Software® Upgrades and Conversions Manual

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Bio-Plex Manager

™

Software 6.1

User Guide

Summary of content (260 pages)

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Bio-Plex Manager™ Software 6.
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BioPlex_6.book Page i Friday, September 23, 2011 11:55 AM Bio-Plex Manager™ Software 6.1 User Guide Bio-Rad Laboratories, Inc.
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BioPlex_6.book Page ii Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide BIO-RAD TECHNICAL SUPPORT DEPARTMENT The Bio-Rad Technical Support Department in the U.S. is open Monday through Friday, 5:00 a.m. to 5:00 p.m., Pacific Standard Time. Worldwide technical support is available on the Web at www.consult.bio-rad.com. Phone: 1-800-424-6723, option 2 Fax: 1-510-741-5802 Email: LSG.TechServ.US@Bio-Rad.com (U.S.) LSG.TechServ.Intl@Bio-Rad.com (International) Web: www.
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BioPlex_6.book Page iii Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide Table of Contents Chapter 1. Bio-Plex Suspension Array System Overview . . . . . 1 Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . For More Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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BioPlex_6.book Page iv Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Table of Contents Disconnecting with Sleep Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Menu and Toolbars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quick Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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BioPlex_6.book Page v Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide Chapter 6. Preparing Protocols . . . . . . . . . . . . . . . . . . . . . . . . . 53 Protocol Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Creating/Opening Protocol Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saving Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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BioPlex_6.book Page vi Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Table of Contents Bead Map Display Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raw Data Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bead Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sampling Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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BioPlex_6.book Page vii Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide Reducing the Size of a Results File . . . . . . . . . . . . . . . . . . . . . . . . . . Results Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Custom Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viewing/Changing the Protocol Settings . . . . . . . . . . . . . . . . . . . . . .
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BioPlex_6.book Page viii Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Table of Contents Deleting a Graph. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exporting Graphs to Other Applications . . . . . . . . . . . . . . . . . . . . . . Adjusting Your Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Printing Graphs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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BioPlex_6.book Page ix Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide Norm Ratio’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Norm Ratio’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Normalized Fluorescence Values (Norm FI) . . . . . . . . . . . . . . . . . . . . Ratio (Simple) and the Grouping Function . . . . . . . . . . . . . . . . . . . . . Basic Concepts . . . . . . . . . . . . . . .
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BioPlex_6.book Page x Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.
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BioPlex_6.book Page 1 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 1 Bio-Plex Suspension Array System Overview The Bio-Plex® suspension array system is a flow-based dual-laser system for simultaneously identifying and quantitating up to 100 different analytes in a single biomolecular assay (xMAP technology).
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BioPlex_6.book Page 2 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Components The system also includes validation and calibration reagents, a selection of cytokine and phosphoprotein assays, sample preparation reagents, and a computer running Bio-Plex Manager™ 6.1 software. New angiogenesis, acute phase, diabetes, cancer and isotyping assays have been introduced with this version of the software. Check www.bio-rad.
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BioPlex_6.book Page 3 Friday, September 23, 2011 11:55 AM Advantages • • • • • • • • • • • • • • • • Bio-Plex reservoir Pentium-class PC, preinstalled with operating system, Microsoft Excel, Microsoft Internet Explorer, Microsoft.
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BioPlex_6.book Page 4 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | For More Information For More Information For more on the principles and concepts of the Bio-Plex suspension array system, see Basic Concepts in the Appendix on page 227. Bio-Rad Technical Support Bio-Rad Technical Support in the United States is open Monday through Friday, 5:00 a.m. to 5:00 p.m., Pacific Time. Worldwide technical support is available on the Web at www.consult.bio-rad.com.
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BioPlex_6.book Page 5 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 2 Bio-Plex Manager™ Software Overview Bio-Plex Manager™ 6.1 software runs on a computer installed with the Windows XP or Windows 7 operating system, and requires a hardware protection key, also known as a HASP key, installed on either the computer itself or the computer network system. The software features a standard Windows interface, with pulldown menus, toolbars, and keyboard shortcuts.
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BioPlex_6.book Page 6 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Software Licenses NOTE: Bio-Plex Manager Security Edition must be installed on the Windows XP Professional or Windows 7 operating system for full security and functionality.
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BioPlex_6.book Page 7 Friday, September 23, 2011 11:55 AM General Workflow ANALYZING LUMINEX DATA IN BIO-PLEX MANAGER Bio-Plex Results Generator 3.0 converts CSV Output files from xPONENT 3.1 and 4.0 into Bio-Plex Manager data files. You can analyze the converted results on any computer running Bio-Plex Manager 4.0 or later. You can make the files available to an entire workgroup by placing them on a public server.
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BioPlex_6.book Page 8 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | General Workflow Quick Guide The Quick Guide, which displays automatically at startup, is designed to guide you through the typical Bio-Plex Manager workflow, from startup and calibration through shut down. Commands, such as Start up & Calibrate, open dialog boxes that contain choices to guide you through the procedures. Figure 3.
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BioPlex_6.book Page 9 Friday, September 23, 2011 11:55 AM Types of Files Types of Files There are two main types of files created and used by Bio-Plex Manager: • Protocol files (see page 53) contain the settings and controls for reading a microplate • Results files (see page 143) contain the data from each reading, and tools and reports for analyzing that data The application database file (bioplexdata.mdb) is installed on your hard drive or file server when you install the software.
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BioPlex_6.book Page 10 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.
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BioPlex_6.book Page 11 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 3 Software Installation Install, or reinstall, Bio-Plex Manager™ 6.1 software as described in the following section. If you need to upgrade your software from a previous version, see the “Bio-Plex Manager 6.1 Software Upgrade and Configuration Guide” to determine the part number of your upgrade kit.
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BioPlex_6.book Page 12 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Software Installation Required Screen Resolution Your computer screen resolution must be set to at least 1024 x 768 pixels for correct display of the Bio-Plex Manager interface. The status bar and some dialog boxes will not display properly at lower resolutions. If your display is currently set to a lower resolution: 1. Go to the Windows Start menu, select Settings, and select Control Panel. 2.
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BioPlex_6.book Page 13 Friday, September 23, 2011 11:55 AM Installing the Software Installing the Software To install or reinstall the software, insert the Bio-Plex Manager CD into the CD-ROM drive on your computer. NOTES • Before installing Bio-Plex Manager 6.1, we recommend that you first uninstall any existing version of Bio-Plex Manager on your computer • We recommend that you turn off any antivirus protection software before installation.
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BioPlex_6.book Page 14 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Software Installation NOTE: The bioplexdata.mdb file is not compatible with versions of Bio-Plex Manager earlier than 4.0. If you have an earlier version of the software, installing 4.0 or later will copy the data from your existing database file (bioplex.mdb) into the new database. A copy of your old database will remain in the application folder.
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BioPlex_6.book Page 15 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 4 Starting the System Before starting Bio-Plex Manager™ 6.1 software, make sure the Hardware Protection Key is attached to your computer, and switch on the array reader, the HTF (if one is being used), and the microplate platform. This turns on the optics inside the array reader and enables communication between the array reader and software.
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BioPlex_6.book Page 16 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Starting Bio-Plex Manager Connecting with the Array Reader and Microplate Platform The computer running Bio-Plex Manager software is connected to the array reader by a USB cable, and to the microplate platform by a serial cable. See the figure below for a diagram of the cable connections.
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BioPlex_6.book Page 17 Friday, September 23, 2011 11:55 AM Starting Bio-Plex Manager Communication between the Array Reader and Microplate Platform Communication between the computer and the array reader and microplate platform is automatically established when you open Bio-Plex Manager. If Bio-Plex Manager is unable to communicate with the instrument, a dialog box prompts you to check the cable connections. Figure 6.
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BioPlex_6.book Page 18 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Starting Bio-Plex Manager If you do not want to automatically connect to the array reader and platform when you open the software, select the Do not connect at startup checkbox. This is useful if you are not connected to an instrument or want to use the software without connecting to the instrument. To re-enable automatic connection at startup, select Reconnect from the Instrument menu (see next section).
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BioPlex_6.book Page 19 Friday, September 23, 2011 11:55 AM Menu and Toolbars Disconnecting with Sleep Mode When your computer goes into sleep mode, the instrument is disconnected from Bio-Plex Manager. When you awaken your computer from sleep mode, you will not be able to reestablish the connection to the instrument using the Reconnect option on the Instrument menu, and you will need to restart BioPlex Manager. Bio-Plex Manager overrides any sleep mode settings on your computer.
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BioPlex_6.book Page 20 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Quick Guide The Bio-Plex Manager software status bar is below the instrument status bar, and provides information about the command under the cursor. This information is continuously updated as you move your cursor over the software window.
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BioPlex_6.book Page 21 Friday, September 23, 2011 11:55 AM Sample Needle Adjustment Sample Needle Adjustment See the Bio-Plex 200 System Hardware Instruction Manual or the Luminex manual for instructions on installing the sample needle. WARNING: To protect hands and fingers, keep them out of the microplate platform when the needle is not in the down position. The height of the sample needle must be adjusted when the microplate type has changed, and/or when the sample needle is replaced.
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BioPlex_6.book Page 22 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Sample Needle Adjustment 8. Select the plate type to adjust the needle height appropriately. Choose Standard Plate if you are using a Bio-Plex Pro™ flat-bottom microplate for magnetic beads or the Millipore filter plate. Your other choice is PCR Plate. 9. In the Adjust Needle window, click on the Up/Down button. The needle will move to the down position. 10.
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BioPlex_6.book Page 23 Friday, September 23, 2011 11:55 AM Sample Needle Adjustment 17. Perform a Wash Between Plates step to remove any air introduced into the lines.
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BioPlex_6.book Page 24 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.
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BioPlex_6.book Page 25 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 5 Controlling the System This chapter covers the Bio-Plex Manager™ 6.1 software commands that control basic system functions, such as instrument start up, warm up, calibration, validation, and shut down. NOTE: The startup, warm up, and calibration functions must be performed prior to running an assay with Bio-Plex Manager, all of which together take about 45 minutes.
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BioPlex_6.book Page 26 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Bio-Plex MCV Plate IV Bio-Plex Manager 5.0 and later requires the use of the Bio-Plex MCV (Maintenance, Calibration and Validation) Plate IV. This plate contains wells marked for the different types of fluids used in validation, washing, calibration, and other functions. The Bio-Plex MCV Plate IV has been modified from the previous MCV plate to work with Bio-Plex Validation Kit 4.
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BioPlex_6.book Page 27 Friday, September 23, 2011 11:55 AM Start Up Start Up Startup is a series of fluidic functions that prepares the array reader to acquire data. This process requires the Bio-Plex MCV Plate IV, distilled water, and 70% isopropanol. Startup takes approximately 10 minutes. NOTE: The startup procedure can be performed while the optics are warming up, but no validations, calibrations or runs should be performed before the optics are warmed up. This takes an estimated time of 30 minutes.
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BioPlex_6.book Page 28 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Optics Warm Up and Shut Down To ensure accurate and reproducible results, the optics (that is, the lasers) in the array reader must warm up for at least 30 minutes prior to calibration, validation, and reading assays. Optics warm up begins when you first turn on the array reader.
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BioPlex_6.book Page 29 Friday, September 23, 2011 11:55 AM Calibration Calibration Calibration of the array reader is essential for optimal performance and dayto-day reproducibility of results. Calibration is required: • Each day after the startup procedure is complete and the optics have warmed up • If the array reader temperature changes by more than 2ºC during the course of the day.
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BioPlex_6.book Page 30 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Opening the Calibration Dialog Box To begin calibration, click the Calibrate button on the main toolbar or select Calibrate from the Instrument menu. The Calibrate dialog box opens. Figure 15. Calibrate dialog At the top of the dialog box, enter your name in the field.
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BioPlex_6.book Page 31 Friday, September 23, 2011 11:55 AM Calibration Select existing control numbers from the pulldown list, and the target values for the control numbers appear in the appropriate fields. When you receive a new Bio-Plex Calibration Kit, you must add the new CAL1 and CAL2 control numbers and target values to the Calibrate dialog. These numbers are printed on the bottles containing the beads. NOTE: It is critical that you enter the correct target values for your CAL1 and CAL2 calibrators.
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BioPlex_6.book Page 32 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Next, enter the DD, CL1, and CL2 target values for the control number, as printed on the bottle, in the appropriate fields. NOTE: Use only the DD target value on the Bio-Plex CAL1 bottle. Using Bio-Rad’s calibrators is highly recommended, because other manufacturers’ DD target values have not been validated. Click Add to close the dialog box and save your changes.
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BioPlex_6.book Page 33 Friday, September 23, 2011 11:55 AM Calibration Setting Up the Calibration When you have specified the control numbers of your calibration microspheres and specified the type of calibration you want to perform (CAL1 & CAL2, CAL1 Only, or CAL2 Only), click OK in the Calibrate dialog. Another dialog box lists step-by-step instructions for preparing the Bio-Plex MCV Plate IV. Figure 19.
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BioPlex_6.book Page 34 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Performing the Calibration Click the Eject/Retract Plate button to eject the microplate platform plate carrier. Place the MCV Plate IV in the carrier with the arrow facing toward the platform, and then retract the plate. Click OK to start the calibration process.
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BioPlex_6.book Page 35 Friday, September 23, 2011 11:55 AM Calibration To open the Calibration Log, go to the View menu and select Calibration Log. The Calibration Log viewer opens. Figure 21. Calibration Log viewer Each calibration is listed by date and time. If you are using Bio-Plex Manager Security Edition in Secure Mode, the User and Access Level columns list information about the user who was logged into the application when each calibration was performed.
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BioPlex_6.book Page 36 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System To print the calibration report, select Print from the File menu. Print Preview displays the report as it will appear in a printout. Print Setup allows you to select some standard print settings.
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BioPlex_6.book Page 37 Friday, September 23, 2011 11:55 AM Start Up and Calibrate Start Up and Calibrate Each time you use Bio-Plex Manager, the array reader must go through the start up, warm up, and calibration procedures. The Start up & Calibrate command allows you to perform these steps with a single command. Click the Start up & Calibrate button on the Quick Guide or main toolbar, or select the command from the Instrument menu.
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BioPlex_6.book Page 38 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Wash Between Plates You should wash the fluidics lines between each plate reading to prevent traces of sample or other debris from building up inside the system. Click the Wash Between Plates button on the Quick Guide or main toolbar, or select the command from the Instrument menu. A dialog box guides you through the steps for preparing the MCV Plate IV for a wash procedure.
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BioPlex_6.book Page 39 Friday, September 23, 2011 11:55 AM Remove Air Bubbles Remove Air Bubbles Microscopic air bubbles in the cuvette may cause a sudden shift in the bead regions during an assay reading. If the array reader detects such a shift, the reading stops and you are prompted to perform an alcohol wash to force air bubbles out of the system. Click the Remove Bubbles button on the main toolbar or select the command from the Instrument menu.
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BioPlex_6.book Page 40 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Unclog If the array reader detects an unusually low bead count during a reading, you are prompted to perform an Unclog operation to remove possible obstructions from the fluidics lines. 1. Click the Unclog button on the main toolbar or select the command from the Instrument menu. An instruction box guides you through preparation of the MCV Plate IV. Figure 24.
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BioPlex_6.book Page 41 Friday, September 23, 2011 11:55 AM Validation Validation Validation of the array reader is a formal process for documenting that the instrument is fit for its intended use and that it is kept in a state of maintenance and calibration. NOTE: Validation is performed after startup and calibration have been performed. Validation is dependent on successful calibration for accuracy.
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BioPlex_6.book Page 42 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Setting Up a Validation Run To set up a validation reading, click the Validation button on the main toolbar or select the command from the Instrument menu. The Validation dialog box opens. Figure 25. Validation dialog Enter your user name in the top field. If you are using the Security Edition of the software in Secure Mode, your user name is listed and grayed out.
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BioPlex_6.book Page 43 Friday, September 23, 2011 11:55 AM Validation The Add/Remove Control Numbers dialog lists all the available validation control numbers. To make control numbers available in the Validate dialog pulldown list, add them to the Selected List. To select all the numbers in the Available List, click the Add All>> button. To add control numbers individually, double-click them, or select multiple numbers using Shift + Click and Ctrl + Click key combinations, and click the Add>> button.
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BioPlex_6.book Page 44 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Performing a Validation Run When you have made your selections in the Validation dialog, click OK. The dialog box for the selected type of validation opens. Figure 28. Dialog box for performing All Validation procedures See the Bio-Plex Validation Kit 4.
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BioPlex_6.book Page 45 Friday, September 23, 2011 11:55 AM Validation The Validation Results dialog box includes tabs for each type of validation. If you selected and performed All Validations, all the tabs will be available. Otherwise, only the tab for the validation you performed (Optics, Fluidics, Reporter, or Classify) will be available and displayed. Click Create Report to generate and display a Microsoft Excel spreadsheet showing the validation results.
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BioPlex_6.book Page 46 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System The validation results are listed by date and time in the upper window, and the specifications associated with each Control Number are listed in the lower window. If you are using Bio-Plex Manager Security Edition in Secure Mode, the User and Access Level columns list information about the user who was logged into the application when each validation was performed.
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BioPlex_6.book Page 47 Friday, September 23, 2011 11:55 AM Validation A Microsoft Excel spreadsheet is automatically generated and displayed showing all the validation data for that run. Note that Microsoft Excel 2000 or higher must be installed on your computer for this function to work. Figure 31.
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BioPlex_6.book Page 48 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System Platform Heater The microplate platform has a heater for warming samples. The heater has an operating range of 35ºC to 60ºC (95ºF to 140ºF), and can be controlled through Bio-Plex Manager. To access the heater controls, select Platform Heater from the Instrument menu, or click the button on the main toolbar . Figure 32.
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BioPlex_6.book Page 49 Friday, September 23, 2011 11:55 AM Instrument Information Instrument Information The array reader continuously monitors its internal temperature, voltage, pressure, and other systems for diagnostic purposes. You can access this information by clicking the Information button selecting Information from the Instrument menu. on the main toolbar or Figure 34. Instrument Information dialog Click the tabs in the dialog to access information about the instrument.
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BioPlex_6.book Page 50 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System NOTE: Major fluidics functions, such as Wash Between Plates, Remove Bubbles, and Unclog, consist of combinations of the following functions. To perform each of the following functions, select it from the Additional Functions submenu of the Instrument menu and follow the steps in the dialog box that appears.
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BioPlex_6.book Page 51 Friday, September 23, 2011 11:55 AM Shut Down Cancel Operation The Cancel button, located in the status bar at the bottom of the Bio-Plex Manager window, can be used to cancel many of the instrument functions, including calibration, validation, or any of the fluidics functions. Click the Cancel button to cancel the current operation. An alert box prompts you to confirm the cancellation. Figure 35.
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BioPlex_6.book Page 52 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Controlling the System NOTES: If you install Bio-Plex Manager 4.0 or later, the data from your existing database (bioplex.mdb) copies into the new database (bioplexdata.mdb). A copy of your old database will remain in the application folder. This Instrument Operations Log did not exist in Bio-Plex Manager versions before 4.0, so if you are installing Bio-Plex Manager 6.1 over versions earlier than 4.
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BioPlex_6.book Page 53 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 6 Preparing Protocols Protocol Files A protocol file contains the parameters of a Bio-Plex Manager™ 6.1 software reading. It specifies the analytes used in the reading, the plate wells to be read, sample information, the values of standards and controls, and instrument settings.
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BioPlex_6.book Page 54 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Standard protocol files have the file extension *.pbx. Secure protocol files, created using Bio-Plex Manager Security Edition, have the file extension *.spbx. Multi-assay Standard Edition Protocols (see page 56 for more information) have the file extension *.mpbx, and those saved with the Security Edition have the file extension *.spbx.
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BioPlex_6.book Page 55 Friday, September 23, 2011 11:55 AM Protocol Files NOTE: We strongly recommend keeping a copy of the original protocol file with the raw bead event data intact. These data are necessary for changing the DD gate range in the Results file, and can also be exported in XML file format from the Results file for further analysis. In the Save As dialog box, select the Compressed mode checkbox to save the protocol file without the bead map and histogram data. All other data are preserved.
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BioPlex_6.book Page 56 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Multi-Assay Protocols Bio-Plex Manager 6.1 has the ability to run multiple assays at one time. You describe each assay, using Describe Protocol, in the same way as describing single assays. If you have chosen New Multi-Assay Protocol from the File menu, the information you enter for each assay becomes its own tab, as outlined in red in the figure below. Figure 39.
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BioPlex_6.book Page 57 Friday, September 23, 2011 11:55 AM Multi-Assay Protocols Creating a Multi-Assay Protocol 1. Choose New Multi-Assay Protocol from the File menu. This action creates two tabs on the left pane, called Assay 1 and Assay 2. 2. Enter the protocol settings for Assay 1, then save. 3. Click on the Assay 2 tab, currently located at the bottom of the left pane. It will jump to a position under the tab for Assay 1. 4.
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BioPlex_6.book Page 58 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Protocol Window The Protocol window steps you through a series of windows.. Figure 40. Protocol window The buttons are numbered to guide you through defining your protocol: 1. Describe Protocol (optional) 2. Select Analytes 3. Format Plate (see page 59). (see page 68). 4. Enter Standards Info 5. Enter Controls Info (see page 59). (see page 78). (see page 96). 6.
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BioPlex_6.book Page 59 Friday, September 23, 2011 11:55 AM Protocol Window Click each setting button and enter the necessary information, then select the next setting. After all the settings have been specified, you are ready to run the protocol. Step 1. Describe Protocol The Protocol description window displays when you first open a protocol. (Otherwise, click the Describe Protocol button to display this window.) Entering a description of your protocol is optional.
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BioPlex_6.book Page 60 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols NOTE: During a reading, the array reader always detects all the analytes in the sample, including any you have not selected. However, analytes that were detected but not selected will, by default, not appear in the final reports and tables.
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BioPlex_6.book Page 61 Friday, September 23, 2011 11:55 AM Protocol Window Each analyte is listed by name and region number. The region number refers to the region of a fluorescent color map used to identify the analyte's bead set. Each bead set is embedded with specific quantities of two fluorescent dyes; the combination of these fluorochromes, as detected by the array reader, places the bead set within a unique region on the color map, thereby identifying the set and its associated analyte.
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BioPlex_6.book Page 62 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols CREATING A NEW PANEL OF ANALYTES Click the Add Panel button panel of analytes. in the Select Analytes toolbar to create a new Figure 43. Creating a new panel of analytes Enter a name for the new panel in the top field. Select the matching Assay type from the Assay drop-down menu, which is used to ensure the correct defaults. Then click Add to add the analytes.
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BioPlex_6.book Page 63 Friday, September 23, 2011 11:55 AM Protocol Window Click an analyte in the list to select it, then click the appropriate arrow to move it up or down in the list. Figure 45. Sorting analytes You can also remove an analyte from the list by selecting it and clicking the Remove button, or you can remove all analytes, by clicking the Remove All button. To edit an analyte in the list, select it, then click the Edit button.
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BioPlex_6.book Page 64 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols 3. Import this CSV file into Bio-Plex Manager by choosing the import button in the Add Panel dialog. Figure 47. Add Panel Dialog Box 4. Navigate through your file system to where you have saved the CSV file. 5. Click on the file to highlight, and press the Open button. Figure 48.
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BioPlex_6.book Page 65 Friday, September 23, 2011 11:55 AM Protocol Window 6. The new panel will be added to the list of panels in Bio-Plex Manager. Figure 49.
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BioPlex_6.book Page 66 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols EDITING A PANEL OF ANALYTES To edit an existing panel of analytes, first select it from the Panel pulldown menu in the Select Analytes window. Then click the Edit Panel button The Edit Panel dialog box opens. . Figure 50. Editing a panel Use the New, Edit, Remove, Remove All, and Sort buttons as described on the preceding pages to change the analytes in the panel.
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BioPlex_6.book Page 67 Friday, September 23, 2011 11:55 AM Protocol Window DELETING AND RENAMING CUSTOM PANELS To delete a custom panel of analytes: 1. Select it from the Panel pulldown menu in the Select Analytes window. 2. Click the Remove Panel button want to delete the panel. . You are asked if you are sure you Figure 51. Delete Panel Alert To rename a custom panel of analytes: 1. Select it from the Panel name pulldown menu. 2. Click the Rename Panel button . 3.
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BioPlex_6.book Page 68 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Step 3. Format Plate After you have selected analytes, you are ready to specify the format of your 96-well microtiter plate using the plate template in the Protocol window. The formatting in the plate template tells the array reader which wells to read, and tells Bio-Plex Manager how to analyze the different sample types in each well.
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BioPlex_6.book Page 69 Friday, September 23, 2011 11:55 AM Protocol Window PLATE FORMATTING To format the well types on the plate, select the Plate Formatting tab. Then use the buttons on the toolbar above the plate template to define the wells on the microtiter plate. These formatting commands are also located on the Format Options menu. Figure 54. Plate formatting tools (Plate Formatting view).
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BioPlex_6.book Page 70 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Note that the cursor changes to an Unknown Sample cursor as you move it over the template. As you select each well, it is marked with a square and a number at the center. The square identifies the well as containing an unknown sample, and the number identifies the specific sample. The Set Number of Unknown Samples command is available only in the Security Edition of the software.
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BioPlex_6.book Page 71 Friday, September 23, 2011 11:55 AM Protocol Window DEFINING A REPLICATE GROUP To define multiple wells as a replicate group, select Turn Off Autofill from the formatting toolbar or Format Options menu, then drag your cursor over several wells that contain the same sample. The wells are labeled with the same number. Figure 57.
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BioPlex_6.book Page 72 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols CHANGING A REPLICATE GROUP To change the replicate group of a well or group of wells: 1. Click the Select tool , and click or drag the wells you want to change. The wells appear highlighted. 2. Right-click the selection, and select Set Replicate Group from the context menu. 3. In the pop-up box, select the desired replicate group number from the pulldown list. 4. Click OK.
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BioPlex_6.book Page 73 Friday, September 23, 2011 11:55 AM Protocol Window To define the wells containing standards in your microtiter plate, click the Standard button , then click or drag the wells in the template. Use the Autofill buttons as described on page 70 to define multiple standard wells and/or replicate groups of standards. Figure 61. Defining a row of standard wells NOTES: • The cursor changes to a Standard cursor as you move it over the template.
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BioPlex_6.book Page 74 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Note that the cursor changes to a Control cursor as you move it over the template. Control wells are marked in the template by an octagon with a number inside it. NOTE: After you have defined the control wells, you are ready to enter the concentrations of the controls as described on page 96.
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BioPlex_6.book Page 75 Friday, September 23, 2011 11:55 AM Protocol Window Plate Groupings After you have formatted the wells (see previous section), you can organize them into groups and identify one member of each group as the Reference, or Primary member. You can then calculate the ratio of each member's fluorescence intensity to the fluorescence intensity of its Reference. This type of analysis is similar to that performed in Western blotting.
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BioPlex_6.book Page 76 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols DEFINING A GROUP To define an assay group, click the Group button across the wells you want to group. and drag the cursor NOTES: • Wells must be defined as Unknown samples, Standards, Controls, or Blanks before they can be grouped. • You can group across well types (for example, a single group can include Standards, Unknown samples, Controls, and/or Blanks).
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BioPlex_6.book Page 77 Friday, September 23, 2011 11:55 AM Protocol Window CONTEXT MENU Right-click and drag over a number of formatted cells to bring up a context menu, that allows you to change several settings, including dilution factor, replicate group, and sample description. Figure 67. Right-click context menu DEFINING THE REFERENCE To change the Reference member of the group, click the Reference button and click the desired Reference member in the group.
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BioPlex_6.book Page 78 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Step 4. Enter Standards Info This section describes how to enter information about your standards. To enter information for standard wells defined on the current plate, make sure the Standards Info tab is selected. Standards are analytes of known concentration. They are used to generate a standard curve of values using one of the several regression methods built into Bio-Plex Manager.
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BioPlex_6.book Page 79 Friday, September 23, 2011 11:55 AM Protocol Window Click the Enter Standards Info button in the Protocol Settings pane to enter information about your standards. Figure 68. Entering information about standards The Enter Standards Info window allows you to define standards on your current microtiter plate, and/or import external standards data from another plate. Use the tabs above the data fields to select between standards on the current plate and external standards.
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BioPlex_6.book Page 80 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols The Standard Concentration Lot window is at the top of the screen. Figure 69. Standard Concentration Lot window LOADING A STANDARD LOT INTO YOUR PROTOCOL If you have previously saved a standard lot or it is otherwise present in your current version of Bio-Plex Manager, you can quickly load it into your protocol by using the load function. Figure 70.
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BioPlex_6.book Page 81 Friday, September 23, 2011 11:55 AM Protocol Window MANAGING STANDARD LOTS You can import new lots, export lots to collaborators, or delete lots on your Bio-Plex Manager by using the Manage Standard Lots function. Figure 72. Manage Standard Lots Pressing the Manage Standard Lots button brings up a dialog that allows you to import, export and delete standard lots. Figure 73.
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BioPlex_6.book Page 82 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols ENTERING STANDARD LOTS MANUALLY If your lot is not preloaded in the software or you cannot find it on the Bio-Rad web site, you can enter the information manually from the product insert. You can save any manually entered standard information as a new standard lot, as described above.
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BioPlex_6.book Page 83 Friday, September 23, 2011 11:55 AM Protocol Window 2. Once each analyte’s starting concentration is entered, apply the dilution factor which is appropriate for your experiment. Figure 74. Applying the Dilution Factor 3. The software will calculate all the concentrations for each analyte at each dilution, as seen below. Figure 75.
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BioPlex_6.book Page 84 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols When starting concentrations are the same across all analytes—follow the following steps. 1. Check the box next to Apply same concentration to all analytes. This is not the default setting. 2. Enter the starting concentration in the box indicated in the image below.
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BioPlex_6.book Page 85 Friday, September 23, 2011 11:55 AM Protocol Window 3. Enter the dilution factor and press calculate. 4. The software calculates all the concentrations for each analyte and at each dilution, as seen below.
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BioPlex_6.book Page 86 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols DELETING STANDARDS To delete your Standards entries, click in the spreadsheet title called “Std” to highlight all entries as shown below. Press the Delete button on your keyboard. Figure 76.
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BioPlex_6.book Page 87 Friday, September 23, 2011 11:55 AM Protocol Window SELECTING EXTERNAL STANDARDS This section describes how to select a Results file from which to import external standards data into a protocol. Click the Select External Standards tab. In the window, click the Fill Available List button, navigate to the directory containing the Results file with your standards of interest, and select the file. (Note that the Open dialog displays only Results files.) Figure 77.
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BioPlex_6.book Page 88 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols NOTE: Each external standard you select must match the region of a selected analyte in the protocol. For example, if you selected analytes in regions 19, 32, 52, and 73 in the Select Analytes window (page 59), you can select only external standards in those same regions. (Note that the analyte name does not have to match, only the region.
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BioPlex_6.book Page 89 Friday, September 23, 2011 11:55 AM Protocol Window NOTE: The Standards Info and External Standards Info tabs contain the same fields, but you cannot alter the concentrations under the External Standards Info tab (they appear grayed out), as shown in Figure 79. Figure 79.
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BioPlex_6.book Page 90 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols The analytes that you selected in the Select Analytes window appear in the Assign Standards Information table. The most efficient method for entering standard concentrations is to enter them directly into the table. Enter the highest concentration into the first row for each analyte. Figure 80.
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BioPlex_6.book Page 91 Friday, September 23, 2011 11:55 AM Protocol Window ENTERING CONCENTRATIONS (FOR CURRENT STANDARDS ONLY) To calculate them automatically from a dilution series, select Enter Automatically. Enter the high standard concentration (S1) into the first row of the table, for each analyte. Then select the Dilution Factor, check the Apply dilution to all analytes box, and click Calculate. The table fills with the standard concentrations.
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BioPlex_6.book Page 92 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols CALCULATING CONCENTRATIONS AUTOMATICALLY If a dilution series was created for the standards, the software can automatically calculate the concentrations of each standard. 1. Select the appropriate Most Concentrated Standard well number. (The most concentrated standard must be in either the first standard well or the last.) Figure 83. Entering standard concentrations automatically 2.
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BioPlex_6.book Page 93 Friday, September 23, 2011 11:55 AM Protocol Window CONCENTRATION UNITS You can enter the units of concentration in the Concentration Units field (units are not required and do not affect calculations). These units appear in your reports. If all your analytes have the same concentration units, select the Same Units for All Analytes checkbox.
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BioPlex_6.book Page 94 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols LOGISTIC WEIGHTING If you select a logistic curve fitting method (Logistic-5PL or Logistic-4PL), the software automatically weights the points in the curve. Click the Logistic Weighting button to access the weighting algorithm controls. Figure 86.
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BioPlex_6.book Page 95 Friday, September 23, 2011 11:55 AM Protocol Window RECOVERY PERCENTAGE RANGE Because the standard curve (page 168) is critical for calculating the concentrations of your unknowns, Bio-Plex Manager includes a mechanism for assessing the fit of the curve to the standards. This is the recovery percentage. See Standard Curve Optimizer on page 199 for how Bio-Plex Manager incorporates Recovery Range data automatically.
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BioPlex_6.book Page 96 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Step 5. Enter Controls Info (Optional) Controls are samples of known concentration whose values are measured during a reading in the same manner as the unknown samples. You can then compare the calculated concentrations of the controls after a reading with the expected concentrations to determine the overall accuracy of the assay.
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BioPlex_6.book Page 97 Friday, September 23, 2011 11:55 AM Protocol Window The table lists your control wells (C1, C2, C3, etc.). These are the wells that you defined in the Format Plate window (page 68). Enter the concentration of the selected analyte in the Conc column for each well. Figure 89. Entering concentrations of the controls If the Same Conc. Values for All Analytes checkbox is selected, the concentration values you enter are applied to all the available control analytes.
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BioPlex_6.book Page 98 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Preparing Protocols Step 6. Enter Sample Info (Optional) Before entering information about your unknown samples, select the analytes you are analyzing in the reading, as described on page 59, and then define the plate well(s) containing your unknown(s), as described on page 69. NOTE: Only formatted wells are read by the array reader.
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BioPlex_6.book Page 99 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 7 Running Protocols After you have prepared the Protocol as described in the previous chapter, you are ready to run it. Click the Run Protocol button settings and run the Protocol.
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BioPlex_6.book Page 100 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols Run Protocol Window The Run Protocol window has controls for performing the run, and two views for displaying the status of the run: the Raw Data table view and the Histogram/Bead Map view.
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BioPlex_6.book Page 101 Friday, September 23, 2011 11:55 AM Bead Count Bead Count The Beads field in the Run Protocol window lists the number of beads to be detected before a reading is complete. Note that this number can be beads detected per region or total beads detected, depending on the pulldown selection. If you select per region as the counting method, you can enter a number between 1 and 10,000 in the Beads field.
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BioPlex_6.book Page 102 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols Reservoir Functions The Bio-Plex Manager™ 6.1 software reservoir functions allow you to incorporate standard maintenance processes directly into the Run Protocol. This enables you to perform the Run Protocol without needing to replace the microplate with the MCV plate.
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BioPlex_6.book Page 103 Friday, September 23, 2011 11:55 AM Reservoir Functions Skip Functions The Run Protocol - Reservoir Functions window contains a drop-down menu that allows you to skip reservoir functions built into existing Protocols when they are not necessary. For instance, if you have paused to troubleshoot before a plate run, and know that the Protocol calls for the instrument to perform a function you performed manually, you may choose to skip that function in order to save time.
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BioPlex_6.book Page 104 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols Standard Curve Optimization When Protocols are run, by default, the software will optimize the fit of the standard curves. This can be turned off from the Run Protocol window, if optimizing is not desired. In Security mode, only users with permissions are allowed to change this setting. For more information on standard curve optimization see Standard Curve Optimizer on page 199.
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BioPlex_6.book Page 105 Friday, September 23, 2011 11:55 AM Advanced Run Settings Sample Size The Sample Size is the amount of sample that will be aspirated from the microplate well by the reader. The default sample size of 50 μl is recommended. You can specify a sample size as small as 10 μl or as large as 200 μl. NOTE: To avoid air uptake, make sure the sample needle is properly aligned (see page 21) and that each well to be read contains at least 125 μl of sample.
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BioPlex_6.book Page 106 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols Select the Pause Run If Error Occurs checkbox to select all the possible error conditions, or select/deselect the individual error condition checkboxes. By default, these are not selected (that is, the run will not pause for any error condition). Figure 96.
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BioPlex_6.book Page 107 Friday, September 23, 2011 11:55 AM Plate Loading Guidelines Doublet Discriminator Gates During a reading, a doublet discriminator (DD) channel measures the amount of light scatter from particles that flow past the red laser. The light scatter is directly proportional to particle size, and an internal DD “gate” is designed to identify particles smaller or larger than a single microsphere, including microspheres that are clumped or aggregated.
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BioPlex_6.book Page 108 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols WARNINGS: • Protect your assay microspheres from light. Once photobleached, the beads are no longer usable. Proper care of microspheres must be observed to maintain the benefits of your product warranty • When using a filter plate, avoid touching the bottom of the wells when wrapping the plate.
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BioPlex_6.book Page 109 Friday, September 23, 2011 11:55 AM Running the Protocol 3. In the Save As dialog, enter the name of the Results file, and click Save. If you selected reservoir functions, the Run Protocol Reservoir Function window opens. If you did not select any reservoir functions, skip to Step 5. 4. In the Run Protocol Reservoir Function window, follow the steps described and choose any functions you want to skip from the Skip Functions drop-down list. Click Next. 5.
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BioPlex_6.book Page 110 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols Status Bar As the sample is analyzed, the status of the reading displays in the status bar at the bottom of the Bio-Plex Manager window. Figure 98. Status bar during a reading The status bar provides a running count of both the Number of beads and the Beads per second.
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BioPlex_6.book Page 111 Friday, September 23, 2011 11:55 AM Histogram and Bead Map To generate a new Results file from a Protocol, select New Results from Protocol from the Run Protocol toolbar or the File menu. Histogram and Bead Map The histogram and bead map provide a graphical display of the raw data from a reading. These graphs are continuously updated during the reading in the Run Protocol window of a Protocol file.
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BioPlex_6.book Page 112 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols When viewing the histogram and bead map, you can choose to display all analytes, each analyte separately, or all beads that pass through the discriminator gate. Make your selection from the pulldown menu above the histogram. Figure 100. Selecting the analyte(s) to display To print the histogram and/or bead map, click the Print Histogram/Bead Map button .
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BioPlex_6.book Page 113 Friday, September 23, 2011 11:55 AM Histogram and Bead Map DOUBLET DISCRIMINATOR GATE RANGE The Doublet Discriminator (DD) range specified under the Run Protocol Advanced Settings (see page 107) is shown on the histogram as two red lines perpendicular to the xaxis. In the Protocol window, you can change the DD gate range by positioning your cursor over the red lines and dragging them to the right or left. This also changes the range as listed in the Advanced Settings dialog.
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BioPlex_6.book Page 114 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols • • • The Reporter 1 channel measures fluorescence from the reporter molecules bound to the analytes on each bead as they pass through the green laser. The amount of fluorescence per bead type is directly proportional to the amount of analyte present in the assay.
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BioPlex_6.book Page 115 Friday, September 23, 2011 11:55 AM Histogram and Bead Map Click the Maximize button to maximize the histogram portion of the window within the larger Protocol window. Click the Restore button return the histogram to its default state. to Figure 103. Histogram maximized in the Protocol window Bead Map The bead color map is a density dot plot of the events in a reading for a selected well.
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BioPlex_6.book Page 116 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols The white areas on the map indicate the expected regions of the selected analytes. If you move your cursor over these areas, the name of the analyte appears in a pop up window. The data point clusters should fall within these regions. Figure 104.
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BioPlex_6.book Page 117 Friday, September 23, 2011 11:55 AM Histogram and Bead Map If you are developing your own assay, you may want to plot the Reporter 1 channel against the Doublet Discriminator channel to evaluate the distribution of the reporter signal. This yields information about the specificity of the antibodies in the assay. Figure 105. Changing the channel displayed on the x-axis SCALING THE BEAD MAP DATA The default view of the data is log scale.
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BioPlex_6.book Page 118 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols DENSITY FILTERING Each colored dot in the bead map represents a certain number of events measured by the reader at that data point. Different colors are used to distinguish among different numbers of events. You can adjust the display so that data points with low numbers of events are not shown, or to set the number of events at before which the data point color changes. Figure 106.
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BioPlex_6.book Page 119 Friday, September 23, 2011 11:55 AM Raw Data Table Raw Data Table The Raw Data table displays the raw numerical data of a reading, as well as all instrument settings and information related to the system hardware and software used in the reading (version numbers, voltage, temperature, gain, etc.). NOTE: The instrument hardware and software information included in the Run Info window of previous Bio-Plex Manager versions is now provided in the Raw Data table.
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BioPlex_6.book Page 120 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols Bead Statistics To display statistical data for the individual beads as well as the entire well, click the Show/Hide Bead Statistics Columns button on the table toolbar. The number of beads collected for each analyte displays in parentheses after the analyte fluorescence intensity. Figure 108.
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BioPlex_6.book Page 121 Friday, September 23, 2011 11:55 AM Raw Data Table Error Code Indication 3 Bead classification efficiency problem detected in the well 4 Region selection problem detected in the well 5 Platform temperature problem detected Raw Data Display Options To select the columns to display in the Raw Data table, and other display settings, click the Raw Data Display Options button command from the Table Options menu. or select the Figure 110.
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BioPlex_6.book Page 122 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Running Protocols Set Number Format In the Raw Data Display Options dialog, click the Set Number Format button to select the number format for each column in the Raw Data table. Figure 111. Set Column Number Format dialog In the dialog, select the column from the pulldown list, then specify the number of decimal places for the value of that column in the Decimal Places field.
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BioPlex_6.book Page 123 Friday, September 23, 2011 11:55 AM Rerun/Recovery Mode Rerun/Recovery Mode After a reading has stopped—because of an error or user intervention, or because the reading is complete—the Rerun/Recovery Mode checkbox in the Run Protocol window will be selected. This allows you to rerun some or all of the samples. With Rerun/Recovery Mode selected, checkboxes appear next to each well number in the Run Protocol table. Select the checkboxes next to the wells you want to read again.
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BioPlex_6.book Page 124 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.
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BioPlex_6.book Page 125 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 8 Using the Security Edition Bio-Plex Manager™ Security Edition Bio-Plex Manager™ 6.1 Security Edition software works in conjunction with the built-in security features of the Windows XP Professional or Windows 7 operating systems to provide a secure environment for the maintenance, verification, and tracking of all electronic records generated by the application.
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BioPlex_6.book Page 126 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Bio-Plex Manager™ Security Edition Background on 21 CFR Part 11 Effective August 20, 1997, the United States Food and Drug Administration (FDA) released Part 11 “Electronic Records; Electronic Signatures” of Title 21 of CFR.
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BioPlex_6.book Page 127 Friday, September 23, 2011 11:55 AM Installing and Starting Bio-Plex Manager Security Edition System Requirements Bio-Plex Manager Security Edition has the same system requirements as the Standard Edition, except that the security functions are not supported under the Windows XP Home Edition operating system. The software must be installed on a computer running the Windows XP Professional or Windows 7 operating system for full Secure Mode functionality.
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BioPlex_6.book Page 128 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.
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BioPlex_6.book Page 129 Friday, September 23, 2011 11:55 AM Enabling and Disabling Secure Mode Enabling and Disabling Secure Mode Only users with Administrator-level access can enable or disable Secure Mode. Enabling or disabling Secure Mode requires that you close all documents and restart the application. When Secure Mode is enabled, the status bar displays the locked symbol. Figure 114. Secure Mode disabled (left) and enabled (right) To enable Secure Mode, go to the Tools menu and select Preferences.
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BioPlex_6.book Page 130 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Electronic Records Users are required to enter their user name and password when they: • Start the application • Start a reading • Unlock the application • Sign a document, such as a Protocol or Results file Different users may authenticate operations.
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BioPlex_6.book Page 131 Friday, September 23, 2011 11:55 AM Calibration, Validation, and Instrument Operations Logs Calibration, Validation, and Instrument Operations Logs These logs provide a permanent chronological record of all calibration, validation, and instrument operations events. You can view, copy, and print the data from these logs. They are available from the View menu in Bio-Plex Manager.
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BioPlex_6.book Page 132 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Calibration, Validation, and Instru- If you make changes to a signed Protocol or Results file and save them as a new file, the new file will not be signed; however, the audit trail (see page 137) will be preserved in the new file and the file will remain a secure file.
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BioPlex_6.book Page 133 Friday, September 23, 2011 11:55 AM Calibration, Validation, and Instrument Operations Logs When you click OK, a Save As dialog opens. Figure 117. Save As dialog for Secure Protocol files Enter a name for the file or use the default name and click Save. After a file has been signed, it is flagged as read-only and cannot be modified. Any subsequent changes must be saved as a new file.
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BioPlex_6.book Page 134 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Calibration, Validation, and Instru- Document ID Number and Signature Each signed Protocol and Results file has a unique document identifier (Document ID) number. To view this number, go to the File menu and select Document Properties. Figure 118. Document Properties dialog The Document Properties dialog also provides information about the signature.
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BioPlex_6.book Page 135 Friday, September 23, 2011 11:55 AM Calibration, Validation, and Instrument Operations Logs All printouts and exports of a signed document also include the signature and Document ID number. Figure 120. Document ID and signature in an Excel export file Adjusting the Number of Unknown Samples in a Protocol A Supervisor-level user can create a “master” Protocol file with a defined plate format, including a set number of unknown sample wells.
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BioPlex_6.book Page 136 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Calibration, Validation, and Instru- In the Set Number of Samples dialog, the maximum number of unknown samples defined in the plate template will be noted next to the field. Figure 121. Set Number of Unknown Samples dialog The maximum number of unknown samples is determined by the number of formatted unknown sample wells and the number of wells per replicate group defined in the template.
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BioPlex_6.book Page 137 Friday, September 23, 2011 11:55 AM Audit Trail NOTE: In Secure Mode, each reading, including reruns of plate wells, generates a new Results file that cannot be overwritten. Figure 122. Electronic Signature required for reading Results file You are also prompted to sign and save the Results file if you select the New Results from Protocol command from the File menu (see page 143).
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BioPlex_6.book Page 138 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Audit Trail Each change you make to a signed Protocol or Results file or a new file saved from a signed file must be documented in the Reason for Change dialog. The information in this dialog is used to generate the audit trail. The dialog opens automatically whenever you make a change and either save the document or switch among subwindows in the Protocol or Results file. Figure 123.
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BioPlex_6.book Page 139 Friday, September 23, 2011 11:55 AM Audit Trail Viewing the Audit Trail To view the audit trail for a Protocol or Results file, go to the View menu and select Audit Trail. The audit trail is displayed in a subwindow of the Protocol or Results file. Figure 124. Audit trail for a Protocol file Figure 125.
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BioPlex_6.book Page 140 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Audit Trail The audit trail also notes each time the file has been signed. In the example below, note that each time the file was signed and made read-only, the audit trail was continued in the new file. Figure 126.
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BioPlex_6.book Page 141 Friday, September 23, 2011 11:55 AM Locking Bio-Plex Manager Locking Bio-Plex Manager If you are stepping away from the computer and want to prevent anyone else from using Bio-Plex Manager Security Edition, you can lock the application. Once locked, the application can only be unlocked by the user who locked it or a Supervisor-level user. NOTE: Locking Bio-Plex Manager will not shut down the application or end any instrument operations that are in progress.
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BioPlex_6.book Page 142 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.
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BioPlex_6.book Page 143 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 9 Analyzing the Results Results Files After a reading is complete, a Results file generates and opens automatically. The Results file contains the data from a reading, the Protocol parameters used to collect that data, and analysis tools for interpreting the data.
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BioPlex_6.book Page 144 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Opening a Results File To open an existing Results file, click the Open Results button on the Quick Guide or main toolbar, or select Open Results from the File menu. Locate and select the file using the usual Windows Open dialog. Standard Edition Results files have the file extension *.rbx. Secure Edition Results files have the extension. *.srbx.
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BioPlex_6.book Page 145 Friday, September 23, 2011 11:55 AM Results Files In the Save As dialog box, select the Compressed Mode checkbox to save the Results file without the bead map and histogram data. All other data are preserved. Figure 128. Save As dialog for Results files NOTE: This compressed file option is not available for Secure Protocol files generated using the Security Edition of the software.
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BioPlex_6.book Page 146 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Results Window The Results window is divided into subwindows, each containing different options for viewing, formatting, and calculating your data. Figure 129.
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BioPlex_6.book Page 147 Friday, September 23, 2011 11:55 AM Results Files Custom Reports The custom report function is located in the view menu and is active when you are viewing a results file. Figure 130. Custom Reports Choose from available report templates by using the pull down menu, or by navigating to a folder location. Figure 131. Custom Reports Dialog Bio-Rad plans to release custom report templates for specialized applications periodically via the web. Bio-Plex Manager 6.
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BioPlex_6.book Page 148 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Viewing/Changing the Protocol Settings In the Results window, you can review and in some cases change the plate formatting, analyte selections, sample concentrations, etc., from the original Protocol file to display different data or recalculate data in the Results file tables. These controls are located in the toolbar across the top of the Results window.
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BioPlex_6.book Page 149 Friday, September 23, 2011 11:55 AM Results Files Changing the Doublet Discriminator Gate Range You can change the doublet discriminator (DD) gate range (see page 107) in the Results file and recalculate the data based on the new range. The original DD gate range set in the Protocol file and used during the reading is preserved and can be displayed in the Raw Data table. To reset the DD gate range, go to the Edit menu, open the Reanalyze Results submenu, and select Change DD Gates.
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BioPlex_6.book Page 150 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Raw Data The Raw Data window in the Results file includes the same histogram, bead map, and numerical Raw Data table as the Run Protocol window. These features are described in the previous chapter on the following pages: • Histogram and bead map—see page 111 • Raw Data table—see page 119 Figure 133.
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BioPlex_6.book Page 151 Friday, September 23, 2011 11:55 AM Raw Data In the dialog, enter the plate ID number in the Plate ID field. If you want to associate the same ID number with all the wells in the reading, select Apply Plate ID to All Wells checkbox. If you want to associate different ID numbers with different wells, select the well for this number from the Well pulldown list. Click OK to save the changes. The plate ID number is displayed for each well in the Raw Data table.
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BioPlex_6.book Page 152 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Raw Data Table Error Codes If data cannot be measured or a sampling error occurs in a well, it will be flagged in the Raw Data table. The possible error conditions in the table are listed below.
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BioPlex_6.book Page 153 Friday, September 23, 2011 11:55 AM Report Table Report Table The Report Table provides detailed information about the analytes in your samples. Click the Report Table button to open the Report Table window. Toolbar The toolbar allows you to make formatting changes to your samples, and to change your report table in many ways. The figure below shows the toolbar functions. Figure 136.
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BioPlex_6.book Page 154 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Display Options Click the Report Table Display Options button, or select Report Table Options from the Table Options menu, to select columns to display in the Report Table. This dialog box also contains many other display settings, which are described below. Figure 137.
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BioPlex_6.book Page 155 Friday, September 23, 2011 11:55 AM Report Table PREDEFINED REPORT SCHEMES These selections display different sets of columns. Select Quantitative or Qualitative in the Report Scheme field to choose among predefined Report Table displays. Use the Norm Qualitative column if your data are normalized. NOTE: The predefined Norm Qualitative column is designed to be used with gene expression.
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BioPlex_6.book Page 156 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results ORGANIZING BY TYPE OR GROUP Click the Organize by Type button or go to the Table Options menu and select Organize Table by Sample Types, to highlight the divisions between the unknowns, standards, controls, etc. in the report.
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BioPlex_6.book Page 157 Friday, September 23, 2011 11:55 AM Report Table Click the Display Help for Table Legends button definitions for any error conditions. on the toolbar, to get SETTING NUMBER FORMATS Click the Set Number Format button in the Report Table Display Options dialog to set the number format for any column in the Report Table. The Set Column Number Format dialog opens. Figure 141.
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BioPlex_6.book Page 158 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Report Table Column Descriptions The columns in the table show the analyte data for each well or replicate group of wells. The available Report Table columns are defined below: • Type — sample type (unknown, standard, blank, etc.
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BioPlex_6.book Page 159 Friday, September 23, 2011 11:55 AM Report Table • • • • • • • • • • • • • • • • • • • • Conc in Range — observed concentration that falls within the range of standard concentrations, given a specified recovery range. Values outside the range are flagged as ***. See page 160 for how Conc in Range relates to the Obs/Exp * 100 Column Obs Conc — observed concentration of the analyte, calculated from its fluorescent intensity and the standard curve.
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BioPlex_6.book Page 160 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Obs/Exp * 100 Column Because the standard curve (see page 168) is critical for calculating the concentrations of unknowns, the Obs/Exp * 100 column in the Report Table allows you to assess how well the curve fits the standards. This column is also used with controls (page 96) to determine the overall accuracy of an assay.
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BioPlex_6.book Page 161 Friday, September 23, 2011 11:55 AM Report Table For example, if only standard concentrations from 1.9 to 8,100 g/ml fall within the specified recovery range, then only unknown concentrations that fall within this range are displayed in the Conc in Range column. Refer to Standard Curve Optimizer on page 199 for information about how the Conc in Range column can help you optimize your Standard Curve fit.
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BioPlex_6.book Page 162 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results If you flag a replicate group of wells as an outlier, data for the entire group are excluded. If you click the Show Replicates button , you can flag each well in the group as an individual outlier. If you do this, the outlier checkbox for the entire group appears checked and grayed out, indicating that part of the group has been flagged.
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BioPlex_6.book Page 163 Friday, September 23, 2011 11:55 AM Report Table Sorting Report Table Data To sort the table data alphanumerically, click the heading of the column that you want to sort, then click the Sort button order. . Clicking twice will invert the Copying the Report Table You can copy the contents of the table to the Windows clipboard. Select rows, columns, cells, or the entire table, then select Copy from the Edit menu. The copied values can be pasted into other Windows applications.
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BioPlex_6.book Page 164 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Export Format Select Single Analyte Layout to display all the data for each analyte in individual worksheets. Figure 148. Example of an Excel workbook, single analyte layout Select Multiple Analyte Layout to display one or more columns of data (for example, FI) for all analytes in a single worksheet. Figure 149.
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BioPlex_6.book Page 165 Friday, September 23, 2011 11:55 AM Report Table Note that this selection exports only a single column of data at a time. Figure 150. Example of an Export worksheet, 96-well layout Click the Advanced Export Options button to access additional options. In this section, choose a subset of data to export from the Report Table These are Preference Settings governing where and how your Report Table is exported Figure 151.
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BioPlex_6.book Page 166 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results If you have selected Single Analyte Layout, All Analytes is the only choice available. Export Preferences Select Excel Workbook to export the data to a Microsoft Excel file, as described above, or Text File (Tab Delimited) to export to a tab-delimited text file (see Figure 152 for an example). Figure 152.
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BioPlex_6.book Page 167 Friday, September 23, 2011 11:55 AM Report Table Use Multiple Worksheets If you selected Excel Workbook and are generating multiple tables of data, select this checkbox to export each table to a different worksheet within the workbook. Otherwise, all tables appear on the same worksheet. Choose from the following settings, based upon how you want your data presented: Single Analyte—This choice generates a workbook with one worksheet for each analyte.
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BioPlex_6.book Page 168 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Standard Curve The Standard Curve window displays the standard curve for each standard analyte in the Results file, and allows you to change the regression model as well as various curve display controls for each analyte. (Curve settings are initially selected in the Enter Standards Info window in a Protocol; see page 78.
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BioPlex_6.book Page 169 Friday, September 23, 2011 11:55 AM Standard Curve Use the Error Bars pulldown list to select from several types of error bars (such as 1-Std Dev, 2-Std Err) to plot for each point in the curve. If there is only one replicate for a point, the bar will have a value of zero. If both external and current standards are displayed in the graph, error bars will be displayed for the external standards only.
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BioPlex_6.book Page 170 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Regression Methods There are seven regression types for calculating the standard curve: • Logistic-5PL • Logistic-4PL • Linear • Cubic spline • Point-to-point • Linear (semi-log) • Point-to-point (semi-log) The minimum number of nonzero standards required for each regression method type is shown in the following table.
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BioPlex_6.book Page 171 Friday, September 23, 2011 11:55 AM Standard Curve LOGISTIC-5PL This version of the five-parameter logistic equation has been developed by Brendan Scientific, creator of StatLIA immunoassay software. The Logistic5PL regression type yields the best results for most assays; therefore, this is the default in Bio-Plex Manager. However, there may be cases in which the Logistic-4PL equation gives slightly better results.
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BioPlex_6.book Page 172 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results LINEAR The Linear equation is: y  a  bx where a is the intercept and b is the slope of the line. The simplest method for determining concentrations from a standard curve is to construct a plot using the linear portion of the response curve. The R2 integrity value may be used to determine the overall goodness of the linear fit.
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BioPlex_6.book Page 173 Friday, September 23, 2011 11:55 AM Standard Curve Linear vs. Logistic Regression Methods In terms of performance, the primary difference between linear and logistic regression models is that the linear range in which concentrations can be accurately predicted is much smaller. In most cases, the overall response of the assay is best modeled using a logistic fit and the Logistic-5PL model will yield the best results.
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BioPlex_6.book Page 174 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results You can also copy the regression method equation from below the standard curve by dragging your cursor over the equation to highlight it, and then selecting Copy from the Edit menu or right-click Context menu. Figure 157.
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BioPlex_6.book Page 175 Friday, September 23, 2011 11:55 AM Graphing Function Graphing Function Click the Graphs button to display your Results data as graphs. You can choose one of two default graphs from the Graphs drop-down menu: All Unknowns & Controls, or All Samples for single or multiple analytes. The y-axis for each default graph is the FI Result value, but you can change to a different parameter by choosing from the Result (y-axis) drop-down menu.
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BioPlex_6.book Page 176 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Figure 160. All Samples graph with Std Dev y-axis Perform any data normalization functions before displaying your graphs. If you want to see ratios among samples, or other normalization options, click the Normalization Settings button for more information. 176 .
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BioPlex_6.book Page 177 Friday, September 23, 2011 11:55 AM Graphing Function Choosing Graph Options VIEW Select the Graph Options button to choose the type of graph that best displays your Results data. These options are in the View area.. Figure 161. Graph Options dialog SAMPLE LABEL You can identify samples of the graph either by their Type (X1, C1, S1) or by their description (which you enter for each sample).
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BioPlex_6.book Page 178 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Under the Graph Options dialog, you can choose among four basic views, as shown in the four figures below. Click the appropriate button in the View field to display your choice. • Samples across a single analyte—In this view a single analyte is represented for each sample and the selected result (such as FI, Norm ratio, etc.) is used as the y-axis parameter. Figure 162.
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BioPlex_6.book Page 179 Friday, September 23, 2011 11:55 AM Graphing Function • Analytes across a single sample—In this view a single sample is represented for each analyte and the selected result (such as FI, Norm ratio, etc.) used as the y-axis parameter. Figure 164. Analytes across a single sample view • Analytes across multiple samples—Analytes grouped by sample along the x-axis and the selected result (such as FI, Norm ratio, etc.) used as the y-axis parameter. Figure 165.
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BioPlex_6.book Page 180 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results To gain room for your graphs, you can click the Hide Legends checkbox from the Graph Options menu. To adjust y-axis values so that the data will be comparable throughout a series of graphs, click the Adjust data to same y-axis scale checkbox. To change the y-axis display from linear to logarithmic, click the Logarithmic y-axis checkbox.
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BioPlex_6.book Page 181 Friday, September 23, 2011 11:55 AM Graphing Function You can zoom into any graph by dragging a marquee around specific bars. Click the Zoom out button on the toolbar to return to the previous magnification. Figure 168. Marquee to zoom into a graph Right-clicking any results graph displays a context menu to access many functions described in this chapter. Figure 169.
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BioPlex_6.book Page 182 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Adding New Graphs To create additional graphs, click the Add Graph button on the toolbar. Figure 170. Selecting elements from Add Graph dialog Select samples and analytes by clicking individual checkbox es, or with the Select All button. You can also click and drag over a range of samples, and select only those samples, by choosing Select from the right-click context menu.
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BioPlex_6.book Page 183 Friday, September 23, 2011 11:55 AM Graphing Function After you choose options, name and save this graph, it is added to your Saved Graphs List under the Graph drop-down menu. Figure 172. Saved graphs list in the Graphs drop-down menu Editing an Existing Graph The Edit Graph button is available only when viewing graphs with choices created by the user. The button will be grayed out while viewing either of the default graphs.
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BioPlex_6.book Page 184 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Deleting a Graph Delete a graph from your Saved Graphs List with the Delete Graph button on the toolbar. You can also delete a graph by right-clicking any graph to bring up the Context menu. Exporting Graphs to Other Applications You can export your data to a spreadsheet application such as Excel, for its expanded graphing capabilities, or to match existing graphs.
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BioPlex_6.book Page 185 Friday, September 23, 2011 11:55 AM Graphing Function Adjusting Your Graphs If you open a graph that represents a very large number of data points, you may see black lines, as in the figure below. Figure 175. Graph containing many data points Choose fewer analytes or samples, and your columns will widen, so that the analyte color codes can be represented. Zoom into your graph by clicking and dragging over any area of interest, as shown in Figure 168.
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BioPlex_6.book Page 186 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Document Export Options In addition to the export options available in the raw data and results table section of the data file, there are a number of document export options. These options are chosen from the Document Export Properties dialog box shown below, which is available from the File menu.
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BioPlex_6.book Page 187 Friday, September 23, 2011 11:55 AM Document Export Options All these export options can be set to occur automatically at the end of a run, by choosing the Auto export after run checkbox from the Run Settings dialog. The Advanced Run Settings dialog box launches from the Run Protocol screen, during the process of setting up a Protocol. Figure 178. Advanced Run Settings Bio-Plex XML Export You can export all the information in a Results file as an XML-formatted text file.
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BioPlex_6.book Page 188 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results To export the Results data in XML format: 1. Select Document Export Properties from the File menu. 2. Choose the Bio-Plex XML option. 3. Choose the location to save the file. XML SCHEMA FILE An XML schema file named Bio-Plex_5.xsd is included in the main Bio-Plex Manager application directory, for those who want to write an importer for Bio-Plex Manager results files.
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BioPlex_6.book Page 189 Friday, September 23, 2011 11:55 AM Document Export Options Output CSV File Format This option exports an output CSV file similar to those generated by IS 2.3 software. It can be opened in third-party applications designed to analyze IS 2.3 files. You have the option of changing analyte labels, well labels, and analyte sort order in the resulting CSV (comma-separated values) file. There are several export options available in the CSV format Content field. Figure 179.
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BioPlex_6.book Page 190 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results ANALYTE SORT ORDER You can indicate the left to right sort order in the output CSV file. None—sorts as the analytes are shown in the panel entry screen of Bio-Plex Manager Analyte Name—sorts alphanumerically by analyte name Analyte Region—sorts by region number These sort options are shown in the Figure 180. Both plate location and sequential Figure 180.
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BioPlex_6.book Page 191 Friday, September 23, 2011 11:55 AM Document Export Options Using Stylesheets Stylesheets allow unlimited customization of exported data. Click the “Use the following stylesheet” button to navigate to one of the default stylesheets. Stylesheets describe the format and content of the resulting export. Using stylesheets allows you to format data in any way desired, provided you have experience developing stylesheets. Some default stylesheets are included for guidance.
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BioPlex_6.book Page 192 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Command Line Export This is an advanced option for customers who use custom-designed applications to analyze their data.
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BioPlex_6.book Page 193 Friday, September 23, 2011 11:55 AM Document Export Options Exporting the Document After you make the choices described above in the Document Export Options dialog box, click OK. Choose the Document Export option from the File menu. The Document Export window uses the properties previously set in the Document Export Properties dialog and allows you to export. Your choices can also be changed in this dialog. Figure 183.
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BioPlex_6.book Page 194 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results Importing Luminex xPONENT Output Files From Bio-Plex Manager, you can launch Bio-Plex Results Generator and import a Luminex xPONENT 3.1 or 4.0 CSV output file into Bio-Plex Manager. 1. From the File menu, select Import from xPONENT Output CSV. Bio-Plex Results Generator is launched and the Bio-Plex Results Generator window displays.
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BioPlex_6.book Page 195 Friday, September 23, 2011 11:55 AM Exporting Results Data to Bio-Plex Data Pro™ Exporting Results Data to Bio-Plex Data Pro™ You can seamlessly export your data from Bio-Plex Manager and import them into Bio-Plex Data Pro, where you can do an extensive and in-depth analysis of your results. To use this feature, you must have both products installed on your computer, though you do not have to have Bio-Plex Data Pro running at the time.
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BioPlex_6.book Page 196 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Analyzing the Results 2. In the Export to Bio-Plex Data Pro dialog, enter a name for the data set you are exporting. By default, the name of the results file is displayed. 3. Click Export. The Bio-Plex Data Pro application is launched if it is not already running. The Import Data Sets dialog in Bio-Plex Data Pro automatically appears.
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BioPlex_6.book Page 197 Friday, September 23, 2011 11:55 AM Exporting Results Data to Bio-Plex Data Pro™ 4. Select an existing project from the Import Into drop-down list or create a new project into which to import the data. Additional data sets can be added to the Data Set Files list by using the Browse button. You may also return to Bio-Plex Manager to export another data set. 5. Click Import. When the data have been imported, the Status displays Done.
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BioPlex_6.book Page 198 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.
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BioPlex_6.book Page 199 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 10 Standard Curve Optimizer Bio-Plex Manager™ 6.1 software contains a new function to optimize the fit of standard curves. A standard practice used to improve the fit is detecting and removing outliers by using nonlinear logistic (4PL or 5PL) equations when fitting data points.
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BioPlex_6.book Page 200 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Background Refer to Determining Outliers on page 201 for more information and instructions. Background Standard Curve Recovery Bio-Plex Manager calculates an observed concentration for each standard point from the standard curve. This is divided by the expected concentration (entered by the user in the Enter Standards Info window) and multiplied by 100 to give a recovery percentage.
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BioPlex_6.book Page 201 Friday, September 23, 2011 11:55 AM Determining Outliers Concentration in Range Concentration in range (Conc in Range) reports which standards, controls, and unknown concentrations are reliable, based on the recovery range of the standards. The Conc in Range column shows only observed concentration values that fall within the range of valid standards, as defined by acceptable recovery ranges. Values that do not fall in the range are displayed as < OOR or OOR >. Figure 186.
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BioPlex_6.book Page 202 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Determining Outliers Standard Curve Optimizer Report Once a data set has been optimized, a report is generated that will indicate any standard curves which should be reviewed by the user. Figure 187. Standard Curve Optimization Report Two types of information presented for each analyte; Results and Comments.
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BioPlex_6.book Page 203 Friday, September 23, 2011 11:55 AM Determining Outliers COMMENTS Comments are included to point out particular issues that may be of interest. • Low Bead Counts Detected (Low Bead)—This message will be presented if fewer than 20 beads are detected.
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BioPlex_6.book Page 204 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Determining Outliers Residual Variance and Fit Probability Other statistical parameters that can be used to evaluate and adjust goodness of fit include spiked recovery, residual variance, fit probability and weighting. Residual variance measures how well a curve fits the individual data points. Fit probability provides statistical evaluation of residual variance to determine acceptability.
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BioPlex_6.book Page 205 Friday, September 23, 2011 11:55 AM Determining Outliers Alternatively, users can specify a different coefficient. Click the Curve Options menu and select Logistic Weighting to display the dialog and deselect the Auto calculate coefficient a checkbox. Weighting coefficient b has a default value of 1.8. Users can enter a different value to adjust curve fitting. Figure 189.
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BioPlex_6.book Page 206 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Determining Outliers These symbols can be excluded from the exported file by checking the box for Exclude table error codes, as shown in the figure below. Sampling errors related to instrument, reagent and sample preparation can also be monitored by checking the box for Sampling Errors under Report Display Options. Figure 190.
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BioPlex_6.book Page 207 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide 11 Data Normalization There are two main reasons to use data normalization: • You may need to account for differences in the amount of material loaded in each sample.
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BioPlex_6.book Page 208 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Data Normalization Selecting Internal Controls/Housekeeping Genes The Internal Control/Housekeeping Gene field in this dialog allows you to account for differences in the amount of sample material. This function requires that the sample contain analytes whose concentrations will not vary from sample to sample (for example, housekeeping genes, reference genes, and internal controls).
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BioPlex_6.book Page 209 Friday, September 23, 2011 11:55 AM . Figure 192. Selecting Multiple Internal Controls Double-click an analyte, or select and click Add. The housekeeping genes populate the Selected field of the dialog box. Bio-Plex Manager™ allows you to add up to 10 housekeeping genes. NOTE: The normalized value for each sample is calculated by dividing the mean fluorescence intensity (MFI) of the analyte of interest by the MFI of the analyte used for normalization.
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BioPlex_6.book Page 210 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Data Normalization Figure 193. Assigning a Control Sample NOTE: For the formulas used to calculate control sample ratios, see Normalization Formulas on page 223. Report Table Options To display normalization results, click the Report Table Options button, and select the columns you want to display from the Report Table Display Options dialog.
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BioPlex_6.book Page 211 Friday, September 23, 2011 11:55 AM Figure 194. Report Table Display Options dialog The Report Table displays columns for data normalization. The column titles associated with the normalization calculations begin with Norm Ratio or Norm FI. Predefined Normalization Report Schemes Select Norm Qualitative in the Report Scheme field. The Norm Qualitative column is designed to be used with gene expression.
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BioPlex_6.book Page 212 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Data Normalization Normalization Definitions The following columns and their associated calculations are provided when performing data normalization. Here are definitions for these columns: • Norm Ratio—normalized ratio.
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BioPlex_6.book Page 213 Friday, September 23, 2011 11:55 AM The following table is based on the selections made in Normalization Options.
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BioPlex_6.book Page 214 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Data Normalization Normalization Factor Calculations Normalization factors are determined in the following ways. Table 1. Case Factor Definition A Control sample present for normalization Average of (FI-Bkgd) for the external control for all analytes. Factor = Average [ (FIcontrol-Bkgdcontrol) analyte1, (FIcontrol-Bkgdcontrol) analyte2, (FIcontrol-Bkgdcontrol) analyte3, etc.
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BioPlex_6.book Page 215 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide Troubleshooting Follow these suggestions to troubleshoot your Bio-Plex system. Message/Problem Cause “Bio-Plex Manager 6.1 More Likely: software has detected Too few beads in the assay a problem with low (2,500 per region recommended). bead number” Plate not shaken for 30 seconds before analysis. Solution Check bead number calculations in assay. Remove plate from array reader and shake for 30 seconds.
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BioPlex_6.book Page 216 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Troubleshooting Message/Problem Cause “Bio-Plex Manager has detected a possible problem due to microbubbles in the cuvette or photobleached beads” More likely: Solution Calibration was performed before the array reader was warmed up. Perform 30-minute warm up and recalibrate. Calibrated with incorrect CL1 or CL2 target values.
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BioPlex_6.book Page 217 Friday, September 23, 2011 11:55 AM Message/Problem Cause “Check Link” in status Array reader or microplate bar of software platform is not turned on. Software is not communicating with array reader. Solution Turn on array reader and microplate platform. Close and restart Bio-Plex Manager. Cables between computer and Check cables for proper connections. array reader or microplate platform See System cable connections on are loose/not connected. page 16.
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BioPlex_6.book Page 218 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Troubleshooting Message/Problem Cause Solution The calibration was unsuccessful. Please repeat calibration. If calibration fails a second time, follow these steps. Calibration procedure failed. 1. Make sure CAL1 beads and CAL2 beads are placed in the appropriate wells (CAL1 in red well and CAL2 in green well). 2.
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BioPlex_6.book Page 223 Friday, September 23, 2011 11:55 AM Normalization Formulas Normalization Formulas Bio-Plex Manager uses the following formulas to calculate normalization values. • All calculations are done first on a per well basis • All fluorescence values are adjusted mean fluorescence (Fl – Background), unless no blanks are provided in the experiment • All calculations are done on a per gene level, except where indicated • The following equations are written for gene expression studies.
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BioPlex_6.book Page 224 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Appendix Norm Ratio The calculation is a simple division of each sample’s calculated value by the control sample’s calculated value.
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BioPlex_6.book Page 225 Friday, September 23, 2011 11:55 AM Normalization Formulas Normalized Fluorescence Values (Norm FI) (This calculation requires internal controls to be chosen.) Normalized Fluorescence Values (Norm FI) are fluorescence values adjusted to account for experimental sample to sample variations (loading differences). Norm FI can be used to compare results across experiments to get a clearer picture of experimental variability.
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BioPlex_6.book Page 226 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Appendix Ratio (Simple) and the Grouping Function If you are not using the grouping function present in the plate formatting section of the software, this does not apply to you. When using this function, remember that the Ratio value presented is only relevant for making comparisons within a group.
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BioPlex_6.book Page 227 Friday, September 23, 2011 11:55 AM Basic Concepts Basic Concepts The following is a brief overview of the basic principles of the Bio-Plex suspension array system. For more information, see "Practical Flow Cytometry", 3rd edition, by Howard M. Shapiro, M.D. (New York: Wiley-Liss Inc., 1995). Microspheres xMAP microspheres are highly uniform, 5.5-micron polystyrene beads that have been crosslinked during polymerization for physical and thermal stability.
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BioPlex_6.book Page 228 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Appendix Fluidics There are two fluidic paths in the array reader. The first path includes a syringe-driven mechanism that controls the sample uptake. This mechanism permits small sample uptake volumes from small reaction volumes. The syringe-driven system transports a user-specified volume of sample from a microplate well to the cuvette.
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BioPlex_6.book Page 229 Friday, September 23, 2011 11:55 AM Microsphere Handling Microsphere Handling Microsphere Dispersion xMAP microspheres will settle and aggregate if left undisturbed. You should always ensure that they are evenly distributed before dispensing. Gentle vortexing is the most effective method of mixing for most coated microsphere preparations; however, sonication can also be used to separate aggregated microspheres prior to coupling.
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BioPlex_6.book Page 230 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Appendix Enumeration of Microsphere Suspensions xMAP microspheres are provided at standard concentrations. The microsphere yield after a coupling process may be less than the starting concentration due to loss during wash steps. The microsphere loss can vary according to operator technique, coupled reactant properties, and scale of coupling.
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BioPlex_6.book Page 231 Friday, September 23, 2011 11:55 AM Microsphere Handling Microsphere Agitation During Assay Agitation of Bio-Plex assays with a plate shaker during incubation steps prevents microspheres from settling during extended incubation periods. The benefit of agitation may not be discernible for some assay conditions and should be evaluated according to the requirements of a given application. It is important to resuspend the beads in an assay for 30 seconds before performing a reading.
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BioPlex_6.book Page 232 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Appendix Software Warranty Bio-Rad Laboratories warrants that Bio-Plex Manager™ software shall substantially conform, in all operational features, to Bio-Rad's current specifications as published in Bio-Rad's user and installation guides and that, when properly installed, it will be free of material defects which affect system performance.
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BioPlex_6.book Page 233 Friday, September 23, 2011 11:55 AM Reporting Problems to Bio-Rad Reporting Problems to Bio-Rad Included in your Bio-Plex Manager installation is a program called Solobug. You can use this program to request features and design changes, or report noncritical problems. NOTE: For critical problems, contact Bio-Rad Technical Support at the numbers listed on the inside front cover of this manual.
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BioPlex_6.book Page 234 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Appendix Report Table Error Codes Certain data that cannot be measured or calculated will be flagged in the Report Table. Columns and their possible error codes are listed below.
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BioPlex_6.book Page 235 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide Glossary Term Definition Accuracy The closeness of a measured value to the actual value or the difference between expected and measured values. Using the Reporter Validation Kit, accuracy is defined as the percent difference between the measured regression curve and the expected or actual data points. Aggregated microspheres Microspheres that have associated.
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BioPlex_6.book Page 236 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Glossary Term Definition Concentration in range A measure of unknown, standard, and control concentrations that are reliable, based on the recovery range of valid standards. Values outside the range are flagged as >OOR or
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BioPlex_6.book Page 237 Friday, September 23, 2011 11:55 AM Term Definition Group A group of wells defined on a plate. You can calculate the ratio of the fluorescent intensities of the group's member wells to the fluorescent intensity of a Reference well in the group. Immunofluorescence A technique that uses a covalently linked fluorochrome-antibody complex to detect or quantify a particular antigen.
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BioPlex_6.book Page 238 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Glossary Term Definition PE See phycoerythrin. Phosphoprotein An enzyme that functions to phosphorylate specific proteins. Phosphoproteins are responsible for the activation of a variety of proteins through the process of phosphorylation. Photobleaching A chemical reaction caused by exposure to light, in which a fluorochrome is converted into a differently fluorescent or nonfluorescent compound.
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BioPlex_6.book Page 239 Friday, September 23, 2011 11:55 AM Term Definition Reporter A fluorescent molecule that is incorporated into an assay in such a way that the fluorescence intensity is directly proportional to the amount of analyte in an assay. Reporter channel The channel of the array reader that detects the fluorescent signal of the reporter molecule (phycoerythrin). Reporter system Any combination of molecules in solution that serves to quantitate the analytes in a particular assay.
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BioPlex_6.book Page 240 Friday, September 23, 2011 11:55 AM Bio-Plex Manager Software 6.1 User Guide | Glossary Term Definition Slope Defined as the rise over the run when plotting a series of points to form a line. Using the Reporter Validation Kit, the slope of the response is related to the dynamic range of the array reader and yields information about the response of the photomultiplier tube (PMT). Spectral address The unique fluorescent emission spectra of a microsphere.
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BioPlex_6.book Page 245 Friday, September 23, 2011 11:55 AM Sorting Report Table Data 137 Standard Concentration Lots 73 Standard Curve 142 Copying 147 Linear vs.
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Bio-Rad Laboratories, Inc. Life Science Group 10022815 Rev B Web site www.bio-rad.