 Load Reference Image If you have previously saved a group of reference events we can load them in the diagram
94. he user will have to choose which of the Advance Analysis sections wants to follow In this version only BCR ABL is available and in future versions more analysis will be included in this part When the BCR ABL is selected click Next CY XX 090507 Rev 002 Issue date April 29 2010 Page 124 of 127 STEP 2 SELECT CONTROLS AND SAMPLES 4 Steps Negative Control 1 Select an analysis NEGATIVE CONTROL fcs 2 Select controls and Positive Control samples x p POSITIVE CONTROL fcs 3 Merge info Samples 4 BCR ABL report ibcr abl fes 2bcr abl fcs 3bcr abl fcs Select Files 4 QJ Main Menu re Previous Next 5 In this step the user has to Select a negative control sample Select a Positive control sample and finally in the Samples box the User can Select as many files as he she wants to analyze When negative positive and at least one sample is uploaded then it is possible to select the button Next to continue the analysis STEP 3 MERGE INFO It is showed a matrix with info about the files and the parameters In this step it is possible to deselect parameters if you consider that are not necessary 7 Advanced Analysis hin y 4 Steps Select Parameters Esc FSc 4 S9 55C 4 Beads parameter PE A 1 Select an analysis 2 Select controls and samples 3 Merge info File Preview 4 BCR ABL report W FSC 4 SSC 4 FITC 4 PE A PerCP Cy5 5 4
95. ing his her own model of printed report in a very easy way consisting of handling different elements such as graphics statistics charts text pictures and all the different elements the user wants to include 5 1 How to create a Report Go to File menu and click Print A report page opens see figure with different components with standard information about the analyzed populations and the data included in the file Each component can be deleted just right clicking on the component and choosing Remove Component Report File Sheet ge SAMPLE REPORT DATA OF THE SAMPLE Name PATIENT 000000111114 Last name ooooo00000 Sample Type PERIPHERAL BLOOD Hospital UNIVERSITY HOSPITAL Staining protocol LYMPHOCYTOSIS SCREENNG TOTAL CELULARITY Population Population Files Total Partial 8 3 25 83 2 10 4 CD4 T lymph 67 6 26 4 28 25 CD56 KAPPA B cells x ag 1 1 1 NK CELLS cD8 LAMBDA IMMUNOPHENOTYPING OF PATHOLOGIC CELLS Population File 2 Mean SD Comments Blast cells Pathological clonal cells CD22 FITC A FITC A 36 6 20 0 POSITIVE CD23 PE A PE A 31 5 38 1 DIM POS POSITIVE CD19 PerCP Cy5 5 A PerCP Cy5 156 0 78 6 Cb20 ERTER 356 9 332 7 POSITIVE Add component CD103 FITC A FITC A 27 26 NEGATIVE CD25 PE A PE A 90 93 NEGATIVE DIM POSITIVE CD11C APC A APC A 34 47 NEGATIVE Copy FMC FITC A FITC A 13 3 30 3 NEG DIM POSITVE CD24 PE A PE A 222 5 125 6 OISTIVE CD34 APC A AP C A 2
96. iously stored as a reference with a new sample that we are analyzing In this sense basal conditions of a sample cells at diagnosis or normal patterns can be saved as a reference guide to have it available whenever we want to analyze the same type of sample but in different conditions 4 9 1 Save Reference Image Any time we want to save the cells on a given dot plot as a reference just go to Profile Save Reference Image in the Manual Analysis Window Menu Bar and all the events visualized will be stored in this reference image Therefore pay attention on saving as a reference image only the populations of interest instead the whole sample events 4 9 2 Load Reference Image Whenever the user is interested in recover a Reference Image to compare with the current sample just go to Profile Load Reference Image in the Manual Analysis Window Menu Bar and choose the one you want to apply Infinicyt will automatically check if the current parameters where you are applying the reference Image are the same than the parameters in which you had previously saved the Reference Image If there are discrepancies in the parameters the following dialogue will be shown W Check parameters Reference Image normal B cells Concordances Previous Groups Parameters concordance File Parameters Concordance ASCA Jooo Check Labels If the light is green this parameter matches it means that the label is exactly the same and the referen
97. istogram Graphical display of tabulated frequencies We can choose NEW DIAGRAM Histogram C DotPlot Parameter Band gt APS diagram gt MultiDimensional gt Stat diagram Number of parameters CY XX 090507 Rev 002 Issue date April 29 2010 Page 29 of 127 Histogram Graphic representation of one or more parameters in the horizontal axis The number of events is represented in the auto scaled vertical axis There is a possibility of a manual configuration of the scale by right clicking on the Histogram and choosing Diagram Configuration Histogram Configuration 100 0 Max histogram percent Smooth Histogram 163 183 164 165 PerCP Cy5 5 A CD19 LOGICAL Smooth Histogram 1s a tool to improve the visualization of the peaks Auto regulated Histogram you can auto regulate the maximum value to show on the Y axes This is a useful option when the high values on the Y axes don t let to clearly visualize the lower values of the other peaks We can choose the number of dimensions to be represented in the same histogram For example we can overlap two parameters of the same population in two different ways as shown in the next figure O oE PB HG BAXIL RE A KAPPA Logic e e o e Lad o o r o u t M N e o o 100 0 1E2 1E3 PE A KAPPA Logic FITC A LAMBDA Logic The first graphic is a Parameter Band Histogram of two dimensions CY XX 09050
98. ization of these populations in the conventional 2D graphics Usually 2D graphs can not give a good separation of clusters and a big number or diagrams with different combinations is needed to get the separation of all the groups APS shows different views By default Infinicyt shows APS 1 view which is the best combination for population separation The APS 1 view shows the first versus the second Principal Component In order to change to a different combination of parameters to obtain the second one or third view right click on the axes and choose a different one The number of possible APS views is the same as the number of parameters in the file ag aps Jog WARNING if you are working with a merged file in which you have null values parameters not measured in all the tubes in some tubes for some parameters these events won t be shown The correct way of showing the APS view in a merged file is configuring it only with common markers The software will automatically do it but it is CY XX 090507 Rev 002 Issue date April 29 2010 Page 35 of 127 possible to change the configuration of the parameters nght click Diagrams Configuration APS parameter Configuration APS Diagram Info Interesting information of the APS is to know how is the contribution of each of the parameters to the separation of the clusters You can get this information right clicking in he graph and choosing Diagram Info You will obtain th
99. kground color size and font settings Some default values are shown in this option so the user can change the appearance for each new graph Configure EJ I Profile Configuraton d coba Config raton Diag ams List U Meiereoitra o Ansivss Background Colar e Dagan Black 0 White Contes Statetcs Global Derensions Velual Paaretes Width wols Hesght es d Commersa tions Gaits and Analyse Strateoes Font Settings Reteexe P a Z Show legend rom ConSgure APS Pa aneters 7 Show score Font Properties 9 Toss Smooth Histogram Configuration Previous Gous Smooth histogram Denmsily Configuration Density representation v Coritour fines Density mcrenent Mor 7 Maw Rod 100 0 Giohal Dots 117886 Events 2 Gobel percentace 0 01 100 Global number of events Value shows al events OK Cance Some changes in the Profile Configuration of the Graph may generate a totally different display of the same diagram i PE A KAPPA L FITC A LAMBDA Logic Changes in this part of the profile will apply to all the graphs If the user wants to change the appearance in only one of he diagrams it 1s possible to do it one by one in the graph right click menu see chapter 4 7 1 Diagram Configuration The different options to configure the diagrams in the profile are Background Color black or white is available Global Dimensions of the diagrams Font Settings for legends and scales S
100. lations 5 FCS file an ae ib cecieeeeaiies Show a message when there is a discrepancy in the classification d Negative Visibility Configure APS Parameter Properties Show a message when you don t save the current analysis 4 13 1 2 Open Next File Choosing this option Ctrl N the software will automatically load the next FCS file stored in the same folder as the previous file until you reach the last file of the folder When this occurs and the user tries to open the next one the following message appears Warning A You have reached the last File of this Folder 4 13 1 3 Open Previous File Choosing this option Ctrl P the software will open the previous FCS file in the folder until it reaches the first one and the following message will appear Warning A You have reached the First File of this Folder 4 13 1 4 Open Analyzed File As described on chapter 4 11 Infinicyt software allows saving the files analyzed containing the information of the population tree classification statistics graphics report and store them in the hard disk These files will be stored as a cyt extension CY XX 090507 Rev 002 Issue date April 29 2010 Page 60 of 127 Whenever the user wants to open a file already analyzed then the cyt file must be opened from the option Open Analyzed File The software will warn you that it will upload a previously saved profile This occurs because the software
101. lect File 1 2 3 CY XX 090507 Rev 002 Issue date April 29 2010 Page 127 of 127
102. lumns previously described except these statistics columns will be visible Nevertheless at any time clicking with the right button of the mouse this information can be hidden or shown J 0000 001 Manual ANALYSIS BAX File Edit Diagrams Statistics Profile Help Population Show Events Column unclassified events Show Total Column f RATIO AJB 0 00 Show Partial Column Other unclassified events 209 0 42 0 6 Show Visible Column 2572 5 14 5 Show Comments Column 1910 3 82 3 6 Show Reference Column 27028 54 06 54 0 Show Statistics 1389 2 78 2 78 0 00 16892 33 78 33 78 0 00 Gate 0 events Total 0 00 visibility 0 00 OOO When the user activates show statistics there will appear as many columns in the dialogue box as the number of parameters measured in the data file The columns can be closed lt 4 or opened to visualize the data about Mean SD and CV or to visualize the comments if they exist 4 5 7 Review Column Rev On Infinicyt v1 4 you can review your gating strategy Any time an analysis is performed it will appear a lens that you can click to review the regions applied to get that population See chapter 4 10 1for more info BE FF uw Oee s e a ee ee ee ee eea SE Exp S50 Low i Fst LINEAR he a FITC ALAMEDA FITC A LOGARITHMIC LLL ia FSC A Exp 330 L v a a a E el oak ERS MRT LESS Port P E yi
103. median value or a combination of some of them dots and Mean as you can see in the following picture amp lasd d FL2 H icZAP70 PE LOGARITHMIC 1t4 1 181 162 183 t4 FL3 H CD19 PERCPCYS S LOGARITHM Jag W Configure Visualization Data Visualization Date Dots v Means of thes Oo medians or fias 4 Denaty Configuration De riity type No dansdy O Total Qenety C Popedation OK Cancel If you draw a gate around a mean or median dot all the events contributing to this value will be selected Moreover as shown in the following picture clicking on the median or mean dot the entire population is selected as well FL2 H cZAP70 PE LOGARITHMIC 1E4 1E3 1E2 1E1 1 FL3 H CD19 PERCPCY5 5 LOGARITHM olga oa Wiles gt Mean B cells 1E1 1E2 1E3 1E4 FL2 H cZAP70 PE LOGARITHMIC 1 1E1 1E2 1E3 1E4 FL3 H CD19 PERCPCY5 5 LOGARITHM Density Configuration Density type CY XX 090507 Rev 002 Issue date April 29 2010 Page 42 of 127 Whenever it is necessary the user can change the 2D Plot into a density plot by only activating this function in Configure Visualization Data The Plot will change the appearance automatically No density this is the default choice Unless the users change it the graphs appear always without any density representation Total Density This choice will consider all the events visualized to perf
104. mooth Histogram Configuration this option will show the same histograms but reducing the resolution of the data Density Configuration only when density is selected in some of the diagrams right click visualization options density 1s possible to see the changes in this part Default CY XX 090507 Rev 002 Issue date April 29 2010 Page 78 of 127 density is with dots in a colors scale but if the user wants to show density lines as well it is possible to do it clicking on this option of contour lines Also the Density increment can be configured in this box Global Dots Percentage in this option you can reduce both the number or percentage of events to display in all graphs A second tab in the Diagrams Configure shows the list of diagrams to show in this profile Configure IL Profile Configuration Preferences Analysis Diagrams D Default desktop Controls Statistics 2D lt FMC7FITC A FITC A LOGARITHMIC SSC A Exp SSC Low gt valid Virtual Parameters 2D lt CD20 APC A APC A LOGARITHMIC SSC A Exp SSC Low gt valid Stored Compensations 2D lt CD22 FITC A FITC A LOGARITHMIC CD 19 PerCP Cy5 5 A PerCP Cy5 5 A LOGARITHMIC Gates and Analysis Strategies 2D lt CD11C APC A APC A LOGARITHMIC SSC A Exp SSC Low gt valid Reference Image 2D lt CD19 PerCP Cy5 5 A PerCP Cy5 5 A LOGARITHMIC SSC A Exp SSC Low gt valid z3 FCS file Calculation desktop Configur
105. n the box displayed it 1s possible to choose the name the variables and the mathematic calculation between them Add Function X Name New Function Clear All Result Type Constant Value Function Population Statistic Parameter Statistic Summation Clicking in the first part of the formula different Type of data will be displayed so the user can choose the appropriate one The data may be Constant value The user will choose a Value Function To get a more complex function the user can click on this choice and it will appear as a new function in the first part of the formula This option can be repeated as many times as needed For each component of the sub function the user will choose the type of data Population statistic When the data consist of one of the parameters shown in the Population Tree regarding events percentage The user has to choose the data the population involved and the files considered for that calculation in case of merged files 8 Add Function Name hise hrenn ear Al Sonia CY XX 090507 Rev 002 Issue date April 29 2010 Page 81 of 127 Parameter Statistic In this part the user can choose the statistic value of the Parameter Data Mean SD CV for a Selected Parameter and for a Selected Population WY Add Function fa Marne hew Furcteges Gee Al Parameter Statistic Mean ar Par ire er F See faea Pepali Galle TE Praia Tae
106. nd populations analyzed If you choose a Text a dialogue box will ask you about the text you want to write color font and alignment CY XX 090507 Rev 002 Issue date April 29 2010 Page 91 of 127 Choose a tent Alignment G Left Center O Right Finally choosing Date a box with the current date will appear All these components can be moved and resized in the report and will update with new data as soon as the user change the data file in the analysis While the report is open no gates or operations can be done in the analysis 5 2 Export Report to XML For each of the components of the report you can choose whether to export or not to XML files This can be done by clicking twice on the component to configure and in the advanced tab you can select or not the export option You can also write a name for this component so any system will identify the component Population es SSCS Options Advanced Export options Export Id GLOBAL CELULARITY The XML files generated in this way can be read by customized external programs that may allow import data to LIS CY XX 090507 Rev 002 Issue date April 29 2010 Page 92 of 127 6 FILE MERGE 6 1 Introduction and merge conditions With this function the user can select several FCS files and merge its information in a single file The file resulting from the merger will contain all the events of all the files selected for the merge
107. not the software will try to adjust the scales and will show you a warning CY XX 090507 Rev 002 Issue date April 29 2010 Page 93 of 127 The appropriate marker panel design is essential in order to obtain satisfactory results with merger mode The design of the necessary antibodies panel must be done taking into account that in each tube of the panel should appear o A series of common parameters that allow to unequivocally identifying the population of interest for our particular study The more common parameters we use the more precise the identification of the population of interest will be e g to identify the population of B lymphocytes from a peripheral blood sample FSC SSC and CD19 at least could be used as common parameters o Itis necessary to include in each tube all the markers that will allow us to identify the population of interest common parameters e g if our population of interest was the helper inducer T cells all the tubes in the panel must have at least CD3 and CD4 markers for their correct identification o The common markers used in the panel must be marked with the same fluorochrome o In fluorescences not occupied by common parameters the rest of the antibodies to be used in our study will be included Infinicyt includes an internal control system that verifies if the aforementioned conditions apply See chapter 6 5 CY XX 090507 Rev 002 Issue date April 29 2010 Page 94 of 127 6 3 Merge
108. nts fields Hea Stored Compensations Gates and Analysis Strategies ions 4 Reference Image Show a message when you assign events associated with two or more populations 5 0 FCS file be Configure APS Parameters is Properties Show a message when there is a discrepancy in the classification Show a message when you don t save the current analysis Previous Groups CY XX 090507 Rev 002 Issue date April 29 2010 Page 76 of 127 To automatically activate highlighting in any defined population the user can Define the maximum population percentage in which any population with lower percentage of events will be shown in highlight Default value is less than 1 The user can configure a default option to always make populations visible each time you assign events to it By default it is deactivated Show in analyzer will be used to choose how to identify the different tubes merged in a file iL 4 Visibility Options t Define maximum population percentage for highlight 1 00 npa Make population visible when you assign events to it Controls Statistics Erte number laa Virtual Parameters Number of comments fields 1 File De Stored Compensations File name Gates and Analysis Strategies Warnings ile comment Reference Image Show a message when you assign events associated with two of more populations _ 3 FCS file ee i ee Configure APS Par
109. of this parameter in the different files This image difference will result in a warning message 1f there are at least two files for which the difference between the mean values of this parameter plus the difference between the standard deviations relative to the total parameter range exceeds the value set by the user for the threshold value Minimum difference for each parameter required for warning which has a currently predefined value of 5 In the analysis window of a merged file we will find a CONTROL button for POPULATION If we click on it a window with the Population differences will be listed in case there are CY XX 090507 Rev 002 Issue date April 29 2010 Page 102 of 127 Controls Population a EOSINOPHILS A Selected events absent in some files amp Coherence Warning FileDitterences 014 LLC Fe r 24 19 34 0g LLC K L 19 5 12 LLC 22 25 19 20 013 LLE 105 25 19 110 This button will appear red Coherence warning whenever any population of the Population tree is over the fixed threshold of coherence In the right part of this box we will see the files and parameters with incoherence This button will appear yellow Selected events absent in some files whenever the selection performed in common parameters does not contain events from all the tubes as could be expected when working on a dot plot with common parameters If we click in the Manual Analysis Window Menu Bar option Profile Confi
110. onal axes to represent density of the populations J MD 3D Density Joe Ar aa N 5 i ow ygsc 1 85C HeTgnt EXP Sse Pop Say 4 6 5 Statistical Diagrams BoxPlot A new statistical representation available is the Box Plots If you click on Diagram New Diagram on the Manual Analysis Menu Bar you can choose the box plot in the Stat Diagrams option NEW DIAGRAM Histogram Box Plot DotPlot APS Diagram Multidimensional Stat Diagram Number of Parameters Then you will obtain a diagram when you represent in the X axe all the populations visible in that moment in the population tree and in the Y axes you can choose the parameter to represent 1E4 1E3 aon lt D Lic a 9 4 o a lt 1E2 0 1E2 All Files CY XX 090507 Rev 002 Issue date April 29 2010 Page 37 of 127 The box itself contains the middle 50 of the data The upper edge hinge of the box indicates the 75th percentile of the data set and the lower hinge indicates the 25th percentile The range of the middle two quartiles is known as the inter quartile range The line in the box indicates the median value of the data If the median line within the box is not equidistant from the hinges then the data is skewed The ends of the vertical lines or whiskers indicate the minimum and maximum data values unless outliers are present in which c
111. orm a density display om Lt a 1 360 450 600 S00 en ee ee nd SSC H SSC Height Exp SSC Low SSC H SSC Height Exp S C Low e z2r 366 Jia coo 4 s j l j 2 an e j Fi ie i ri E DAs f i tis 4 4 102 1e ver 2 FLIH CD4 PECYS Log FLI HCD4 PECYS Log Population Density This choice will consider the populations already classified to maintain the colors for the density representation The density will be represented degrading the chosen color aa ns ow Dae SSC HiSSC Height Exp 550 Low qa0 za Jw on SSC MiSSC Height Exp b3 Lew EEE Hia goe dia dob 1 104 VOR 100 i 104 10 FL EEA PECyS Log FLI 4 C D4 PEC YS Log Density Configuration Density representation The user can represent also contour lines In the following figure you can visualize the same diagram with total density and with total density and contour lines 2D Density Jie 2D Density Joe 100 z Ea z o a 2 x q o a 7 Density increment Using this scroll the density of the graphs can be configured in order to choose the best contrast increment between the colors in it CY XX 090507 Rev 002 Issue date April 29 2010 Page 43 of 127 4 7 3 Local Visibility Initially the populations represented in a diagram are the ones which are selected in the Vis column of the Manual Analysis window The user can go to this option to have a special visu
112. ot remove the drivers associated with the license USB key because they may be used by other programs The drivers can be manually uninstalled from Control Panel Add or Remove Programs 2 7 Support Services The standard support service included in the price of the first year of license and renewable for an annual fee allows the user to refer to us his or her technical problems or questions by e mail The standard e mail address to which technical user requests should be addressed is supportinfinicyt cytognos com The standard way of reply will also be e mail although Cytognos technicians may choose other communication methods if available and considered more appropriate for the case Users with special support needs for whom the above coverage might be insufficient can consult us about the possibility of improving this coverage at our website www infinicyt com by e mail to supportinfinicyt cytognos com or through their usual commercial representative Cytognos also offers a variety of training courses that can be tailored according to the customer s needs Please contact us for more information about availability and pricing of these courses 2 8 Upgrades Users will receive newsletters with new possible updates in the address registered in the Users Area of the website www infinicyt com Please enter your data once you ve received the license with your login user and password for further communications From the Users Area
113. pondence Check parameters Analysis Strateg Concordances Previous groups Parameters concordance File Parameters Concordance SSC JOK PerCP Cy5 5 A CD19 p Check parameters Analysis Strat tegy n 1 L Concordances Previous groups a Previous groups H a FSC A a 55C A a CD19 PerCP Cy5 5 A PerCP Cy5 5 A a FSC A la 55C A a F5C A esac seco ca acs CY XX 090507 Rev 002 Issue date April 29 2010 Page 55 of 127 Clicking OK the strategy will be applied in the other file CES Ea g o E E E Et g oi Tuen Manual ANALYSE i i set ts ii i ree File Edit Diegas Statisticy Profile Help E i p i eC x z Be z Sii s f Bes 12 H 57M VE af wiar 13 FA a Fa uma N A b 4 J S wi W ui Hii is ts rT oe ie k a H a feel ee HPA law fi oe re teens HP iE wae ga ae i ia 3s IA FSC LINEAR PerGP Cy5 5 AiCID9 LOGICAL teri ms APC A CD45 LOGICAL Se 2 isle 2 ij GICAL EI tha 4l 400 PercP cy a CD019 LO ww cytognos EuroFlow After applied it is possible again to click on the lens of the population to Review the gates It will appear the gate saved on the diagrams made and with the same visibility If the gate does not fit on the new file just click on it and modify Review the population Leucocytes SSC Exp
114. ppear with the file name instead of the file number you can change it in Profile Configure Preferences Analysis Show in analyzer The expression NA non applicable will appear in the statistics in case there is no way of giving a logic calculation of a percentage of a population included in different files 3 Possibility of select gating per file Select File when the user is interested only in gating all the events from one of the original tubes See more information in chapter 4 13 2 4 The new merged files contain the new FILE NUMBER parameter which will allow us to separate the events corresponding to the original files using a FILE NUMBER representation In this sense if a parameter is common to all files it will be represented with all the bands and a parameter which has only been measured in a file will be represented only in that band In the next figure the parameters SSC Height and TCRgd FITC have been measured in the 7 files and are considered by the software as common parameters while the parameter TNFa PE has only been measured in file number 5 and only those events are represented CY XX 090507 Rev 002 Issue date April 29 2010 Page 105 of 127 File Number File Number File Number 1 1E1 TNFa PE Log 1 SSC Height SSC H Exp SSC Low TCRgd FITC FL1 H Log In this representation the user can also perform a quick gate on all events of each tube with the option Selection Select File or Ctrl 1 Ctrl 2
115. ptions that can be found on the Edit Menu in the Manual Analysis Window Menu Bar Reset Analysis Ctrl F Delete All Selections Ckri 0 Select File b 4 13 2 1 Undo Redo These options apply exclusively to the operations performed during the analysis Other actions regarding configuration of the diagrams or application settings cannot be undone Undo Ctrl Z By using the first of these options it is possible to undo the last Operation performed in the event selection procedure whether this operation was a drawing step or a modification of the classification of events or any of the operations correcting the selection procedure Redo Ctrl Y Performs the inverse operation i e it redoes any operation undone by the Undo function Both options become active only when there are operations that can be undone or redone and in this case the text appearing in both buttons includes the description of the type of operation that will be undone or redone by using the corresponding option These Undo Redo options of this menu are the same options we can find if we click in the middle of the plot with the right button of the mouse opening a pop up window which includes these options among many others referred to the diagram There is an unlimited number of undo and redo actions in memory CY XX 090507 Rev 002 Issue date April 29 2010 Page 63 of 127 4 13 2 2 Reset analysis Choose this option Ctrl R to clea
116. r all the analysis gates and go back to the initial situation with all the events classified as Events WARNING Reset analysis will apply to clear all colors in the analysis but not to any change made on the options concerning the profile 4 13 2 3 Delete All Selections Choose this option Ctrl 0 to delete the consecutive regions performed in several diagrams before classifying a population 4 13 2 4 Select File This option applies only when working with merged files Chapter 6 Through this option the user can select gate all the events corresponding originally to file number 1 or Ctrl 1 file number 2 or Ctrl 2 etc without having to draw the region with the mouse du 3 o E E F z 9 a Mee en File Number ane es RS 1E2 1E2 TCRgd FITC FL1 H Log TCRgd FITC FL1 H Log TCRgd FITC FL1 H Log Selection Select File File 1 Selection Select File File 3 Selection Select File File 5 WARNING Any other selection previously done without assignation of color will be lost if we perform this type of selection of the whole tube CY XX 090507 Rev 002 Issue date April 29 2010 Page 64 of 127 4 13 3 Diagrams In this section we described all the options that can be found on the Diagram Menu in the Manual Analysis Window Menu Bar Mew Diagram Ctrl D Tile Ctrl T All Diagrams to Front Ctrl 4 Close All Diagrams Ctrl B Apply Default Values to All Diagrams 4 13 3 1 New dia
117. rams before the label assignation this diagram will display the message GATE in the title as shown in the next figure CY XX 090507 Rev 002 Issue date April 29 2010 Page 21 of 127 s9C H 55C Height ssC T FSC H F5C Height SPN 4 LINEAL FL3 H CD4 PECy5 LOGARITHMIC On Histograms 2D DotPlots Multigraphs or APS view Automatic Population Separator it is possible to draw regions with the mouse The 3D Graphic is the only representation in which it is not possible to draw or select regions Each one of the operations you perform in the manual analysis can be Undone or Redone through these commands available in the Edit option of the Analysis Window menu 4 13 2 1 4 4 2 Resize a region Before clicking on the corresponding label to give a color to a region it can be resized in the same DotPlot just by clicking inside the region Then you can drag make it bigger or smaller or move the region with the mouse Then click again outside of the gate 00 300 400 600 300 SSC H SSC Height Exp SSC Low 100 2 FSC H FSC Height Lin 4 4 3 Redraw a region Only one region can be drawn on each diagram Therefore if you draw a second region in a same plot it will be considered a redraw and only the second one will apply Once a region is closed on a DotPlot it can be redrawn just drawing a new one The last one will replace the initial one CY XX 090507 Rev 002 Issue date April 29 2010 Page 22 of 127 4 4 4
118. rategy NA NA w z Analysis Info Gos i T 3 3 90 FA a Configure Ctrl G 16 0 74 Gate 0 events Total 0 00 Visibility 0 00 23 of 1013 MB used ryp H xX Analysis Strategy name Strategy n 1 Name the strategy This new Strategy has been stored in the profile To check the strategy go to Profile Configure in the Manual Analysis Window Menu Bar and a new window will be open CY XX 090507 Rev 002 Issue date April 29 2010 Page 53 of 127 In the new window select Gates and Analysis Strategy Ip Load Profile Save Profile Save Reference Image Load Reference Image iF o 13 Save Analysis Strategy 8 Unload Reference Image Load Analysis Strategy i2 Unload Analysis Strategy NA Analysis Info 15 Configure Ctrl G teho Gate 0 events Total 0 00 visibility 0 00 iz of 1013 MB used 4 10 3 Apply Strategies Open a new file and Go to Profile Load Analysis Strategy in the Manual Analysis Window Menu Bar and select the previously saved strategy 01 Tutorial Manual ANALYSIS caL a File Edit Diagrams Statistics Help Load Profile Save Profile Population Partial Save Reference Image Load Reference Image Load Analysis Strategy 00 00 100 00 p an NA NA Save Analysis Strategy NA N Unload Reference Image Analysis Strategy name Strateg
119. register a parameter as common to the different tubes the software must recognize exactly the Same name in the parameter description in the FCS file of each different original tube In case there are differences for example regarding capital letters dots hyphens commas script etc the software may not identify the corresponding parameters as common Therefore it is fundamental to check the spelling of the parameters that are going to be common 8 2 Available commands in the editor When you click in the Editor you have to select first the file you want to edit and then this window is opened File Editor Oo Fie CAQ08 LLC K L 19 5 DataSet 1 Parameter Value 5Y5 Windows xF 5 1 XFORT USER MAME Administrator FITC E 4 5 TE A LAMED FIT 4 KAPPA PE A PercP Cy5 5 4 CO19 PercP cy5 5 4 Parameter PPN Show All File Parameters CY XX 090507 Rev 002 Issue date April 29 2010 Page 117 of 127 Show All File Parameters will show us all the parameter values even the ones that we cannot modify If it remains unselected the software will show only the ones which can be modified The values that cannot be modified are shown in grey Help document with the User s Manual chapter of this Module will be displayed With the Select File button the user selects the FCS file that needs to be modified Once the editor has opened the file it displays a table with a line for each text parameter in the TEXT sec
120. rg Some instruments such us Coulter generates FCS files with two different data sets Dataset 1 generated in version FCS 2 0 and Dataset 2 generated in version FCS 3 0 This new version of Infinicyt is able to open FCS files which includes two or more datasets Once the file is selected Infinicyt warns about this situation The software asks the user which of the Datasets wants to open This file has two or more datasets E g Coulter files contain Dataset 1 FCS 2 0 Dataset 2 FCS 3 0 For further information click on the help button _ Do not show this tip again Clicking on OK the second dialogue box configures how to use these files during the whole session lt Select DataSet Fae C ponmi aed Set lies 0 1 LS Phalaruels available PCS Vemi 4 0 Events 33000 Parameters 2 EE Paraencbers Compermation FES Inia Pareceter Scale PSAS U T las BE SS LAJ yp Ric Low PLInpersure Loge Aig re Lage Fc Loge PLURAUS Loge Flic Por Langer an pope 1 The software as default uses the Dataset 2 FCS 3 0 but Dataset 2 not always displays information of the labels and compensation 2 Click on this tip to choose the Parameters labels and Compensation information from the Dataset 1 3 Use the same configuration for all the files you open in the same session to avoid repeating this message CY XX 090507 Rev 002 Issue date April 29
121. rocess however the Infinicyt version which will be installed contains both languages Infinicyt will be configured to run using the selected language during the installation process The language can be later changed in order to do that double click to open the file named Infinicyt ini placed in your Infinicyt installation folder usually c Program Files Cytognos Infinicyt and add the following line If you want to select English language en If you want to select Spanish language es If no language is selected Infinicyt will use the language selected in the Windows language bar If Spanish is selected in the language bar Infinicyt will run using Spanish if any other language is selected then Infinicyt will run using English 2 6 Uninstallation The program can be uninstalled through the Windows normal uninstallation process Control Panel Add or Remove programs Alternatively the program can also be uninstalled by double clicking on the file Uninstall Infinicyt exe localized in the folder Uninstall_Infinicyt inside the Infinicyt installation folder CY XX 090507 Rev 002 Issue date April 29 2010 Page 11 of 127 In some cases the uninstallation program may inform the user that it was not possible to remove some of the files that were installed along with the program In that case the user should eliminate these files by hand It should also be noted that the Infinicyt uninstaller does n
122. rofile is the configuration of the workspace as well as other internal configurations of the software In this sense the user can save different profiles where will define the diagrams in the screen the population tree the model of report and other detailed configurations The detailed configurations can be found on the Manual Analysis Window Menu bar in the option Profile Configure In this window different choices for changing options can be reviewed All the changes made here will apply to the program and will be part of the profile when saved Save a profile Once you have defined the diagrams population tree report and the other internal configurations go to Profile in the Manual Analysis Window Menu Bar and choose Save Profile Automatically the software will ask you where to save it and the extension applied will be inp Infinicyt profile Open a profile Before or after opening a sample you can always go to Profile Open Profile and the software will ask the user to select the profile of interest Then choose Select The software will warn that when applying a saved profile the current one will be lost unless you want to save it first Warning This action will load a previously saved profile The current profile will be lost if unsaved Do you want to continue CY XX 090507 Rev 002 Issue date April 29 2010 Page 48 of 127 4 9 Reference Image Reference image is a useful tool to compare events prev
123. s return to the saved default settings clicking this option Information about default values is available on Profile Configure Graphs CY XX 090507 Rev 002 Issue date April 29 2010 Page 68 of 127 4 13 4 Statistics In this section we described all the options that can be found on the Statistics Menu in the Manual Analysis Window Menu Bar Show Statistics Export Statistics Configure These options refer to the visualization and configuration of the main statistical data of the different populations For each analyzed population the program always displays some default information about its relative numbers with reference to the total cells or to partial populations which can be shown in the Manual Analysis Window 4 13 4 1 Show Statistics The analysis window shows by default information about the events number and percentages related to total cells or partial populations Apart from this basic information the user can also obtain statistic values of the populations The option Show Statistics generates more columns one per each parameter Each column corresponds to the statistics for each parameter The columns can be closed lt 4 but clicking on them they are opened to visualize the data so the user only has to keep open the columns needed Then it will appear by default the Mean SD and CV for each population To obtain more statistic functions they need to be configured in the profile See chapter 4 13 5 10 to
124. s with this option When we click Load Reference Image we will be able to choose among the different groups we have previously saved in this profile The reference events will appear in a new label with the same name and we can hide or show them clicking on the VIS box n Relapse 004 Man OJEJ File Edit Diagrams Statistics Profile Help Population 7 Edit Belerence Image EJ unclassified events Meee Hit Other unclassified events MELTROPHILS aS ae events Total 0 00 Visibility 0 00 CY XX 090507 Rev 002 Issue date April 29 2010 Page 72 of 127 With a right click on the new label the user can edit the appearance of these reference events For example they can be displayed as highlight dots a contour line etc amp N Sax TD Bex P 1 1E1 1E2 163 164 1 161 162 163 164 FL1 H CD10 LOGARITHMIC FL1 H CD10 LOGARITHMIC Reference Image with Lines Reference Image with Dots FL2 H CD20 LOGARITHMIC FL2 H CD20 LOGARITHMIC 164 182 163 1 4 13 5 5 Unload Reference Image Click this option to unload the reference events If you don t unload a reference image and you try to analyze a different sample with other parameters in which you can not load the same reference image the software will try to load it and 1f the parameters don t match it will show you the a warning for example like the following figure where none of the parameters
125. stallation of the program This user s guide assumes that the operator has a basic knowledge of flow cytometry techniques as well as data interpretation for a correct use of this software CY XX 090507 Rev 002 Issue date April 29 2010 Page 15 of 127 4 MANUALANALYSIS 4 1 Introduction The manual analysis mode allows the multidimensional study of single FCS files files generated by new generation cytometers as well as n dimensional files generated after the Merge Calculate Data process The software includes a wide variety of analysis tools plots and graphics as well as printed reports which allow user friendly interpretation of obtained results The identification of different cell populations is carried out by drawing regions on the computer screen which are assigned different names and colors allowing classification as a hierarchic tree The analysis panels are completely configurable by the user Results can be shown in Histograms 2D plots and multidimensional plots with different choices of transformation of the original parameters logarithmic compensated non compensated or even SSC expanded Statistics are exportable to different types of data bases and reports may be personally designed by the user following the requirements of each study 4 2 Getting Started on Manual Analysis Once you have installed Infinicyt software on your computer you are ready to use it Here is a short description of how to us
126. sted by significance O06 LLC K L 19 5 O135 LL0 103 25 19 110 014 LLC FMC 24 1 9 34 In the analysis window of a merged file we will find a CONTROL button for MERGE process m If it is shown on red and click on it a window with the Population differences will be listed If there are no differences it will remain green CY XX 090507 Rev 002 Issue date April 29 2010 Page 101 of 127 6 5 2 Population control This second control evaluates the similarity of cell clusters from different files that we consider within the same population This control operates once the files are merged and the user starts to analyze the populations To activate this control check the Population Control option in this part w Configure Profile Configuration Threshold parameters for coherence controls Freferences Analysis na Merge Control Diagrama Minimum difference of global aspect required for warning Controls Statistics Virtual Parameters fp Population Control Stored Compensations Gates Show message when differences are detected in visible populations Reference Image Minimum difference for each parameter required for warning 5g Calculation Data Controls Disease Categories Configuratio Lg This validation process operates on each defined population in a merged data file the process evaluates for each common parameter of the population the possible image differences between the one dimensional distributions
127. ta Controls Maximum distance in the population space to exclude an event ko a Minimum distance in the population space to apply heterogeneity cortrol 90 Value for heterogeneity control 90 Maximum distance in the population space to exclude an event considering 100 the maximum distance possible to find it we can reduce this distance to exclude any event as valid for being the nearest equivalent neighbour Minimum distance in the population space to apply heterogeneity control considering 100 the maximum distance the user can reduce the area in which any event is valid as neighbour and the events out of this region but inside the maximum distance control will need to apply heterogeneity control Value for heterogeneity control for those values inside of the first value and out of the second value the user can establish how heterogeneous the populations are CY XX 090507 Rev 002 Issue date April 29 2010 Page 116 of 127 8 FILE EDITOR 8 1 Introduction With this module the user may perform limited changes on FCS files such as correcting typing errors in the parameter labels occurred during acquisition in case they may interfere in the merger process The program allows only the modification of text values of the FCS file and only of those whose modification would not affect the coherence of the data for example the long names of the cytometric parameters can be modified Please note that to
128. te background for presentations This action applies only to the selected graphic In the Profile Configure Graphs menu 4 13 5 10 the user has the option to change the default background color as a default for all the graphics 4 7 8 3 Invert Gate Selection It is possible to draw a region around a population and invert the gate to select all events excluded in the region This can be done in the right click menu of the graph and choosing Invert and then Invert Gate Selection GATE 2D Joe S gt aax Diagram Configuration b Configure Visualization Data 200 300 800 800 1000 Local Visibility Number of Events Displayed 100 Window Manipulation SSC H SSC Height Exp S SSC H SSC Height Exp S Undo Redo d b d Assign Population b b b Invert Invert Axes FL1 H CD3 FITC LO Diagram Info Invert Diagram Color FL1 H CD3 FITC LOGARI Invert Gate Selection 4 7 9 Diagram Info This option allows the user to obtain the information about the type of diagram and the dimensions included This information is especially useful in the APS view so the user may know which parameters were relevant being the principal components in the separation of populations It is especially relevant for the merged data files where the user can check which parameters are included in the data file CY XX 090507 Rev 002 Issue date April 29 2010 Page 47 of 127 4 8 Profiles The p
129. tion of the FCS file For each of these parameters the program shows the key at the left of the line and the value at the right The keys cannot be modified and the text that can be modified is shown in white in the right column To modify one of them the user only has to double click its cell and type the new text Save As Once the user has performed the appropriate modifications it is possible to save the result to disk through this option It 1s strongly recommended that the user saves it to a new file instead of rewriting the original one Non standard parameters are not guaranteed Main Menu At any moment the user can return to the Main Menu of the program by using this option CY XX 090507 Rev 002 Issue date April 29 2010 Page 118 of 127 9 FILE EXPORT 9 1 Introduction In this module there are several options available to save to disk the results of the program possibly with some additional information in export formats readable by external analysis or database software to facilitate data analysis There is also an access to File Export option from the Manual Analysis menu which is more flexible to export only a group of visible gated events see section 4 13 1 6 9 2 Available commands in the File Export window When you click in the Export you have to select first the file you want to export and then the Export window is opened BASIC FUNCTIONS File Export Joe _ File C Documents and Settings
130. tunity of recovering the original data file values PnR This value indicates the range of this parameter PnB This value indicates the size of each value in the hard disk in bits Exp SSC Low If you click this checkbox a mathematical transformation will be applied to the parameter chosen of your export file to improve the separation between populations in the lower part of the scale 9 3 Export a population from the Manual Analysis From the Main Screen the File Export option will consider the export of a whole file If the user is interested on exporting only one or more populations then the Export option must be open from the Analysis Window in the option File Export Ctrl E In case the user wants to export only the events included on a given population the export must be done from the Manual Analysis and once the population is classified and visible go to the option File Export In this dialogue box you will find the option export only visible populations which is not present in the export window obtained from the main menu Through this option only visible events in the file analyzed will be exported to the new data file File Export File Only visible populations Export Parameter FSC A o I _ Simuted Time O O o rn CY XX 090507 Rev 002 Issue date April 29 2010 Page 121 of 127 9 4 Export files n XML format The Extensible Markup Language XML is a standard format of files compatibl
131. ue date April 29 2010 crnm7tmmonoarp FITC A CD22 PE A CD23 PerCP Cy5 5 4 CD19 PE Cy7 amp APC A CD20 APC Cy 7 A Pacific Blue A AmCyan A Page 85 of 127 NEGATIVE VISIBILITY These values represent the percentage for negative data range in the LOGICAL scale which is shown in the graph It corresponds to the part of the negative axes in relation to the positive axes amp Profile Configuration Py Preferences H Analysis Graphs Controls se Statistics Stored Compensations i Gates FCS file H Current Compensation Negative Visibility sod Configure APS Parameters i Properties Negative Visibility Percentage A Automatic 1 10000 negativity Parameter FSc 4 55C A FITC 4 CD22 PE A CD23 PerCP Cy5 5 4 CD19 PE Cy7 A APC 4 CD20 APC Cy 7 4 Pacific Blue 4 AmCyan 4 Apply to all se ss The percentages shown for each parameter can be moved to improve the visualization Depending on this percentage the negative part of the scale of the parameter will be less represented The maximum value is 20 Parameter FSC 4 55C CD7 FITC A CD4 PE 4 1 88 CD19 PerCP Cy5 5 4 10 35 CD56 PE Cy7 A 2 92 CD3 APC 4 I CD20 4PC Cy 4 3 6 CD45 Pacific Blue A 2 09 CD7 FITC A LOGICAL CDZAPC A LOGICAL Parameter Yo 0 01 0 01 0 01 m
132. ulation p Exp SSC Low Configiration Legend Scale Configuration SSC A Exp SSC Low b Undo Redo gt gt Kiai Invert 1E3 0 1E3 Diagram Info APC A CD34 LOGICAL SSC A FITC A CD15 PE A CD16 PerCP Cy5 5 A CD45 PE Cy7 A APC A CD34 APC Cy7 A Pacific Blue A AmCyan A Apply to alk 4 7 1 3 Virtual Parameter Configuration Though this option you obtain the Virtual Parameter Configuration window in the profile See chapter 4 13 5 10 in the section of Virtual parameters In this window you can create new parameter as a combination of others and you can choose which calculation you want to represent e g dividing a parameter by a constant number rest one parameter from another CY XX 090507 Rev 002 Issue date April 29 2010 Page 40 of 127 4 7 1 4 Compensation This option shows the information of the compensation values which is stored in the data file These values can be changed manually and be saved as but you can always restore the original values It is also possible to choose a stored compensation and change the values to modify it 4 7 1 5 EXP SC Low Configuration This option gives the possibility to expand the SSC parameter with the values desired by the user When clicking on it the SSC plot will appear with two scrolls in the lower part of the graph to regulate the part of the scale you want to amplify Configurable Enp 54E Low Ed SH
133. ulations with data from all the markers of the panel within each cell in each tube see following figure q0ufo08 HPI dad Poo SSC HEIGHT SSC T CD34 APC e m l f Li wW a e In order for the calculation algorithm to work correctly the FCS files that you want to calculate have to meet the following requirements Regarding the panel design the common parameters of the population to be calculated have to exhibit different characteristics 1 e markers expressed differently in the same cell populations should be in the same tube The acquisition settings have to be the same for each acquired tube to be merged The software is able to harmonize the FSC SSC characteristics in surface and CY XX 090507 Rev 002 Issue date April 29 2010 Page 108 of 127 cytoplasmic staining to search for the same selected cells in the different files Other common parameters apart from FSC and SSC can also be harmonized Infinicyt includes an internal Calculate process control system that verifies if all the previously mentioned conditions apply although it is always possible to carry out a control of the Calculate Data comparing the calculated results with a real file WARNING Validation of Calculated data mode has to be done for each user with its own panels 7 2 Limitations This module has been validated under conditions where identical cells can be unequivocally selected with the Common markers included in the panel
134. ve percentage of the populations visualized at that moment in the analysis the value 100 representing the sum of all shown populations 4 5 5 Comments column By clicking on each of these labels from this column the user may write a comment about the expression of a parameter in a population Comments B Parameters Comments This comment will be saved and will appear any time you click on each colored label and it will be saved to the printed report Po rr G J Comments Lymph B sa Sample 1025 Manual ANALYSIS File Edit Diagrams Statistics Profile Help ParametersComments Normal Patologica an aA spre ar l a SSC lt a K Population Wifler IEUCUCYLIES T a gt z T Neutrophils a T F z T z Monoc yt es Ne K K gt z T T gt zZz gt r zZ T Z gt zZz gt z i CD19 Pe f a ESE T Events tse veep vee fet fa fee es Total Partial yan tK K K EA Il Gate 0 events Total 0 00 Visibility 0 00 The number of comment columns can be chosen as explained on chapter 4 13 5 10 in the section of Analysis CY XX 090507 Rev 002 Issue date April 29 2010 Page 27 of 127 4 5 6 Statistics columns As a default all the co
135. w B Cells Gate 3 Leucocytes 2D Leucocytes FSC 4 Lin SSC 4 Exp SSC Low 2D LeucocytesPerCP Cy5 5 4 CD19 Logic 5SC 4 Exp 59 NK Cells Gate 4 Leucocytes mW p With the different colors we can see the populations analyzed and for each of them the different steps performed to analyze them The steps can be positive if gated events were included or negative if gated events were excluded CY XX 090507 Rev 002 Issue date April 29 2010 Page 75 of 127 4 13 5 10 Configure In this chapter we will review all the elements that are part of the Profile so the user may decide how to personalize a profile with the different internal parameters Profile Configuration 5 Freferences Diagrams Controls H 4 Statistics co Virtual Parameters 4 Stored Compensations Gates and Analysis Strategies 4 Reference Image 5 23 FCS file Current Compensation a Negative Visibility fen Configure APS Parameters i i Properties J Tools H Previous Groups ANALYSIS In this option the user can change some Visibility Options and Warnings during the analysis Ke Configure x ian Profile Configuration q Visibility Options B Preferences a m Eee Define maximum population percentage for highlight 1 00 Diagrams Make population visible when you assign events to it Controls S Statistics Show in analyzer File number v 2 Virtual Parameters Number of comme
136. y nl Load Analysis Strategy NA NA P Unload Analysis Strategy NA NA Analysis Info NA NA s NA NA i Configure Ctrl G NA NA Gate 0 events Total 0 00 visibility 0 00 56 of 1013 MB used The saved analysis has been applied on the new file just check by clicking on the lens of each population q gt E 1 Teese y Monae ANALYST te i Guerre IANAO Prove Hee ty a 1 v w ve s s Te Ya m mm omh Vam hie pes nam 2a c w M 3 ITPA Der Aan 7 ee ee ee Pereh CyFF2COr LOGHAL im ipa iam Jx Dh DS Sele Severs Tad C0 Web STOR oF Re coed Nt EuroFlow k _ _ a a e CY XX 090507 Rev 002 Issue date April 29 2010 Page 54 of 127 4 10 4 Matching of Parameters Infinicyt will only apply Saved Strategies if the parameters are coincident in both files for example same parameters with same writing In case the labels of the parameters were not exactly the same Infinicyt will suggest you an option to match the parameters Filel where we have created the strategy File 2 the file over we apply the strategy Fac a ssc Ss FITC A CDS PE A CD56 PerCP CyS S AiCDIS PE Cy7 A APC A CD45 APC Cy7 A Pacific Blue A AmCyan A Notice that FSC and SSC are not written in the same way in both files When we apply the strategy on file 2 the software shows us a box for the parameter corres
137. y o 0 00 All the columns can be moved or their order changed by just clicking and dragging the heading of the column to the desired place The column Population contains the name or key that identifies the population whose data appears in the line The color of this field coincides with that of the dots or lines representing this population in the graphics The user can edit the populations chart CY XX 090507 Rev 002 Issue date April 29 2010 Page 23 of 127 4 4 6 Create labels To create a new population right click on the first label of Events and you will have the option to create a population which will depend on the total cells If you want your new populations to be a subset of a pre existing one then right click on the pre existing population and create new one depending from the previously selected population 0000 001 Manual ANALYSIS BAX Fie Edit Diagrams Statistics Profile Help E 2 2 Fi i 5 Pay T Population 5 5 be VIS i E i kA 5 E gt WwW unclassified events Other unclassified events 0 42 0 42 0 00 5 14 5 14 57 39 3 82 3 82 42 61 54 06 54 06 0 00 0 13 0 25 0 00 23 37 43 23 0 00 30 56 56 53 0 00 2 78 2 78 0 00 33 78 33 78 0 00 MOO OBS Add Population Add Function Edit Population Up Population Remove Population Remove All Populations ate events Total 0 00 Visibility 0 00 J 0000 00

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