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FCAP Array™ Software v3.0 User`s Guide

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1. Plex creation The workflow of creating a plex includes these stages workflow Stage Description 1 Selecting beads and analysis model page 51 2 Selecting instrument settings page 53 3 Assigning beads to clusters page 55 4 Using debris filtering page 57 5 Using manual clustering page 59 6 Defining standards and QC page 64 7 Defining controls page 72 8 Viewing standard curves page 74 More information Workflow overview page 24 e Adding plexes page 31 Chapter 5 Working with plexes 51 Selecting beads and analysis model Introduction This topic describes the process of adding beads to a plex and selecting the analysis model Before you begin If necessary add beads to the bead library When you first install FCAP Array the bead library is empty For BD CBA reagents we recommend adding beads to the bead library by importing an XML file See Importing a bead group page 107 Selecting beads for To select beads for a plex a plex 1 Click Beads and Model in the Plex navigation panel to define the beads needed for your plex The Selected Beads panel opens ree Bead Group Group Description Al Beads Bead Name Lot Number Catalog Number Name Model 2nd Reporter AT 9 B6 c4 o7 Panence Mouse Thi Th2 Cytokine Kit 551287 BD CBA Kit Cell Signaling BD CBA Flex Sets Human Enhanced Sensitivity E5 BD CBA Flex Sets Human Immunoglobulin BD C
2. 1 Starting the software page 17 2 Creating a new experiment from BD FACSuite files page 96 3 Evaluating the experiment page 98 4 Viewing analysis results page 81 5 Viewing reports page 89 Chapter 7 Using the BD FACSuite workflow 95 About CBA specific To use the BD FACSuite workflow most efficiently it is important keywords that CBA specific keywords be added to the experiment while it is being run in BD FACSuite software See the Guide to Using BD FACSuite Software with BD Cytometric Bead Array Products to learn how to perform this setup Go to bdbiosciences com cbasetup for more BD CBA information Keyword Description CBA Plex Name Identifies the specific plex from the FCAP Array software plex library If a match is found in the plex library the FCS files are automatically associated with that plex CBA Type Identifies whether the tube or well contains a standard sample or control FCAP Array software lists analysis results in the following order standards samples controls CBA Standard e ID of the standard contained in the tube or well The value can also be ID Pos or Neg to identify positive and negative for qualitative BD CBA assays e Standards will be arranged for the standard curve in alphabetical order by the value of the CBA Standard ID keyword Assign this keyword appropriately to ensure that standards are plotted in the correct order on the standard curve Use l
3. 13 This page intentionally left blank 2 Installing and starting the software This chapter covers the following topics e Installing FCAP Array software page 16 e Starting the software page 17 e Obtaining the bead library file page 19 e Uninstalling FCAP Array software page 20 16 FCAP Array Software User s Guide Installing FCAP Array software Introduction Caution Procedure Next step This topic provides instructions for installing FCAP Array software version 3 0 Caution Do not install the FCAP Array version 3 0 A security key until after step 6 in the procedure The software and drivers must be installed first FCAP Array version 3 0 can exist concurrently with FCAP Array version 1 0 on the same PC See Limitations page 10 for more information To install FCAP Array software 1 Ensure that no other programs are running 2 Insert the FCAP Array software installation disc into the DVD ROM drive If the installer does not start up automatically use Windows Explorer to view the disc contents then double click the Setup exe icon 3 Click Next on the installer Welcome screen 4 Read the license information then select I Agree and click Next 5 Note the destination folder for FCAP Array software and click Install We recommend that you accept the default location 6 Click Finish to exit the installer 7 Install the FCAP Array security key in a USB port Proceed to St
4. Forgot password Your software administrator can reset your password Possible causes Recommended solutions Database is corrupted Restore a database backup or delete the database file and FCAP Array will regenerate the database at the next run Possible causes Recommended solutions User manual file was not installed with the software Re run the FCAP Array installer and select Repair Possible causes Recommended solutions Experiment is locked Make a copy of the experiment by click the Save As icon in the Experiment group Possible causes Recommended solutions Bead name and analyte already exist in the bead library Make corrections to the bead identifiers 118 FCAP Array Software User s Guide Unexpected analysis results Possible causes Recommended solutions Re using saved fitting curves Calculate new standard curves for every experiment Software extrapolation leads to inaccurate values Calculate new standard curves for every experiment Single color and dual color beads are in the same experiment Single color and dual color beads require different instrument setups Experiment sample layout does not match actual sample layout Correct the experiment layout redo the file assignment and re analyze Incorrect bead information in the bead library Correct the information I
5. Selecting FCS files To select FCS files 1 Navigate to your FCS files from the File Explorer tab on the File Assignment panel The FCS files are listed in the lower window File Assignment File Explorer E Research In Motion a i O PS RSIGuard CHES Soft Flow FCAP Array v3 Fea FCS files TechSmith E ThinPrint Client F Uninstall Information ALES WaohEv File types All Files X Name Date 4 Std02_001 Plex 8 fcs 8 5 2004 1 32 06 PM Std03_001 Plex 8 fes 8 5 2004 1 32 34 PM Std04_001 Plex 8 Fes 8 5 2004 1 33 00 PM Std05_001 Plex 8 fes 8 5 2004 1 33 26 PM Std06_001 Plex 8 fes 8 5 2004 1 33 51 PM StdO7_001 Plex 8 fes 8 5 2004 1 34 17 PM Std08_001 Plex 8 fcs 8 5 2004 1 34 42 PM Std09_001 Plex 8 Fes 8 5 2004 1 35 09 PM Std10_001 Plex 8 Fes 8 5 2004 1 35 38 PM Group A Spike Sample_x256_001 Plex 8 8 5 2004 1 36 05 PM Group A Spike Sample _x64_001 Plex 8 fcs 8 5 2004 1 36 33 PM Group A Spike Sample _x8_001 Plex 8 fcs 8 5 2004 1 37 00 PM Group A Spike Sample_x2_001 Plex 8 fcs 8 5 2004 1 37 28 PM 2 Filter by file type in the File types list If you do not see your files listed change the File types to All Files This displays files that do not have a fcs or lmd file extension 3 Files are displayed in ascending order by acquisition date and time in the default settings You can change the order for both name and date from ascending to de
6. To modify sample information using the Sample List tab 1 Click the Sample List tab on the File Assignment panel Using the Sample List tab enables you to view all the samples at once and to enter data more rapidly File Assignment File Explorer Sample List r gt Sample Name Dilution Results File Name stdoo2 Std02_001 Plex 8 Fcs Std003 Std03_001 Plex 8 fcs Std004 Std04_001 Plex 8 fcs Std005 Std05_001 Plex 8 fes Stdo06 Std06_001 Plex 8 fes Std007 Std07_001 Plex 8 fcs Std008 Std08_001 Plex 8 fcs Std009 Std09_001 Plex 8 fcs Std010 Std10_001 Plex 8 fcs Test001 Group 4 Spike Sample_x256_001 Plex 8 fcs Test002 Group 4 Spike Sample _x64_001 Plex 8 fcs Test003 Group 4 Spike Sample_x8_001 Plex 8 fcs Test004 Group 4 Spike Sample_x2_001 Plex 8 fcs ma oia oaa oaa oan moa oan aa ee roe roe res Select a sample by clicking in the Sample List Edit the name and press Enter Select a sample by clicking in the Dilution field O a E Edit the value and press Enter Next step Proceed to Working with experiment information page 43 Chapter 4 Creating a new experiment 43 More information Adding samples page 35 e Workflow overview page 24 e New experiment overview page 28 Working with experiment information Introduction This topic describes how to enter information about an experiment in the data sheet You can also add or remove users f
7. corresponding row Organizing the data in the statistics table Setting up custom filtering in the statistics table Chapter 6 Results and reports management 85 You can organize the order of the data in the columns in the statistics table to decreasing or increasing To organize data 1 2 Click the column header Click the arrow in the header to change the order of the data You can set the criteria for data that is displayed and hide rows that you do not want to show by using the custom filter You can then export this filtered data To set up custom filtering 1 Click the filter icon in the right corner of the column header to open the menu I Analyte Name Event mEt a can a7 Human IL 6 Non blanks 48 Human IL 7 282 203 B6 Human IL 8 292 See C4 Human Angiogenin 203 es D7 Human LT alpha 288 292 301 Select Custom The Custom Auto Filter dialog opens Custom AutoFilter Show rows where Analyte Name like 3 Complete the fields to filter the data 86 FCAP Array Software User s Guide Exporting results Printing Next step More information 4 Click OK The filter is displayed at the bottom of the table where you can modify inactivate or delete the filter The data from results tables can be exported as different file formats including Microsoft Excel XLS PDF RTF HTML and CSV The file format is set to XLS by default Cli
8. To change back to auto clustering for selected samples 1 Clusters Seon oon P seo 1608 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A 6 6 A Clear the Use Manual Clustering checkboxes for your selected samples in the list 3 0 E 3 E pl gt fy 1000 1000 Navigating the sample list Selecting failed files for manual clustering Selecting all files for manual clustering Reverting to auto clustering for all files Applying manual clustering to selected files 62 FCAP Array Software User s Guide To navigate the sample list do one of the following e Click a row in the sample list to show the file s sample data e Click the Next File icon to move down the sample list and show the next file s sample data e Click the Previous File icon to move up the sample list and show the previous file s sample data To select failed files for manual clustering 1 Click the Use Manual Clustering for Failed Files icon You can now edit the failed files indicated by red colored text Adjust scatter parameters adjust clustering gates and assign beads To select all files for manual clustering 1 Click the Use Manual Clustering for All Files icon You can now edit all files Adjust scatter parameters adjust clustering gates and assign beads To revert back to automatic clustering for all files 1 Click the Use Auto Clusteri
9. Units mL 2 Do one of the following e Select one of the options from the menu Specify your own parameters by clicking Custom completing the fields and clicking OK Custom AutoFilter Show rows where Analyte Name like And Or Cancel The filter is displayed at the bottom of the table where you can modify inactivate or delete the filter 102 FCAP Array Software User s Guide Using the experiment library Introduction This topic describes how to work with experiment files in the experiment library About experiment The available experiments are organized in the experiment library functions The functions are located in the Experiment group on the ribbon Open Experiment v Import New Experiment Delete X A Export Experiment Experiment fy Creating an To create an experiment experiment 1 Click the lower half of the New Experiment icon 2 Select an experiment type A dialog opens 3 Enter any experiment details and click OK For more detailed instructions see Creating a new experiment page 29 Opening an To open an experiment experiment 1 Select an experiment from the experiment table Name CreatedBy Creation Date Comment Last Save D Last Save By Locked Experiment Administrator 3 7 2011 3 7 2011 Administrator No i 2011 2 Click the Open Experiment icon Alternatively you can double click the selected experiment to open it Viewing experiment prop
10. library The functions are located in the Bead Library group on the ribbon ef SL ModfyBed ge A New Bead Delete Bead s Mew Bead Import Export Group 6 X Bead Library i Adding beads Modifying beads Deleting beads Bead group functions Chapter 8 Data management 105 To add beads 1 Click the New Bead icon The New Bead dialog opens 2 For each bead specify the Bead Name Analyte Second Reporter Parameter if applicable and Catalog Number optional 3 Click OK To modify beads 1 Select a bead from the library 2 Click the Modify Bead icon 3 Change the details in the Modify Bead dialog 4 Click OK To delete a bead 1 Select a bead from the list in the left panel 2 Click the Delete Bead icon The Confirm bead deletion dialog opens 3 Click OK Creating bead groups allow you to manage a set of beads that can be used for specific purposes and makes it more efficient to handle them as a block rather than individually 106 FCAP Array Software User s Guide Creating a bead group Modifying a bead group Deleting a bead group Adding a bead group Removing a bead from a group To create a bead group 1 Click the New Bead Group icon The New Bead Group dialog opens 2 Enter group name and description 3 Click OK To modify a bead group 1 Select a bead group 2 Click the Modify Bead Group icon The Modify Bead Group dialog opens 3 Edit
11. 99 BB Standard 5000 E test Control E contr 4000 J 2 686 66 am 2 461 82 2000 1 265 73 1000 4 616 56 688 77 24315 38 18 19 38 04 75 54 155 22 a 18 49 87 94 D a a a Ss Fs SF Fs FS FSF SF EF EF Sf SES SESESEESESESEEESE 1000 Chapter 6 Results and reports management 83 Results per sample This view shows the list of samples in the left table and the statistics for the selected sample in the right table Use the horizontal scroll bar in the right table to see all of the statistics columns In the following figure the plot shows results for the analyte selected from the left table Sample Name Clustering Results File E Analyte na Event Nominal CC Ftedc Finale Recovery Dision OCR std001 Auto Std04 004 CS Ee Std02_001 Human 301 25 48 11 49 43 54 NjA 20 81 pg ml 20 81 pg N A NjA stdo03 Auto Std03_001 AB Human 282 16 25 8 36 50 48 NjA 16 24 pgjmL 16 24 pg N A 0 NjA Stdo04 Auto Std04_001 B6 Human 292 14 99 8 40 56 82 NjA 21 07 pgjmL 21 07 pg NjA 0 NjA Stdoos Auto Std05_001 C4 Human 203 47 40 16 19 36 73 NjA 17 75 pgjmL 17 75 pg NJA 0 NjA Stdoo6 Auto std06_001 D7 Human 288 9 82 7 80 63 01 NjA 7 41 pgfml 7 41 pg ml NjA 0 N A stdoo7 Auto Std07 001 stdoos Auto Stdo8_001 Staoo9 Auto Std09_001 stdo10 Auto Std10_001 restoot Auto ___Groww Ai al A m E Parameter X Red A bd Std00
12. FACSuite software to simplify your workflow A standard dilution calculator Debris filtering and manual clustering tools Graphical reporting of results Exporting of reports in multiple formats including PDF Limitations page 10 System requirements page 11 24 FCAP Array Software User s Guide Workflow overview Introduction Standard workflow description Standard workflow stages This topic describes the two workflows for using FCAP Array software e Standard workflow Analyzing FCS files from standard cytometry software including BD CellQuest Pro software BD FACSArray software and BD FACSDiva software e BD FACSuite workflow Analyzing FCS files from BD FACSuite software for an optimized workflow The standard workflow is used when analyzing FCS files from most cytometry software applications You design an experiment in FCAP Array software that reflects the data contained in the FCS files that you are importing The process of designing this experiment can be greatly simplified if you first design a plex which defines beads standards analysis models and instrument settings that are common to multiple experiments After designing the experiment you proceed to analyze the FCS files The results are calculated and displayed in the same manner for both the standard workflow and BD FACSuite workflow The standard workflow includes these stages Stage Description 1 Starting the softw
13. Name Department E mail Contact Info Password and Administrator checkbox Click OK Note that you cannot delete your own user account and a minimum of one administrator account is required by the software To delete a user 1 2 Select a user from the user table Click the Delete User icon The Confirm user deletion dialog opens Click Yes Chapter 8 Data management 113 Using the units library Introduction This topic describes how to manage the measurement units that are used in experiment analysis when selecting the concentration unit for standard and control samples Measurement unit The measurement functions are located in the Units group on the functions ribbon S A L My New Unit Delete Unit Units ty Adding a unit To add a unit 1 Click the New Unit icon The New Unit dialog opens 2 Enter the unit name and unit description 3 Click OK Modifying a unit To to modify a unit 1 Select a unit in the unit table Name Description T ng mL pg mL fg mL Units mb 2 Click the Modify Unit icon The Modify Unit dialog opens 3 Edit the unit name and unit description 4 Click OK 114 FCAP Array Software User s Guide Deleting a unit To delete a unit 1 2 Select a unit in the unit table Click the Delete Unit icon The Confirm unit deletion dialog opens Click Yes Troubleshooting This chapter covers the following topic e Troubleshooting page 116 11
14. ade twee HS ai a a su ete Sank 48 Selecting beads and analysis model 0 cece cece eee eens 51 Selecting instrument settings srren esop srr cc cee eee teen een S 53 Assigning beads to clusters 0 e cee cece cence nent nenes 55 Using debris filtering 2 0 0 0 cc cette ene e nee 57 Using manual clustering 0 cece eee eee e en nnes 59 Defining standards and QC oo ccc tenet tenes 64 Defining controls en eyi ie loy hed ars le eee BE BE eM A ae leh ebb arte le a A 72 Viewing standard curves 000 ce ec cece ec eee ence eee e tee ennees 74 Chapter 6 Results and reports management 79 Overview of results and reports 0 0 0 ce cece eee nen eeees 80 Viewing analysis results ssc 00 64 caw bee eee belie eeWededan seeders 81 Customizing charts 2 0 ccc ccc cece ene n nee nee 87 NIE WINE TEPOLtS sagire DEN ease dea Ria Gila atte Net aed ey VA aie eae 89 Completing anvexperiment 25 6 Ges eee wees Rae Pete ek jee WM 91 Chapter 7 Using the BD FACSuite workflow 93 BD FACSuite workflow overview 00 cece eee eee e eee n ees 94 Creating a new experiment from BD FACSuite files 00 5 96 Evaluating the experiment 0 0 cece cece eee ence enn 98 Chapter 8 Data management 99 Working with libraries 0 0 eet tent nees 100 Using the experiment library 2 0 0 ccc cee ee eee eee 102 Using the bead library 24 0053 48 egean ekia E eos Beg end eet Gow Shenk ba 104 Using the plex tem
15. an analyte from the Analytes list or by double clicking a plot The fitted results statistics are displayed below the plot Note that in an experiment with multiple replicates of a standard sample the results table shows the average values first and the values for the replicates in the rows below Standard curve options Changing the axis scaling Changing the fitting type Chapter 5 Working with plexes 75 You can modify the parameters of the calculated standard curves by using the features displayed in the Standard Curves Options group XAxis CC Logarithmic Fitting Type 5 Parameter Log Recovery Threshold 20 Y Axis MFI Logarithmic Weighting No Weighting Force Through Zero Chart Color The Trees Fitting Accuracy 98 Hide Blank Standard Standard Curve Options The scales of the x axis and y axis are set to Logarithmic by default To change axis scaling 1 Click Linear in the X Axis or Y Axis menu The fitting type is 5 Parameter Logistic by default To change the fitting type 1 Select another type from the Fitting Type menu The recalculation of the curves runs automatically and the plots refresh to show the new data You can find the equation of each fitting type in the list Fitting Type 5 Parameter Log Recovery Threshold 20 0 Weighting 5 Parameter Logistic Fitting Accu 76 FCAP Array Software User s Guide Changing the weighting type Chang
16. chapter covers the following topics e Overview of results and reports page 80 e Viewing analysis results page 81 e Customizing charts page 87 e Viewing reports page 89 e Completing an experiment page 91 80 FCAP Array Software User s Guide Overview of results and reports Introduction This topic describes the stages in the workflow for viewing analysis results and creating reports 8 Results per Analyte 9 Results per Sample 10 Report Results and reports The workflow for viewing results creating reports and completing workflow an experiment includes these stages Stage Description 1 Viewing analysis results page 81 2 Customizing charts page 87 3 Viewing reports page 89 4 Completing an experiment page 91 More information New experiment overview page 28 e Workflow overview page 24 Chapter 6 Results and reports management 81 Viewing analysis results Introduction Viewing results This topic describes how to review the results of an analysis by inspecting the analysis statistics and histograms Two types of results views are available e Results per analyte e Results per sample To view results 1 Click one of the views in the Plex navigation panel The results data is displayed in two tables at the top and in charts and plots below For replicate samples the black lines that are shown on the bar chart depict a range bar It extends from the h
17. clustering page 59 Using manual clustering Introduction This topic describes how to use manual clustering You can use this function to resolve problems caused when the automatic clustering process fails to locate your bead clusters Automatic bead clusters can also be modified with this method 60 FCAP Array Software User s Guide The functions located in the Manual Clustering group can be used to navigate up or down the sample list and to perform actions on multiple samples at once s z y A a t a z y Ya a Clear Cluster Previous Next File Apply to All Manual Use Manual Clustering Use Manual Clustering Use Auto Clustering Clear All Assignment File Clustering Files for Failed Files for All Files for All Files Bead Clusters Manual Clustering T Applying manual To apply manual clustering to selected samples clustering to 1 Click Manual Clustering in the Plex navigation panel selected samples The list of samples opens on the left and two plots open on the right 2 Review the Clusters column in the sample list table where you can find out if manual clustering is needed for any of the samples The text turns red for any tube that fails auto clustering 3 Select a sample from the list Data according to the scatter parameter selected in the Instrument Settings view is shown in the histogram Data corresponding to the clustering parameters is shown in the dot plot In the dot plot you can check which cluster was not f
18. ignore standard points on the standard curve 1 Go to the detailed view 2 Clear the checkbox in the results table of the standard point you want to ignore The recalculation runs automatically and the results and the plots reload with the new data AS Human IL 4 R2 100 00 A 2 601 B 2 943 C 7 737 D 12 555 E 0 056 Fitting type 5 Parameter Logistic std010 1000 Std009 4141 78 stdoos 2206 73 Std007 1154 78 100 Std006 552 32 stdoos 268 96 re stdoo4 129 80 j 65 52 10 stdoo2 38 20 23 93 04 T T 10 100 1000 Concentration Sample Name Event MFI sD CV MFT Nominal CC Fitted CC Recovery W stdooz 285 23 93 9 84 44 09 19 53 pgjmb 17 89 pgjmL 91 61 W stdoos 319 38 20 13 90 36 60 39 06 paml 38 46 pgjmL 98 45 Stdoo4 305 65 52 21 13 30 32 78 13 pgfmL 75 40 pajml 96 51 W stdoos 314 129 80 44 30 35 92 156 25 pg ml 155 30 pgjmL 99 39 W Stdoo6 ati 268 96 72 95 30 11 312 50 pg mL 314 01 pgjml 100 48 W stdoo7 263 552 32 160 06 26 80 625 00 pg ml 618 98 pg mL 99 04 W stdoos 285 1 154 78 332 96 29 57 1 250 00 pg mL 1 263 68 pg mL 101 09 W stdoo9 346 2 206 73 566 92 25 93 2 500 00 pgjml 2 464 51 pgjml 98 58 W stdoio 299 4 141 78 1 120 63 26 10 5 000 00 pg mL 5 027 83 pg mL 100 56 Next step e Proceed to Overview of results and reports page 80 More information Plex creation workflow page 50 Results and reports management This
19. is greater than the user defined multiplier times the MFI of the negative standard Negative The MFI of the sample is less than or equal to the MFI of the negative standard Indeterminate The MFI of the sample is greater than the MFI of the negative standard but less than the user defined multiplier times the negative standard Error Calculation error Verify that you have the correct file s selected when performing this procedure Calculations are based on the selected negative standard To define sample based QC for an individual analyte 1 Select the Show Individual Analytes checkbox 2 Click a column header of an individual analyte 3 Click the Customize Settings icon in the Quality Control Settings group The Sample Based QC Definition dialog opens 4 Select your negative standard from the Negative MFI of Selected Sample menu 5 Specify the multiplier for your negative standard in the Positive MFI of Negative x field 72 FCAP Array Software User s Guide 6 Click Apply to Selected Next step Proceed to Defining controls page 72 More information Plex creation workflow page 50 e Selecting beads and analysis model page 51 Defining controls Introduction This topic describes how to add control samples to a plex for quantitative and qualitative analysis About defining You can specify the parameters of the control samples and then use controls them in a plex to verify th
20. is used to calculate the concentration values for each of the measured analytes in a sample Qualitative analysis Quality control analysis What s new in version 3 0 More information Chapter 3 Overview 23 Qualitative analysis determines whether a specific type of analyte exists in a sample based on known controls FCAP Array software can determine analyte existence for many analytes beads per sample Experiments can include positive controls negative controls or both FCAP Array software reads the FCS data files from an experiment locates clusters to which analytes have been assigned and then determines the MFI of the detector antibody for each analyte The control data is used to determine MFI cutoff values for each analyte Test sample results are based on the cutoff values For beads that have two reporter parameters the quality control analysis model is used FCAP Array software compares the MFI of a sample to a user defined range or a threshold determined from a control Values that are out of range are flagged in the results and reports Quality control analysis can be customized to calculate the MFI for a negative standard by multiplying that MFI by a user defined multiplier This calculated cut off value is used to determine whether a specific analyte or analytes exist in a sample FCAP Array version 3 0 has the following new features Support of FCS 3 0 files The ability to utilize CBA keywords from BD
21. of Intel Corporation BD BD Logo and all other trademarks are property of Becton Dickinson and Company 2011 BD Regulatory information For Research Use Only Not for use in diagnostic or therapeutic procedures History Revision Date Change made 23 11472 00 Rev 01 7 2011 New document Chapter 1 Introduction About the documentation 00 00 eee Technical assistance 0 cece eee eee ee Limitations nerens ta peri ead dda ee eee bees System requirements 0 eee eee eee eee Basi terms e e a e Ree th es WE A Rs Chapter 2 Installing and starting the software Installing FCAP Array software 2005 Starting the software 0 0 cece eee eee Obtaining the bead library file Uninstalling FCAP Array software 4 Chapter 3 Overview Software overview 1 0 cece eee ees Workflow overview 0 c ce eee ee ee ee eee Chapter 4 Creating a new experiment New experiment overview 2 008 Creating a new experiment 000 Adding plexes esset c eeni waits E KEE ade Setting the sample layout 00 000s Adding samples 0 0 c cece cece ee eens Modifying samples in the layout view Contents iv FCAP Array Software User s Guide Working with experiment information 00sec eee ee eee eee 43 Chapter 5 Working with plexes 47 Plex OVERVIEW occvec dh ccgae oe bute
22. plex reports and experiment reports You can view and print both types Report documents contain all the defined data for analysis and results The plex report contains all the details of the selected plex such as sample layout beads and model selection standard and quality control definition control definition instrument settings standard curves and results The experiment report contains all the plex reports merged into a single document To view a plex report 1 Click Report in the Plex navigation panel To view an experiment report 1 Click Report in the Experiment navigation panel The options for working with reports are shown on the ribbon The groups include e Print e Page Setup e Navigation e Zoom e Page background e Export You can modify the layout and the format of the report by clicking one of the icons To see a description of the icon hover the pointer over it to see a tooltip dialog 90 FCAP Array Software User s Guide Using report You can show or hide specific parts of a report content options To toggle report content options 1 Click the Options icon in the Page Setup group The Report Options dialog opens Report Options Plex Definition V Visual Plate Report V Plex Components Report Y Control Definition Report V Instrument Settings Report Y File Assignment Report tandards V Standard Curves Report Standard Curve Tables V Standard Definit
23. the group name and description 4 Click OK To delete a bead group 1 Select a bead group 2 Click the Delete Bead Group icon The Confirm bead group deletion dialog opens 3 Click OK To add bead to a group 1 Select one or more beads from the bead list 2 Drag and drop them onto the bead group To remove a bead from a group 1 Select one or more beads from the bead group 2 Click the Remove Bead from Group icon Importing a bead group Exporting a bead group More information Chapter 8 Data management 107 To import a bead group 1 Click the Import icon The Import dialog opens Select the XML file you want to import Click Open You can export either one single bead group or all groups including all beads from the library To export a bead group 1 Select the bead group 2 Click the Export icon and select an export option Export All Bead Groups Export Selected Bead Group Export All Beads The Export Bead Data dialog opens 3 Enter a file name 4 Click Save Selecting beads and analysis model page 51 108 FCAP Array Software User s Guide Using the plex template library Introduction This topic describes how to work with plex template files in the library All saved plex templates are stored in the library Plex template You can select a plex template to be used in an experiment The functions functions are located in the Plex Template Library group o
24. to vary slightly if comparing version 3 0 to a previous version e Technical assistance page 9 e System requirements page 11 System requirements Introduction Minimum PC configuration Software requirements Chapter Introduction 11 This topic specifies the system requirements for using FCAP Array software FCAP Array software requires a computer with the following minimum configuration Item Specification CPU Intel Pentium 4 2 GHz or equivalent RAM 512 MB or higher Video RAM 16 MB or higher Hard drive space Monitor resolution Monitor color 50 MB for installation XGA 1024 x 768 pixels or higher 1280 x 1024 recommended 16 bit color or higher DVD ROM drive Required for software installation USB port Required for security key FCAP Array software requires the following e Microsoft Windows XP or Windows 7 32 bit and 64 bit versions e Microsoft NET 3 5 framework e Microsoft SQL Server Compact Edition 3 5 The installer will install NET framework and SQL Server if they are not already on the computer 12 FCAP Array Software User s Guide More information e Technical assistance page 9 e Limitations page 10 Basic terms Introduction Terms This topic describes the basic terms used throughout the manual to indicate specific constructs procedures or concepts as they apply to FCAP Array software The basic terms use
25. workflow page 50 e Defining standards and QC page 64 74 FCAP Array Software User s Guide Viewing standard curves Introduction Viewing the standard curves This topic describes how to view the standard curves and how to modify the display to optimize the views for an experiment To view the standard curves 1 Click Standard Curves in the Plex navigation panel The window is separated into two panels curves on the left and the analytes table on the right The following figure shows a portion of a set of standard curves Bead Name A5 Human IL 4 A7 Human IL 6 Analyte 100 00 A 2 255 B 4 767 C 10 988 D 11 657 E 3 9 99 97 A 2 119 B 20 000 C 28 766 D 9 180 E 23 44 jal fal Fitting type 5 Parameter Logistic Fitting type 5 Parameter Logistic AS Human A7 Human m 10000 stdo09 t 10000 4 Stdo10 ptdo09 stdo10 4255 07 6367 78 AS Human 2206 73 i 4141 78 StdOO B6 Human 1000 00 1000 4 1000 Stdoos C4 Human Stdoos stdoos std008 205 35 1980 96 D7 Human 129 80 1154 78 SF 5td006 stdoo3f Sg stdoos Std003 469 76 100 q 38 20 268 96 100 43 71 stdoo4 stdoo4 66 12 palira 10 104 Stdoo2 23 93 25 48 Std001 Std001 aan po 8 20 14 14 1 5 SS L U o ER 0 10 100 1000 o 10 100 1000 Concentration Concentration 2 If needed zoom in on the standard curve and statistics for a specific analyte either by selecting
26. 2 Final CC Paramter Y NIR A bd 20 51 21 07 2l 18 19 18 16 24 15 12 9 6 3 0 2 hi D y y 5 5 5 5 x PS Y x Ts 4 To S 1000 10000 Column headers 84 FCAP Array Software User s Guide The statistics table has columns for each of the categories of data The following table describes the meaning for each column Column Description Position The location of the sample on the plate in the experiment design view Results file Assigned FCS data file Clustering Type of clustering used manual or auto Event The number of events in the gated cluster MFI The median fluorescence intensity SD The standard deviation of the reporter MFI for the bead cluster CV Coefficient of variation as a percentage of the reporter MFI of a cluster Nominal CC Nominal concentration of the standard or control samples Fitted CC Sample concentration calculated from the fitting curves Final CC Sample concentration calculated from the fitting curve with a dilution factor applied Recovery Applicable to standard sample only It represents the comparison of the nominal CC to the fitted or final CC Dilution Dilution factor QC Quality Control analysis result message Qualitative Qualitative analysis result message Message Shows error message for the sample in the
27. 2 Type in your old password and new password 3 Click OK Resetting a forgotten password Changing user preferences Viewing the user tracking log Chapter 10 Reference information 123 If you forget your password an administrator can reset your password To reset a user password 1 Log in as an administrator 2 Click the User Management icon 3 Double click a user in the user table The Modify User dialog opens 4 Enter a new password for the user 5 Click OK To change user preferences 1 Click the Preferences icon The User Preferences dialog opens 2 Change the Default Measurement Unit Default Fitting Accuracy or the default FACSuite Directory for your BD FACSuite FCS files 3 Click OK To view the user tracking log 1 Click the User Tracking Log icon The user tracking log shows the following events software startup software close user login user logout experiment open experiment close experiment create experiment save experiment delete experiment export and experiment import 124 FCAP Array Software User s Guide Using the Clustering Test Tool Use this tool to check for correct clustering without needing to go through the plex creation workflow To use the Clustering Test Tool 1 Click the Clustering Tool icon The Clustering Test Tool window opens Navigate to your FCS files in the File Explorer Select your scatter parameter number of peaks and clu
28. 6 FCAP Array Software User s Guide Troubleshooting Introduction This section describes tips to help you troubleshoot problems with FCAP Array software If you have problems with using FCAP Array you can find help included in the software The Help menu is located on the right side of the ribbon Calculation error The following table lists calculation error messages in the statistics messages table Message Description NaN Acronym for not a number Can be the result of zero divided by zero or no calculation done negative infinity Negative number divided by zero positive infinity Positive number divided by zero FCAP Array does not start Possible causes Recommended solutions Too many software Close unnecessary applications instances are running on the PC The security key is Connect the FCAP Array security key to an not connected available USB port on the computer Missing program Reinstall the software files User cannot log in User unable to open or edit data User manual does not open from Help menu Experiment cannot be edited New bead cannot be added to the bead library Chapter 9 Troubleshooting 117 Possible causes Recommended solutions User name is incorrect Check the caps lock key Re type your user name Forgot user name Contact your software administrator Password is incorrect Check the caps lock key Re type your password
29. 7 label 77 88 Polar 88 saving 77 88 type 87 88 clustering about 12 22 assigning 55 128 FCAP Array Software User s Guide automatic 53 59 manual 59 63 120 clustering tool 124 coefficient of variation CV 76 84 completing experiment 91 92 computer 11 concentration 64 quantitative analysis 22 specifying 64 65 72 unit 113 contact information 9 control data 23 definition 72 73 89 creating plex 48 49 50 reports 89 90 customizing charts 77 87 88 cutoff value 23 67 71 CV 76 84 cytometer flow 22 D data sheet 43 45 type 87 database 125 debris filtering 57 118 defining controls 72 73 89 standards 64 72 design window 30 dilution 37 42 65 dongle 11 16 116 E error messages 116 event number 84 experiment about 12 completing 91 92 data sheet 43 45 group 44 91 102 library 102 104 locked 45 91 117 new 29 31 owners 44 public 45 security 45 F FACSArray software 24 FACSDiva software 24 FACSuite See BD FACSuite FCAP Array software installing 16 starting 17 uninstalling 20 version 10 16 23 104 FCS file 22 25 53 96 119 File Explorer 35 39 filtering 57 85 101 118 final CC 72 84 fitted CC 84 fitting accuracy 76 119 123 type 75 flow cytometer 22 Flow Cytometry Standard FCS file 22 25 53 96 119 fluorescence 12 force zero 76 G gate 53 63 graphs See chart H help 116 117 histogram 53 58 60 81 installer 16 117 instrument settings
30. 98 FCAP Array Software User s Guide Evaluating the experiment Introduction About generating experiment results Verifying and adding information Solving problems More information This topic describes the process of evaluating the experiment that is created after opening the BD FACSuite FCS files FCAP Array automatically generates plexes samples plates and their layout based on the information contained in the keywords in the BD FACSuite FCS files FCAP Array creates a number of plexes to match those defined in the keywords If the automatic analysis runs successfully based on the information the experiment results are generated without defining any additional settings To verify or add information 1 Check the positions and types of samples in the experiment design view by clicking a well to see if it matches your corresponding BD FACSuite FCS file Sample Properties panel While evaluating the created experiment FCAP Array provides the option to select and add more samples files or plexes to it You can also modify the properties of the previously added files For further information see Working with plexes page 47 For cases where there is missing or an undefined plex name you must define additional information To learn more about how to solve this issue see Working with plexes page 47 or Troubleshooting page 115 e BD FACSuite workflow overview page 94 e Workflow overview page 24 Data ma
31. BA Flex Sets Thi Th2 Cyto Kit 550749 BD CBA Kit Thi Th2 Cytokine Kit II 551609 BD CBA Kit Th1 Th2 Th17 Cytokine Kit 5 BD CBA Kit BD CBAKE ine BD CBA Kt jin Isotyping Kit BD CBA Kt 52 FCAP Array Software User s Guide Selecting the analysis model 2 Select one or more beads from the Library list and do one of the following e Drag and drop them onto the Selected Beads window e Click Add to Plex in the Bead and Model Selection group on the ribbon e If you have a bead group defined in the bead library you can add all beads of the selected group in one step by adding the group to the plex Select the group by clicking its name then drag and drop onto the Selected Beads window There are three analysis models e Quantitative e Qualitative e Quality Each bead has one analyte and optionally a second reporter parameter The analysis model for an analyte is set to Quantitative by default To select the analysis model do one of the following e Select the analysis model from the menu in the Model column This changes the analysis model for the selected bead only Analyte Name Model v 2nd Reporter ERIE ee Quantitative Pe No Human Angiogenin Quantitative E No Human IL 8 Qualitative No Human IL 7 Qualty No Human IL 6 Quantitative No Human IL 4 Quantitative No e Select the analysis model from the menu in the Select Model For All on the ribbon Chapter 5 Working with p
32. FCAP Array Software Version 3 0 User s Guide For Research Use Only M bdbiosciences com 23 11472 00 Rev 01 7 2011 Becton Dickinson and Company BD Biosciences BD Biosciences European Customer Support San Jose CA 95131 Tel 32 2 400 98 95 Tel 877 232 8995 Fax 32 2 401 70 94 Fax 408 954 2347 help biosciences europe bd com ResearchApplications bd com Copyrights 2011 Becton Dickinson and Company All rights reserved No part of this publication may be reproduced transmitted transcribed stored in retrieval systems or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without prior written permission from BD Biosciences The information in this guide is subject to change without notice BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments Although this guide has been prepared with every precaution to ensure accuracy BD Biosciences assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information BD Biosciences welcomes customer input on corrections and suggestions for improvement Trademarks Microsoft Excel and Windows are registered trademarks of Microsoft Corporation FCAP Array is a trademark of Soft Flow Hungary Ltd Pentium is a registered trademark
33. FCAP Array keeps the original experiment unmodified and saves your copy with the completed changes as well To use save experiment as 1 Click the Save As icon in the Experiment group The Save As dialog opens 2 Enter a name for the experiment 3 Click Save 92 FCAP Array Software User s Guide Saving a plex as a template Closing an experiment More information You can reuse the saved template for your next experiment and streamline your workflow To save a plex as a template 1 Click the Save Plex as Template icon in the Experiment group The Save As dialog opens 2 Enter a name for the plex 3 Click Save To close an experiment 1 Click the Close icon in the Experiment group The experiment is closed automatically The program returns to the main window showing the experiment library e Overview of results and reports page 80 e Workflow overview page 24 Using the BD FACSuite workflow This chapter covers the following topics e BD FACSuite workflow overview page 94 e Creating a new experiment from BD FACSuite files page 96 e Evaluating the experiment page 98 94 FCAP Array Software User s Guide BD FACSuite workflow overview Introduction This topic describes the workflow for creating an experiment from BD FACSuite FCS files Workflow stages The following table shows the stages in a typical workflow when using FCS files from BD FACSuite software Stage Description
34. FI values of the negative standard You can define your standard as negative positive or both a negative and a positive standard by clicking the corresponding tab in the Sample Based QC Definition dialog The results for your analysis can be found in the QC Result column of the statistics table in the report view Use the following method for the analysis of the BD CBA Mouse Immunoglobulin Isotyping kit To define sample based QC Definition 1 Click the Customize Settings icon in the Quality Control Settings group The Sample Based QC Definition dialog opens 2 Select your negative standard from the Negative MFI of Selected Sample menu 3 Specify the multiplier for your negative standard in the Positive MFI of Negative x field We recommend a value of 3 for the multiplier 4 Click Apply to All Sample Based QC Definition Negative MFI of Selected Sample Positive MFI of Negative x 3 Apply to All Apply to Selected j Cancel Defining sample based QC for an individual analyte Chapter 5 Working with plexes 71 5 Click the Reporter 2 icon and repeat steps 1 to 4 Verify that you have the correct Reporter icon highlighted on the ribbon while performing this procedure Toggling between the Reporter icons will toggle between the two Quality Control Definition tables The software provides results for each analyte and reporter Result Definition Positive The MFI of the sample
35. Windows 7 folder C ProgramData Soft Flow FCAP Array v3 To restore the database from a saved database file 1 Click the Restore Database icon The Open dialog is displayed Navigate to the location of your database file Click Open To close FCAP Array software 1 Click the exit icon X in the top right corner of the application If you have an experiment open you will need to click the exit icon twice This page intentionally left blank Numerics 3D bar chart 88 4 parameter logistic 119 5 parameter logistic 75 119 96 well plate 30 A access rights 43 44 45 active plex 31 33 adding plex 31 32 algorithm 10 54 57 60 76 alignment direction plate 34 analysis models 51 52 qualitative 23 52 67 73 84 quality control 23 52 68 71 84 89 quantitative 22 52 64 72 results 81 88 statistics 81 86 analyte 12 22 65 71 74 82 antibody 12 22 assigning clusters 53 56 assistance technical 9 automatic clustering 53 59 B backup database 125 bar chart 88 range 81 Index BD CBA keyword 23 25 95 BD CBA Mouse Immunoglobulin Isotyping kit 70 BD CellQuest Pro software 24 BD FACSArray software 24 BD FACSDiva software 24 BD FACSuite software 18 workflow 24 25 94 98 bead about 12 51 55 104 assay 8 12 22 group 105 106 107 ID 55 117 library 51 104 107 beads and analysis models BMP 77 88 51 52 C CBA keyword 23 25 95 CellQuest Pro software 24 chart bar 88 data type 8
36. a new experiment You can use this workflow for analyzing data from multiple flow cytometry systems If you use BD FACSuite software you can use either this workflow or the BD FACSuite workflow which provides better optimization New experiment The following table shows the stages in a typical new experiment workflow workflow Stage Description 1 Creating a new experiment page 29 2 Adding plexes page 31 3 Setting the sample layout page 33 4 Adding samples page 35 5 Working with experiment information page 43 More information Workflow overview page 24 e Basic terms page 12 e Working with plexes page 47 e Modifying samples in the layout view page 40 Creating a new Introduction Chapter 4 Creating a new experiment 29 experiment This topic describes how to create a new experiment Procedure To create a new experiment 1 Click the lower half of the New Experiment button on the application toolbar and select one of the following Empty Experiment Select this option to create a new experiment From Existing Experiment Select this option to use a saved experiment From FACSuite Files Select this option only if you want to use the BD FACSuite workflow See Using the BD FACSuite workflow page 93 A3 Home Settings Open Experiment m i Import Delete iy A Export Experiment Empty Experiment E From Existing Experiment From FaCSuite File
37. are page 17 2 Creating a new experiment page 27 3 Working with plexes page 47 4 Results and reports management page 79 Chapter 3 Overview 25 BD FACSuite The BD FACSuite workflow is optimized for BD FACSuite workflow software but you can use the standard workflow as well When description you use BD FACSuite software to set up an experiment and run an acquisition you add CBA specific keywords which will be included in the exported FCS files FCAP Array software reads the FCS files and creates a new experiment based on the keywords The workflow is streamlined and you are able to view analysis results quickly BD FACSuite The BD FACSuite workflow includes these stages workflow stages Stage Description 1 Starting the software page 17 2 Creating a new experiment from BD FACSuite files page 96 3 Evaluating the experiment page 98 4 Results and reports management page 79 This page intentionally left blank Creating a new experiment This chapter covers the following topics e New experiment overview page 28 e Creating a new experiment page 29 e Adding plexes page 31 e Setting the sample layout page 33 e Adding samples page 35 e Modifying samples in the layout view page 40 e Working with experiment information page 43 28 FCAP Array Software User s Guide New experiment overview Introduction This topic describes the workflow for creating
38. arting the software page 17 Chapter 2 Installing and starting the software 17 More information System requirements page 11 e Starting the software page 17 e Uninstalling FCAP Array software page 20 Starting the software Introduction This topic describes how to start the software Procedure To start the software 1 Double click the FCAP Array V3 0 desktop icon As the software starts the FCAP Array Login dialog opens 2 Type your user name and password User names and passwords are case sensitive The password can be blank When FCAP Array software is installed there is a default user with the user name Administrator password welcome and administrative rights The Administrator password should be changed immediately and accounts created as needed 18 FCAP Array Software User s Guide 3 Click Login As the software starts the main window opens with the experiments library displayed Start Page FCAP Array V3 Creation Date Comment Last Save Date Last Save By Locked Properties EEN EEN 29 2011 4 29 2011 Administrator No Experiments 2 Administrator logged in Starting from BD FACSuite software Next step More information When FCAP Array and BD FACSuite software are installed on the same computer you can start FCAP Array from the menu bar in BD FACSuite software See the BD FACSVerse System Reference for setup instructions Proceed to Obtaining the bead libr
39. ary file page 19 e New experiment overview page 28 e BD FACSuite workflow overview page 94 Chapter 2 Installing and starting the software 19 Obtaining the bead library file Introduction Procedure More information This topic describes how to obtain the bead library file for FCAP Array software This file is used to populate the bead library with information about BD CBA reagents Note that the bead file for FCAP Array software version 3 0 has a different format than for version 1 0 To download and import the bead library file 1 2 Navigate to bdbiosciences com cbasetup Download the bead library file for FCAP Array software version 3 0 After the download is complete unzip the file Click the Bead Library icon Click the Import icon The Import dialog opens Select the downloaded XML file Click Open Working with libraries page 100 20 FCAP Array Software User s Guide Uninstalling FCAP Array software Introduction This topic describes how to uninstall FCAP Array software Procedure To uninstall FCAP Array software 1 Select Start gt Programs gt FCAP Array V3 gt Uninstall More information System requirements page 11 e Limitations page 10 e Installing FCAP Array software page 16 Overview This chapter covers the following topics e Software overview page 22 e Workflow overview page 24 22 FCAP Array Software User s Guide Software overview Int
40. ck the arrow on the Export icon to change it To export results 1 Click the Export icon in the Results group The Save As dialog opens 2 Select a destination to save the file 3 Click Save To print the results tables 1 Click the Print icon in the Results group 2 If necessary edit the printer and page setup in the Print Preview dialog 3 Click the Print icon Proceed to Customizing charts page 87 e Overview of results and reports page 80 e Workflow overview page 24 Chapter 6 Results and reports management 87 Customizing charts Introduction Displaying different sample types in the results per analyte view Displaying the different data types This topic describes how to customize the display of results data in the graphical charts You can select a row in the left table to display the data in the chart When the view is set to Results per Analyte the chart displays the data for each sample for a given analyte When the view is set to Results per Sample the chart displays the data for each analyte for a given sample To change the sample type view on the chart 1 Click the corresponding icons Standard Test or Control in the Results group The data type displayed in the chart is set to Final CC by default To change the data type 1 Select a data type from the Data Type menu in the Chart group The menu contains all the listed data types from the results table Data Ty
41. clustering 53 54 120 reporter 53 55 scatter 53 54 J JPEG 77 88 K keyword 23 25 95 L layout sample 33 35 library about 100 bead 105 107 experiment 102 104 plex template 108 110 units 113 114 user management 111 112 limitations 10 linear scaling 75 locked experiment 45 91 117 log user tracking 123 logarithmic 75 login 17 111 117 122 M manual clustering 59 63 120 measurement unit 64 67 Index 129 median fluorescence intensity MFI 22 23 68 84 messages error 116 MFI 22 23 68 84 Microsoft Windows 8 model selection 51 52 Mouse Immunoglobulin Isotyping kit 70 N negative standard 67 71 nominal CC 72 84 notes 45 P password 17 111 117 122 123 PDF 23 86 plate 96 well 30 plex about 13 24 48 49 adding 31 32 assigning bead to clusters 55 56 beads and analysis models 51 52 controls 72 73 defining standards 64 70 instrument settings 53 template 108 110 workflow 50 plots See chart PNG 77 88 polar chart 88 positive standard 67 71 public experiment 45 Q QC 23 52 68 72 84 89 qualitative analysis 23 52 67 73 quality control QC 23 52 68 71 84 89 130 FCAP Array Software User s Guide quantitative analysis 22 52 64 72 R R square 76 range bar 81 ReadMe file 8 recovery 77 84 replicate 13 35 36 74 reporter antibody 22 parameter 52 53 55 64 second 52 55 64 69 71 reports about 80 creating 89 90 restoring database 125 results a
42. d throughout this user guide are defined in the following table Term Definition bead e An analyte specific capture particle with distinctive discrete fluorescence characteristics All instances of an individual bead bind to the same analyte and have the same fluorescence characteristics e An FCAP Array software construct that specifies the analyte binding and fluorescence characteristics of a bead bead assay A method of using beads and reporter antibodies to detect or measure the concentration of analytes in a sample cluster A population of events in a bead assay data file Each cluster corresponds to a bead population Therefore the events in a cluster have distinctive discrete fluorescence characteristics experiment e A bead assay e An FCAP Array software construct that defines the plex es standards controls and test samples for a bead assay Chapter Introduction Term Definition plex e One or more beads used in a bead assay e An FCAP Array software construct that specifies a set of standards beads analysis models instrument settings and standard curves replicate s One or more copies of a sample When more than one replicate of a sample is used the replicate results are averaged to determine the result reported for the sample sample A solution containing analytes to be measured by a bead assay A sample can be a test a standard or a control
43. e accuracy of the measurement and the calculations Controls added to the plex are displayed in the quantitative or qualitative analysis table corresponding to the selected analyte model Defining controlsin To define controls in a quantitative analysis a quantitative 1 Click Control Definition in the Plex navigation panel analysis 2 Specify the concentration of your control samples by clicking in the Nominal CC field and entering the value The measurement unit matches the one defined in Standards and QC 3 If needed change the Acceptance Range value It is set to 20 by default Final CC 20 Enter a value between zero and one to change it Chapter 5 Working with plexes 73 Values that are out of range are indicated in the results and reports Range ol P Range Setting i Control Samples of Quantitative Analysis Name Nominal CC Range Control001 Controloa2 0 20 00 Defining controlsin To define controls in a qualitative analysis q qualitative 1 Click Control Definition in the Plex navigation panel analysis 2 Define the control samples as negative or positive by clicking the appropriate sign in the Negative Positive column Values that are out of range are indicated in the report and the results Control Samples of Qualitative Analysis I Name Negative Positive fone Controloo2 Next step Proceed to Viewing standard curves page 74 More information Plex creation
44. eading zeros in the CBA Standard ID and CBA Control ID keywords This ensures that your files can always be sorted in order 01 02 03 as opposed to 1 2 3 CBA Control ID e ID of the control contained in the tube or well The value can also be Pos or Neg e Controls will be arranged in alphabetical order by the value of the CBA Control ID keyword CBA Dilution e Used to specify the dilution of the sample The default dilution value is 1 00 e Note that the dilution factor can be entered as a keyword or it can also be entered into FCAP Array software More information Workflow overview page 24 96 FCAP Array Software User s Guide Creating a new experiment from BD FACSuite files Introduction Procedure This topic describes how to create a new experiment using FCS files created in BD FACSuite software Note that you cannot create a plex using the standard workflow and then try to use that plex in the BD FACSuite workflow The keywords in the BD FACSuite FCS files are used to generate the plex es for the BD FACSuite workflow To create a new experiment from FCS files created in BD FACSuite software 1 Ow Click the New Experiment button in the Home tab and select New Experiment gt From FACSuite Files A3 Home Settings Open Experiment L Import J Delete ie A Export Experiment fy Empty Experiment From Existing Experiment Name The New Expe
45. erties Deleting an experiment Importing an experiment Chapter 8 Data management 103 To view experiment properties 1 Select an experiment from the experiment table The properties table displays the details for the selected experiment To delete an experiment 1 Select an experiment from the experiment table 2 Click the Delete Experiment icon The Confirm experiment deletion dialog opens 3 Click Yes Experiments can be shared between different computers using the experiment import and export functions To import an experiment 1 Click the Import icon The Import Experiment dialog opens 2 Navigate to your experiment file and click Open 104 FCAP Array Software User s Guide Exporting an experiment More information To export an experiment 1 Select an experiment from the experiment table 2 Click the Export icon The Export Experiment dialog opens Name your experiment file and click Save Ow e Creating a new experiment page 29 Using the bead library Introduction Bead functions This topic describes how to work with beads and bead groups in the bead library Bead files from FCAP Array Version 1 0 cannot be used with Version 3 0 The converse is true also Bead files from version 3 0 cannot be used with version 1 0 The bead files for BD CBA reagents can be found at bdbiosciences com cbasetup All the available beads and bead groups are organized in the bead
46. gate in the left plot The drawn gate s applies to all samples and is saved with the plex The Apply Filter icon in the Debris Filtering group toggles the filter on and off in the plot on the right side To use debris filtering 1 Click Debris Filtering in the Plex navigation panel 58 FCAP Array Software User s Guide A list of samples and two plots are displayed Select a sample from the list The histograms are displayed according to the selected parameter of the selected sample file If needed change the selected parameters by selecting from the menus above the histograms You can use different parameters in the two plots On the left histogram hold down the mouse button and move the mouse left or right to draw your gate You can draw multiple gates The histogram on the right shows the filter results Optional Gates can be moved resized and deleted e Move a gate by selecting it and dragging it to another position e Resize a gate by grabbing one of the sides e Delete a gate by selecting it and clicking the Remove Selected Gate icon You can also press the Delete key Chapter 5 Working with plexes 59 6 The debris filter removes your assignment of beads to populations so you need to re assign them in the Instrument Settings or Manual Clustering views srk Number Next step Proceed to Assigning beads to clusters page 55 More information Plex creation workflow page 50 e Using manual
47. hapter Introduction 9 Technical assistance Introduction This topic describes how to obtain technical assistance If you require technical assistance contact your local BD Biosciences technical support representative or supplier Contact When contacting BD Biosciences have the following information information available e Version of FCAP Array software you are using e Name and version of the acquisition software you are using e Any error messages e Details of recent cytometer system performance For FCAP Array software support from within the US and Canada call 877 232 8995 Customers outside the US and Canada contact your local BD representative or distributor More information Troubleshooting page 116 10 FCAP Array Software User s Guide Limitations Introduction Limitations Compatibility with previous versions More information This topic describes the limitations for use of the software For Research Use Only Not for use in diagnostic or therapeutic procedures FCAP Array version 3 0 cannot use experiment plex or bead files from FCAP Array version 1 0 The security key for version 1 0 does not work with version 3 0 Therefore if you want to run both versions on the same computer you must insert both security keys in the computer The gating and curve fitting algorithms for previous versions of FCAP Array software are different The user can expect bead clusters and standard curves
48. hreshold percentage recovery threshold 1 pereentag Change the Recovery Threshold value in the Standard Curve Options group either by entering a new number or using the spin button and then pressing Enter Saving the chart When displaying a single curve the Save Chart icon is active You can adjust the image size to change the proportions of the saved chart The saved chart image will appear the same way it does on the screen To save the chart 1 Click the Save Chart icon to save the active chart to an image file 2 Select a file format from the menu The choices are JPEG PNG and BMP The Save As dialog opens 3 Designate a file location and name 4 Click Save Changing the chart To change the chart color color 1 Select a chart color from the menu on the Standard Curve Options group When saving a curve the image file corresponds to the selected color Changing the chart To change the chart labels option labels option 1 Click the Chart Labels icon on the Standard Curve Options group to toggle the display of standard point descriptors standard point name and respective MFI value on the chart 78 FCAP Array Software User s Guide Ignoring standard You can view the standard curves and ignore one or more standard points points in case they do not correctly fit the curves Clearing the checkbox of a standard point ignores the standard point from all curves not just the one being viewed To
49. ighest point to the lowest point 82 FCAP Array Software User s Guide Results per analyte statistics for the selected analyte in the right table Use the horizontal scroll bar in the right table to see all of the statistics columns This view shows the list of analytes in the left table and the In the following figure the plot shows results for the analyte selected from the left table Analyte Name Sample Name Position Clustering Results File Event MFI Nominal cc Fittedcc ES ase Hunan Ts sooi CE PE EC ooo palme 0 00 palmt A7 Human IL 6 stdoo2 1 Auto Std02_001 23 93 9 84 44 09 19 53 pg mL 18 19 pafmL AB Human IL 7 stdoo3 1 c1 Auto a oL ve 319 38 20 13 90 36 69 39 06 pg mL 38 04 pg mL B6 Human IL 8 Std004 1 D1 Auto Std04_001 302 66 12 21 48 30 42 78 13pgjmL 75 54 pgjml C4 Human Angiogenin stdoos 1 E1 Auto std05_001 314 129 80 44 30 35 92 156 25 pg mL 155 22 pg D7 Human LT alpha Stdo06 1 F1 Auto Std06_001 311 268 96 72 95 30 11 312 50 pg mL 315 38 pg Std007 1 G1 Auto Std07_001 254 547 37 154 95 26 83 625 00 pg mL 616 56 pg stdoos 1 H1 Auto Std08_001 285 1 154 78 332 96 29 57 1 250 00 p 1 265 73 stdoo9 1 A2 Auto std09_001 343 2 206 73 579 38 26 05 2 500 00p 2 461 82 Std010 1 82 Auto Std10_001 294 4 141 78 1 120 63 26 32 5 000 00 p 5 030 99 Bg i 7 Parameter X Red A gt A5 Human IL 4 Final CC Parameter Y NIR A 9 5 030
50. ing the fitting accuracy percentage Changing the force through zero option Hiding the blank standard FCAP Array calculates standard curves without weighting by default There are two weighting models available based on event numbers and on event numbers CV values To change weighting 1 Select a weighting model from the Weighting menu After you select one of the models the recalculation runs automatically and the plots refresh the data If the R square of the fitting algorithm result is lower than the value of the fitting accuracy you receive an error message regarding the related analyte Fitting accuracy is set to 98 by default To change the fitting accuracy percentage 1 Change the Fitting Accuracy value in the Standard Curve Options group either by entering a new number or using the spin button and then pressing Enter If you have a blank standard sample 0 pg mL in the plex you have an option to ignore the data in the fitting calculation The Force Zero option in the Standard Curve Options group is enabled by default To change the force through zero option 1 Click the Force Zero button on the Standard Curve Options group To hide the blank standard on the standard curves 1 Click the Hide Blank Standard icon The blank standard is hidden on all curves but the corresponding file is still present in the analysis Chapter 5 Working with plexes 77 Changing the To change the recovery t
51. ion clustering fails or incorrect number of clusters are found More information 120 FCAP Array Software User s Guide Possible causes Recommended solutions Data file from the wrong experiment was loaded Load a data file from the correct experiment Wrong scatter or clustering parameters specified Specify the correct parameters Incorrect instrument settings Correct the instrument settings so that clusters are in the expected locations Cluster overlap in data files Use manual clustering e Technical assistance page 9 10 Reference information This chapter covers the following topic e Using settings functions page 122 122 FCAP Array Software User s Guide Using settings functions Introduction Logging out Changing your password This topic describes the functions of the Settings tab in the main menu Click the Settings tab to display the settings functions p a of Logout Change Preferences User Trackinglog Backup Restore Clustering Password Database Database Tool Settings wj Maintenance Tools To log out from FCAP Array 1 Click the Logout icon The application closes and the Login window opens When using FCAP Array for the first time the initial password is created by an administrator After logging in you can change it at any time To change your password 1 Click the Change Password icon The Change Password dialog opens
52. ion Report Results Sample Statistics Report V Analyte Statistics Report Sample Quantitative Report Analyte Quantitative Report Sample Qualitative Report Analyte Qualitative Report OK lr Cancel 1 2 Select or clear the options as necessary Any options that are selected are included in the report You can turn options on or off at any time Note that the selected options will be applied to the current and future reports 3 Click OK Next step Proceed to Completing an experiment page 91 More information Overview of results and reports page 80 e Workflow overview page 24 Chapter 6 Results and reports management 91 Completing an experiment Introduction Saving an experiment Saving an experiment as This topic describes the process of completing an experiment You can finish your work during any phase of the analysis There are several options for completing an experiment These functions are available from any window or view of the experiment If you are working with a locked experiment saving is not active and modifications are also disabled To save an experiment 1 Click the Save icon in the Experiment group on the ribbon FCAP Array saves all changes to the experiment When working with a locked experiment use this option to save a copy of it The new copy of the locked experiment is now editable and your modifications can be saved Using the Save As function
53. lexes 53 This changes the analysis model for all beads Select Model For All Quantitative X Quantitative Qualitative Quality ction Next step Proceed to Selecting instrument settings page 53 More information Workflow overview page 24 e Using the bead library page 104 e Plex creation workflow page 50 Selecting instrument settings Introduction This topic describes how to apply instrument settings This process includes selecting a sample data file based on the defined plex and specifying the scatter clustering and reporter parameters Requirements To complete the instrument settings and cluster assignment process you must have a FCS file that was acquired for the experiment and assigned to a sample in your plex About instrument For common instruments parameter options scatter clustering settings reporter are loaded into the menus automatically based on the selected file Parameters can also be selected manually When you have selected the parameters and change the FCS file the scatter histogram and the clustering plot refresh according to the selected file 54 FCAP Array Software User s Guide Selecting To select instrument settings instrument settings 4 Click Instrument Settings in the Plex navigation panel 2 Select any of the assigned files from the Selected File menu ow The scatter peak is set to one by default You can change the value according to the number of differe
54. lowing e Click the Public icon in the Security group This enables editing of the experiment by all users e Click the Owners Only icon in the Security group This enables editing of the experiment only by the respective owner s and administrators e Click the Locked icon in the Security group This locks the experiment from any editing Click the Locked icon again to allow editing To add notes to an experiment 1 Click the Notes icon in the Experiment panel The Notes dialog opens 2 Type in your notes and click OK Your notes are shown when you select an experiment in the experiment library Proceed to Working with plexes page 47 e Workflow overview page 24 e New experiment overview page 28 e Modifying samples in the layout view page 40 This page intentionally left blank Working with plexes This chapter covers the following topics e Plex overview page 48 e Selecting beads and analysis model page 51 e Selecting instrument settings page 53 e Assigning beads to clusters page 55 e Using debris filtering page 57 e Using manual clustering page 59 e Defining standards and QC page 64 e Defining controls page 72 e Viewing standard curves page 74 48 FCAP Array Software User s Guide Plex overview Introduction This topic describes plexes plex templates and the stages in the workflow for creating a plex About plexes An FCAP Array experiment contains one or more plexe
55. lue in the Highest Concentration field Ow ma Chapter 5 Working with plexes 65 Enter a value in the Dilution Factor field Click Use Blank if you need a zero concentration point Click Apply If the icon is not available click the column header The program calculates the standard dilution series and fills in the concentration values You can also type in the values manually Reporter Parameter 1 Standard Samples of Quantitative Analysis Std001 Std002 Sstdoo3 Std004 Std005 Stdo06 Std007 stdoos Std009 Std010 L Show Individual Analytes Adding concentration settings for an individual analyte Standard Sample Concentration 0 00 pg mL 19 53 pg mL 39 06 pg mL 78 13 pg mL 156 25 pg mL 312 50 pg mL 625 00 pg mL 1 250 00 pg mL 2 500 00 pg mL 5 000 00 pg mL You can modify individual concentrations manually by editing or using the following procedure To add concentration settings for an individual analyte 1 2 3 4 5 6 Select the Show Individual Analytes checkbox Click in the column header for an individual analyte Enter a value in the Highest Concentration field Enter a value in the Dilution Factor field Click Use Blank if you need a zero concentration point Click Apply Only the selected analyte s concentration column is calculated 66 FCAP Array Software User s Guide 7 Repeat this process to define the concentrations for each analyte Reporter Paramete
56. mple type by clicking the icon shown in the selected well of the sample type you wish to assign To change the sample type the sample must have a file associated with it Removing asample To remove a sample do one of the following e Right click the sample you want to remove and select Remove Sample e Click the well then press the Delete key When removing multiple samples all selected wells become empty Clearing the file To clear the file association from a sample do one of the assignment following e Right click a well and select Remove file association e Click the Clear All File Associations icon in the Plate group e Right click a well and select Clear all file associations Viewing sample To view sample properties do one of the following properties e Click the Properties tab to view data about a selected sample e Click the File Header tab to see FCS file header keywords for a selected sample The File Header table is empty when no well is selected Sample Properties Properties ie Header E o Dilution 1 42 FCAP Array Software User s Guide Modifying the Samples are named automatically with a default setting when they sample name and are imported When using the standard workflow the prefix for dilution factor standards is Std for test samples it is Test for controls it is Control and for background it is Background You can modify the name of the sample and its dilution factor
57. mport the bead list from an XML file to avoid entry errors Incorrect file assignment Review the acquisition sample order Correct the file assignment and re analyze Too much debris in one or more samples Use Debris Filtering to define gated data that excludes debris Unable to fit standard curves Cannot analyze data file Chapter 9 Troubleshooting 119 Possible causes Recommended solutions One or more standard samples failed Remove bad data points in the Standard Curves window and re analyze Acquire another standard sample series Standards are fitted out of sequence Correct the file assignment in the Experiment Design view Choice of fitting curve does not model the standard sample data Choose another fitting curve 4 Parameter and 5 Parameter Logistic curves are recommended for most beads Fitting accuracy is too high Lower the fitting accuracy value in the Standard Curves window Sample preparation error Prepare and acquire a new standard sample set Experiment design does not match the actual layout Correct the experiment layout redo the file assignment and re analyze Possible causes Recommended solutions Corrupt or incorrect FCS file format Use FCS 2 0 or 3 0 list mode data files only Re export list mode data files from the instrument system in FCS 2 0 or 3 0 format During plex definit
58. n the ribbon JS amp E Import Export Delete New Experiment from Template Plex Template Library Creating anew To create a new experiment from a plex template experiment froma 41 Select a plex template from the plex template table plex template Drag a column header here to group by that column Name Creation Date Created By Number of Standard Samples 3 18 2011 Administrator 2 Click the New Experiment from Template icon The New Experiment From Template dialog opens 3 Enter an experiment name 4 Click OK Viewing plex template properties Renaming a plex template Importing a plex template Chapter 8 Data management 109 To view plex template properties 1 Select a plex from the plex template table The properties table displays the details for the selected plex Instrument Settings Fitting To rename a plex template 1 Click in the name field for a template 2 Edit the name 3 Press Enter To import a plex template 1 Click the Import icon The Open dialog opens 2 Navigate to your plex template file 3 Click Open 110 FCAP Array Software User s Guide Exporting a plex To export a plex template template 1 Click the Export icon The Save As dialog opens 2 Enter a name for the plex template 3 Click Save Deleting a plex To delete a plex template template 1 Select a plex template from the plex template table 2 Click the Delete ic
59. nagement This chapter covers the following topics e Working with libraries page 100 e Using the experiment library page 102 e Using the bead library page 104 e Using the plex template library page 108 e Using the user management library page 111 e Using the units library page 113 100 FCAP Array Software User s Guide Working with libraries Introduction About libraries Editing data Sorting data This topic describes how to work with the libraries The available data from different parts of the application are organized in libraries The default library in the main window is the experiment library Use the Libraries panel to view the different libraries Libraries Experiments Bead Library L Plex Templates amp User Management u Units In each library the data is displayed in tables and the corresponding management icons display on the ribbon Some of these functions are also available by right clicking a table To edit data 1 Click a cell in the library table 2 Edit the data 3 Press Enter To sort data 1 Click the column header 2 Click the arrow in the header to change the order of the data Chapter 8 Data management 101 Filtering data To filter data 1 Click the filter icon in the right corner of the column header to open the menu Name H inti Cs a Bam Blanks ngfm Non blanks pg mL fgjmL fgjmL nam pg ml Units mL ug
60. nalysis 81 86 S sample about 13 adding 35 39 layout 33 35 list tab 42 modifying 40 42 scaling linear 75 scatter parameter 53 peak 54 SD 84 second reporter 52 55 64 69 71 security key 11 16 116 rights 45 settings functions 122 125 software BD CellQuest Pro 24 software BD FACSArray 24 software BD FACSDiva 24 software BD FACSuite See BD FACSuite software FCAP Array See FCAP Array software solution 13 standard curve 22 74 78 89 118 119 defining 64 70 negative 67 71 positive 67 71 qualitative analysis 23 52 67 73 quality control 23 52 68 72 84 89 quantitative analysis 22 52 64 72 workflow 24 28 standard deviation SD 84 statistics 81 83 85 system requirements 11 T technical assistance 9 template 32 108 110 threshold 68 troubleshooting 115 U units 64 67 113 114 user management 111 112 tracking log 123 username 17 44 117 W weighting 76 workflow BD FACSuite 25 94 98 new experiment 28 overview 24 plex 50 standard 24 Index 131 xX XML 19 51 107 118
61. ng Parameters Red A NIR A fx 1000 E Reporter Parameter 1 Yellow A X Selected Beads Bead Analyte AS Mana AT Human IL 6 as Human IL 7 B6 Human IL 8 c4 Human Angiogenin D7 Human LT alpha 1000 10000 Removing bead To clear the bead cluster assignments do one of the following assignments Click the Clear Cluster Assignment icon on the ribbon to clear all bead cluster assignments e To remove individual bead cluster assignments right click the individual cluster and select Remove Bead Assignment Next step More information Chapter 5 Working with plexes 57 If the automatic clustering is satisfactory then go to Defining standards and QC page 64 If you wish to adjust your gates then go to one of the following e Using debris filtering page 57 e Using manual clustering page 59 e Plex creation workflow page 50 e Viewing standard curves page 74 Using debris filtering Introduction Procedure This topic describes how to use debris filtering You can use this function to filter out debris that could affect the automatic clustering algorithm and lead to incorrect clustering results b Ig Vt Remove Remove Apply Previous Next File Selected Gate All Gates Filter File Debris Filtering fy To filter out debris from a plot you draw a gate in the left plot Before Filtering plot around the area that you want to keep in the plot The right plot After Filtering plot displays the results of the
62. ng for All Files icon All parameters are restored to automatic clustering To apply manual clustering to selected files 1 Select one or more files from the sample list by clicking the corresponding checkboxes 2 Draw a clustering gate s adjust scatter parameter s or assign beads 3 Click the Apply to All Manual Clustering Files icon All your parameter changes are applied to all selected files Chapter 5 Working with plexes 63 Clearing the bead To clear the bead assignments assignments 1 Select a file from the sample list by clicking the corresponding checkbox 2 Click the Clear Cluster Assignment icon All the bead assignments for the selected file are removed The scatter parameter and clustering gates remain unchanged Note that if you select multiple checkboxes in the sample list only the sample in the highlighted row is affected when you click the Clear Cluster Assignment icon Clearing allbead To clear all bead assignments and all clustering gates assignments andall 4 Select a file from the sample list by clicking the corresponding clustering gates checkbox 2 Click the Clear All Bead Clusters icon All bead assignments and all clustering gates are removed The scatter parameter remains unchanged Note that if you select multiple checkboxes in the sample list only the sample in the highlighted row is affected when you click the Clear All Bead Clusters icon Next step Proceed to Defining
63. nt bead sizes used in your assay The applied algorithm searches for the number of scatter peaks you defined All BD CBA assays use one scatter peak If the algorithm fails to find the defined number of peaks FCAP Array notifies you about the problem The clustering parameters are used to locate the beads and the recommended clustering parameters can be found in the reagent manual The clustering algorithm finds the defined clusters In case of failure FCAP Array notifies you about the problem Selected File ents and Settings adetranm De Instrument Data FaCSArray 1 Scatter Parameter SSC 4 v Scatter Peaks 1 bg Clustering Parameters Red A NIR A e Reporter Parameter 1 Yellow 4 m Select a parameter from the Scatter Parameter menu The scatter diagram shows the data Select a number from the Scatter Peaks menu Select the first and optionally the second clustering parameter from the Clustering Parameters menus according to the fluorescence of the beads For kits that use only one clustering parameter you can clear the second clustering parameter selection by clicking the red X icon next to the menu Chapter 5 Working with plexes 55 6 Select a parameter from the Reporter Parameter 1 menu for the bead analyte 7 If your plex contains a second reporter parameter select a parameter from the Reporter Parameter 2 menu You can clear the second reporter parameter selection by clicking the red X icon ne
64. ogged in Next step Proceed to Adding plexes page 31 Chapter 4 Creating a new experiment 31 More information Workflow overview page 24 e New experiment overview page 28 Adding plexes Introduction This topic describes how to add plexes About new and An experiment always contains one plex as the default setting but saved plexes you can have multiple plexes in an experiment By default a plex named New Plex is added to a new experiment Saving a plex as a template streamlines your workflow by creating a flexible template which does not have restrictions on required content Using a saved template reduces manual data entry in subsequent experiments See Plex overview page 48 The plex functions are accessed from the groups of buttons on the ribbon as shown in the following figure AY Home Settings gt Y Number of Replicates 1 A 6 Ey io A T WG OG se 4 Frename S Number of Samples 1 Active Plex Save SayeAs SavePlex Close Standard Test trol Background Undefined DEA Add Plate oat All File Na All Horizontal Print Add New Delete Add New from on as Template Factor 1 0 New Plex Gi Associations aa lignment Alignment Template Experiment A New Sample a Plex a Adding a new plex To add a new plex 1 Click Add New in the Plex group The Add New Plex dialog opens 2 Enter a name for the plex and click OK The new plex will be the active one in the experiment 32 FCAP Array Soft
65. on The Confirm template deletion dialog opens 3 Click Yes More information Plex overview page 48 Chapter 8 Data management 111 Using the user management library Introduction User management functions Adding a new user This topic describes how to manage users and user privileges Only a user with administrator rights can modify the users list and the rights of users If the administrator name and password are successfully entered in the Login dialog the user management function is available The functions are located in the User Management group in the ribbon se amp New User Modify Delete User User User Management fy Note that a user with administrator rights can view and edit all experiments To add a new user 1 Click the New User icon The New User dialog opens 2 Enter details into the fields Fields include User Name Full Name Department E mail Contact Info Password and Administrator checkbox Only the User Name and Full Name are mandatory fields 3 Click OK 112 FCAP Array Software User s Guide Modifying a user Deleting a user To modify a user 1 Select a user from the user table User Name Full Name E Mail user C E Administrator Click the Modify User icon The Modify User dialog opens You can also double click the user from the user table to open the Modify User dialog Edit user details in the fields Fields include User Name Full
66. ound by the algorithm 4 Change the auto cluster to manual cluster by selecting the Use Manual Clustering checkbox in the row of the selected sample 5 On the histogram hold down the mouse button and move the mouse left or right to draw your gate You can draw multiple gates 6 Optional Gates can be moved resized and deleted e Move agate by selecting it and dragging it to another position e Resize a gate by grabbing one of the sides e Delete a gate by selecting it and pressing the Delete key Clearing manual clustering from selected samples Use Manual File Name Std02_001 P std03_001 P Std04_001 P Std05_001 P Std06_001 P Std07_001 P Std08_001 P Std09_001 P Std10_001 P Group A Spik Group 4 Spik Group A Spik Group A Spik Sample Name Std002 Std003 Std004 Std005 Std006 Std007 stdoos stdoo9 Std010 Test001 Test002 Test003 Test004 Chapter 5 Working with plexes 61 Use the mouse to draw a new gate around the cluster in the dot plot Do one of the following e Hold the mouse button while drawing your freehand gate e Sequentially click to draw the sides of a polygon gate Optional To resize a dot plot gate hold Ctrl drag one of its sides After defining all the clusters assign beads to them a Select the bead you want to assign from the Selected Beads table b Double click the cluster that you want to assign the bead to
67. pe MFI MFI Event Count SD alyte Name c nen Nominal CC Fitted CC Human IL Final CC 88 FCAP Array Software User s Guide Changing the chart type Changing the chart labels option Saving a chart Next step More information To change the chart type 1 Click the Chart Type icon in the Chart group 2 Select one of the following Bar Chart 3D Bar Chart or Polar Chart When you select 3D Bar Chart after clicking the chart you can rotate it with your mouse and zoom in or out with your mouse wheel To change the chart labels option 1 Click the Chart Labels icon on the Chart group to toggle the display of descriptors on the chart To save a chart 1 Click the Save Chart icon in the Chart group The Save As dialog opens You can adjust the image size to change the proportions of the saved chart The saved chart image will appear the same way it does on the screen The format is set to JPEG by default BMP and PNG formats are available as well 2 Select a destination to save the file 3 Click Save Proceed to Viewing reports page 89 e Overview of results and reports page 80 e Workflow overview page 24 Chapter 6 Results and reports management 89 Viewing reports Introduction Viewing plex reports Viewing experiment reports Report layout and format This topic describes how to work with reports Two types of report documents are available in FCAP Array
68. plate library 20 0 ccc ccc eee eee eee 108 Using the user management library 00 ccc cece cee eee 111 Using the units libeaty ixj cscs satay ed eee ey ness Pe ee REY ale a blbees 113 Chapter 9 Troubleshooting Troubleshooting 00s cece eee eens Chapter 10 Reference information Using settings functions 000 cece ee eee Index Contents v Introduction This chapter includes the following topics e About the documentation page 8 e Technical assistance page 9 e Limitations page 10 e System requirements page 11 e Basic terms page 12 8 FCAP Array Software User s Guide About the documentation Introduction Documentation contents ReadMe file More information This topic describes the contents of the guide The FCAP Array Software User s Guide describes how to install and operate Flow Cytometric Analysis Program FCAP Array software The software facilitates data analysis for multiplex bead assays This guide assumes you have a working knowledge of the basic Microsoft Windows operating system If you are not familiar with the operating system of your computer see the documentation provided with your system Before using FCAP Array software review the ReadMe file by clicking the link in the Start menu The ReadMe file contains important information not covered in this user s guide e Technical assistance page 9 e Limitations page 10 C
69. r 1 Standard Samples of Quantitative Analysis Sample Name D7 Human LT alpha C4 Human Angiogenin B6 Human IL 8 A8 Human IL 7 A7 Human IL 6 45 Human IL 4 Stdo01 0 00 pg mL D OO pg mL 0 00 pg mL 0 00 pg mL 0 00pg mL 0 00 pg mL Std002 19 53 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Std003 39 06 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Std004 78 13 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Std005 156 25 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Std006 312 50 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Std007 625 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Std008 1 250 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Std009 2 500 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Std010 5 000 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL 0 00 pg mL Specifying the measurement unit About qualitative analysis Specifying positive and negative standards Chapter 5 Working with plexes 67 When defining concentrations the active measurement unit will be valid for your analysis To change the measurement unit 1 Select a Measurement Unit in the Concentration Settings group 2 Click the Concentration column header to highlight it 3 Click Apply The selected measurement unit is displayed in the results and report documents Note that using different meas
70. riment from FACSuite files dialog opens Navigate to the directory containing the appropriate FCS files The FACSuite Experiment folder defined in Preferences is selected as the default in the FACSuite Experiment Selection browser The list of experiments in the right panel is the list of all unique PROJ keyword values extracted from all FCS files contained in the folder The new experiment will be populated with all the FCS files that share the selected PROJ value Select the experiment name in the Experiments field to use the same name for the FCAP Array experiment Chapter 7 Using the BD FACSuite workflow 97 A custom name can also be entered into the Experiment Name field You can also enter a comment New Experiment from FACSuite files Experiment Properties Experiment Name CBA Standard Assay UD Comment FaCSuite Folder Selection H P 23 11781 00_FACSClearCount_ifu PS Experiments PS 23 12943 00_FACSuite_CBA_im CBA Standard Assay UD S F build 1047 GES build 1081 HES CQC bead lot and FCS file O PS FCAPv3_beta5_Naen_Setup CAER FCS files for testing 425 Framemaker Fonts X Layout KRA if S96 cee OK J Cancel 4 4 Click OK The new experiment opens with the layout as defined by the keywords in the BD FACSuite FCS files Next step Proceed to Evaluating the experiment page 98 More information BD FACSuite workflow overview page 94 e Workflow overview page 24
71. rking with plates Adding a plate This topic describes how to set up the sample layout in an experiment Note that when samples have been acquired manually in tubes they still need to be arranged on a plate for use in the software An experiment always contains one plate as the default setting but you can have multiple plates in an experiment The controls for working with plates are located in the Plate group on the ribbon You can also right click in a plate to open a menu to perform some of the functions g ra Add Plate Clear All File Clear All Y Associations Plates A Plate iP To add a plate 1 Click Add Plate in the Plate group The new plate is added below the current plate If you cannot see the plate scroll down and make sure the new plate was added 34 FCAP Array Software User s Guide Changing the plate alignment Removing a plate Clearing a plate Clearing all plates Next step The alignment direction for the samples on a plate is set to horizontal by default When selecting a direction make sure it matches your acquisition order The selected direction determines the order when adding multiple samples or assigning multiple files in one motion File assignment should match the order and the direction of added samples The selected direction also influences the order of samples in the following screens debris filtering manual clustering standards and QC results per analyte re
72. roduction Overview Quantitative analysis This topic gives an overview of FCAP Array software version 3 0 and describes the quantitative qualitative and quality control analysis methods FCAP Array software facilitates data analysis of bead assays These assays can detect the presence of or determine concentrations for multiple analytes for example proteins and peptides in a sample Samples are acquired with a flow cytometer and acquisition software capable of saving data in flow cytometry standard FCS 2 0 or 3 0 file format FCAP Array software analyzes the FCS files to find bead populations by clustering Reporter antibody fluorescence within a population measures analyte specific binding Quantitative analysis determines analyte concentrations based on the known concentration values of a set of standards FCAP Array software can determine analyte concentration for many analytes beads per sample The number of concentration levels used to calibrate the standard curve and the number of sample and standard replicates are easily adjusted FCAP Array software reads the FCS data files locates clusters to which analytes have been assigned and then determines the MFI of the detector antibody for each analyte From the MFI of the standards the software calculates a curve that describes the relationship between MFI and concentration The curve used to fit the data is selected from several mathematical models A standard curve
73. rom the list of experiment owners to change access rights to the experiment You can also change the security rights for the experiment Entering To enter information in the experiment data sheet information 1 Click the Data Sheet icon in the Experiment panel 2 Click in the fields next to each field type and enter information about the experiment Comment Responsible Institute Operator Conditions Plate Direction Instrument Type FACSArray Instrument Serial Number Li 44 FCAP Array Software User s Guide Changing users in an experiment You can add or remove users from an experiment in the Experiment Owners table Users added to the Experiment Owners list can open read and modify the shared experiment To add a user 1 Select a user name from the All Users list 2 Click Add Selected User in the Security group on the ribbon The selected user is added to the Experiment Owners list To remove a user 1 Select the name from the Experiment Owners list 2 Click the Remove Selected User icon in the Security group on the ribbon The selected user is removed from the Experiment Owners list Changing user security rights in an experiment Adding notes to an experiment Next step More information Chapter 4 Creating a new experiment 45 Note that a user with administrator rights can view and edit all experiments To change user security rights in an experiment select one of the fol
74. s e If you have plex template files in the plex template library you can create a new experiment from that location Select a template then click the New Experiment from Template icon Enter a name and an optional comment in the New Experiment dialog Select a size for the sample layout 30 FCAP Array Software User s Guide The default is a 96 well plate Also use this layout if samples are acquired manually in tubes New Experiment Experiment Name 4 Click OK The design view opens C test FCAP Array V3 aes as Template e e Ry x Qe ve Number of Samples 1 Active Plex Save SaveAs SavePlex Close Undefined MO numberof Repicates 1 le amp i Ck 7 we E Adapa Gew Alnis Ge AI Horizontal Print AddNNew 6 Delete Add New from Dilution Factor 1 0 New Plex 5 Alignment Template New Sample 5 zji Expermert Design 2 Data Sheet Notes i Report NewPlee Beads and Model Instrument Settings Debris Filtering Manual Clustering Standards and Qc Control Definition Standard Curves Results per Analyte Results per Sample Report Layout 1 7 GH Techsmith HE ThinPrint Client GHB Uninstall Information E WebEx F4 E Windows Media Connect 2 Je wiodows Macia pay Fle types fcs md hae oas D m Administrator l
75. s A plex is created or modified within an experiment Plexes can be saved to the plex templates library to be reused or exported to other users Plex navigation Creating a plex is typically done in the order shown in the Plex panel navigation panel as shown in the following figure New Plex Beads and Model Instrument Settings Debris Filtering Manual Clustering Standards and QC Control Definition Standard Curves Results per Analyte oo n oOo Oo SF WS ND Results per Sample o Report Chapter 5 Working with plexes 49 Plex template The plex template stores the following information Parameter Content Standard Samples Number name concentration number of replicates Beads and Model Selected beads lot numbers analysis models Instrument Settings Scatter and clustering parameters cluster positions bead cluster assignment Debris Filtering Defined debris filtering gates status of the filtering active or not Standards and QC Uniformity concentrations measurement units quantitative model positive negative standard selection qualitative model MFI threshold values QC model Standard Curves Axis types linear logarithmic force through zero weighting type fitting type fitting accuracy The plex template is flexible There are no restrictions regarding the information required to save a plex as a template 50 FCAP Array Software User s Guide
76. scending by clicking the column header 4 You can select multiple files by Ctrl clicking the files You can also select by Shift clicking the first file while clicking the last file Next step Proceed to Working with experiment information page 43 40 FCAP Array Software User s Guide More information e Modifying samples in the layout view page 40 e Setting the sample layout page 33 e Workflow overview page 24 e New experiment overview page 28 Modifying samples in the layout view Introduction Selecting samples Moving samples Changing the sample type This topic describes how to modify and move samples once they have been added to a plate Note that the well counters for the numbering of the sample name do not reset unless you delete all samples of a given type To select a sample 1 Click on its well You can select multiple samples by Ctrl clicking the desired wells or by drawing a rectangle around the desired wells The background color of the wells indicates the selection When removing samples all selected wells become empty To move samples 1 Select one or more samples then drag the samples to the new position Samples cannot be moved to a position on a different plate To change the sample type 1 Right click the sample you want to change and select Change Sample Type The changed sample is now undefined Chapter 4 Creating a new experiment 41 2 Redefine the sa
77. shold values for all analytes 1 Click a column header 2 Type values into the Negative Standard s MFI and Positive Standard s MFI fields 3 Click Apply The columns are filled with the minimum and maximum values for all samples Specifying MFI values for an individual analyte Chapter 5 Working with plexes 69 If you have a second reporter parameter click the Reporter 2 icon and repeat steps 1 to 3 Quality Control Definition Reporter Parameter 1 Sample Name Std001 Std002 Std003 Std004 Std005 Std006 Std007 Std008 Std009 Std010 Show Individual Analytes 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 Neg MFI Value Pos MFI Value 100 00 100 00 100 00 100 00 100 00 100 00 100 00 100 00 100 00 To specify quality control MFI threshold values for individual Select the Show Individual Analytes checkbox Click the column header for an individual analyte Type values into the Negative Standard s MFI and Positive Repeat this process to specify the MFI threshold values for analytes 1 2 3 Standard s MFI fields 4 Click Apply 5 each analyte 6 If you have a second reporter parameter click the Reporter 2 icon and repeat steps 1 to 5 70 FCAP Array Software User s Guide About sample based QC Defining sample based QC for all samples Using the sample based QC definition enables the minimum MFI values to be derived from the M
78. standards and QC page 64 More information Plex creation workflow page 50 e Using debris filtering page 57 64 FCAP Array Software User s Guide Defining standards and QC Introduction Viewing standards About quantitative analysis Specifying the standard concentration Reporter This topic describes how to define standards for quantitative qualitative and quality analysis It also describes how to use the sample based QC method for the BD CBA Mouse Immunoglobulin Isotyping kit To view standards 1 Click Standards and QC in the Plex navigation panel The content appears corresponding to the selected beads and their analytes analysis models When your plex contains a second reporter parameter the window is divided into two panels When defining standards for quantitative analysis you specify these items e Concentration values e Measurement unit You can use uniform concentrations for all analytes default or specify different concentrations for each analyte To specify the standard concentration 1 Click Ascending or Descending in the Concentration Settings group to define the order for standard concentrations The typical choice here is ascending since most reagent instructions recommend running from lowest to highest concentration Highest Concentration 5000 Dilution Factor 2 Measurement Unit pg mL Descending Use Blank Concentration Settings 2 2 Enter a va
79. ster parameters The autoclustering is shown in the plots Clustering Test Tool File C Documents and Settings adetranm Desktop FCS files for testing FCS files For testing Instrument FACSAria 1 Scatter Parameter S5C A Number of Scatter Peaks 1 Clustering Paramters APC A APC Cy7 A 4cluster s found File Explorer FS 23 11472 00_FCAP_SW_ug G E 23 11781 00_FACSClearCount_ifu FS 23 12943 00_FACSuite_CBA_im build 1047 GF build 1081 S F CQC bead lot and FCS file F FCAPv3_beta5_Ngen_Setup 2 FCS files For testing FCS files for testing Clustering amp Debris 25 FCS files From Soft Flow E FCS files with FACSuite keywords O PS Framemaker Fonts Q pdf 1000 File types Fs Imd File Name Date 9 plex fes 8 11 2010 1000 10000 Backing up the database Restoring the database Closing FCAP Array Chapter 10 Reference information 125 To back up the database 1 Click the Backup Database icon The Save As dialog opens Designate a path and filename for your backup file Click Save The backup database function does not save your settings The settings include last used instrument settings parameters report header logo images and FCS data files To back up your database and settings go to one of the following folders in Windows and manually make a copy of the FCAP sdf file Windows XP folder C Documents and Settings All Users Application Data Soft Flow FCAP Array v3
80. sults per sample and report To change the plate alignment 1 Click the Vertical Alignment or Horizontal Alignment icons in the Plate group To remove a plate 1 Right click a plate and select Remove Plate The selected plate and all samples within the selected plate are removed from the experiment To clear a plate 1 Right click a plate and select Clear Plate All samples are removed from the selected plate in the experiment To clear all plates 1 Right click a plate and select Clear All Plates All samples are removed from all plates in the experiment Proceed to Adding samples page 35 More information Chapter 4 Creating a new experiment 35 e Workflow overview page 24 e New experiment overview page 28 e Adding plexes page 31 Adding samples Introduction About sample types Methods for adding samples This topic describes how to add samples to a plate There are five different sample types Standard Test Control Background and Undefined The control and background sample types are not used in BD CBA assays Note that a sample type is always selected and that clicking the sample layout creates the highlighted sample type Each type has a different color and icon in the ribbon OOA ss Test Control Background Undefined There are three methods for adding samples e Adding samples by designing the layout With this method you add samples first and then assign FCS files
81. to the selected samples Use this method when adding samples with replicates e Adding files with sample types With this method you select the sample type and the corresponding FCS files from the File Explorer window and add them to the plate When the files are in the correct order in the File Explorer the assignment of files to the plate can be done in one step e Adding undefined samples With this method you add all files to the experiment and then define the sample types one by one 36 FCAP Array Software User s Guide Adding samples by designing the layout Standards are arranged for the standard curve in alphabetical order by sample name Std001 Std002 Std003 etc Confirm that names are assigned in such a way that standards will be plotted in the correct order Wells containing assigned sample files have a floppy disc icon To add samples by designing the layout 1 Select one of the four sample types Undefined is not applicable with this method from the New Sample group by clicking the icon 2 Define the number of samples number of replicates and the dilution factor Number of Replicates 1 X Number of Samples 1 X Dilution Factor 1 0 3 Select the first well you want to start with and click it According to your settings the sample type icon and the number of replicates appear on the plate number of replicates multiplied by number of samples If the number of replicates exceeds the available
82. urement units gives slightly different results for your standard curves We recommend not using units that will lead to a concentration of lt 1 When using qualitative analysis you can select positive and negative standards You can use positive and negative standards for all analytes default or specify standards for each analyte All analytes should have a positive or negative standard To specify positive and negative standards 1 Select negative standards in the negative row of the qualitative table by selecting a standard sample name from the Standard Sample menu Standard Samples of Qualitative Analysis Qualitative Type Standard Sample Negative Positive Std002 Stdo01 Stdo02 Std003 Std004 stdo0s Stdo06 Std007 2 Use the same method for positive standards 68 FCAP Array Software User s Guide About quality control analysis Specifying MFI values for quality control With quality control analysis you have the option to check if MFI values are within the specified range Values that are out of range are indicated in the results and reports The quality control range is uniform for all analytes by default In addition to using the following procedures you can manually edit each value am Negative Standard s MFI 0 I 4 1 I ter Reporter Positive Standard s MFI 100 Customize ie Settings Reporter a Quality Control Settings fy To specify quality control MFI thre
83. ware User s Guide Adding a new plex from a template Changing the active plex Deleting the active plex Saving the active plex as a template Next step To add a new plex from a template 1 09 Click Add New from Template in the Plex group The Add New Plex from Template dialog opens From the Plex Template menu select a template from the library In the New Plex Name field enter a name then click OK The new plex will be the active one in the experiment To change the active plex 1 Click a plex from the Active Plex list The selected plex becomes the active one in the experiment New samples are added to the active plex Samples belonging to other plexes appear in a pale shade Options for the active plex are shown in the navigation panel on the left To delete the active plex 1 Click Delete in the Plex group The Confirm plex deletion dialog opens Click Yes to confirm To save the active plex as a template 1 Click Save Plex as Template in the Experiment group The Save Plex Template dialog opens Enter a name for the plex template in the Plex Template Name field and click OK Proceed to Setting the sample layout page 33 More information Chapter 4 Creating a new experiment 33 e Workflow overview page 24 e New experiment overview page 28 e Creating a new experiment page 29 e Working with plexes page 47 Setting the sample layout Introduction Wo
84. wells then a new plate is automatically added to the experiment 4 Select FCS files from the File Assignment panel and drag and drop them onto the plate For more detailed instructions on selecting FCS files see Selecting FCS files page 39 Adding files with sample types Chapter 4 Creating a new experiment 37 To add files with sample types 1 2 Click one of the four sample type icons except Undefined If test samples were diluted define the Dilution Factor It is not necessary to define dilution factors for standards Select FCS files from the File Assignment panel and drag and drop them onto the plate When your files are in the correct order in the File Explorer you can assign your files in one motion For more detailed instructions on selecting FCS files see Selecting FCS files page 39 The following figure shows a typical plate layout at this stage Layout 1 eeeeeeeeee 38 FCAP Array Software User s Guide Adding undefined samples To add undefined samples 1 ow Click the Undefined sample type icon in the New Sample group Define the Dilution Factor if necessary Select FCS files from the File Assignment panel and drag and drop them onto the plate For more detailed instructions on selecting FCS files see Selecting FCS files page 39 Define the sample types a Select one of the four colored circles in the well Ja HP Chapter 4 Creating a new experiment 39
85. xt to the menu Next step Proceed to Assigning beads to clusters page 55 More information Plex creation workflow page 50 e Defining controls page 72 Assigning beads to clusters Introduction This topic describes how to assign the plex beads to clusters About assigning You have to assign plex beads to the corresponding clusters in the beads to clusters clustering plot To simplify assignment first click the Bead column header in the Select Beads table to sort by bead ID Note that BD CBA kits are clustered left to right in one dimension Bead 1 on the left most position is the dimmest bead You can assign only one bead to one cluster at a time Assigning beads to To assign beads to clusters clusters 1 Select the bead you want to assign from the Selected Beads table Sort by bead location and assign from the top left cluster Assign beads going left to right and top to bottom 56 FCAP Array Software User s Guide 2 Select the cluster by doing one of the following e Double click the cluster that you want to assign the bead to The label of the analyte is displayed on the cluster e Drag and drop the selected bead onto a cluster 3 Repeat this process for all of the clusters The following figure shows the clustering plot after beads have been assigned Selected File C Documents and Settings adetranm Deskta Instrument Data FACSArray 1 Scatter Parameter S5C A Scatter Peaks 1 Clusteri

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