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Bio-Plex Manager Software 6 - University of Iowa Health Care

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1. o V CN CN CS C8 I V NA MZ A NA NM VOY COO M ULL UM e AAAAAA WOU Serial port MN CN CN CN CS P8 o YUYYY YY Computer OOO0000000000 oooooooooooo Qo o oOooooooo0ooo nummuuumuuuu Array Reader Serial port Figure 5 System cable connections 14 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 Connection Error e Unable to establish communication with the Bio Plex system 1 Make sure both the reader and microplate platform are powered on 2 Check cable connections to both reader and microplate platform 3 Click Reconnect to re establish communication with the system If you are unsuccessful in re establishing communication try rebooting the computer and cycling the reader and microplate platform power Thi
2. 1 Describe Protocol 04 Oct 2007 12 33PM BP SupervisorName Supervisor Modify Sample s Info WAS Dilution Changed 5 0 0 528 1 0 06 04 Oct 2007 12 33 PM BP SupervisorName Supervisor Format Wells WAS Removed samples 5 0 0 528 1 0 0 6 l 2 Select Analptes 04 Oct 2007 12 31 PM BP SupervisorName Supervisor Sign File Protocol From Results DETAILS Accepted 0 0528 1 0 06 3 Format Plate 4 Enter Standards Info 5 5 Enter Controls Info 6 Enter Sample Info 7 Run Protocol Figure 180 Audit trail for a Protocol file R Gene Expression Read Only Audit Trail lt signed by BP SupervisorName gt Q 3s e js Uc ix e E Ba Resus Raw Data Access Level 04 Oct 2007 12 37 PM BP SupervisorName Supervisor Description Sign File Uncontrolled Document DETAILS Reason 1 Version Bring it to the security 5 0 0 528 1 0 0 6 iti Report Table JB Standard Curve ni Graphs Figure 181 Audit trail for a Results file The audit trail notes the date and time of each action the user the access level the action a description of the action automatically generated by the software the reason for the action entered by the user and the version of the software 181 Bio Plex Manager Software User Manual 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 a
3. Std Regression Curve Regression T ype 2 Select Analytes Logistic 5PL Lot Expiration Date Assign Standards Information Axis Transformation Std Description HuL 2 9 HumL 4 52 Hui s 33 Hutl 10 56 HulL f2 pT0 75 St 0 00 0 00 0 00 0 00 3 Format Plate e 1 4 Enter Standards Info Le iJ 5 Enter Controls Info i 6 Enter Sample Info Loaf x Linear y Same regression type for all analytes Concentration Units Same units for all analytes Units don t impact calculations Acceptable Recovery Range 70 130 v Same recovery range for all analytes 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 Calculate Concentrations Most Concentrated Standard 51 Apply dilution to all analytes 7 Run Protocol Pl Protocol2 Enter Standards Info Protocol Settings Standards Info Extemal Standards Info Select Extemal Standards DS v Apply same concentrations to all analytes Ose Concentration of 1 50000 Hu IL 2 38 Clear All Concentrations Enter the starting concentration in the box indicated in the image below 1 Describe Protocol 2 Select Analytes 3 Format Plate 4 4 Ente
4. Result Y axis FI v Analyte Mo IL 1 53 v Mo IL 1a 53 Chart Name I lt All Unknowns amp Controls gt Result Y axis Fl File Name C Program Files Bio Rad Bio Plex Manager 5 0 Example Files Mouse Cytokine 23 Plex Standard PMT rbx Acquisition Date 09 Jun 2005 04 34 PM Reader Serial Number LX10004167313 Plate ID x2 x3 X4 x5 x6 X7 X8 Mo IL 1a 53 21631 5 168162 2 12049 5 5821 7 1866 8 537 5 179 7 81 8 Figure 159 Exporting graphs to Excel You can also export the graphs as bmp files so they can be used in word processing presentation or other text based applications Click the toolbar File menu or right click the graph and a menu appears Choose Save as bitmap and the Save As dialog appears so that you can name the file and place it anywhere in your file system 157 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 158 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 Fl h3 e e e o p Figure 160 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 153 To return to the original view click Zoom Out from the toolbar Another option is to print your
5. E 10 Well Type Ra IL 1a 21 Ra IL 1b 35 Ra IL 2 18 Ra IL 4 32 Ra IL 6 55 Ra IL 10 20 11 D4 xi 548 383 606 3332 40 25 5 12 E4 x1 549 407 635 3340 38 28 5 13 F4 x1 572 427 652 5 3583 5 38 28 Figure 176 Document ID and signature in an Excel export file Clinician 2 Users 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 A Clinician 2 level user can then open the Protocol and adjust the number of unknown samples to be read in a particular reading This enables Clinician 2 level users to use the same Protocol file in multiple readings with different numbers of samples NOTES Clinician 2 level users do not have access to the other plate formatting tools in Protocols See Defining Unknown Sample Wells page 63 for information on defining unknown sample wells and replicates in Protocols With the Protocol file open and the Format Plate window displayed click the Es Set Number of Unknown Samples button X in the formatting toolbar or select the command from the Format Options menu 177 Bio Plex Manager Software User Manual Calibration Validation and Instrument 178 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 Set Number of Unknown Samples Number of Unknown Samples Max
6. A standard that has been imported from a different file A statistical measure of goodness of fit that incorporates the residual SSE sum of the square of the error A fit probability of 1 indicates a perfect fit while a fit probability of O indicates no fit The process of verifying the well to well carryover of beads in the array reader The emission of light that occurs when the electrons of a fluorochrome drop to a lower energy state A fluorescent molecule An electronic method of discriminating or eliminating aggregated microspheres in the analysis of an assay A gate separates singlet beads from aggregated beads Term Group Immunofluorescence Instrument threshold Kinase Laser Linearity Logistic regression Lower limits of Quantitation Median fluorescent intensity Microparticle Microsphere set Microspheres Multianalyte or multiplex Operational qualification Optics validation Outlier Panel Definition 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 A technique that uses a covalently linked fluorochrome antibody complex to detect or quantify a particular antigen The instrument threshold is a measure of system noise and is the reporter channel signal from a blank bead that contains no reporter dye An enzyme that functions to phosphorylat
7. Add Panel Panel name Assay Bio Plex Pro Assay Magnetic Bio Plex Pro Assay Magnetic Region Bio Plex MagPlex Beads Magnetic Bio Plex Assay Non Magnetic 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 dropdown menu which is used to ensure the correct defaults Then click Add to add the analytes The Add Name Analyte dialog box opens Figure 44 Adding a new analyte Add Analyte Analyte Region Add Cancel Enter the bead region number of the first analyte in the Region field and the analyte name in the Name field NOTE The bead region number must be correct for proper detection of analytes Confirm this number is correct before proceeding Click Add Continue to add the analyte to the panel and continue adding more analytes When you have entered your last analyte click the Add button to add it to the list and close the Add Analyte dialog After you add several analytes to the panel you are creating you can use the Sort buttons at the bottom of the Add Panel dialog to sort them Protocol Window Click an analyte in the list to select it then click the appropriate arrow to move it up Add Panel Panel name SW panel or down in the list Assay Bio Plex Region Analyte 13 H IL 6 Figure 45 Sorting analytes 3 H GM CSF 38 H IL 2 You can also remove an analy
8. Tanat MAINE FI Linky Fluidics Validation Specifications 01 Jun 2006 08 May 2007 23 Nov 2006 23 Nov 2006 23 Nov 2006 23 Nov 2006 23 Nov 2006 23 Nov 2006 23 Nov 2006 23 Nov 2006 23 Nov 2006 12 Apr 2007 4 fine WANT 2 3 1 6 0 7 1 1 1 2 1 1 1 4 1 0 1 4 0 9 0 796 3 696 Taw Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed 39 Bio Plex Manager 6 0 Software User Manual Controlling the System 40 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 Use the buttons in the upper left corner of the window to view the different validation types Optics ES Fluidics is Heporter Es and Classify Fd To print the Results window click the Print Report Results button E To print the Specifications window click the Print Specifications button s23 To show or hide the specifications data in the Validation Log viewer click the Show Hide Specifications Window button En To create a spreadsheet report for a particular validation run click the row for the run in the Results window It will appear selected as will the specifications associated wi
9. 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 Entering incorrect values results in an incorrectly calibrated array reader which adversely impacts assay results To add a new CAL1 control number click the Add button under CAL1 Control Number in the Calibrate dialog The Add New CAL1 Control Number dialog box opens Add New CAL1 Control Number Enter Control Number Cancel Expiration Date Help None Enter Target Values CALI CAL2 DD Target i CLI Target CL2 Target l Low RP1 Target Figure 16 Adding a new CAL 1 control number In the dialog enter the control number from the CAL 1 bottle in the Enter Control Number field If the expiration date is printed on the bottle select the option button next to the date field under Expiration Date and click the pulldown button next to the field to open the calendar selection box Expiration Date ONore 24 Jul 2007 July 2007 EM Sun Mon Tue Wed Thu Fri 12 3 4 5 8 9 10 11 12 14 2 Target 15 16 17 18 19 22 23 25 26 29 30 31 j Today 7 24 2007 Figure 17 Calendar selection box Scroll through the calendar using the scroll buttons at the top Click to select a particular date
10. 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 R integrity value may be used to determine the overall goodness of the linear fit A linear regression with an R value of greater then 0 99 is considered a very good fit The primary advantage of this method is that it is extremely simple The primary disadvantage is that the linear range of concentrations is small compared to that which may be obtained using nonlinear regression The linear curve is available in linear and semi log versions CUBIC SPLINE Cubic spline curves are smooth curves that go through every data point The model is a cubic polynomial on each interval between data points In some cases a spline curve can work well as a standard curve for interpolation However because the curve is calculated individually for every pair of points it does not correspond to any single equation The cubic spline curve is applicable to a wide range of assays however there are no built in biochemical restrictions on the curve shape and values cannot be extrapolated PoINT TO POINT No single equation is available for the point to point method The slope of each segment of the curve between data points is calculated independently The Point to Point curve is available in linear and semi log versions The Immunoa
11. 100 Conc in Range Group Ratio Dilution M Select All Save settings as default for new protocol Layout table by 5 Single Analyte CO Multiple amp nalytes Organize samples by O None Type Group Expand Replicate Info Exclude table error codes Set Number Format Figure 122 Report Table Display Options dialog SELECTING COLUMNS TO DISPLAY See Report Table Column Descriptions on page 132 for definitions Use the checkboxes to select which columns to display in the Report Table Display Options dialog 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 SINGLE OR MULTIPLE ANALYTE LAYOUT You can view the Report Table either in Single Analyte and Multi Analyte view and you can also toggle between them Fy Click the Single Analyte Layout E in the toolbar or choose Layout Table with Single Analyte from the Table Options menu Select an analyte to review from the Analyte pulldown list above the table Select analyte from the pulldown list Ri Human Cytokine 1 7 Plex Standard PMT Read Only Report Table Bs 4 Us UC
12. Drain Back flush Sanitize Alcohol wash Start up Shut down Hemove bubbles Skip Functions The Run Protocol Confirmation window contains a drop down menu that allows you to dismiss instrument functions built into existing protocols when they are not necessary Reservoir Functions 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 You can choose to skip the following functions e Functions that will happen Before a Plate Run e Functions that will happen After a Plate Run e All Instrument Functions NOTE If you are running multiassay protocols the reservoir functions can be run between the assays as well NOTE Other MCV functions and operations such as Calibration Validation Alcohol flush and Unclog require using the MCV plate The available volume in the reservoir limits the number of times each function can be performed with a protocol The total allowable number of repetitions for each function includes the total of functions selected Before Plate Run between and After Plate Run tabs The following table shows the total allowable number of repetitions for those individual functions that use reagent from the reservoir Reservoir Function Max of Operations Wash 20 Alcohol Wash Sanitize To access these functions select Instrument from
13. File Security and Validation The files used by Bio Plex Manager Security Edition cannot be opened or edited using other programs Bio Plex Manager checks the integrity and validity of a file each time it opens If a file becomes damaged or shows signs of tampering with an outside application it will not open in Bio Plex Manager 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 NOTE If the application is in Secure Mode each event in a log includes the name and access level of the user who performed authorized the action If the application is in Standard Mode the event is flagged as having occurred outside Secure Mode and the access level is labeled as unrestricted These logs are stored in a database file called bioplexdata mdb which by default is saved in the main Bio Plex Manager application folder on your computer you can specify a different location during installation NOTE The bioplexdata mdb database is not compatible with versions of Bio Plex Manager earlier than 4 0 If you have an earlier version of the software installing version 4 0 or later will copy the data from your existing database bioplex mdb into the new databa
14. Regi Analyt Add gt Reni Analyt egion nalyte i egion nalyte Mo IL 1a Mo IL 1b zx Remove Mo IL 2 Ma IL 3 Mo IL 4 Ma IL 5 Mo IL 6 Ma IL 9 Ma IL 10 Mo IL 12 p40 Ma IL 12 p70 lt 2 Remove All Figure 193 Selecting Multiple Internal Controls 194 Normalization Settings 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 When multiple analytes are used for normalization the geometric mean of the normalization analytes MFI is used Assigning a Control Sample The Control Sample field in this dialog allows you to express all gene sample measured values relative to an assigned control The control sample has a value of 1 and other samples are expressed as ratios for example 2 fold higher compared to mRNA or 5 fold higher protein compared to control Brackets around analytes in any list indicate they are used as internal controls To assign a control sample click the Single button in the Control Sample field Click None to disable your choice Normalization Settings Internal Control Housekeeping Gene s Control Sample 9 None None O Single single x1 Sample 1 Multiple
15. Region Analyte The Internal Control Housekeeping Gene Field accounts for differences in the amount of sample material The Control Sample field allows you to express all samples as a ratio to the assigned control Update Selection For more detail click Help Figure 194 Assigning a Control Sample NOTE For the formulas used to calculate control sample ratios see Normalization Formulas in the Appendix 195 Bio Plex Manager Software User Manual Normalization Settings 196 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 Ell Report Table Display Options Report Scheme Select columns to display Layout table bu Display Column j Type 5 Single Analyte Vell CO Multiple amp nalytes Outlier Description Organize samples bu Fl O None FI Bk Std Dey Type Std Err Group WEY Norm Ratio Expand Replicate Info Morm Ratio Std Dev Morm Ratio Std Err Morm Ratio 95 CV Morm FI Morm Fl Std Dev Morm Fl Std Err Morm FI 95 CV Obs Conc Obs Conc Std Dev Obs Conc Std Err Obs Conc WEW Exp Conc Obs Exp 100 Conc in Range Group Ratio Dilution Set Number Format Esclude table error codes A d C Select All C Save settings as default for new protocol Figure 195 Report Table Display O
16. X Reservoir Functions Advanced Settings Rerun Recovery Mode Histogram Bead Map 100 region Well Al aiGaed J I5 Lu lz e B 1000 10000 100 Classification 2 1 0 100 1000 10000 1 10 100 1000 10000 4335 10000 Doublet Discriminator Classification 1 10 10 11 11 13 13 14 14 15 15 46 16 17 17 18 18 Sampling Errors 12 12 ji 3 S4 cc T Figure 97 Run Protocol dialog 2 Check that the needle height is set for the type of plate you are using either Standard or PCR plate 3 Enter your user name in the field If you are using the Security Edition of the software in Secure Mode your user name will be entered and cannot be changed 4 f your microplate has a barcode number or other type of ID number enter it in the Plate ID field 5 Click Eject Retract Plate to insert the plate into the reader and click OK to begin the reading If the Auto Save After Run checkbox is selected you are prompted to enter a name for the Results file to be generated The following conditions may delay the start of a reading e Ifthe array reader optics have not warmed up the reading is delayed until warm up is complete e lf the pressure is too low in the fluidics system you receive a warning message prompting you to check the sheath fluid level hos
17. atl Charts 100 N c 2 n c 9 i S o 100 1000 10000 100 1000 10000 Doublet Discriminator Classification 1 177 5 1081 0 1073 5 70 0 338 0 Figure 114 Results window Navigate among the different subwindows using the buttons along the left side of the Results window e Raw Data see page 124 e Report Table He see page 127 e Standard Curve le see page 142 e Graphing Functions see page 149 120 Custom Reports Results Files The custom report function is located in the view menu and is active when you are viewing a results file Bio Plex Manager Multi Allele example File Edit RYE Instrument Table Options Window Help O wv Toolbar w Status Bar v Quick Guide Describe Protocol Select 4nalytes Format Plate Enter Standards Info Enter Controls Info Enter Sample Info Normalization Settings Raw Data Report Table Standard Curve Calibration Log Validation Lag Instrument Operations Lag Refresh Calculations Figure 115 Custom Reports Graphs t Custom Reports gt aS mmi Description Fi 1599 2953 0 420 1843 0 3516 0 1933 0 View Custom Report fi 1309 1930 792 0 563 1931 111 0 118 t 1932 17250 1496 1933 2632 0 2403 0 Choose from available report templates by using the pull down menu or by navigating to a folder location M Custom Reports Report Template Folder D Pr
18. 74 C Mo G CSF 54 6 Mo GM CSF 73 7 Mo IFN a 34 n T T Graph Options ue ee j Figure 155 Selecting elements from Add Graph dialog Select samples and analytes by clicking individual checkboxes 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 Samples Ht Standard 3 Standard 4 d Type Description Wells Grou Grou a 1 43 44 45 Sample 2 Sample 3 Sample 4 Sample 5 Figure 156 Selecting elements from context menu Click the Graph Options button from the Add Graph dialog to choose a view and other options for displaying your data as described on page 150 155 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 156 After you choose options name and save this graph it is added to your Saved Graphs List under the Graph dropdown menu Ri New Sample 23 Plex Standard PMT Graphs g oS 4 iis ic ix E raphs Samp X1 Std6 Analyte 10 v Y fa tg d Samp X1 Std6 Analyte 10 Result Y axis FI Figure 157 Saved graphs list in the Graphs dropdown 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 This dialog displays checked boxes for the elements already c
19. Bio Plex 200 System Hardware Instruction Manual available for download from the Bio Rad website The Bio Plex suspension array system includes the following components e Array reader e Microplate platform e Bio Plex MCV Plate IV required for Bio Plex Manager 5 0 and later Advantages Bio Plex reservoir Pentium class PC preinstalled with operating system Microsoft Excel Microsoft Internet Explorer Microsoft NET and Bio Plex Manager Instrument Control software Software package containing Bio Plex Manager CD ROM and a hardware protection key Instrument manual for array reader and microplate platform Sheath fluid bottle Sample needles two long Protective shield Waste bottle Communications cable to connect the reader to the computer Communications cable from microplate platform to the computer Computer monitor Power cords Computer keyboard Computer mouse HTF High Throughput Fluidics if selected Communications cable from the reader to the HTF if HTF is present The Bio Plex suspension array system comes with these reagents Bio Plex Calibration Kit CAL1 and CAL2 Bio Plex Validation Kit 4 0 optics fluidics reporter and classify components Sheath fluid Advantages With the Bio Plex suspension array system you can Simultaneously quantitate up to 100 different analytes from culture media and serum samples as small as 50 ul Automatically analyze all the samples in a 96 well microtiter
20. Enter Sample Info Optional x Before entering information about your unknown samples select the analytes you are analyzing in the reading as described on page 53 and then define the plate well s containing your unknown s as described on page 63 NOTE Only formatted wells are read by the array reader Be sure to define all your unknown sample wells before running a Protocol Click the Enter Sample Info button in the Protocol window to enter information about your unknown samples Pl human cyokines 001 Enter Sample Info Protocol Settings Assign S ample Information 1 Describe Protocol Sample Description _ ne 2 Select Analytes 3 Format Plate 4 Enter Standards Info i B Enter Controls Info 6 Enter Sample Info Run Protocol X XS X15 X22 Dilution Factor 1 Set All Dilution Factors Example 4 sample diluted 1 in 4 has a dilution factor of 4 Figure 91 Enter Sample Info dialog The information table lists all the unknown sample wells as defined in the plate template for the reading page 63 You can enter a description and a dilution factor for each unknown in the appropriate columns Note that a sample diluted 1 in 2 would have a dilution factor of 2 To enter the same dilution factor for all unknowns enter the factor in the Dilution Factor field and click the Set All Dilution Factors button NOTE You can use
21. Supervisor Figure 169 User Information dialog 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 Platform Heater Off NA Platform Heater Off NA Steve E NUM BPSupervisor NE NUM Figure 170 Secure Mode disabled left and enabled right To enable Secure Mode go to the Tools menu and select Preferences In the Preferences dialog select the Enable Secure Mode checkbox to enable Secure Mode or deselect the checkbox to disable it If your user name and password are set up on a Windows network server enter the network domain where they reside in the Domain to Be Used for Authentication field Contact your Windows system administrator if you are unsure of your domain NOTE Local groups can be set up on the local machine and authenticated using domain users Click the box to use local user groups for establishing user security levels When you click OK you are prompted to sign in as an Administrator level user to authenticate the change You then need to restart the software User Authentication Bio Plex Manager Security Edition requires user authentication entering a user name and password at key points during operation User authenticat
22. TroubDIlesHoolirig 23 325 oboe a ee kei See oe dd 201 GIOSSANY 3 ues aO ed oe dede an el Uu bird o a dd 205 viii Bio Plex Manager 6 0 Software User Manual ADHERO acuracue wax ris a Oe OE e d teste a A cta A 211 Security Edition User Access by Function 00000 e eee eee 211 Normalization Formulas l l a a a i L eee 215 NOM RAIO 4 Gut oos tea e aac td MC dre aei ees ae ee a ee oes 216 NOMA AHO PH TM 216 NOT RAIO eee fo rx TEE CRESCE 216 Normalized Fluorescence Values Norm Fl lu s 217 Ratio Simple and the Grouping Function LL 218 Basic CONCEDIS s isn saa Ie eaa a eb TUR d aue dg dae au 219 WMIICFOSDIIBI GS 7 os Laud Eo aoa ROREM E ERE teehee ae EAE eee a 219 Reporter Fluorochromes cler 219 PWV CG Sie arse a ees P RPM ERE 220 EXC IATI OPN s d eror ua 208 quet 6nd oes a sit tN ce es ta ee ae he Oe A 220 VICKOSDNCKE MaANGIING 4 sitos tate taeda a S S Pee a barca se QUAM 221 Microsphere Dispersion llle 221 Probe SONICO ac 6262 6o OSEE AULAE Reto Bp E A ee tes 221 Bath Soni6altor 52 uri er NU ente RAP ERU dun oS sa iic CER mad 221 Enumeration of Microsphere Suspensions 222 Microsphere Separation Methods 2 0 00 eee ee eee 222 Microsphere Agitation During Assay 000 0c eee ee es 223 Microsphere Stability and Storage 2 000 eee eee 223 Software Warranty cece eee ee
23. e E 10 00 100 00 1000 00 10000 00 Concentration pg ml Standard O Partial Outlier 6 Outlier Figure 141 Copying graph to clipboard Exporting the Standard Curve To export a standard curve to Excel follow the steps below 1 Select the Export Standard Curve icon ze from the toolbar 2 Select your choice of exporting an individual analyte or all analytes in the Export Standard Curve dialog Legends the standard curve equation fit probability and residual variance display below the chart 147 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 148 See the Export Options section starting on page 124 for more information 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 Bi Standard O Partial Outer EJ Outlier Std Curve Fl 33 4808 20730 5 33 4808 FitProb 0 8629 ALI Figure 142 Copying regression method equation Printing the Standard Curve You can print the standard curve for the selected analyte using the Print command on the main toolbar or File menu You can print the curves for all analytes using the Print All Analytes command on the main toolbar or File menu When you select a print command a dialog box will open allowing you to specify the page layout of the printed curve The layout diagrams
24. s 20715 221 73 2210391 2634700 168628 168628 160 16 304199 659675 16988 1168 0 8s 455 Figure 187 Concentration in Range and Observed Concentration columns Determining Outliers The Standard Curve Optimizer maximizes the usable range of an analyte s standard curve based on the percent recovery range selected the recommended recovery range is 70 130 The optimization program evaluates several parameters including tests for bead count CVs of replicates hook effect recovery value and points in the noise region of the curve flatness at the low end Additionally the optimization program will favor the fit at the low or high end of the curve depending upon the number of unknowns falling in the respective regions Recommended Use We recommend you review the standard curve fit optimizer report after running an optimization and review the Results and Comments material outlined below These are designed to help you review your data quickly for issues that may concern you We also recommend that you pay particular attention to those analytes with comments or those that return a Check Data state 187 Bio Plex Manager Software User Manual 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 Ri Human Diabetes 12 Plex Read Only Standard Curve e e us Uc ux oe Labels
25. 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 e 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 Mo IL 3 18 Figure 147 Samples across a single analyte e Samples across multiple analytes Samples are grouped by analyte along the x axis and the result such as Fl Norm ratio etc used as the y axis parameter Figure 148 Samples across analytes view 151 Bio Plex Manager 6 0 Software User Manual Analyzing the Results e 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 A a p D m jS ES n m a S NS I er a e sS V cat S d eo e qo e ee QU eR Figure 149 Analytes across a single sample view e Analytes across multiple samples Analytes grouped by sample along the x axis and the selected result such as Fl Norm ratio etc used as the y axis parameter LL Mp IL 1a 53 EM 11 3 18 Eo IL 6 38 Mo IL 1b 19 mo IL 4 32 mo IL 9 33 Mo IL 2 36 Mo IL 5 52 Figure 150 Analytes across multiple samples view To choose between plain or 3 dimensional graph bars choose from the Graph Type dropdown menu 152 Graphing Function
26. 00 0 00 T Logistic Weighting 0 00 i 0 00 j 0 00 0 00 1 0 00 y 0 00 0 00 0 00 v Same regression type for all analytes 0 00 0 00 0 00 0 00 4 Enter Standards Info 0 00 j 0 00 i 0 00 0 00 Concentration Units 0 00 i 0 00 0 00 0 00 2 Select Analytes e 5 Enter Controls Info v Same units for all analytes Units don t impact calculations e 1 Acceptable Recovery Range 70 1302 v Calculate Concentrations Most Concentrated Standard 51 Oss Apply dilution to all analytes Dilution Factor Calculate Bir nun Prao C Apply same concentrations to all analytes Clear All Concentrations 6 Enter Sample Info Same recovery range for all analytes Figure 74 Applying the Dilution Factor 3 The software will calculate all the concentrations for each analyte at each dilution as seen below Pi Protocol2 Enter Standards Info Protocol Settings d Standards Info External Standards Info Select External Standards Standard Lot Lot Expiration Date Analyte 1 Describe Protocol Hu IL 13 51 Std Regression Curve Regression Type Assign Standards Information Logistic 5PL z Su emnon tn On Hu IL 4 52 Hu IL 5 33 HulL 0 56 HulL t2 pT0 75 Axis Transformation 33054 00 40750 00 10736 00 23445 00 18653 00 EK 00 Loaf Linear y v 8263 50 10
27. 12096 means that the observed concentration of a standard should be within 80 120 of the expected concentration Concentrations of standards and unknowns that fall outside this range are flagged as unreliable A well within a group of wells defined on a plate that is used as a reference You can calculate the ratio of the fluorescent intensity of the Reference well to the fluorescent intensities of the other wells in the group The region of a fluorescent color map used to identify a particular 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 Term Reporter Reporter channel Reporter system Reporter validation Residual variance Results file rbx or srbx Sample Sampling error Sandwich immunoassay Secure file Sensitivity Signal Signal to noise ratio Singlet s Definition 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 The channel of the array reader that detects the fluorescent signal of the reporter molecule phycoerythrin Any combination of molecules in solution that serves to quantitate the analytes in a particular assay A standard method for verifyin
28. 17 Plex St Add All 53 Hu MCP I MCAF Human Cytokine 17 Plex St IRA Lh Lu lt o mm me Pablo 17 Dlan Ct Clear Available List Figure 77 Selecting the Results file that contains external standards The Available External Standards list will be populated with the standards from the selected file To add additional standards repeat the procedure selecting a different file To delete all standards from the Available list click the Clear Available List button To make the available external standards accessible in the current Protocol you must add them to the Selected External Standards list right hand pane To add all of the standards in the Available column click the Add All gt gt button To add available standards individually double click them or select multiple analytes using Shift click and Ctrl click key combinations and click the Add gt gt button Use the lt lt Remove and lt lt Remove All buttons to deselect them 81 Bio Plex Manager 6 0 Software User Manual 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 53 you can select only external standards in those same regions Note that the analyte name does not have to match only the region Also each analyte must come from a different region When you a
29. 2 3 4 5 6 7 8 39 10 11 12 A 31 00 31880 00 364 00 27041 00 32043 00 3221450 3204550 32735 00 32666 00 32735 00 32735 00 32735 00 B 33 00 2921350 380 00 2620500 31977 50 31616 00 31204 00 32735 00 30125 00 29382 00 27725 00 30507 50 C 29 00 29646 00 46 00 20294 00 20248 00 15367 00 23669 00 30275 00 4824 50 3128 00 3249 00 3843 00 D 28781 00 45 00 1370 50 1441 50 2077 00 1830 00 3452 50 268 50 206 50 205 50 257 00 E 30630 00 6489 00 47 00 113 00 87 00 119 00 140 00 267 50 38 50 31 00 31 00 37 00 F 31636 50 6372 00 20 00 11 00 13 00 24 00 14 00 38 00 18 00 18 00 15 00 10 00 G 31561 50 6606 00 20 00 4 00 1 00 3 00 5 50 19 00 4 00 4 00 0 50 1 00 H 31243 00 372 00 20 00 7 00 7 00 8 00 5 50 0 00 32735 00 32735 00 32735 00 32735 00 Figure 137 FI Bkgd data multiple analytes 96 well plate format exported to a text file If you are exporting to Excel Microsoft Excel automatically launches when you click OK and a worksheet appears If you are exporting to a text file a Save As dialog will open when you click OK Select the desired directory and name and click Save to complete the operation 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 presen
30. 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000 0210 Sig 1109
31. 5 micron non magnetic polystyrene beads the gates are 4335 10000 whereas the gates for the 6 5 micron magnetic beads are 5000 25000 Some Bio Rad assays are built on a larger 8 1 micron magnetic bead which uses DD gates of 5000 32000 If you are using different sized beads or want to change the default settings you can change the size of the gate by selecting the Override Gates checkbox and entering new values in the Low and High fields You can also change the range by dragging the red lines on the Run Protocol window histogram see page 123 Plate Loading Guidelines NOTES The values in the Low and High fields are channel numbers corresponding to the amount of light scatter from a particle If you want to manually change the range you need to know the approximate channel numbers corresponding to the size of the beads you are using You can change the DD gate range in the Protocol before a reading or in the Results file after a reading See page 106 If you change the DD gate range in the Results file a record of the range during the reading is preserved You can set the gates on only the DD channel not the RP1 CL1 or CL2 channel For Bio Plex assays or Luminex type assays from other manufacturers the default DD gate settings should work well However if you are developing your own assay the position of the DD peak may shift and thus the DD gates may need to be shifted accordingly Note that you can adjust the DD gate
32. 50 PM 29 Aug 2007 12 44 PM 29 Aug 2007 12 39 PM 28 Aug 2007 02 14 PM 17 Aug 2007 04 48 PM 17 Aug 2007 04 43 PM 03 Aug 2007 03 05 PM 03 Aug 2007 02 38 PM 03 Aug 2007 02 36 PM lt User SRINISri SRINISri IPD DOMAINTBPSupervisor IPD_DOMAIN BPSupervisor IPD_DOMAIN BP Supervisor IPD_DOMAINisnallan IPD_DOMAINisnallan IPD_DOMAINisnallan IPD_DOMAINisnallan IPD_DOMAINisnallan IPD_DOMAINisnallan IPD_DOMAINisnallan IPD_DOMAIN BP Supervisor IPD_DOMAINisnallan IPD_DOMAINisnallan SI LX200 John Pocekay SI LX200Vohn Pocekay SI LX200Vohn Pocekay Access Level Unrestricted Unrestricted Supervisor Supervisor Supervisor Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Supervisor Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted CAL1 Expiration Date CAL1 A5449 NA CAL1 A5449 CAL1 A5449 NA CAL1 A5449 NA CAL1 A5449 CAL1 A5448 CAL1 A5449 CAL1 A5449 NA CAL1 A5449 CAL1 A5013 CAL1 A5013 CAL1 A5013 CAL2 CAL2 A5450 CAL2 A5450 CAL2 A5450 CAL2 A5450 CAL2 A5450 CAL2 A5450 CAL2 A5450 CAL2 A5450 CAL2 A5450 NA CAL2 A5450 NA NA CAL2 A5450 DETECTOR VOLTAGE DD RP1 CL1 CL2 Yoana Tana an an cn 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 in
33. Bio Plex assays most Bio Plex Pro Assays and Bio Plex COOH beads A 25 region map is also available The 25 region map includes select regions from the default 100 region map The result is that the regions are larger and more spread out The 25 region map is not available when creating a new panel as it only applies to a specific subset of pre installed Bio Rad assays It will be applied correctly to these preinstalled assays 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 ul is recommended You can specify a sample size as small as 10 ul or as large as 200 ul NOTE To avoid air uptake make sure the sample needle is properly aligned see page 18 and that each well to be read contains at least 125 ul of sample The maximum sample volume per well is approximately 140 ul The array reader rinses an additional 160 ul into each well after a reading is complete the maximum well capacity is 300 ul Advanced Run Settings Save Options You can automatically save the Results file generated by the reading With the Auto Save After Run checkbox selected when you click Start to begin a reading you are prompted to enter a name for the Results file NOTE In Bio Plex Manager Security Edition Secure Mode this checkbox is selected and cannot be deselected If the Auto Save After Run checkbox is selected you can also select the Auto Expor
34. Classify Control Number 9999991 Low Date amp Time 04 Oct 2007 12 53 PM Result Passed Calculated Results Parameter DD Median CL1 Median CL1 Cvz CL2 Median CL2 C RP1 Median RP1 CY Specification Measured Value 4774 5533 3134 4067 2 00 7 00 3190 4142 3 00 8 00 3260 4231 4 00 10 00 5363 3633 3 80 3724 7 85 3733 9 67 PK Create Report Figure 29 Validation Results dialog 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 Note that Microsoft Excel 2000 or higher must be installed on your computer for this function to work Excel comes preinstalled on all computers supplied with the Bio Plex Suspension array system See the Bio Plex Validation Kit 4 0 Instruction Manual part 171 203001 for interpreting the results of a validation run Validation Log Each validation run is recorded in the Validation Log This log is stored in a secure database file called bioplexdata mdb By default this file is saved in the main Bio Plex Manager application folder on your computer NOTE If you have an earlier version of Bio Pl
35. Labels Sequential 1 2 3 Analyte Sort Order Analyte Name O Use the following stylesheet C Exclude Raw Bead Event Data Destination File Change File C Documents and Settings csetine Desktop New Sample 23 Plex Standard PMT csv Command line Figure 168 Document Export window 166 Bio Plex Manager Software User Manual 9 Using the Security Edition Bio Plex Manager Security Edition Bio Plex Manager 6 0 Security Edition software works in conjunction with the built in security features of the Windows XP Professional operating systems to provide a secure environment for the maintenance verification and tracking of all electronic records generated by the application These records include Protocol and Results files and the Calibration Validation and Instrument Operations logs The tools provided in the software include e Access controls and authority checks via the use of user identification codes and passwords e Electronic record security via the use of protected directories e Time and date stamped audit trails e Electronic signature of electronic records user authentication When properly configured and administered these tools ensure compliance with the rules for secure handling of electronic records as outlined in Title 21 Part 11 of the Code of Federal Regulations CFH 167 Bio Plex Manager Software User Manual Bio Plex Manager Security Edition 168 Background on 21 CFR P
36. Lot Expiration Date v Figure 70 Load Standard Lot button This will result in the following window which will display all the lots loaded into your version of Bio Plex Manager Choose the lot you want to use and press OK This will load the concentration and dilution information associated with this lot W Load Standard Lot Select a Standard Lot to load Lot Mame Expiration Date 5005432 Mouse Group Cytokines Mone Figure 71 Load Standard Lot dialog 74 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 Standard Lot Lot Expiration Date L v 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 W Manage Standard Lots Lot Name Expiration Date C 5005432 Mouse Group I Cytokines None Import Figure 73 Manage Standard Lots screen Importing Standard Lots Standard lots will be added continually to the Bio Rad web site which can be accessed in the help menu of Bio Plex Manager Download the lot file which is in XML format from the web site to your PC and then choose Manage Lots above and press the Import button from the resulting window You can import all or a subset of the lots present in this file Exporting Standard Lots You can export all or a
37. Lot Analyte r ABCB4 CP65 7 Lot Expiration Date o amp Le Std Regression Curve Regression Type Assign Standards Information Std Description ABCB4 CP65 7 HFKB1 CP25 26 RELA CP63 44 CDKH1A CP42 37 IFHG CP36 61 1 i 0 00 0 00 0 00 0 00 Log x Linear v Logis Linear y e Logistic 5PL Axis Transformation r7 0 00 Logistic Weighting istic Weighting 3 gung i 0 00 Same regression type for all analytes 0 00 Concentration Units Same units for all analytes Units don t impact calculations Caise Connerbahane Acceptable Recovery Range 70 130 v Most Concentrated Standard 1 Concentration of 1 A ABCB4 CPB5 7 Same recovery range for all analytes Apply dilution to all analytes Dilution Factor X Calculate Appl ti ll analyt z C Apply same concentrations to all analytes E gt 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 NOTE You can define standards on the current plate and import external standards in the same protocol This is useful if you want to run standards for some analytes on the current plate but include external standards for other analytes in your concent
38. Manual Locking Bio Plex Manager Security 184 Bio Plex Manager Software User Manual 10 Standard Curve Optimizer Bio Plex Manager 6 0 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 Bio Plex Manager uses the percent recovery of individual standard points to assess standard curve recovery and assess the goodness of fit The optimizer tools are bordered in red on the Standard Curve figure below iz Cyto21pGrpll Standard Curve 4 GS Uc Ux dR Labels lt NONE gt v EtorBars lt NONE gt Results ELO EET Analyte Hu IL 1a 53 x Regression Type Logistic 5PL Y Axis Transformation Log x Linear y Logistic W eighting v Same regression type for all analytes Swap XY Axes Show Conc Range Lines Show Unknown Samples Show Control S amples Fluorescence Intensity F1 Curve Fit Optimize Clear Outlier Apply across all analytes v Show report after optimization Optimization Report Hu IL 1a 63 ULOQ 1759 387 LLOQ 2 033 1 00 10 00 100 00 1000 00 10000 00 Concentration E Standard O Partial Outlier CJ Outlier Std Curve Fl 34 9736 37292 3 34 9736 1 Conc 4 18451 0 324582 10 27 FitProb
39. Plex Manager versions before 4 0 so if you are installing Bio Plex Manager 6 0 over versions earlier than 4 0 no Instrument Operations data will be copied To view this log go to the View menu and select Instrument Operations Log The log viewer opens Instrument Operations Log Viewer mE Fie view Help amp Bio Plex Instrument Operations Log Date Time User Access Level Operation Status 04 Oct 2007 10 21 AM LSGXP 01005334Mohn Test Unrestricted Run Sample Completed 04 Oct 2007 09 44 AM GlobahBPSupervisor Supervisor Run Sample Completed 04 Oct 2007 09 43 AM GlobahBPSupervisor Supervisor Run Sample Completed 04 Oct 2007 09 38 AM GlobahBPSupervisor Supervisor Run Sample Completed 04 Oct 2007 09 28 AM GlobahBPSupervisor Supervisor Platform Heater Heater Off 04 Oct 2007 09 25 AM GlobahBPSupervisor Supervisor Run Sample Completed 04 Oct 2007 09 24 AM GlobahBPSupervisor Supervisor Platform Heater Heater On 04 Oct 2007 09 20 AM GlobahBPSupervisor Supervisor Wash Completed 04 Oct 2007 09 20 AM GlobahBPSupervisor Supervisor Run Sample Canceled 04 Oct 2007 09 14 AM GLOBAL jpoceka Unrestricted Wash Completed 04 Oct 2007 09 07 AM GLOBAL jpoceka Unrestricted Run Sample Completed 04 Oct 2007 09 03 AM GLOBAL jpoceka Unrestricted Warm Up Canceled 03 Oct 2007 05 10 PM LSGXP 01006334 John_Test Unrestricted Wash Completed 03 Oct 2007 05 07 PM LSGXP 01005334Vohn Test Unrestricted Run Sample Completed 03 Oct 2007 05 06 PM LSGXP 01008334
40. Print command on the main toolbar or File menu You can print the Report Table for all analytes when in Single Analyte Layout using the Print All Analytes command on the main toolbar or File menu Print Preview displays the table as it will appear in the printout Exporting the Report Table To export the data in the Report Table to an Excel workbook or tab defined text file click the Export Report Table button are in the toolbar The Table Export Options dialog opens Table Export Options Export Format Single Analyte Layout Cancel CO Multiple Analyte Layout C2 SB well plate Help Advanced Export Options gt gt Figure 132 Table Export Options dialog 137 Bio Plex Manager 6 0 Software User Manual Analyzing the Results Export Format Select Single Analyte Layout to display all the data for each analyte in individual worksheets EJ Microsoft Excel ARR t File Edit view Insert Format Tools Data Window Help Adobe PDF Type a question for help 3 i ara z 0 z B A AAEE 55 3 4 20 iE PE C D E E G H File Name C Program Files Bio Rad Bio Plex Manager 5 0 Example Files New Sample 23 Plex Sta Analyte Mo IL 1a 53 Acquisition Date 09 Jun 2005 04 34 PM Reader Serial Number LX10004167313 Plate ID RP1 PMT Volts 647 13 RP1 Target 3832 Analyte Type Well FI FI Bkgd Mo IL 1a 53 B Ab Bb5 C5 D6 61 6 61 6 Mo IL 1a 53 51 A1 A2 20287 20225 3 Mo IL 1a 53 52 B1 E2 18336 18274 3
41. Standard S1 Concentration of 1 24332 HulL 2 38 v Apply dilution to all analytes Dilution Factor 4 x Calculate v i ll t 7 v App ly same concentrations to all analytes ceset Figure 76 Deleting Standards Entries 80 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 Fill Available List Look x O Example Files Ne xternal Standards Fie name Fies of type Bio Plex Result Documents stoc ibx C Open as jesd ony P human_cyokines_001 Enter Standards Info EuSUURSUSCILAPIS Standards Info External Standards Info Select External Standards Available External Standards Selected External Standards Analyte Ext Std Source Analyte Ext Std Source 18 Hu MIP 1b Human Cytokine 17 Plex St 13 HulL 5 Human Cytokine 17 Plex St 21 Hu IFN g Human Cytokine 17 Plex St 32 HulL 1b Human Cytokine 17 Plex St 33 HulL 5 Human Cytokine 17 Plex St 34 Hu GM CSF Human Cytokine 17 Plex St 36 Hu TNF a Human Cytokine 17 Plex St 38 Hu IL 2 Human Cytokine 17 Plex St 51 HulL 13 Human Cytokine 17 Plex St 52 HulL 4 Human Cytokine
42. Status Bar The Instrument Status Bar shows the current state of the instrument Calibration Warm Up Ready Pressurizing etc It includes a Cancel button for canceling the current operation see page 45 Sytem Sistus Bury Command Wash Between Plater Time Aena 1 mn 5 sec Pistes Heater ey 27 7 C Figure 8 Instrument status bar When the array reader performs an operation the time remaining for the operation shows in the instrument status bar 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 The software status bar also shows the current user and if you are using the Security Edition of the software whether you are in Secure Mode shown as a locked symbol m or Standard Mode shown as unlocked See page 5 of this manual and the Bio Plex Manager 6 0 Software Upgrade and Configuration Guide for more information about the Security Edition The software status bar also shows the Caps Lock Scroll Lock and Num Lock keyboard status Quick Guide When Bio Plex Manager first opens a Quick Guide toolbar appears in the upper right corner of the screen You can use the Quick Guide to guide you through the typical workflow from start up and calibration through shut down Quick Guide Ms ik KY Start up W ash Between Plates
43. Testign images For Beta test Key Drive Back up 9 22 09 Key drive backup Launch Package template lt System Folc System Folc System Folc File Folder File Folder File Folder File Folder File Folder File Folder File Folder File Folder File Folder File Folder File Folder gt File name New Panel csv Save as type Csv Comma delimited csv Template xlt Text Tab delimited Ext Unicode Text Ext Microsoft Excel 5 0 95 Workbook xls Microsoft Excel 97 Excel 2003 amp 5 0 95 Workbook xls CSV Comma delimited csv Figure 46 Saving a CSV File in Excel Cancel of Bio Plex Manager 6 0 Software User Manual Preparing Protocols 3 Import this CSV file into Bio Plex Manager by choosing the import button in the Add Panel dialog 2 Protocol Select Analytes is Add Panel x Remove Panel 53 Edit Panel 3 Rename Panel Panel Bio Plex Pro Hu Group Cytokine 27 Plex w 1 Describe Protocol 2 Select Analytes 3 Format Plate 2 1 4 Enter Standards Info n 1 5 Enter Controls Info Le 1 Available Add Panel ME HulL 1b Hu lL fra Panel name de Assay Bio Plex Pro Assay Magnetic Hu IL 5 Hu IL 6 Region Analyte Hu IL 7 Hu IL 8 Hu IL 9 Hu IL 10 Hu IL 12 p70 Hu IL 13 Hu IL 15 Hu IL 17 Hu Eotaxin Hu FGF basic Hu G CSF Hu GM CSF Hu IFN g Hu IP 10 Hu M
44. The spread in individual data points for example in a replicate group to reflect the uncertainty of a single measurement The standard deviation of a set of replicates divided by the square root of the number of replicates A microsphere set that has been chemically modified so that its surfaces are covered with some functional group but that has not been chemically prepared for use as a component in an assay system These base microspheres do not carry biomolecular reactant The reproducibility of a signal over a series of replicate measurements For any given assay this is the highest value Bio Plex Manager will report in the Concentration in the Range column of the data table Values above this range are considered less trustworthy based on either the poor recovery values of standards above this range or saturation of the assay For more details review the definition of Concentration in Range A formal process for documenting that an instrument is fit for its intended use and is kept in an appropriate state of maintenance and calibration A set of reagents and procedures designed to verify the performance of the array reader apart from an assay Bio Plex Validation Kit 4 0 assesses the alignment of the optics assembly measures the performance of the reporter channel evaluates the fluidics system and verifies the classify efficiency of the array reader The mean square deviation of a variable around the average value Bio Pl
45. 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 To adjust legends to accommodate the number of columns in your graph click the x axis labels orientation dropdown menu from the Graphs Options dialog You can select vertical or horizontal legends or legends angled at 30 45 or 60 degrees Viewing Information within Graphs To browse through many analyte graphs click the green arrows to the left and right of the Analytes field in the toolbar This displays a series of analyte graphs without having to search and select from a long list R New Sample 23 Plex Standard PMT Graphs e us Bc Ux Graphs All Samples E Hg P Analyte Mo IL 1a 53 v 89 tg v Result Y axis F Reus Mo IL 1a 53 22000 20000 18000 16000 Figure 151 Viewing a series of analyte graphs Analyte names sample names user defined descriptions and values are always available for view Place your cursor over a specific column for a moment and the data display Analyte Mo IL 5 52 ample lt 3 S ample 3 alue 14602 00 0 DL yh 45 49 40 349 Figure 152 Click to view data from graphs 15
46. a user Once signed this file is a controlled document that cannot be overwritten by Bio Plex Manager and that preserves a built in audit trail of all saved changes If you make changes to a Secure file and save it as a new file the new file will not be signed however the audit trail will be preserved in the new file and the file will remain a Secure file The lowest detectable signal above instrument noise Noise may be attributed to the lasers the detectors and the amplification electronics of the array reader The detectable measurement unit of a reporter molecule The ratio of a specific assay signal to the underlying noise in the assay Signal to noise is typically used to measure the sensitivity of an assay A single microsphere or a population of single microspheres Compare to doublets which are two microspheres associated together 209 Bio Plex Manager 6 0 Software User Manual Glossary Term Slope Spectral address Standard deviation Standard error Unconjugated microspheres Uniformity Upper Limit of Quantitation Validation Validation kit Variance 210 Definition 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 The unique fluorescent emission spectra of a microsphere
47. an electronic signal that can be quantitated PMT settings display as an RP1 number during calibration when using Bio Plex Manager The fluorochrome used as the reporter molecule in Bio Plex assays Excitation 546 nm emission 575 nm See photomultiplier tube A measure of the reproducibility of replicate readings usually represented by the coefficient of variation 96 CV A file containing the settings of a reading including the microplate wells to read the analytes to detect sample size etc To perform a reading you open a Protocol file select the settings and then run the Protocol You can then save the Protocol settings and reuse or modify them After a reading a Results file is created containing the settings from the Protocol as well as the data results The raw data from the most recent reading are also stored in the Protocol file In Bio Plex Manager Security Edition Protocol files may be Secure files in which case they have the file extension spbx Procedures and guidelines that determine conformity to requirements A mechanism for assessing the fit of the standard curve to the actual standards For each analyte standard an observed concentration is back calculated from the standard curve and the fluorescent intensity This is divided by the expected concentration and multiplied by 100 to give the recovery percentage The range of acceptable recovery percentages see above For example a recovery range of 80
48. analyte You can enter additional information about your controls in the description column for each well If you are performing a dilution of your controls you can enter a dilution factor for each control in the Dilution column Note that a control diluted 1 in 2 would have a dilution factor of 2 This feature allows for parallelism studies whereby a control diluted by different dilution factors is analyzed to verify that the final concentration is the same for all samples Analyte Mo IL 1b 13 v Assign Control Information Ctrl Description Conc Dilution C1 3200 00 1 00 C2 1000 00 2 00 Figure 90 Entering Controls with different dilution factors If you are entering different concentrations for each analyte standard repeat the procedure by selecting each analyte from the pulldown menu as described above To enter the same dilution factor for all controls enter the value in the Dilution Factor field and click the Set All Dilution Factors button The dilution factor you enter for one analyte is automatically applied to all analytes NOTE You can use the Cut Copy and Paste commands on the Edit menu to copy your descriptions concentrations and or dilutions between analytes You can also copy and paste from and to other applications such as Microsoft Word or Excel Click a cell row or column in the table to copy and paste 91 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 92 Step 6
49. are applied to all the analyte standards Deselect the checkbox if you want to enter different concentrations for each analyte standard and repeat the steps 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 REGRESSION METHODS Using the Std Regression Curve pulldown list choose from among seven different regression methods to generate the standard curve page 142 e Logistic 5 PL e Logistic 4 PL e Linear e Cubic spline e Point to point e Linear Semi log e Point to point Semi log Figure 84 Selecting a regression method You can change your selection later in the Standard Curve window of the Results file See page 144 for a more detailed discussion of regression methods You can also select the regression curve axis scale from four different combinations of linear and logarithmic scales Select the axis scale from the Axis Transformation pulldown list Axis Transformation Linearty Lo ili a aee e ee pee Figure 85 Selecting a regression curve axis transformation Note that the axis transformation only changes how the curve is displayed not the calculated values NOTE The regression method and axis transformation you select in the Protocol window
50. are used to generate a standard curve of values using one of the several regression methods built into Bio Plex Manager This curve is used to calculate the concentrations of your unknowns The standards you select depend on the performance characteristics of your assay and the type of curve fitting model you want to use Due to variations in assay preparation we recommend using a minimum of eight standard concentrations to generate the standard curve See the appropriate Bio Plex Assay Instruction Manual for recommendations of standard concentrations To see what manuals are available visit www bio rad com bio plex The minimum number of non zero standards required for each type of regression method available in the software is shown in the following table Method Minimum number of standards Logistic 5PL 6 Logistic 4PL 5 Cubic spine 4 Linear linear and semi log 2 Point to point linear and semi log 2 Before entering the concentration values of your standards you must select the analytes that you will be analyzing in the reading as described on page 53 If your current plate contains your standards you must also identify the standard wells as described on page 66 Protocol Window Click the Enter Standards Info button in the Protocol Settings pane to enter information about your standards Pi Protocol1 Enter Standards Info Protocol Settings Standards Info External Standards Info Select External Standards Standard
51. check your data to identify systemic loading issues Also make sure you agree with the algorithm decisions to mark certain standards as outliers e More than one standard all replicates has been marked as an outlier gt 1 Std Removed This message will be displayed if all replicates of more than one standard point has been completely removed that is marked as an outlier You may want to review the data for such standard curves e The upper range of your curve is limited because 2 or more high concentration standards have poor recovery By default the report displays at the end of an optimization You may disable the automatic display by deselecting the checkbox next to Show report after optimization This report can also be viewed at any time by choosing the button below the optimizer controls 189 Bio Plex Manager Software User Manual Determining Outliers 190 Fit Probability and Residual Variance 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 The values of fit probability range from 1 0 perfect fit to O no fit These fit probability and residual variance values are reported by Bio Plex Manager as shown in this figure R Extern
52. data This process requires the Bio Plex MCV Plate IV distilled water and 70 isopropanol Start up takes approximately 10 minutes NOTE The Start up 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 cum Click the Start Up button v on the Quick Guide or main toolbar or select the command from the Instrument menu Follow the step by step directions in the dialog box for preparing the MCV Plate IV Start Up Start up prepares the reader for operation 1 Empty waste bottle 2 Refill sheath battle or verify that sheath cube is not empty 3 Fill 70 isopropanol and DI water reservoirs HCY Plate IV 4 Click Eject then load MCV plate 5 Press OK to start 4 Eiect Retract Plate Figure 14 Start Up dialog Insert the prepared plate into the microplate platform and click OK to begin the start up process 23 Bio Plex Manager 6 0 Software User Manual Controlling the System Optics Warm Up Rup and Shut Down 24 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 You can proceed with the array reader start up procedure described in the previous section while the optics are
53. delivery of the software not including delivery of any subsequent modifications to the software of any defect If the software is found to be defective by Bio Rad Bio Rad s sole obligation under this warranty is to remedy the defect in a manner consistent with Bio Rad s regular business practices For a defect which adversely affects the performance of the software Bio Rad shall use its best efforts to cure such defect as soon as reasonably practicable after receipt of Purchaser s notice For minor defects Bio Rad shall use its best efforts to correct such minor defects in the next release of its software If however Bio Rad is unable to cure a major defect within 90 days of receipt of Purchaser s notice Purchaser shall have the option to cancel this agreement whereupon Bio Rad shall refund only the software fees paid The warranties set forth in this agreement are in lieu of all other representations and warranties expressed or implied including warranties of merchantability and fitness for a particular purpose and any other statutory or common law warranty Bio Rad on its own behalf expressly disclaims and excludes any and all such other representations and warranties Liability of Bio Rad to Purchaser if any for breach of warranty or any other claim relating to this agreement shall be limited to the total amount of software fees paid by Purchaser to Bio Rad In no event shall Bio Rad be liable for incidental or consequential damages lo
54. ensure that system not turned on message disappears Air Compressor not working List for air pump to turn on when Warm Up is selected Contact Technical Support for further assistance Bio Plex Manager has Room temperature has changed Calibrate array reader detected a change in array reader tempera ture Please calibrate before running an assay to ensure accurate results 203 Bio Plex Manager 6 0 Software User Manual Troubleshooting Message Problem The calibration was unsuccessful Please repeat calibration If calibration fails a second time follow these steps Or The calibration was unsuccessful Bio Plex Manager has detected a problem with low bead number Please repeat calibration Optics Validation Procedure shows value s outside of acceptable range s Fluidics Validation Procedure shows value s outside of acceptable range s Reporter Validation Procedure shows value s outside of acceptable range s Classify Validation Procedure shows value s outside of acceptable range s 204 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 Make sure the calibration target values match the target values printed on the CAL1 and CAL2 bottles 3 Replace or clean the needle Or 4 Adjust needle height 5 Run Unclog until it passes If it doesn t pass by the
55. 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 After sonication microspheres generally remain dispersed for about 1 hour Sonication is most effective if applied to a centrifuged pellet of microspheres In order to maintain bead concentration we suggest you centrifuge your stock beads before sonication The following two indirect sonication methods are effective at separating aggregates in closed containers of microsphere preparations Inserting a sonicator probe into preparations is not recommended Probe Sonicator To disperse a microsphere pellet using a probe sonicator 1 Place probe tip in a small bath of water 2 Insert the end of the microsphere tube near the tip of the sonicator probe without touching it Do not immerse the tube closure 3 Adjust sonication for optimal disruption and pulse sonicate until the microsphere pellet is dispersed Bath Sonicator To disperse a microsphere pellet using a bath sonicator 1 Turn on the bath sonicator and examine the surface for an area of maximum disruption 2 Insert the end of the tube into the sonicator bath near the area of maximum disruption 3 Sonicate until the microsphere pellet is dispersed 221 Bio Plex Manager 6 0 Software User Guide Appendix 222 Enumerati
56. etc and number based on plate formatting Well Well number all wells are listed for a replicate group See next section Outlier Contains a checkbox for flagging the well or group of wells as an outlier See page 135 Description Description you entered for each standard control or sample in the Protocol FI Median fluorescent intensity of the analyte FI Bkgd Median fluorescent intensity minus background measured using the defined blank wells of the analyte Std Dev Standard deviation of a replicate group of wells Std Err Standard deviation of a replicate group of wells divided by the square root of the number of replicates CV Coefficient of variation of a replicate group of wells If this value is high you may want to review the individual Fl values and select the appropriate member of the replicate group as an outlier see next section Norm Ratio Normalized ratio See Data Normalization on page 165 for more information on normalizing data Norm Ratio Std Dev Standard deviation of the Norm Ratio values for all the wells within a sample s replicate group Norm Ratio Std Err The normalized standard deviation Norm Ratio Std Dev divided by the square root of the number of replicate wells within the sample s replicate group Norm Ratio CV The normalized standard deviation Norm Ratio Std Dev divided by the normalized ratio of the sample s replicate group Norm Fl N
57. file and you can generate a new Results file from this data Creating Opening Protocol Files To create a new Protocol file click the New button on the main toolbar or Quick Guide or select New from the File menu A new Protocol window opens To open an existing Protocol file click the Open button on the Quick Guide or main toolbar or select Open from the File menu Locate and select the file using the standard Windows Open dialog Standard Protocol files have the file extension pbx Secure Protocol files created using Bio Plex Manager Security Edition have the file extension spbx 47 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 48 Multi assay Standard Edition protocols see page 50 for more information have the file extension mpbx and those saved with the Security Edition have the file extension spbx NOTES e Secure Protocol files can be opened only as read only files using the Standard non Security Edition version of Bio Plex Manager e Protocol files created or modified by the current Bio Plex Manager cannot be opened by earlier versions of the software You can generate a new Protocol file from the settings stored in a Results file see page 117 for information about Results files With the Results file open select New Protocol from Results from the File menu A new Protocol window opens Saving Protocols To save the settings in a Protocol click the Save button m on the ma
58. 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 The Bio Plex Results Generator 3 0 application RBXGenerator 3 exe is included on the Bio Plex Manager 6 0 software CD There is also a help file Bio Plex Results Generator 3 0 Online Help to walk you through installing the Results Generator and setting up Luminex protocols for conversion General Workflow General Workflow To collect analyze and output data using Bio Plex Manager software follow the general steps outlined below Figure 2 Bio Plex Manager software workflow Quick Guide The Quick Guide which displays automatically at rT startup is designed to guide you through the typical 1 3G start up Bio Plex Manager workflow from Start Up and Wash Between Plates Calibration through Shut Down e EM Commands such as Start Up and Calibrate open dialog boxes that contain choices to guide you through the procedures Figure 3 Quick Guide 3 Gb Open Resuts 4 amp stu Down v Show at startup 2 D New Protocol Open Protocol Bio Plex Manager 6 0 Software User Manual Types of Files Types of Files There are two main types of files created and used by Bio Plex Manager e Protocol files see page 47 contain the settings and controls for reading a microp
59. 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 WASH The Wash command performs a single fluidics system wash using distilled or deionized water It can be used to remove traces of sample in the fluidics pathway Note that this procedure is less comprehensive than Wash Between Plates page 32 DRAIN This command completely drains the fluidics system of the array reader for example if you need to move the instrument Make sure you have enough room in your waste fluid container to prevent overflow PRIME This command primes the fluidics system with sheath fluid SANITIZE This command performs a comprehensive cleansing of the fluidics system using 10 bleach ALCOHOL WASH This command performs a single fluidics system wash using 70 isopropanol ALCOHOL FLUSH This command performs a comprehensive cleaning of the fluidics system using 70 isopropanol BACK FLUSH This command flushes sheath fluid through the fluidics system in the opposite direction of sample flow to remove particle obstructions from the cuvette 44 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
60. graph on multiple pages which allows the bars to be much wider See the Printing Graphs section below Printing Graphs Access the controls to print your graphs from the toolbar the right click context menu or the File menu Print Graph Figure 161 Print Graph dialog When the graph containing many data points from Figure 160 is printed onto four pages the color coded columns are visible To preview how your graph will look printed over multiple pages click the Go to Preview button The Print Graph dialog allows you to print your graph on a specific number of pages to choose a landscape or portrait orientation and to preview the printed copy Document Export Options 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 In this section you choose the type of exported file Document Export Properties Content Bio Plex XML Output CSV Format Export Options Use the Following stylesheet Exclude Raw Bead Event Data In this section you direct where the file will be stored Destination File Use Folder Choose Folder C Documents and Settings csetine Desktop Command line used to direct the file to an external application Com
61. have vented caps 93 Bio Plex Manager 6 0 Software User Manual Running Protocols Run Protocol Window 94 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 Click the Show Hide Histogram Bead Map button ne to display only the Raw Data table or the histogram bead map display with the Raw Data table below it maximize the window to see both the table and the display L1 Protocol Run Protocol Beade 10000 pen soie F Run High API Target C Sample Tmeou fect cranes Beerot Functions Aganced tti 1T Select Anales MU Regon Gae Total eAggBeads Samping Errors E17 12 Aid 1o MO e w 1 a Enter Standards Indo E 5 Enter Controls Info Enter Sample Info HEC ATIS ees Figure 92 Run Protocol window Raw Data table view 3 Protecol Run Protocol 4j etj eE lu E P Beade 10000 cerea 0 7 Aunat High PI Target T ee ample Timeout pect mem Bitara Function Agfeammed Setting 2 Select Aratri Hittogr Bead Mace 100 ieg ell 3 Format Plate wahi Ged E uu tj ej al T Erber Standaads info E s e E Enter Controls inio B Enter Sample Info 23 Run Probocol Figure 93 Run Protocol window Histogram Bead Map view The Histogram and Bead Map see page 104 provide graphical data about a run and are continually updated
62. is in the down position Needle height adjustment thumbscrew Figure 11 Sample needle assembly 12 13 14 15 16 17 By holding onto the needle height adjustment thumbscrew on the needle arm manually move the needle so that it just touches the bottom of the needle adjustment well of the MCV plate Move the needle up and down gently a couple of times to verify that the needle is barely touching the bottom of the well Tighten the needle height adjustment thumbscrew so that it is no longer possible to manually move the needle up and down Take care to ensure that the needle does not move while you are tightening the screw Do not overtighten In the Adjust Needle window click on the Up Down button to move the needle up and down Look inside the microplate platform at the MCV plate The needle should just touch the MCV plate at the bottom of the cutout use flashlight for better viewing Readjust the needle height if necessary Save these settings This allows Bio Plex Manager to warn you which type of plate the system will use before it starts a run When the needle is adjusted properly click the Eject button across the top of the title bar Remove the MCV plate from the microplate platform 18 Perform a Wash Between Plates step to remove any air introduced into the lines This chapter covers the Bio Plex Manager 6 0 software commands that Bio Plex Manager 6 0 Software User Manual Controlli
63. next to the Display Above Level field to select the exponent level below which no data points will be displayed For example if the Level Multiplier is set to 2 and the Display Above Level field is set to 3 data points are not displayed until 8 or more events are registered the first color level occurs at 8 events the second color level occurs at 16 events the third color level occurs at 32 events etc The color levels in the display are in ascending order dark blue purple dark green light blue blue light green orange black and brown LOGARITHMIC LINEAR DISPLAY Select whether you want to display the bead map data on a logarithmic or linear scale using these option buttons 111 Bio Plex Manager 6 0 Software User Manual Running Protocols Raw Data Table 112 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 This table is available in both the Run Protocol window and in the Raw Data window of the Results file Note that you can only print and export the Raw Data table from a Results file not a Protocol window The first few lines of this table are visible below the bead ma
64. 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 Beads 100 pes region Advanced Settings _ set FF Renm Riecever Mode Ir aa 51 1745 241 0 146 0 804 0 164 0 2 lee is 2650 2830 1745 1497 0 337 0 Iac s 663 0 5325 275 0 3528 0 995 0 la fols 7108 0 1587 0 BOT D 1216 0 8118 0 IS ID et ss TEST D 7166 0 1948 5 247280 104100 Figure 112 Selecting wells to rerun NOTE To rerun wells in a filter plate you must first vacuum the wells using the vacuum filter apparatus The beads remain on top of each filter plug after vacuuming Resuspend the beads with 125 ul of resuspension buffer in each well See the Bio Plex Cytokine Assay Manual for detailed instructions Each well may be read twice without adversely affecting the results Because there are fewer beads in a well that has already been run once be sure to select 100 beads per region or fewer in the Run Protocol settings see page 95 Also click the Advanced Settings button and deselect the Low bead number error condition checkbox page 98 Otherwise a low bead error may stop the reading Click Start to begin the reading Bio Plex Manager 6 0 Software User Manual S Analyzing the Results Results Files After a reading is complete a Results file generates and opens automatically The Results file c
65. on Calibrate 2 New Protocol gt Open Protocol 3 m Dpen Results 4 f Shut Down Show at startup Figure 9 Bio Plex Quick Guide 17 Bio Plex Manager 6 0 Software User Manual Sample Needle Adjustment By default the Quick Guide opens automatically when you start Bio Plex Manager To disable deselect the Show at startup checkbox at the bottom of the guide To open the Quick Guide after closing select Quick Guide from the View menu Sample Needle Adjustment 18 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 The MCV plate included with your system provides a method for adjusting sample needle height for standard flat bottom plates catalog 171 025001 filter plates Millipore catalog MSBVS1210 or PCR plates Sample Needle Adjustment Follow these steps to adjust the sample needle height 1 2 Turn on the array reader and microplate platform Launch the Bio Plex Manager software Click on Instrument in the menu bar of the software Choose Setup Choose Adjust Needle from the pull down menu The following dialog box appears Needle adjustment provides a met
66. red circles If both types of standards are used in the Results file both will be displayed on the curve Outliers and partial outliers will be displayed on the curve as indicated in the legend below the curve The equation for the selected regression method as well as some curve fit statistics fit probability residual variance will be displayed below the standard curve Equations for standards defined on the current plate are shown in blue while equations for external standards are displayed in dark red Any curve fitting errors will also be displayed below the curve in red Use the Analyte pulldown list to display the curve for a selected analyte Above the curve use the Labels pulldown list to select from a variety of different labels for the points in the curve If both external and current standards are displayed in the graph labels will only be displayed for the external standards 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 Use the Regression Type pulldown list to choose from seven different regression models to generate the standard curve See next section NOTE Changing the regression type will automatica
67. software instructions to identify which settings are suitable for your application Name Only the analyte description is generated in the report as in IL 2 Region Only the region identifier is listed in the column header as in 18 Name Region Both name and region ID are exported as in IL 2 18 WELL LABELS Rows in the CSV file represent well positions There are two ways to indicate well position Well A1 plate location is equivalent to Well 1 Well H1 is equivalent to Well 8 Row H12 is equivalent to well 96 sequential Sequential 1 2 3 Plate Location A1 B1 C1 Document Export Options 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 165 Results Both plate location and sequential Analyte Name only z DataType M Location Hu C peptide Hu Ghrelin Hu GIP Hu GLP 1 Hu Glucag Hu IL 6 Hu Insulin Hu Leptin 1 A1 hu diabetes antigen standard S1 11375 22370 18491 29744 5825 27695 5 23644 23524 2 B1 hu diabetes antigen standard S2 8059 5 14687 16269 30122 5385 5 22177 7253 20203 3 C1 hu diabetes antigen standard S3 1303 7780 149525 2 825 4337 5 75425 1314 12885 5 4 D1 hu diabetes antigen standard S4 127 5 3537 8036 5 1
68. still warming up However if you try to perform calibration validation or an assay reading you will receive a warning message You can cancel the warm up procedure using the Cancel Operation command on the Instrument menu or the Cancel button in the status bar however this is not recommended Results for identical readings may vary if the optics have not reached optimal operating temperature NOTE If you attempt to start a reading during warm up you can eject the plate carrier using the Eject Retract Plate command A on the main toolbar and remove the plate for storage until warm up is complete without canceling the warm up procedure If the array reader is idle for more than four hours the optics automatically power down though the array reader itself remains on Depending on the length of the shutdown a full 30 minute warm up period may be required before more readings can be taken Click the Warm Up button I 1 on the main toolbar or from the Instrument menu to begin warm up after automatic power down If you attempt to perform a reading after automatic power down you receive a warning message and the optics begin warming up Calibration Calibration we Calibration of the array reader is essential for optimal performance and day to day reproducibility of results Calibration is required e Each day after the start up procedure is complete and the optics have warmed up e lf the array reader temperature changes by mo
69. the toolbar or use the Reservoir Functions button from the Run Protocol dialog In the Reservoir Functions dialog select a function in the Available column and move it to the Selected column by using the right arrow button Do the same in the After Plate Run tab To save these settings as the default for future protocols select the Save As Default for New Protocol checkbox You can change the order of the functions by selecting one and moving it up or down with the arrow buttons Click OK 97 Bio Plex Manager 6 0 Software User Manual Running Protocols Advanced Run Settings 98 Click the Advanced Settings button to access additional reader settings and controls Advanced Run Settings Bead Map Save phions v Auto gave after run J e Jn Auto XML export after run DD Gates Sampling Engis C Overide gates Pause run d enor occurs C1 Low bead number _ 2 Aggregated bead 13 Chassiy efficiency L 4 Region selection C5 Platicem bemperature Reporter PMT 1 Qvenide calibration Figure 95 Advanced Run Settings dialog Bead Map Selection Bio Plex Manager software identifies each bead in an assay by its classification region that is the region in the bead map where the bead falls based on its fluorescence as measured by the classification channels in the array reader see page 108 The default bead map is the 100 region map which is appropriate for assays that use xMAP microspheres such as
70. throughout the run The Raw Data table see page 112 displays the raw data of the reading after it is complete The controls for performing the run are described in the following sections 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 For most assays including the Bio Plex cytokine assay we recommend a value of 50 beads per region This ensures that at least 50 beads in each region will be acquired before sample acquisition is complete If you select total as the counting method you can enter a number between 1 and 100 000 in the Beads field Note that if you choose to measure total beads the instrument will stop acquisition as soon as it has acquired the total number of beads in any combination of bead sets If you are detecting multiple analytes this means that some bead sets may be under represented in the final tally NOTE For the Bio Plex phosphoprotein assay we recommend specifying 25 beads per region to avoid triggering a low bead count warning Sample Timeout The Sample Timeout field works in conjunction with the Beads field to ensure that a well reading does not continue for an extended length of time
71. to cancel the current operation An alert box prompts you to confirm the cancellation System Status Busy Command Wash Between Plates Time Remaining 1 min 59sec Platform Heater Off 27 7 T Figure 35 Cancel button in the status bar Shut Down The shutdown procedure consists of a series of functions designed to clean the fluidics lines and prevent a buildup of debris within the system To initiate this process select Shut Down 2 from the main toolbar or Instrument menu and follow the instructions in the dialog box Shut down cleans the fluidics system 1 Fill 10 bleach and DI H20 reservoirs 2 Click Eject then load MCV plate 3 Verify that sheath bottle is at least half full MCV Plate IV 4 Press OK to start 4 Eiect Retract Plate Figure 36 Shut Down dialog Instrument Operations Log Each instrument operation you perform with Bio Plex Manager is recorded in the Instrument Operations Log This log is stored in a secure database file called bioplexdata madb By default this log is saved in the main Bio Plex Manager application folder on your computer 45 Bio Plex Manager 6 0 Software User Manual Controlling the System 46 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
72. v C Same recovery range for all analytes Calculate Concentrations Most Concentrated Standard 51 ose Concentration of 1 3200 Hu IL 1b 32 Apply dilution to all analytes Dilution Factor X C Apply same concentrations to all analytes ET R External Standards Read Only Enter Standards Info g 3 4v s UC UX E Results Standards Info External Standards Info Select External Standards R t Standard Lot ES Ext Std Analyte Feleti Lot Expiration Date E Report T able Hu IL 1b 32 Std Regression Curve Load Bere Manage Standard Lots J Standard Curve 2 Regression Type Assign Standards Information Std Description HuiL 1b 32 Hu IL 5 33 Hu GM CSF 4 Hu THF a 36 HuiL 2 38 HuiL 13 51 HuIL 4 52 Axis Transformation e51 Lot 835 020 Reference St PE 32000 00 32000 00 32000 00 32000 00 32000 00 32000 00 5 e52 Lot 835 020 Reference St PE 8000 00 8000 00 8000 00 8000 00 8000 00 8000 00 Log x Linear y v e53 Lot 835 020 Reference St PE 2000 00 2000 00 2000 00 2000 00 2000 00 2000 00 Logieic weightings a Lot 835 020 Reference St PE 500 00 500 00 500 00 500 00 500 00 500 00 Lot 835 020 Reference St PE 125 00 125 00 125 00 125 00 125 00 125 00 C Same regression type for all analytes pese Lot835 020 Reference S PE 3125 3125 3125 3125 31 25 3125 Lot 835 020 Reference St PE 781 781 781 781 781 781 Con
73. 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 NOTE The DD gate range cannot be changed while the protocol is running Figure 101 Changing the DD gate range 40s 8191 Doublet Discriminabor You can show or hide the gate lines by right clicking on the histogram and selecting Show or Hide from the Gate context menu To reset the gates to their default values click the Restore Default Gates button Hi above the histogram SELECTING THE CHANNEL TYPE You can change the histogram to display different channel types Click the histogram with the right mouse button and select X Axis from the context menu Histogram Bead vem usse 3 fe wife fe ol s 1000 10000 100 10 4095 8191 12087 16383 Douiet Discriminalor 433510000 Figure 102 Histogram right click context menu From the X Axis submenu select from the following channel types e he Doublet Discriminator channel measures the forward light scatter from particles as they pass through the red laser Light scatter correlates to particle size this channel allows you to discriminate single beads singlets from aggregated beads in the histogram This is the default selection Histogram and Bead Map e The Reporter 1 channel measures fluorescence from the reporter molecules bound to the analyt
74. 0 0000 ResVar 43 42 Figure 184 Standard Curve Fit Optimizer tools 185 Bio Plex Manager Software User Manual Background Refer to Determining Outliers on page 187 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 These calculations are automatically performed by the software The reported recovery of the standard curve points are in the Obs Exp 100 column pictured below me mm cm e ne rnm nen 1 S1 A1 A2 43 105142 105142 63188 63 103 S2 B1B2B3 77132 77132 i 15029 73 98 3 S3 C1 C2 3 338558 3355 8 3881 97 102 D1D2D3 10492 1049 2 964 25 101 E1 E2 E3 302 3 302 3 l 229 76 96 F1F2F3 99 6 99 8 59 37 G1 G2 G3 41 5 41 5 15 52 H1 H2 H3 21 0 21 0 3 38 Figure 185 Obs Exp 100 column reports Standard Curve Recovery The recovery percentages of the standard curve points indicate the overall accuracy of the assay The user can specify more or less stringent ranges for individual assays In the Assign Standards Info window Figure 186 use the Acceptable Recovery Percentage Range pulldown menu to select an acceptable recovery range To set different recovery range for different analytes uncheck the box next
75. 0 4801 0 47738 90 51 1 88 S5 ETE 1 00 1609 5 1593 0 67 18 417 208 66 206 00 1932 5 1905 3 12 73 0 66 S6 Fi F2 1 00 450 8 434 3 10 25 227 50 71 51 50 671 0 643 8 25 46 3 79 S G1 G2 1 00 125 0 108 5 5 66 4 53 13 03 12 88 206 8 1795 10 25 4 96 sa H1 H2 1 00 35 0 18 5 1 41 4 04 3 21 3 22 67 0 39 8 1 41 241 x1 A3 A4 A5 1 00 212168 21200 3 379 40 179 39837 06 19112 8 19085 6 605 43 347 B3 B4 B5 1 00 16765 5 16749 0 495 37 2 95 11991 89 14578 2 14550 8 563 98 3 87 C3 C4 C5 1 00 10130 7 10114 2 247 76 245 3044 84 9434 8 9407 6 121 20 1 28 D3 D4 D5 1 00 4830 2 4813 7 325 05 5 73 858 28 4933 5 4905 3 95 08 1 93 E3 E4 65 1 00 1551 2 1534 7 37 65 243 199 84 1958 0 1930 8 32 05 1 64 F3 F4F5 1 00 4105 394 0 12 82 342 45 88 548 8 521 5 17 75 274 G3 64 65 1 00 112 3 958 5 51 4 90 11 63 197 3 170 1 3 79 1 92 e i e e iS cm en e Figure 124 Multiple analyte table view 129 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 130 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 Click the Organize by Group button or go to the Table Options menu and select Organize Table by Sample Groups to highlight the divisions between defined groups see page 69 in the report NOTE Type and Group selections will override the So
76. 0000 00 eee 176 Clinician 2 Users Adjusting the Number of Unknown Samples WL EOLODOL apheresis 177 Generating a Results File from a Protocol File 178 PRUNING GAN tcu eese em ease eleme a esie ae ees grietas dees curas a tua 179 Viewing the Audit Trail 0 0 ce 181 Protected Directories 000 cc ee 182 Locking Bio Plex Manager Security Edition 0000 cee eee 183 LOOGING Oi NEN rM 183 Chapter 10 Standard Curve Optimizer 185 sis KONOUING IT Perm 186 Standard Curve Recovery 2 000 cece eee ee eee 186 Concentration in Range 0 0 cece eee 187 Determining OUUErS i d dedo Se ERU sae owe od eee we nd ee aes 187 Recommended Use 0 00 eee ees 187 Standard Curve Optimizer Report 2 000 eee eee eee 188 Fit Probability and Residual Variance 00 eee ee aes 190 LOGISIG WCIOMING ess ge aov wate had Weta et QU qa eee 191 Chapter 11 Data Normalization 193 Normalization Settings 0 0 eee eee ee 193 Selecting Internal Control s Housekeeping Gene s 194 Assigning a Control Sample llle 195 Report Table Options llle 196 Predefined Normalization Report Schemes LL 197 Normalization Definitions celles 197 Calculations Based Upon Your Settings L 198 Normalization Factor Calculations 0 000 cece eee eee 199
77. 07 Scaling the Histogram Data 107 Selecting the Channel Type 106 Histogram and Bead Map 104 Instrument Information 43 Instrument Operations Log 45 L Logistic Weighting 88 Luminex xPONENT software compatibility with Bio Plex Manager 6 Manually Stopping a Reading 103 Microsphere Handling 221 Agitation During Assay 223 Bath Sonicator 221 Dispersion 221 Enumeration of Microsphere Suspensions 222 Separation Methods 222 Stability and Storage 223 Normalization Definitions 197 Normalization Formulas 215 Normalization Settings 193 Normalized Fluorescence Values Norm FI 217 O Obs Exp 100 Column 134 p Performing a Validation Run 38 Plate Formatting 63 Plate Groupings 69 Plate Loading Guidelines 101 Predefined Normalization Schemes 197 Preparing Protocols Step 1 Describe Protocol 53 Step 2 Select Analytes 53 Step 3 Format Plate 62 Step 4 Enter Standards Info 72 Step 5 Enter Controls Info 90 Step 6 Enter Sample Info 92 Prime 44 Printing Graphs 158 Printing the Plate Format 71 229 Bio Plex Manager 6 0 Software User Manual Raw Data Exporting 125 Printing 126 Setting the Plate ID 124 Table Error Codes 126 Raw Data Table 112 Bead Statistics 113 Copying the Raw Data 115 Display Options 114 Formatting 115 Sampling Error Codes 113 Set Number Format 115 Recovery Percentage Range 89 Regression Methods 87 Report Table Concentration in Range Column 134 Context Menus 136 Copying 137 Display Options 1
78. 155 ST D Giai 257200 111 L3 Re BOSS D EY B42 S 125 5410027 1131540 123 4257 04125 E3 K5 3T6 5 100 1150 1230 81 0 131 754 D 145 451 0 129 Figure 107 Raw data from a reading During a reading the table is filled in line by line as each well is read After the reading is complete all the data are displayed In the table the fluorescence intensities Fl of the analytes you selected are listed for each well number NOTE During a reading the array reader always detects all the analytes in the sample including any that you have not selected However analytes that were not selected in the Protocol are not included in the table even if they were detected After a reading you can go back and change your selections under Select Analytes and the new analytes if detected will appear in the table For online Help information about the Raw Data table click the Display Help for Table Legend button E4l Raw Data Table Bead Statistics To display statistical data for the individual beads as well as the entire well AA 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 Human IL 6 19 Human IFH gamma 21 10744 00 65 11739 00 75 Fluorescent intensity Bead count Figure 108 Analyte fluorescent intensity and bead count With Show Hide Bead Statistics Columns selected the
79. 187 50 2684 00 5861 25 4663 25 6083 00 3 Format Plate 2065 88 2546 88 671 00 1465 31 1165 81 1520 75 2 Select Analytes SEI m 516 47 636 72 167 75 366 33 291 45 380 19 nr Logistic Weighting 129 12 159 18 41 94 91 58 72 86 95 05 i Compra Stuoh Gee for anne 3228 3979 10 48 22 90 18 22 23 76 4 Enter Standards Info 8 07 9 95 2 62 572 4 55 5 94 Concentration Units 2 02 2 49 0 66 1 43 144 1 49 5 5 Enter Controls Info v Same units for all analytes Units don t impact calculations r 1 Acceptable Recovery Range 70 130 v Calculate Concentrations Most Concentrated Standard 51 Oss Concentration of 1 Hu IL 13 51 v Apply dilution to all analytes Dilution Factor X Calculate 7 Run Protocol C Apply same concentrations to all analytes mm B Enter Sample Info Same recovery range for all analytes Figure 75 Bio Plex Manager calculates concentrations f Bio Plex Manager 6 0 Software User Manual 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 P Protocol2 Enter Standards Info puppes Standards Info External Standards Info Select External Standards Standard Lot Analyte 1 Describe Protocol Hu IL 2 38 vj
80. 2 To uninstall Bio Plex Manager from your computer use the Windows Add Remove Programs function Click the Windows Start button select Settings select Control Panel double click Add Remove Programs and follow the instructions for removing the program Bio Plex Manager 6 0 Software User Manual 4 Starting the System Before starting Bio Plex Manager 6 0 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 NOTE Avoid using other applications while Bio Plex Manager is communicating with the array reader Running other applications may interrupt communication between Bio Plex Manager and the array reader Starting Bio Plex Manager es To start Bio Plex Manager click the application icon on your desktop PA or select Bio Plex Manager 6 0 from the Programs directory on your Windows Start menu The software opens and attempts to connect to the array reader and platform 13 Bio Plex Manager 6 0 Software User Manual 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
81. 2 Aggregated beads Bead clumping in the assay sheath fluid is empty waste reservoir is overfull 3 Classify efficiency Microbubbles in the cuvette beads have become photobleached percentage of beads outside the selected bead region is too high 4 Region selection Incorrect beads were selected in the Protocol or assay too few beads are present in the assay 5 Platform temperature Platform temperature has fluctuated 2 C during the reading See Troubleshooting on page 201 for a detailed list of reading errors causes and suggested solutions With the checkboxes selected the reading stops and an alert box describes the problem and suggests steps for correcting it After you have performed the suggested steps you can rerun the reading NOTE If you are rerunning wells in Rerun Recovery Mode you may want to disable the Low bead number checkbox because wells that have already been read once contain fewer beads 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 In the Advanced Run Settings dialog the default DD Gates range for the 100 region map is dependent on the type of bead used For the 5
82. 21105 1147 5 1981 328 5187 5 E1 hu diabetes antigen standard S5 24 1319 2527 1301 158 456 146 1556 b F1 hu diabetes antigen standard S5 15 425 510 224 51 111 99 372 G1 hu diabetes antigen standard S7 11 129 5 253 92 40 40 101 165 5 8 H1 hu diabetes antigen standard S8 12 70 5 182 84 39 29 5 100 123 9 A2 hu diabetes antigen standard S1 12634 22531 5 19942 29294 6916 27019 5 23899 24001 5 10 B2 hu diabetes antigen standard 52 7067 13982 17880 28545 5 5470 21758 5 6543 19714 11 C2 hu diabetes antigen standard S3 1367 5 7600 15055 26967 45925 7339 1213 13429 12 D2 hu diabetes antigen standard 54 118 3348 7424 5 10319 geil 1587 285 5383 13 E2 hu diabetes antigen standard 55 22 1204 2205 1109 5 1315 392 129 1390 5 14 F2 hu diabetes antigen standard S6 14 400 600 232 52 102 101 363 5 15 62 hu diabetes antigen standard S7 13 119 5 251 104 42 36 92 149 5 16 H2 hu diabetes antigen standard 58 14 60 172 Bb 44 22 108 115 5 17 A3 lt B gt 14 56 150 5 56 38 18 Bb 89 5 18 B3 QC1 293 5497 10827 5 23585 2459 5 3394 499 8689 5 19 C3 QC2 33 1830 3589 5 3322 276 5 690 5 171 5 24515 20 D3 hu serum VVTS 159 364 5 1333 101 68 103 5 208 5 1223 AZ 01 FR hi carm WTO 39 Sna 16315 R7 Ra 421 5 110 5 181 M M 4 Human Diabetes 12 Plex lt li gt Figure 165 Well labels and Analyte labels 163 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 164 Using Stylesheets Stylesheets allow unlimited cust
83. 28 Displaying Table Error Codes 130 Exclude Table Error Codes 141 Expand Replicate Info 130 Exporting 137 Organizing by Type or Group 130 Predefined Report Schemes 129 Printing 137 Resizing the Columns 136 Saving Settings 131 Selecting Columns to Display 128 Setting Number Formats 131 Showing Hiding Outliers 135 Single or Multiple Analyte Layout 129 Toolbar 127 Report Table Column Descriptions 132 Reporting Problems to Bio Rad 225 Resequencing Well Numbers 66 Reservoir Functions 96 Run Protocol Window 94 Bead Count 95 Sample Timeout 95 Running Protocols 93 Running the Protocol 102 Status Bar 103 230 S Sample Needle Adjustment 18 Sanitize 44 Security Edition 21 CFR Part 11 168 Audit Trail 179 Calibration Logs 173 Document ID Number 176 Electronic Records 172 Enabling Secure Mode 171 Features 167 File Security and Validation 172 Installing 169 Instrument Operations Logs 173 Locked symbol shows Secure Mode 17 Locking Bio Plex Manager 183 Logging Off 183 Secure Protocol and Results Files 173 Signed vs Unsigned Files 173 Standard Mode vs Secure Mode 168 System Requirements 169 Unlocked symbol shows Standard Mode 17 User Access by Function 211 User Authentication 171 User Information 170 User Level Restrictions 170 Users Passwords and User Levels 169 Validation Logs 173 Selecting Analytes 54 Selecting External Standards 81 Selecting Entering Calibration Control Numbers 26 Software Editions Security Edition 5 S
84. 3 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 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 Hu a li F H l mE EE m a a Figure 153 Marquee to zoom into a graph Right clicking any results graph displays a context menu to access many functions described in this chapter edt OOOO Delete Options Export Copy Save as Bitmap Print Figure 154 Right click context menu Graphing Function Adding New Graphs To create additional graphs click the Add Graph button Add Graph Graph Name Samples pese EE Description Wells Group Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 mmm FI 2 ges 4 7 8 TEE 11 14 LI L E L dl dl C L dl LI L dl Standard 8 A3 A4 A5 B3 B4 B5 C3 C4 C5 D3 D4 D5 E3 E4 E5 F3 F4 F5 G3 64 65 H3 H4 H5 A1 A2 B1 B2 C1 C2 D1 D2 E1 E2 F1 F2 61 62 H1 H2 A6 B6 C6 D6 ali on the toolbar Analytes Analyte Mo IL 1a 53 Mo IL 1b 19 Mo IL 2 36 Mo IL 3 18 Mo IL 4 32 Mo IL 5 52 Mo IL 6 38 Mo IL 9 33 Mo IL 10 56 Mo IL 12 p40 76 Mo IL 12 p70 58 Mo IL 13 37 Mo IL 17 72 Mo Eotaxin
85. 3 4 wis 5 e 2 1a e L 1L TG Figure 56 Defining multiple sample wells with Autofill Note that Autofill Across numbers the wells sequentially left to right then top to bottom while Autofill Down numbers them sequentially top to bottom then left to right Protocol Window DEFINING A REPLICATE GROUP 111 To define multiple wells as a replicate group select Turn Off Autofill e 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 Plate Farmatting Plate Groupings m Figure 57 Defining a replicate group of unknowns For each replicate group a mean value is calculated along with the standard deviation standard error and coefficient of variation AUTOFILLING REPLICATE GROUPS If you have multiple replicate groups of wells in rows or columns you can autofill these by selecting Autofill Across or Autofill Down as before and then entering the number of wells in the replicate group up to 96 in the number field next to the autofill buttons You can also select well numbers up to 10 from the pulldown menu 1134 122 1 o ae I3 WM Figure 58 Entering the number of replicate wells in an Autofill sequence Now when you drag your cursor the sequential sets of replicate wells are defined Plate Formatting Plate Groupings Pa JL 1L TR Figure 59 Autofilling a sequence of rep
86. 46 F storage and allow them to warm to room temperature Vortex each bottle for 30 seconds Proper suspension of the microspheres is essential for efficient calibration e Never dilute the calibration beads and be careful to limit their exposure to light Store at 2 to 8 C immediately following calibration e Load 6 drops of beads approximately 200 ul per reservoir CAL 1 or CAL2 e The DI H2O well of the MCV Plate IV holds about 3 ml of distilled or deionized water 29 Bio Plex Manager 6 0 Software User Manual 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 As calibration proceeds the status bar at the bottom of the Bio Plex Manager window monitors the progress of calibration The number of beads per second should be 100 or higher Fewer beads per second may indicate a problem with the fluidics system Number of beads Region o Gate 31 79 Total 70065 p Beads per second Region o Gate 2g Total 215 Number of beads per second should be 100 or higher Figure 20 Status bar during calibration Because calibration beads are highly concentrated calibration is followed by three wash cycles using distilled water An alert box notifies you whether calibration has succ
87. 5 31 48 83 12 21 12500 00 3125 00 781 25 195 31 48 83 12 21 12500 00 3125 00 781 25 195 31 48 83 12 21 Calculate Concentrations Most Concentrated Standard 51 Apply dilution to all analytes v Apply same concentrations to all analytes Concentration of 1 50000 Hu IL 2 38 Dilution Factor x Clear All Concentrations Ose 19 Bio Plex Manager 6 0 Software User Manual 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 PH Protocol2 Enter Standards Info DER Protocol Settings Standards Info External Standards Info Select External Standards Standard Lot Analyte _ Lot E i mE Expiration Date Hu 7 Hu IL 2 38 Std Regression Curve Load Save Manage Standard Lots Help Regression Type Assign Standards Information Description Hu IL 2 38 Hu 52 Hu IL 5 33 Hu IL 10 56 Hu IL 12 p70 75 Hu IL 13 51 Logistic 5PL Axis Transformation Loa x Linear y 00 f Logistic Weighting Same regression type for all analytes Concentration Units v Same units for all analytes Units don t impact calculations Acceptable Recovery Range 70 130 sv v Same recovery range for all analytes Calculate Concentrations 1 Most Concentrated
88. 5th attempt call Technical Support 6 Be sure to calibrate with freshly vortexed beads and a clean MCV IV plate 7 Repeat calibration and record bead flow optimum flow should be 200 beads second If problem persists record any additional error messages such as temperature problem pressure fluctuation warm up cancellation before calling tech support Calibration procedure failed due to low bead number Problem with optical component of array reader Repeat validation procedure If values are still out of range contact Technical Support Problem with fluidics component Repeat validation procedure If values of array reader are still out of range contact Technical Support Problem with optical component of array reader Repeat validation procedure If values are still out of range contact Technical Support Problem with calibration or optical component of array reader Repeat validation procedure If values are still out of range contact Technical Support Bio Plex Manager 6 0 Software User Manual Glossary Term Accuracy Aggregated microspheres Analyte Antibody Assay Background noise Bead Calibration Calibrators Capture antibody Carboxylated microspheres Classification channels Classification regions Classify validation Clumping Definition The closeness of a measured value to the actual value or the difference between expected and measured values Usi
89. 9 Cancel Figure 177 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 For example a template with 48 wells defined as unknowns and four wells per replicate will have a maximum number of 12 samples Enter the number of samples to use in a particular reading in the field and click OK The plate format will change to indicate the new number and layout of the sample wells Only those wells will be read during a reading NOTE You can specify only the number of unknown samples using this command not standards controls or blanks If you save the Protocol file after changing the number of unknown samples the number of samples you specified will become the new maximum number of samples To preserve the original number of unknown samples save the changed Protocol file under a different name Generating a Results File from a Protocol File The Results file must be signed before a reading can begin In Secure Mode the Auto Save After Run checkbox in the Run Protocol window see page 94 is automatically selected and cannot be deselected You are prompted to sign and save the Results file at the start of each reading Audit Trail NOTE In Secure Mode each reading including reruns of plate wells generates a new Results file that cannot be overwritten Electronic Signatur
90. AVING PretecolS uersum dodici aureae ca dug torte os ae ud ache 48 Reducing the File Size of ProtocolS nanna ce eee ees 48 Multi Assay Protocols tenses anaes cae wanes he whee S RE Rd 50 Uses for Multi Assay Protocols cee ees 50 Creating a Multi Assay Protocol 0 0000 c eee eee ees 51 Changing a Protocol from Single to Multi Assay 51 PrOtOCOI VWVINGOW Pr P a eS a eee ee An eA See ee eee ee 52 Step 1 Describe Protocol cra sanra eieaa N eee 53 Step 2 Select Analytes 0 0 0 0 cc ees 53 Step 3 Format Plate 22s ewes Ge Ra ER ERROR ea SUR ACERO ee 62 Plate GIOUPIPOS s ace 3 os Beet Ru MES se ESU P ER RI EE EE 69 Step 4 Enter Standards Info llle 72 Standard MOUS ecco urs eru See Per E Se ae UR NES S 73 Step 5 Enter Controls Info Optional llle 90 Step 6 Enter Sample Info Optional lll 92 Chapter 7 Running Protocols e 93 RUN FrFotocol VVId OW xod det ose a ARRA E ed aa 94 Bead C OU edu acids etie at ei dra etude it e sede pe Ge 95 Sample TIMEOUT x ord ee ecu d rei aea cae cod my hve ac et tn pe e td n 95 RESEMOMEUNGCIONS wide netetekee die dodo dete io GO se die be de bee wd 96 SKOF UNCION S osa ace Ecran on eB es avis ac d o Uc oe ct E a t i 96 Advanced AUN Serunds sawu i dori uA doo Se put he oso ius ew o owe es 98 Bead Map Selection 2 0 ec eee 98 Sable SIZE son eu erT hee ee amenes R
91. Add 7096 isopropanol and distilled water to the appropriate reservoirs insert the plate in the microplate platform and click OK This procedure performs a series of fluidics operations and takes several minutes After initial system pressurization 5 to 20 seconds the time remaining in the operation displays in the Bio Plex Manager status bar 33 Bio Plex Manager 6 0 Software User Manual Controlling the System Unclog amp 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 E on the main toolbar or select the command from the Instrument menu An instruction box guides you through preparation of the MCV Plate IV MCV Plate IV Unclog removes obstructions from the fluidics 1 Fill 70 isopropanol and DI H20 reservoirs 2 Load 5 drops of CAL1 beads 3 Click Eject then load MC plate 4 Press OK to start 4 Eiect Retract Plate Figure 24 Steps for unclogging the fluidics system 2 Add 7096 isopropanol solution and distilled water to the appropriate reservoirs 3 Add 5 drops of CAL1 beads to the CAL1 reservoir 4 Insert the plate in the microplate platform and click OK This procedure performs a series of fluidics operations and reads a sample of CAL1 beads to verify that the fluidics are operating properly It takes several minutes After initial sys
92. CP 1 MCAF Hu MIP 1a Hu PDGF bb Hu MIP 1b Hu RANTES Hu TNF a 7 Run Protocol Hu VEGF 6 Enter Sample Info 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 m o pbm E New Panel csv Bnew test CSV My Recent Documents ES C File name Cancel CSV Files csv w Files of type Figure 48 Importing the CSV File 58 Protocol Window 6 The new panel will be added to the list of panels in Bio Plex Manager Add Panel Panel name Mew Panel Mame Assay Bio Plex Pra Assay Magnetic w Analyte Analyte 1 Analyte 2 Analyte 3 Analyte 4 Analyte 5 Analyte 6 Analyte Analyte 8 Analyte 9 Analyte 17 Analyte 18 Analyte 13 Analyte 20 Analyte 21 wc J CD on ee to POL Figure 49 Imported Panel DK Cancel Import Add Edit Remove Remove AI E 59 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 60 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 Edit Panel Panel name 30plex IVT panel OGP 2 0 UR Assay Bio Plex Assay Mon M agnetic hl Cancel Region Analyte IL1B CPE TNFASF6E CP41 Add BAD CPB58 CyP2B6 CP32 CSF2 CP53
93. CV The normalized standard deviation Norm Ratio Std Dev divided by the normalized ratio of the sample s replicate group e Norm Fl Normalized fluorescent intensity See Normalization Formulas page 215 in the Appendix for details about the formulas used for this calculation e Norm FI Std Dev Standard deviation of the Norm FI values for all the wells within a sample s replicate group e Norm FI Std Err The normalized standard deviation Norm Fl Std Dev divided by the square root of the number of replicate wells within the sample s replicate group e Norm Fl CV The normalized standard deviation Norm Fl Std Dev divided by the normalized Fl of the sample s replicate group 197 Bio Plex Manager Software User Manual Normalization Settings 198 Calculations Based Upon Your Settings Norm Ratio Normalized Fl Bkgd ratio The following table is based on the selections made in Normalization Options It also describes how the Norm Ratio is calculated Case tilly MS How Norm Ratio is Calculated on MS A Single None Ratio of Fl Bkgd for the analyte and the housekeeping gene Norm Ratio Fl Bkgd Fipk Bkgdhk B m Geometric mean of the normalized FI m a ratios for each housekeeping gene C mE Single Normalized Fl Bkgd ratio Fl Bkgd control ratio for the control as defined in Case A Norm Ratio Norm Ratio single gene Norm Ratio control D Multiple Single Ratio multiple i
94. Delete Rename Edit Panel x X o Functions that Apply to Results File Format Plate View Functions that Apply to Results File Enter Standards Info View Enter Standards Info Description amp Concentration Enter Std Curve Reg Method Enter Std Curve Advanced Settings 213 Bio Plex Manager 6 0 Software User Guide Appendix Enter Standard Concentration X X Units Enter Recovery Range L3 1X 0 Select External Standards FL qo X LX d Functions that Apply to Results File Enter Sample Info View Enter Sample Info Dilution EO WORX CETO i Functions that Apply to Results File Enter Controls Info View Enter Controls Info Desc Conc X X amp Dilution Functions that Apply to Results File Raw Data View Change Display Options Set Clear Outliers Functions that Apply to Results File Report Data View Show Hide Outliers Report Table Options Organize by Type Organize by Group x un Show Replicates Sort Show Help for Table Legend Functions that Apply to Results File Standard Curve View Regression Type BEEMNNE NEN WENN NNNM Same Reg Type All Analytes i Ht RT er n X X WK X X X X Xx x lt x lt x lt x lt x Logistic Weighting Options Labels Error Bars dL e X X X X Xx NOTE System Administrator creates Groups and assigns Users to Groups 214 Normalization Formulas Normalization Formulas Bio Plex M
95. E WI REe v REEF Qd eR Rss 98 5ave ODLIIONS x oun ded oat de So oa bat EXE qne EC e ER Dern E oo d 99 Sampling EN OMS ado uw cots eee eee Meta he eae 99 Doublet Discriminator Gates 0 0000 eee 100 Plate Loading Guidellli6S 1 3 9 cee abeo a eee ace ao e FA o ed 101 RUNNING trie Protocols s siseatacaetc hrs oh No E NIA ee soa Oe oe BUR Boc ee eno a 102 tatus Bahi uut dite sa re hte ae BOGS doe S UM ceo eee 103 Manually Stopping a Reading llle 103 Generating Results from a Protocol l l 103 Bio Plex Manager 6 0 Software User Manual Table of Contents Histogram and Bead Map 00 eee eee 104 PISTO A mE Rr tien of be Bsa each Ree AeA ace we ee oan Meee 105 PCAC VIA uuo ads desque Shee do arto tant eaters et Tear d een d 108 Bead Map Display Options 0 000 ee eee 110 Haw Data Tapie acs cabs a eles quod o deo oe ae ae HARUM Ed 112 PCAC SASH CS ur i dus bac tid d aer eunt f IR va ante tana sat tp as 113 Sampling Enor Codes i uud dice daos Do bw toa Sees Kee heard 113 Raw Data Display Options llle 114 Set Number Format 0 000 cece rs 115 Other Table Formatting 00000 e eee eee eee 115 Copying the Raw Dala sius so deb Ru esi exu EROR ERES 115 Rerun Recovery Mode l l rss 116 Chapter 8 Analyzing the Results 117 RESUS OGL Wie atte el ara Bat teat ee ae pista ae malian eee eee eee ae ae ae 117 Opening a Results
96. E dit TNFSF6 CP56 ILS CP35 PPIB CP54 Bemus VEGF CF29 ABCC CPA Remove All IL10 CP5 GAPD CP33 ABCB1 CP64 CYP354 CP43 SLC22A 7 CPET CyP246 CP71 CYP1A2 CP43 IL2 CP72 PTE23B CP30 ILER CF21 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 If you change the Panel Name the changes are saved under the new name and the old panel will not be overwritten NOTE The preconfigured Bio Plex panels Human Cytokines Mouse Cytokines etc can be edited but not overwritten therefore you must change the panel name to preserve your changes When you are satisfied with your changes click OK 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 x You are asked if you are sure you want to delete the panel Bio Plex Manager This will delete panel Sw panel Do you want to proceed 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 La 3 Enter a new name in the pop up dialog box and click OK Rename Panel New panel name SW panel Figure 52 Rename Panel Dialog COMBINING PANELS OF ANALYTES To combine entire panels of analytes for example Human Cy
97. File cc ee eee 118 Saving a Results File 0 0 cc eee 118 Reducing the Size of a Results File 0 000000 eee 118 Results VUTICIOW x dod Bake aha Sis Se te Stee Dir d se R 120 CUSTOM REDOITS e uie udo anal ave Gd eee eed ae eens An CR S ee ea 121 Viewing Changing the Protocol Settings 0005 122 Changing the Doublet Discriminator Gate Range 123 RaW Dala Cp Site tse ah teat teh fd ose ep es te at cele a nee Deal tae Ne M 124 setting the Plate ID owiehe De eae 124 Exporting the Raw Data 0 cc es 125 Raw Data Table Error Codes 0 0 eee 126 Printing the Raw Data 0 0 es 126 REDON ables 25 hue ie che dee doo pote Auro dS a ute E n ee re AT Du 127 SIO el mE NE E thes eyes Ab ae en He ae Nah BMG ayer eon Hale a a TM 127 DiS Dla yO DONS ape sso aoe death Varii dna anh s atdaed tds Da pe EMI E 128 Report Table Column Descriptions lel 132 ODS EXD 100 COMA uai cad ide ioo aes fcx dear ork dor Red 134 Concentration in Range Column 00000 cece eee eee 134 Showing Hiding Outliers llle 135 Resizing the Columns llle 136 CORLOXE MENUS s c uci oe Diu e bee Dow dg Sisto we dabei d diea pe d 136 Sorting Report Table Data llle 137 Copying the Report Table 0002 eee 137 Printing the Report Table 2 000 eee 137 Exporting the Report Table 0 02 00 c
98. IONS MANUALLY You can enter your concentrations manually The table lists your standard wells S1 S2 S3 etc These are the wells that you defined in the Format Plate window page 62 For the selected analyte enter the concentration for each standard in the concentration column Assign Standards Information 32000 00 a ra e vw NENNEN s 4000 00 000 HE HE ERENEEI Figure 82 Entering standard concentrations manually If the Apply same concentrations to all analytes checkbox is selected the concentration values you enter are applied to all the analyte standards Deselect the checkbox if you want to enter different concentrations for each analyte standard You can also enter additional information about your standards in the description column for each well NOTE You can use the Cut Copy and Paste commands on the Edit menu to Copy your descriptions and concentrations among analytes You can also copy and paste from and to other applications such as Microsoft Word or Excel Click a cell row or column in the table to copy and paste If you are entering different concentrations for each analyte standard repeat the procedure by selecting each analyte from the pulldown menu as described above 85 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 86 CALCULATING CONCENTRATIONS AUTOMATICALLY If a dilution series was created for the standards the software can automatically calc
99. If the specified bead count in the Beads field is not reached for a particular well that is because the well does not contain all of the selected analytes the array reader will continue the well reading until the time limit specified in the Sample Timeout field If no Sample Timeout is specified a default time based on the sample volume is used You can enter a time limit in seconds from O to 200 or make a selection from the pulldown list Select none or enter O if you want no time limit The Sample Timeout value for each well will be noted in the Raw Data table 95 Bio Plex Manager 6 0 Software User Manual Running Protocols Reservoir Functions The Bio Plex Manager 6 0 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 96 The functions below are available to the protocol before the run Before Plate Run after After Plate Run When running the protocol the status bar indicates which function is in process Hesernvoir Functions Esos Piste Bun Alber Plabe Pours replies si Heil Bebesmers Plates f Start Up 2 Free Akoh wach Exsck fushi Remcee Eats Seve os dies aulit For new protocol Figure 94 Reservoir Functions dialog The maintenance processes you can select with the reservoir function windows are Wash Prime
100. Mo IL 1a 53 S3 C1 C2 12515 3 12453 5 han An SP DA T4 nma roar r34nn Figure 133 Example of an Excel workbook single analyte layout Select Multiple Analyte Layout to display one or more columns of data for example Fl for all analytes in a single worksheet E3 Microsoft Excel Book1 SE rea File Edit View Insert Format Tools Data Window Help Adobe PDF Type a question for help amp x i aral 10 B Zz U amp j SL sedi Oe A z J32 Y amp 10 eae C D F G H 2 1 File Name C Program Files Bio Rad Bio Plex Manager 5 0 Example Files Mouse Cytokine 23 Plex Stai 2 Acquisition Date 09 Jun 2005 04 34 PM Reader Serial Number LX10004167313 RP1 PMT Volts 647 13 RP1 Target 3832 B Plate ID 7 Eg 8 Well Type Mo IL 1a 53 Mo IL 1b 19 Mo IL 2 36 Mo IL 3 18 Mo IL 4 32 Mo IL 5 52 Mo IL 38 N ENY 19961 13415 223035 197725 15599 20205 8645 5 B1 18710 5 7561 162055 153785 16538 21662 6389 C1 12875 3432 10588 9734 14598 153566 5 3697 D1 5615 970 4838 4865 7921 7358 5 1191 5 1819 235 1657 1941 5 4349 5 1748 609 5 72 443 5 653 1766 471 4723 4 li 1 md LIE aan 47 16094 mT Ca B ga a m M 4 gt An Mouse Cytokine 23 Plex Standard Ready Figure 134 Example of an Excel workbook Multiple Analyte Layout Select 96 Well Plate to display each analyte in a worksheet layed out like the wells in a 96 well plate rows A H and columns 1 12 138 Report Table No
101. Multiplex Suspension Array Bio Plex Manager Software 6 0 User Manual Bio Plex Manager 6 0 Software User Manual Bio Rad Laboratories Inc 2000 Alfred Nobel Drive Hercules CA 94547 1 800 424 6723 10018879 Rev A Bio Plex Manager 6 0 Software User Manual Bio RAD TECHNICAL SUPPORT DEPARTMENT The Bio Rad Technical Support Department in the United States 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 E mail LSG TechServ US Bio Rad com U S LSG TechServ Intl Bio Rad com International Web www consult bio rad com NOTICE No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopy recording or any information storage or retrieval system without permission in writing from Bio Rad Bio Rad reserves the right to modify its products and services at any time This user guide is subject to change without notice Although prepared to ensure accuracy Bio Rad assumes no liability for errors or omissions or for any damage resulting from the application or use of this information The following are trademarks of Bio Rad Laboratories Bio Rad Bio Plex and Bio Plex Manager Luminex xMAP and xPONENT are trademarks of Luminex Corporation Windows is a trademark of Micr
102. Obs Exp 100 EmorBar NONE v Analyte Hu C peptide 8 Mi Optimization Report Standard Curve Optimization Analyte Result Comments TERE Hu C peptide 8 Successful gt 1 Std Removed Logistic SPL Hu Ghrelin 58 Successful 10000 00 Hu GIP 73 Successful Regression Type Axis Transformation Hu GLP 1 32 Successful Log x Linearly R4 Hu Glucagon 54 Successful gt 1 Std Removed Hu IL 6 19 Successful Logistic Weighting Hu Insulin 77 Successful H Hu Leptin 71 Successful C Same regression type for all analytes ptin 71 C Swap XY Axes Hu PAI 1 50 Successful Hu Resistin 75 Successful 1 Std Removed Show Conc Range Lines Show Unknown Samples 5000 00 Hu TNF a 36 Successful Hu Visfatin 94 Successful 1 Std Removed Show Control Samples ri Exclude successful without comments Curve Fit All Optimized m Clear Outliers LLOGQ 27 138 S6 87 5 102 Apply across all analytes Show report after optimization Concentration 2 b amp gt a 7 o a eo f D o a 5 2 5 w 10000 00 100000 00 Wi Standard O Partial Outlier 6 Outlier amp Unknown A Control Std Curve Fl 1 22299 12365 8 1 22299 1 Conc 6455 05 2 08537 0 881492 FitProb 0 7574 ResVar 0 0954 Figure 188 Standard Curve Optimization Report Two types of information presented for each analyte Results and Comme
103. Rad Technical Support at the numbers listed on the inside front cover of this manual To use Solobug select Solobug from the Start menu enter your information and a description of the request in the Solobug window and click Save This will create a report that you can attach to an email and send to Bio Rad Please send your Solobug files to Bio Rad at LSG TechServ US Bio Rad com in the U S LSG TechServ Intl Bio Rad com International 225 Bio Plex Manager 6 0 Software User Guide Appendix Report Table Error Codes 226 Certain data that cannot be measured or calculated will be flagged in the Report Table Columns and their possible error codes are listed below Column Error Code Cause Fl ie No data present Marked as an outlier Fl Background i No data present negative values if any will be displayed Marked as an outlier SD iind No data present CV96 Hi No data present Obs Conc 4PL un No data present SPL OOR gt Out of Range Above asymptote of equation OOR lt Out of Range Below asymptote of equation Out of Range Math Error Value Above or below standards FI Obs Conc Linear TE No data present Negative extrapolated data Value Positive extrapolated data or Data above below the staqndards FI Exp Conc uo No data entry this value is input by user OBS Exp 100 oe OOR displayed in Obs Conc column Conc in Range OOR lt or Obs Conc is not within the specified recovery range OOR gt of stand
104. Secure Mode e Service Users at this level have full access to all features and functions of the software except that they cannot enable or disable Secure Mode e Clinician 2 Users at this level can perform instrument operations run existing protocols and view Protocol files Results files and log files They can also change the number of unknown samples in the plate format using the Set Number of Unknown Samples command and enter sample descriptions All other access is restricted e Clinician 1 Users at this level can perform instrument operations run protocols and view Protocol files Results files and log files but cannot change any settings All other access is restricted e Reviewer Users at this level can view and sign Protocol and Results files and can view log files All other access is restricted User Level Restrictions If you attempt to select a command or perform an action not allowed by your user level you receive a pop up warning In addition many fields and settings that are restricted by your user level appear grayed out in the application User Information To display information about the current user including user name domain full name and access level go to the Tools menu and select User Info You can also click the user name displayed in the application status bar User Information e BPSUPERVISOR User BPSupervisor Domain IPD_DOMAIN Full name BP SupervisorName Access Level
105. Software User Manual 3 Software Installation Install or reinstall Bio Plex Manager 6 0 software as described in the following section If you need to upgrade your software from a previous version see the Bio Plex Manager 6 0 Software Upgrade and Configuration Guide part 10018402 to determine the part number of your upgrade kit System Requirements Component Recommended Operating system Windows XP XP Professional required Windows XP Professional for running Security Edition Processor Pentium 4 or equivalent 2 8 GHz Core 2 2 6 GHz or higher Hard disk space 80 GB 160 GB Systemmemory je 2GB Screen resolution 1024 x 768 1280 x 1024 Screen colors 24 bit True Color Ports for connecting 1 RS232 serial port and 1 RS232 serial port and 1 USB 2 0 instrument required for 1 USB port port Instrument Control license only Port for connecting 1 USB port 1 USB 2 0 port the HASP key Other software Internet Explorer 6 0 or later Internet Explorer 8 0 NOTE Windows 7 has not been validated with Bio Plex Manager 6 0 Preliminary tests indicate that the Windows 7 operating system is capable of running the application and Bio Rad plans to fully validate Windows 7 in the near future Bio Plex Manager 6 0 Software User Manual 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 statu
106. Std Dev v Analyte lt Mo IL 1a 53 LA Zoom out PS Results Mo IL 1a 53 600 fe Raw Data ma Report Table 500 Wi Standard Curve 400 rp Graphs 300 200 100 0 HPP PPh qe ex dbods ees Se Figure 145 All Samples graph with Std Dev y axis Perform any data normalization functions before displaying your graphs 149 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 150 If you want to see ratios among samples or other normalization options click I E the Normalization Settings button See Data Normalization on page 193 for more information 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 Graph Options view Samples across a single analyte Samples across multiple analytes Analytes across a single sample C Adjust graphs to same Y axis scale Analytes across multiple samples Display X Axis Bar 2D Effect Labels orientation 9 Degrees Sample Y Axis Logarithmic Scale Type Figure 146 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 Choose either Type or Description from the Sample area at the bottom of the Graph Options dialog shown above Graphing Function Under the Graph Options dialog
107. Supervisor Date 25 Sep 2007 04 47 PM Temp Celsius 23 96 Select Calibration type CAL1 amp CAL2 CALI Only CAL2 Only Select control numbers CAL1 Control Number CAL2 Control Number CAL1 A5449 v CAL2 A5450 DD Target CL1 Target CL2 Target Low AP1 Target 5829 3570 3845 3515 Expiration Date Not Assigned Expiration Date Not Assigned Note The displayed target values should match the targets on the CAL1 amp CAL2 bottles Figure 15 Calibrate dialog At the top of the dialog box enter your name in the field If you are using the Security Edition of the software in Secure Mode your user name will be listed and grayed out The time and date of the last calibration using Bio Plex Manager are listed as is the temperature at the time of that calibration If the temperature has not changed by more than 2 C in a single day it is not necessary to recalibrate the instrument Next select the calibration type by clicking on the CAL1 amp CAL2 CAL1 Only or CAL2 Only button You should perform both CAL1 and CAL2 calibration daily Selecting Entering Calibration Control Numbers Under Select Control Numbers in the Calibrate dialog box you can either select existing control numbers for your CAL1 and CAL2 microspheres or enter new control numbers 26 Calibration Select existing control numbers from the pulldown list and the target values for the control numbers appear in the appropriate fields
108. There are two context menus available from the Report Table Right clicking within a header cell brings up a menu that allows you to show hide a column abbreviate analyte names or view the Report Table Options dialog Mo IL 1a 53 amm E E TT A JUI M how Column Abbreviate Analyte Mame Report Table Options TT Bi ee ie 110 34 114 25 Ob _ TTT TL 1 Figure 130 Report Table header context menu Right clicking within a body cell in the Report Table brings up a context menu that allows you to set a Plate ID set a specific analyte as an outlier or view the Report Table Options dialog THFRSF6 CPH 17 Es Fb Horm Ratio Set Plate ID Set Outlier TNFRSF6 CP41 17 Set Outlier All Analytes lt Report Table Options Figure 131 Report Table cell context menu 136 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 ral Clicking twice will invert the order 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 Printing the Report Table You can print the Report Table for the selected analyte or multiple analytes when in Multiple Analyte Layout using the
109. UX E 59 Ag Dr Wan E BIN 77 REESE EN RE RR Standard 1 d i 25193 31 Standard 2 i i j i 7602 28 Standard 3 A 1 2198 71 Standard 4 J i 1 j J 464 69 Standard 5 i j 132 86 Standard 6 p J 31 80 Standard 7 i j i j 747 Standard 6 f i 1 98 D4 E4 F4 Sample 1 15 16 j 23 04 AB G4 H4 Sample 2 113 95 i 127 03 B8 C8 D8 Sample 3 J 156 99 516 44 ojjj i eo ro Figure 123 Selecting an analyte for a Single Analyte Report Table Click the Multiple Analyte Layout button mm in the toolbar or choose Layout Table with Multiple Analytes from the Table Options menu The multiple analyte view shows columns for all analytes Use the horizontal scroll bar at the bottom of the window to scroll sideways through analytes Bio Plex Manager Mouse Cytokine 23 Plex Standard PMT Read Only Report Table ig File Edit View Instrument Table Options Window Help Dies O 3 pz a g suc uxE 5 MS HH Be g Y TEE a a lt a Mois aa A __ type en nies H riora Sew cv obs Cone 03 Cone Obee s Begs Estes cv Obs B A6 66 C6 D6 1 00 165 97 66 273 273 20 77 76 24 31 A1 A2 1 00 221 a 22138 3 5 0 95 56406 33 52736 00 19394 3 19367 0 534 93 276 321 2 B1 B2 1 00 16865 8 16849 3 933 73 554 12266 13 13184 00 15129 8 151025 351 79 2 33 yi s3 C1 C2 1 00 10730 0 107135 200 82 1 87 3442 45 3296 00 9697 0 9669 8 52 33 0 54 2 S4 D1 D2 1 00 4639 3 4622 8 281 07 6 06 809 61 824 0
110. Use the up down arrow keys to highlight and change individual date components 2f Bio Plex Manager 6 0 Software User Manual 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 To add a new CAL2 control number click the Add button under CAL2 Control Number in the Calibrate dialog box The Add New CAL2 Control Number dialog box opens Add New CAL Control Number Enter Control Number A5450 Expiration Date None Enter Target Values CAL1 CAL2 DD Target CL1 Target CL2 Target Low R Eisi arget 3515 Figure 18 Adding a new CAL2 control number In the dialog enter the control number from the CAL2 bottle in the field Select the expiration date if printed on the bottle as described above Next enter the Low RP1 target value for the control number as printed on the bottle in the RP1 field NOTE The CAL2 calibration bottle label lists two RP1 also known as PMT for Photomultiplier tube target values Low RP1 and High RP1 The High RP1 target value cannot be used in Bio Plex Manager 5 0 or higher All calibration is done at the Low RP1 target value which must fall be
111. WE eee hee Ke 160 Output CSV File Format ivi cava dare LEER xac wx EE doe 162 USING Stylesheets 44 222 2 3 o EAE SUE ER cee een CE A PC s 164 Command Line Export 0 cece rns 165 Exporting the DOCUMENTED 3 5 92x DRE PP oV RU ENS ERO EUR 166 Chapter 9 Using the Security Edition 167 Bio Plex Manager Security Edition 0 0 00 167 Background on 21 CFR Part 11 0 0 00 cece eee 168 Standard Mode vs Secure Mode eller 168 System Requirements 0 0000 eee 169 Installing and Starting Bio Plex Manager Security Edition 169 Users Passwords and User LevelS 0 0000 eee eee ee ee eee 169 User Level Restrictions 0 0 ccc ees 170 User Information 2 0 0 cc eee eee 170 Enabling and Disabling Secure Mode 000s eee eee eee eee 171 User Authentication uu wicks cscs Auietie Sica d tt Shara cera weche howe hes wp ew rm od da 171 Electronic HecoFdS seassa ceca hee ated be bed lode dA RE EY 172 vii Bio Plex Manager 6 0 Software User Manual Table of Contents File Security and Validation 0 0 0 cece eee 172 Calibration Validation and Instrument Operations Logs 173 Secure Protocol and Results FileS 0 0000 eee ee eee 173 Unsigned Protocol and Results Files 0000000 ee 173 Signed Protocol and Results Files llle 174 Document ID Number and Signature
112. Xohn Test Unrestricted Run Sample Canceled 03 Oct 2007 05 05 PM LSGXP 01006334Mohn Test Unrestricted Run Sample Canceled 03 Oct 2007 05 04 PM LSGXP 01008334Xohn Test Unrestricted Run Sample Canceled 03 Oct 2007 05 04 PM LSGXP 01006334Xohn Test Unrestricted Warm Up Canceled lt Done Figure 37 Instrument Operations Log Viewer Each instrument operation 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 operation was performed To print the log click the Print button To copy the information in the log to the Windows clipboard and paste it into another document drag your cursor over the rows and columns in the table to select them and use the Ctrl C key command to copy the data Bio Plex Manager 6 0 Software User Manual 6 Preparing Protocols Protocol Files A Protocol file contains the parameters of a Bio Plex Manager 6 0 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 When the reading is complete the parameters in the Protocol file are copied into the Results file along with the data from the reading You can save Protocol files and reuse or modify them for other readings Raw data from the most recent reading is stored in the Protocol
113. able Recovery Range 70 130 Same recovery range for all analytes 3 Format Plate 1 4 Enter Standards Info 1 5 Enter Controls Info Calculate Concentrations Most Concentrated Standard 1 Apply dilution to all analytes v Apply same concentrations to all analytes 6 Enter Sample Info Concentration of 1 50000 Hu IL 2 38 Oss 7 Run Protocol 4 The software calculates all the concentrations for each analyte and at each dilution as seen below PY Protocol2 Enter Standards Info Protocol Settings 1 Describe Protocol 2 Select Analytes 3 Format Plate 4 Enter Standards Info 5 Enter Controls Info B Enter Sample Info 7 Run Protocol Standards Info External Standards Info Select External Standards Standard Lot Lot Assign Standards Information Analyte Std Regression Curve Expiration D ate Regression Type Logistic 5PL 50000 00 50000 00 50000 00 12500 00 12500 00 12500 00 3125 00 3125 00 3125 00 78125 781 25 78125 195 31 195 31 195 31 48 83 48 83 48 83 12 21 12 24 12 24 Concentration Units E 3 05 v Same units for all analytes Units don t impact calculations Acceptable Recovery Range 70 130 v v Same recovery range for all analytes Axis Transformation Log x Linear y Same regression type for all analytes 12500 00 3125 00 781 25 19
114. ager 4 1 1 Example Signed By BP SupervisorMame Sign Date 04 Oct 2007 12 04 PM Document ID C3ES3AD615194BDO0B10BABOSSF3S3 rC C Figure 174 Document Properties dialog The Document Properties dialog also provides information about the signature The full name of the user who signed the document is displayed in the title bar of the File window XI Rat Cytokine 9 Plex High PMT RPMI_001 Read Only Report Table signed by BP SupervisorName Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Sample 1 Sample 2 Sample 4 Sample 3 D4 E4 F4 D8 G4 H4 AB B8 C8 E8F8 G8 Figure 175 Signed and read only status of file shown in title bar Calibration Validation and Instrument Operations Logs All printouts and exports of a signed document also include the signature and Document ID number E3 Microsoft Excel Book1 dE File Edit View Insert Format Tools Data Window Help Type P Q 100 Arial ihe TO Bb a C LH i Snagit Window Al f amp File Name C Program Files Bio Rad Bio Plex Manager 4 1 1 Example A B C High PMT RPMI_001 srbx File Name C Program Files Bio Rad Bio Plex Manager 4 1 1 Example Files Rat Cytokin Acquisition Date 03 Mar 2004 07 27 PM Reader Serial Number LX10000273003 RP1 PMT Volts 578 02 RP1 Target 3885 PI a ID Signed By BP SupervisorName Ss Document ID C3E93AD615194BDO0B10BAB098F9830CC
115. al Standards Read Only Standard Curve g ps pe Ux ope Labels Type v EnorBars 1 StdDev v Regression Type Logistic 5PL Axis Transformation Loaf x Linear y C Same regression type for all analytes CI Swap XY Axes Show Conc Range Lines CI Show Unknown Samples C Show Control Samples Curve Fit T T Cl Apply across all analytes l 1 00 10 00 100 00 1000 00 10000 00 100000 00 Show report after optimization LLOG 1 981 Concentration pg mL W Standard O Partial Outlier E Outlier Std Curve Fl 3 38787 19357 2 3 38787 1 Conc 154 501 0 933497 0 846041 FitProb 0 2653 ResVar 1 3215 Ext Std Curve Fl 28 6726 20793 9 28 6726 1 Conc 904 712 0 705962 1 40794 FitProb 0 6040 ResVar 0 6169 Figure 189 Fit probability and residual variance If you are using Logistic 5PL or Logistic 4PL Bio Plex Manager will automatically weight the points in the curve There are eight models for calculating the variance in the logistic weighting algorithm The default setting for Variance Model is Power Law Variance Weighting coefficient a will be automatically calculated Determining Outliers Logistic Weighting Alternatively users can enter a desired value by deselecting 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 Logistic Weight
116. al exporting and printing functions not available in the Protocol window Setting the Plate ID If you have an identification number for the microplate used in a reading you can specify the ID number in the Results file Right click anywhere in the Raw Data table and you can select Set Plate ID from the context menu ___ Well Type 54 54 73473 Sampling Errors 7 Set Plate ID m ll T 2 ja po MER Set Outlier Well 41 54 544 Plate D 07084 5148 Clear Outlier lappy piate ID to al wets Set Outlier All Analytes Clear Outlier All Analytes Raw Data Display Options Figure 119 Set Plate ID dialog 124 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 Exporting the Raw Data To automatically export your raw data to Microsoft Excel click the Export to Excel button a in the toolbar or select the command from the File menu This command automatically launches Excel and displays a spreadsheet containing your data EJ Microsoft Excel Book dc File Edit View Insert Format Tools Data Window Help Adobe PDF Type a ques i Arial 10 B
117. an_cyokines_001 Enter Controls Info Protocol Settings Analyte PEISE M cv 1 Describe Protocol Assign Control Information ef emt session cone iion 3 Format Plate 4 Enter Standards Info 1 ji 5 Enter Controls Info i 6 Enter Sample Info Run Protocol Same conc values for all analytes Dilution Factor 1 Set All Dilution Factors Example 4 sample diluted 1 in 4 has a dilution factor of 4 Figure 88 Enter Controls Info dialog The analytes you selected in the Select Analytes window are available under the Analyte pulldown menu Select the first analyte for which you want to enter controls 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 62 Enter the concentration of the selected analyte in the Conc column for each well Selected analyte p Anebte MoiL 1b 19 v Assign Control Information Ctr Description Conc Dilution 390909 100 Entered C2 1000 00 1 00 concentrations v Same conc values for all analytes Dision Faso 1 Example A sample diluted 1 in 4 has a dilution factor of 4 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 Deselect the checkbox if you want to enter different concentrations for each control
118. anager To access the heater controls select Platform Heater from the Instrument menu or click the button on the main toolbar pe Platform Heater Settings Turn Heater on Set target temp C Gl Turn Heater off after run Current Temperatures Platform 231 Ambient 240 C Figure 32 Platform Heater dialog In the Platform Heater dialog click the Turn Heater on checkbox to turn on the heater and set the target temperature within the range 35 to 60 C using the scroll buttons To automatically turn the heater off at the end of a run select the Turn Heater off after run checkbox The dialog box and the Bio Plex Manager status bar indicate the current platform temperature The status bar also indicates whether the heater is turned on Platform Heater On 23 8 C Figure 33 Status bar indicating the heater is on If the heater is in the process of warming up or cooling down the temperature is shown in red If the heater is on and has reached the specified temperature the temperature is shown in green If the heater is off and is at ambient temperature the temperature is displayed in black NOTE If you initiate a run before the heater has warmed up or cooled down that is the temperature is shown in red you have the option of waiting until the specified temperature is reached or proceeding with the run immediately Instrument Information Instrument Information The array reader continuou
119. anager 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 You can substitute gene with analyte and use the same calculations for normalizing protein concentrations with internal endogenous controls For these calculations Gene of Interest Analyte of Interest Housekeeping Gene Endogenous Control Protein Variables below are HKG Housekeeping Gene Fl Fluorescence Intensity n number of housekeeping genes Average Flisample Flwetit Flweio Flwetin Norm Ratio is the software s term for normalized expression values It is calculated three different ways depending on the options chosen in the Normalization Settings dialog These are outlined below as Norm Ratio Norm Ratio and Norm Ratio A control sample may be assigned in order to Compare results across multiple plates control sample must be the same biological sample on each plate Simply express all results on the same plate relative to the controls sample 215 Bio Plex Manager 6 0 Software User Guide Appendix 216 Norm Ratio The calculation is a simple division of each sample s calculated value by the contr
120. angiogenesis diabetes isotyping and acute phase assays You can add to these existing panels of analytes or create new panels that contain only the analytes you use in your experiments Bio Plex cytokine phosphoprotein and total target assays are available as custom mixed x Plex assays For repeat sample testing these multiplex assays are offered in one or ten 96 well plate formats Select from the current list of available assays and Bio Rad will mix and quality test the coupled beads and detection antibodies to order You can obtain product information and a catalog number for your custom designed x Plex assay by going to www bio rad com bio plex x plex First select the panel of analytes that you want reported then select the specific analytes SELECTING ANALYTES Go to the Panel pulldown menu and select from the list of preconfigured panels The analytes in the selected panel display in the Available list 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 To move an individual analyte into the Selected list double click it Double click an analyte in the Selec
121. ard Lots Help Regression Type Assign Standards Information Logistic 5PL j Hu IL 2 38 Hum 4652 Hu IL 5 33 _Hutt 10 56 Hu IL 12 p70 75 HuIL 13 6517 Axis Transformation 33054 00 40750 00 Loo Lineary 0 00 0 00 Logistic Weighting Same regression type for all analytes Concentration Units Same units for all analytes Units don t impact calculations Acceptable Recovery Range 70 1302 v Calculate Concentrations Most Concentrated Standard 51 Oss Concentration of 1 _ HuIL 5 33 Apply dilution to all analytes Dilution Factor X Calculate C Apply same concentrations to all analytes CEATA Canana Same recovery range for all analytes 76 Protocol Window 2 Once each analyte s starting concentration is entered apply the dilution factor which is appropriate for your experiment PH Protocol2 Enter Standards Info IUe Standards Info Estemal Standards Info Select External Standards Standard Lot Analyte T Lot Expiration Date 1 Describe Protocol Hu IL 13 51 Std Regression Curve Regression Type Assign Standards Information Logistic 5PL oe aa peseioton Hu IL 2 38 Hu IL 4 52 Hu IL 5 33 Hu IL 10 56 Hu IL 12 p70 75 Hu IL 13 51 Axis Transformation 33054 00 40750 00 10736 00 18653 00 24332 00 Li ge 0 00 0 00 0 00 0 00 3 Format Plate Eat Leer 0 00 0 00 0
122. ards conc the recovery range is specified by the user Sampling Error Low bead number detected in the well Aggregated beads detected in the well Classify efficiency problem detected in the well Region selection problem detected in the well Platform temperature problem detected in the well Index Numerics 21 CFR Part 11 Rule 168 A Adjusting Your Graphs 158 Advanced Run Settings 98 Bead Map Selection 98 Doublet Discriminator Gates 100 Sample Size 98 Sampling Errors 99 Save Options 99 Alcohol Flush 44 Alcohol Wash 44 Autofilling Replicate Groups 65 Autofilling Well Numbers 64 Bio Plex Manager 6 0 Software User Manual Back flush 44 Basic Concepts 219 Excitation 220 Fluidics 220 Microspheres 219 Reporter Fluorochromes 219 Bead Map 108 Density Filtering 111 Display Options 110 Logarithmic Linear Display 111 Magnifying and Resizing 110 Scaling the Bead Map Data 110 Bio Plex assay and reagent website 2 Bio Plex Manager General Workflow 7 Installing 11 Main toolbar 16 Menu bar 16 Quick Guide 17 Required screen resolution 10 Status Bar 17 System requirements 9 Bio Plex Manager software Editions 5 Bio Plex MCV Plate IV 22 22 Bio Plex Manager 6 0 Software User Manual Bio Plex suspension array system Advantages 3 Components 2 Description 1 Disconnecting 14 Reconnecting 14 Bio Rad Technical Support Contact information Website address 4 C Calculating Concentrations Automatically 86 Cali
123. art 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 This rule states the conditions under which the FDA considers electronic signatures and electronic records to be trustworthy reliable and equivalent to traditional handwritten signatures Bio Plex Manager Security Edition is designed to enable organizations and institutions using the Bio Plex suspension array system or Luminex system to comply with the rules laid out under 21 CFR Part 11 It enables system administrators to ensure that Bio Plex Manager operates in compliance with 21 CFR Part 11 within a closed system A closed system is defined as an environment in which system access is controlled by the persons who are responsible for the content of electronic records that are on the system Section 11 3 b 4 NOTES e The security controls built into Bio Plex Manager Security Edition must be properly configured and administered by the system administrator s in your organization Otherwise the system will not be secure and will not be in compliance with 21 CFR Part 11 e Bio Rad makes no claim that Bio Plex Manager Security Edition is CFR compliant in and of itself nor does it guarantee compliance for the user Your organization must establish policies and standard operating procedures that work in conjunction with the tools provided by Bio Rad to ensu
124. atting section of the software defeats any inter and many intra experiment normalization Relative differences among samples within the same group are still valid If you are normalizing your data ratio calculation is a simple ratio of the calculated value of a reference sample of the group compared to the calculated value of each member of that group Data can be presented as member over reference or as reference over member 2 Horm Ratio peample xi Horm Ratio peerence Horm Ratio perence Horm Ratio peample xi If you are not normalizing your data the ratio is calculated using FI values FI sample x FI Fatio reference Ratio et FI ireference j FI Ratio templ x 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 Microspheres are grouped into sets each set is then embedded with specific quantities of two fluorescent dyes The ratio of these two dyes gives each set of microspheres a unique spectral address Each set of microspheres is then conjugated with a different reactant specific for a particular target analyte Reactan
125. bead map and histogram data All other data are preserved Save As _ Of PR Mouse 23 Plex High PMT Setting lot 5001911 pbx 2 Mouse 23 Plex Standard PMT Setting lot 5001911 pbx My Recent 3 Phosphoprotein 11 Plex pbx Documents Save in C9 Example Files Select this checkbox to reduce the file File name MyProtocol pbx size ofa containing Compressed mode without histogram and bead map data reading data Figure 38 Save As dialog for protocols NOTE This compressed file option is not available for Secure Protocol files generated using the Security Edition of the software 49 Bio Plex Manager 6 0 Software User Manual Preparing Protocols Multi Assay Protocols 50 Bio Plex Manager 6 0 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 l Bio Plex Manager Alpha Multi Assay Protocol Describe Protocol Assay Assay Protocol Settings Assay Protocol Settings Author researcher Description Figure 39 New Multi Assay Protocol tabs When an assay tab is clicked the window displays all information connected with the corresponding independent assay The assay number of the assay bein
126. ber Specifications dialog Validation Type Selection In the Validate dialog select the type of validation you want to perform using the Validation Type option buttons All Optics S Fluidics is Heporter Ex and Classify se 37 Bio Plex Manager 6 0 Software User Manual Controlling the System 38 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 All Validation MCV Plate IV This procedure performs Optics Fluidics Reporter and Classify validation 1 Vortex each Validation vial for 30 seconds 2 Load 5 drops of each vial into the specified well 3 Fill DI H20 and 70 isopropanol reservoirs 4 Click Eject then load MEY plate 5 Select OK to start 4 Eiect Retract Plate Figure 28 Dialog box for performing All Validation procedures See the Bio Plex Validation Kit 4 0 manual for information about the bottles in the Validation Kit and instructions on loading the Bio Plex MCV Plate IV for validation When you have prepared the plate according to the instructions click Eject Retract Plate in the dialog box place the plate on the plate carrier and click OK Validation Results After a run the Validation Results dialog box automatically opens You can also view this dialog by going to the View menu and selecting Validation Results Validation Results Optics Fluidics Reporter
127. bration Logging the Calibration 30 Performing the Calibration 30 Setting Up the Calibration 29 Changing a Replicate Group 66 Changing the Doublet Discriminator Gate Range 123 Changing Well Formatting 68 Choosing Graph Options 150 Combining Panels of Analytes 61 Compatibility with Luminex Software 6 Creating a New Panel of Analytes 56 Customizing Analytes and Panels 55 Data Normalization 193 Assigning a Control Sample 195 Calculations Based Upon Your Settings 198 Factor Calculations 199 Predefined Schemes 197 Report Table Options 196 Selecting Internal Control s 194 Defining a Group 70 228 Defining a Replicate Group 65 Defining Blank Wells 68 Defining Control Wells 67 Defining Standard Wells 66 Defining the Reference 71 Defining Unknown Sample Wells 63 Deleting and Renaming Custom Panels 61 Deleting Well Formatting 68 Doublet Discriminator Gate Range Changing 106 Drain 44 Editing a Panel of Analytes 60 Entering Concentration Units 87 Entering Concentrations 85 F Fluidics Functions 43 G Generating Results from a Protocol 103 Glossary 205 Graphing Adding 155 Analytes across a single sample 152 Analytes across multiple samples 152 Deleting 157 Editing 156 Exporting to Other Applications 157 Samples across a single analyte 151 Samples across multiple analytes 151 Viewing Information 153 Graphing Function 149 Hardware protection key use in USB port 12 Histogram Magnifying 107 Resizing the Histogram 1
128. cally recalculated Note that flagging a well as an outlier for the selected analyte does not flag it as an outlier for all analytes To do this right click the outlier checkbox for the well and select Set Outlier All Analytes from the context menu Conc j Dl mam m cm Fl 4 mam Fg DE x dd nuls sj on Hide Column pail 1 00 1 00 Set as an internal control housekeeping gene Ra TNF a 1 00 Report Table Options 1 00 ae H00 1 00 1 00 1 00 Show Column Figure 128 Selecting a well as an outlier for all analytes Now the well data for all analytes are excluded from calculations To remove the outlier flag for all analytes right click the well and select Clear Outlier All Analytes from the context menu 135 Bio Plex Manager 6 0 Software User Manual 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 Type Well Outer Group checkbox appears aie gray and selected Figure 129 A single well in a replicate group flagged as an outlier Resizing the Columns To automatically size the columns to fit the width of the text choose Autofit from the Table Options menu in the toolbar Context Menus
129. cedure Wash Between Plates This procedure washes the fluidics before running an assay 1 Fill 70 isopropanol and DI H20 reservoirs 2 Click Eject then load MEY plate 3 Press OK to start MCV Plate IV 4M Eiect Retract Plate Figure 22 Steps for washing between plate readings Add 7096 isopropanol and distilled water to the appropriate reservoirs insert the plate in the microplate platform and click OK This procedure performs a series of fluidics operations and takes several minutes After initial system pressurization 5 to 20 seconds the time remaining in the operation displays in the Bio Plex Manager status bar 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 IE ep Click the Remove Bubbles button onthe 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 the procedure Remove Bubbles This operation removes air bubbles from the fluidics 1 Fill 70 isopropanol and DI H20 reservoirs 2 Click Eject then load MEY plate 3 Press OK to start MCV Plate IV 4 Eiect Retract Plate Figure 23 Steps for removing air bubbles from the fluidics system
130. centration Units pg mL Lot 835 020 Reference St PE 1 95 1 95 1 95 1 95 1 95 1 95 C Same units for all analytes Units don t impact calculations Li Graphs Logistic 5PL Acceptable Recovery Range 70 130 v lt C Same recovery range for all analytes Caed Roncentrstions Most Concentrated Standard eS1 e58 Concentration of eS1 32000 Hu IL 1b 32 Apply dilution to all analytes Dilution Factor 4 Calculate Apply same concentrations to all analytes Cl I C trati Liear All Loncentrations Figure 79 Standards Info window top and External Standards Info window bottom 83 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 84 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 Assign Standards Infomation Std Description 040 1109 12 12 1303 o NE e oe oe ss oof oof oof 9 s oof 90 9 99 ss 90 90 oof 9 ss 9 9 9 99 Figure 80 Selecting a standard analyte NOTE For the settings described on the following pages you can select different settings for each analyte or select the same settings for all analytes Use the contr
131. ces are covered with molecules that bind with the target analytes in an assay system An immunological protein that can signal a response in the immune system An antibody used in capture sandwich immunoassays that binds a captured analyte to a fluorochrome allowing for quantitation of the protein Detection antibodies may be biotinylated which allows for addition of a streptavidin bound fluorochrome or the detection antibody may be directly labeled with a fluorochrome Two microspheres that have associated The resulting signal of the associated microspheres is twice that of a single microsphere singlet A channel that measures the amount of light scatter from particles that flow past the red laser Light scatter is directly proportional to particle size and the channel is designed to identify particles that are smaller or larger than a single microsphere including microspheres that are clumped or aggregated The calculated number of decades covered by the reporter channel scale of the array reader Range is calculated using the Reporter Validation Kit The wavelength range of light emitted by an excited fluorochrome when its electrons fall from a higher to a lower energy state Expressed in nanometers nm A common way of reporting a molecule s excitation and emission wavelengths for example Ex Em 488 520 nm The wavelength range that excites a molecule s electrons to a higher energy state Expressed in nanometers nm
132. ck OK a Save As dialog opens Save in o Example Files v Mouse 23 Plex High PMT Setting lot 5001911 spbx 2 d Phosphoprotein 8 Plex_001 spbx My F Recent d Phosphoprotein 8 Plex_002 spbx Documents Desktop My Documents 3 My Computer amp File name Prorom 8 Plex Tim My Network Save as type Secure Protocol spbx Figure 173 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 Also any changes made to a file after it has been signed will generate an audit trail documenting each change as described starting on page 179 A new file generated from a signed file can be signed again to verify and lock in any changes made to it As before the new signed file is flagged as read only and any subsequent changes must be saved under a new file name 175 Bio Plex Manager Software User Manual Calibration Validation and Instrument 176 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 Document Properties GR Rat Cytokine 9 Plex High PMT RPMI 001 srbx Type Bio Plex Secure Results Document Location C Program Files Bio Rad Bio Plex Man
133. ck 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 Set Column Number Format x Decimal places Reset Defaults Scientific Notation Sample 1234 0 Same number format for all columns Figure 126 Set Column Number Format dialog In the dialog select a column from the pulldown list then specify the number of decimal places for the value of that column in the Decimal Places field Select the Scientific Notation checkbox to display the number in scientific notation Select the Same Number Format for All Columns checkbox to use the selected number format for all columns Click OK to make the changes or Reset Defaults to return all columns to their default number formats NOTE Changing the number format changes only how the numbers are displayed and printed and does not affect calculations in any way SAVING SETTINGS AS A NEW PROTOCOL To use the selections in this dialog as the default in Results files generated from new Protocols select the Save Settings as Default for New Protocol checkbox 131 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 132 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
134. ckbox Also you can select a specific analyte all gated analytes or all analytes from the pulldown menu above the histogram Then either drag the red dashed lines in the histogram display to adjust the DD gates or enter new values in the DD Low and or DD High fields Click the Update Gates button When you have made your changes click OK The data in the Raw Data table see next section recalculate based on the new settings 123 Bio Plex Manager 6 0 Software User Manual 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 e Histogram and bead map see page 104 e Raw Data table see page 112 R New Sample 23 Plex Standard PMT Raw Data CVs 4 Us Uc UXE Ae dim Wie A Histogram Bead Map 100 region EY wer ar AMGeed wj t e 2 nl Raw Data 10000 e om Report T able 1000 100 Standard Curve n Classification 2 100 1000 10000 1000 10000 Doublet Discriminator Classification 1 Mo IL 2 36 Mo IL 3 18 MolL 4 32 Mo IL 5 52 Moll 13415 0 223035 197725 155990 20205 0 7561 0 16205 5 153785 16538 0 21662 0 12875 0 3432 0 10588 0 9734 0 14599 0 153665 Figure 118 Raw Data window in Results file The Raw Data window in the Results file includes addition
135. curve is then used to calculate the concentrations of your unknowns Protocol Window To define the wells containing standards in your microtiter plate click the Standard button S then click or drag the wells in the template Use the Autofill buttons as described on page 64 to define multiple standard wells and or replicate groups of standards Plate Formatting Plate Groupings Figure 61 Defining a row of standard wells NOTES e The cursor changes to a Standard cursor as you move it over the template Standard wells are marked in the template by a circle with a number inside it e After you have defined the standard wells you are ready to enter the concentrations of the standards as described on page 85 DEFINING CONTROL WELLS Control wells contain samples of known concentration the expected vs Observed concentrations of the controls can be calculated at the end of the reading To define the wells containing controls in your microtiter plate click the Control button then click or drag the wells in the template Use the Autofill buttons as described on page 64 to define multiple control wells and or replicate sets of controls Plate Formatting Plate Groupings 1 2 d Figure 62 Defining a row of control wells 67 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 68 Note that the cursor changes to a Control cursor as you move it over the template Control wells are marked in th
136. d 4 from the toolbar or Format Options menu then click or drag the well s you want to clear To clear the formatting from all wells in the template select Clear Plate from the Format Options menu To change the formatting of a well or group of wells simply select the appropriate tool and overwrite the old formatting as if you were defining the wells for the first time 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 Click the Plate Groupings tab to display the plate grouping tools Pg human cyokines 001 Format Plate k Eo amp X Ratio Member Reference Protocol Settings rere EUER arme A SHEE USES EN ES EIS E Syn E QOG Z90919 e OILL L LL Figure 64 Plate Groupings view The buttons on the toolbar are used to group the wells on the microtiter plate diagram and identify the Reference well s These formatting commands are also located on the Format Options menu xv 1 140 9 4 3 Tools Windo v Select Select All Clear All Groups Group Set Reference Members Ungroup Set Sample Group OOX Figure 65 Tools in the Plate Groupin
137. d method an error message will be displayed in red below the standard curve Standard Curve LOGISTIC 5PL This version of the five parameter logistic equation has been developed by Brendan Scientific creator of StatLIA immunoassay software The Logistic 5PL 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 The Logistic 5PL equation is a d o y d where X is the concentration y is the response a is the estimated response at infinite concentration b is the slope of the tangent at midpoint c is the midrange concentration or midpoint d is the estimated response at zero concentration and g is an asymmetry factor LocisTIC APL In certain cases in which the curve is a perfectly shaped S the Logistic 4PL regression may give better results However the Logistic 5PL regression has been shown to be the most robust routine for most Bio Plex assays The Logistic 4PL equation is a d where X is the concentration y is the response a is the estimated response at infinite concentration b is the slope of the tangent at midpoint c is the midrange concentration or midpoint and d is the estimated response at zero concentration 145 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 146 LINEAR The Linear equation is y a bx
138. d number 12 Aggregated beads 13 Classity efficiency L 4 Region selection C5 Platicem temperature Reporter PHT 7 Ovenide calibcation Figure 163 Advanced Run Settings Bio Plex XML Export You can export all the information in a Results file as an XML formatted text file This export option generates a comprehensive export of individual results files in an XML format This format is useful for import into LIMS databases and to perform data analysis using other applications NOTE XML is a self describing universal file format compatible with a wide range of applications The XML document generated by Bio Plex Manager includes all the data stored in the Results file including but not limited to e Run parameters including DD gate settings instrument temperature settings data limits bead regions etc e Run conditions including time stamps per well error conditions during the run actual temperature during acquisition by well etc e Complete data values and calculations mean median standard deviation coefficient of variation etc per well e Complete protocol information including analytes selected plate formatting standard values run settings etc e Raw bead event data when selected e Standard curve information including mathematical models used how well the data fit the curve etc e Instrument and software information including version numbers e All logged events associated with the da
139. dards 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 R External_Standards Read Only Enter Standards Info g 3 ev Us UC ix EJ Eee Resuits Standards nfo Esana Standards info Select Enana Standards A Raw Data Standard Lot Analyte Lot Expiration Date J v il Report Table Hu IL 1b 32 v Std Regression Curve Manage Standard Lots Regression Type Standard Curve Assign Standards Information HuiL 1b 32 _Huit 2 38 HulL 4 52 Hum 5 33 HulL 6 19 HuIL T T4 Humea Hum 10 56 Hui Axis Transformation 3200 00 3200 00 3200 00 3200 00 3200 00 3200 00 3200 00 3200 00 Uem F j 800 00 800 00 800 00 800 00 800 00 800 00 800 00 800 00 i 200 00 200 00 200 00 200 00 200 00 200 00 200 00 200 00 I 50 00 50 00 50 00 50 00 50 00 50 00 50 00 50 00 Logistic Weighting I 1250 12 50 12 50 12 50 12 50 12 50 12 50 12 50 C Same regression type for all analytes 343 3 13 343 343 3 13 3 13 3 13 3 13 078 078 078 078 078 078 078 078 Concentration Units pg mL i 0 20 0 20 0 20 0 20 0 20 0 20 0 20 0 20 ni Graphs Logistic 5PL C Same units for all analytes Units don t impact calculations Acceptable Recovery Range 70 130
140. dy used in sandwich immunoassays that binds or captures a specific analyte for quantitation Microspheres with chemically modified surfaces covered with carboxyl functional groups COOH that allow covalent biomolecular attachment Fluorescent detection channels of the array reader that detect the fluorescent intensities of two dyes embedded in each microsphere If the signals from the two dyes place the bead within a specified classification region the bead is identified as a unique analyte in a sample Defined regions in a dot plot bead map that are used to identify beads in an assay based on the fluorescent signals from their embedded dyes A validation procedure for measuring the ability of the array reader to efficiently classify assay beads into specified regions The aggregation of two or more microspheres 205 Bio Plex Manager 6 0 Software User Manual Glossary Term Concentration in range Conjugated microsphere Cytokine Detection antibody Doublet Doublet discriminator Dynamic range Emission spectrum Ex Em Excitation spectrum External standard Fit probability Fluidics validation Fluorescence Fluorochrome Gate 206 Definition 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 gt OOR or lt OOR A microsphere set that has been chemically modified so that its surfa
141. e d Please enter the username and password of a valid account on the domain IFD_DOMAIN Please enter your reason Far signing the document Figure 178 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 117 Audit Trail Any changes made or actions performed after a Protocol or Results file has been signed automatically generates an audit trail documenting each change action If you make changes to a signed read only file and save it as a new file the audit trail is preserved in the new file with the signature of the previous file noted as part of the audit trail Also the audit trail for a Protocol file is preserved in the Results file generated from that Protocol All major actions and changes are audited Examples of auditable actions include Signing a file Changing Protocol settings plate format analyte selection etc Changing the standard curve regression type variance model etc Flagging outliers Performing readings audited in the Results file Minor changes that affect only the display such as selecting different columns in the Report Table or changing the axis transformation in the Standard Curve are not audited 179 Bio Plex Manager Software User Manual Audit Trail Each change you make to a signed Protocol or Results file or a new file saved from a signed fi
142. e Control Sample always None or Single 193 Bio Plex Manager Software User Manual Normalization Settings Selecting Internal Control s Housekeeping Gene s 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 To select a single internal control housekeeping gene click the Single radio button and select a housekeeping gene from the pulldown list Normalization Settings Internal Control Housekeeping Gene s Control Sample O None 9 None Single 53 Mo IL 1a O Singe Multiple 53 Mo IL 1a Region The Internal Control Housekeeping Gene Field accounts For differences in the amount of sample material k The Control Sample field allows you to express all Mo IL 6 i Mo IL 9 samples as a ratio to the assigned control Mo IL 10 es ees For more details click Help Mo IL 13 Mo IL 17 Mo Eotaxn Mo G CSF Mn GM CSF Mo IFN g Mo KC Mo MCP 1 Mo MIP 13 Mo MIP 15 Mo RANTES Figure 192 Selecting a Single Internal Control To select multiple internal controls housekeeping genes click the Multiple radio button and select which housekeeping genes to use from the following dialog box Select Internal Housekeeping Gene Controls Available Selected
143. e 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 Save As Save in Example Files id External Standards rbx a Gene Expression rbx My Recent a Human Adipsin SinglePlex rbx Documents 8 Human Cytokine 17 Plex High PMT rbx 2 Human Cytokine 17 Plex Standard PMT rbx a Human Diabetes 2 Plex rbx a Human Diabetes 12 Plex rbx a Human Isotyping 7 Plex rbx a Mouse Adiponectin SinglePlex rbx a Mouse Cytokine 23 Plex Standard PMT rbx a Phosphoprotein 11 Plex rbx 2 Precision Pro Human Cytokine 10 Plex rbx 2 Rat Cytokine 9 Plex High PMT RPMI rbx 2 Total Protein 10 Plex rbx Select this Eene Expression sb checkbox to reduce the size of the Results fie Figure 113 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 119 Bio Plex Manager 6 0 Software User Manual Analyzing the Results Results Window The Results window is divided into subwindows each containing different options for viewing formatting and calculating your data R Human Cytokine 17 Plex Standard PMT Read Only Raw Data s e Us UC UX E 59 4 FS ud m Results Histogram Bead Map 100 region Raw Data i i Ai 4 isl Hi Report T able 40000 Standard Curve 1000
144. e connections etc e lf the platform heater is not at the specified temperature a warning message will appear and you will be prompted to wait for the temperature to change 102 Manually Stopping a Reading When the reading begins the sample needle lowers draws sample from a plate well and retracts to its original position The sample is drawn from the needle into the sample loop and the reading is taken After initial system pressurization 5 to 20 seconds a typical well reading may take up to 2 minutes in most cases individual wells are read in a matter of seconds 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 Number of beads Region o3 Gate 215 Total pm a S Beads per second Region mo Gate 4 Totat zn 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 Within these categories the array reader keeps track of the following information e The Region fields monitor only those beads that fall within one of the regions you selected in the Protocol page 54 e The Gate fields monitor all beads that pass an internal discriminator gate DD gate range designed to eliminate particles smaller or larger than a microsphere including microspheres that are clumped or aggregated e The Total fields report all particles measured by the arra
145. e cursor changes to a hand symbol D when you position it over the histogram Drag the hand cursor to view different magnified regions of the histogram 107 Bio Plex Manager 6 0 Software User Manual Running Protocols 108 Click the Maximize button o to maximize the histogram portion of the window within the larger Protocol window Click the Restore button rH to return the histogram to its default state El Price os Ban Protecd m t esl elau al a helo Wig 1 za rare IH uu B w 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 Different colored dots in the map represent different numbers of events at those data points The number of available regions in the bead map 25 or 100 is listed above the map and is based on the selection in the Advanced Settings dialog see page 98 In the default bead map the x axis is the Classification 1 channel and the y axis is the Classification 2 channel These channels measure the embedded fluorochromes in each bead which are used to identify the bead set and corresponding analyte The resulting data point clusters in the map represent the different bead sets in the assay and their associated analytes NOTE Each bead set generates a cluster of data points rather than a single point because of minute variations in fluorochrome levels and intensity among the beads in a set H
146. e specific proteins Kinases are responsible for the activation of a variety of proteins through the process of phosphorylation A highly purified source of light used to excite fluorochrome electrons An acronym for Light Amplification by Stimulated Emission of Radiation A measurement of the coefficient of determination or R value of a set of standard beads An R value of 0 995 or greater is an acceptable linearity value using the Bio Plex Validation Kit A regression model for binary dichotomous outcomes The data are assumed to follow binomial distributions with probabilities that depend on the independent variables For any given assay this is the lowest value Bio Plex Manager will report in the Concentration in Range column of the data table Values below this range are considered less trustworthy based on either the poor recovery values of standards below this range or background of the assay For more details review the definition of Concentration in Range A relative measurement of fluorescent intensity based on the median a robust statistical measure A solid substance with a diameter in the micrometer range Often used as a synonym for a microsphere A set of multianalyte microspheres containing a unique mixture of two distinct fluorochromes to distinguish them from other multianalyte microspheres Latex spheres with a diameter in the micrometer range Also called beads An analysis of several assays or tests pe
147. e 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 90 DEFINING BLANK WELLS In certain types of assays such as the Bio Plex phosphoprotein assay it may be useful to subtract the assay background from the readings of standards controls and unknown samples To do this you can prepare blank wells containing all the assay components except sample Wells prepared as blanks are read along with the rest of the assay and then Bio Plex Manager subtracts the mean background reading of these wells from the fluorescence intensity values of the wells containing standards controls and unknowns To format blank wells in the plate template click the Blank button e then click or drag the blank well s in the template Note that the cursor changes to a Blank cursor as you move it over the template Plate Formatting Plate Groupings ESTE s COCE D mnn Figure 63 Defining a row of blank wells Blank wells are marked in the template with a diamond shape containing the letter B Multiple blank wells in a template are treated as a single replicate group A mean value standard deviation standard error and coefficient of variation are calculated for the group and the mean is used as the background value DELETING OR CHANGING WELL FORMATTING To delete the formatting from a well or set of wells select Undefine
148. e than 1 hour Needle stuck in down Protective assay plate covering See hardware manual for procedure for position was not removed raising needle stuck in down position Then remove cover from assay plate Needle guide is not screwed all Tighten needle guide by turning tube the way in clockwise until tight Sample needle is bent Replace bent needle with a new needle see hardware manual for procedure Optics Fluidics Beads not resuspended Resuspend beads and repeat validation Reporter or Classify procedure If procedure still fails Validation Procedure contact Technical Support fails due to low bead Cio in instrument Perform Unclog operation and repeat number validation procedure If procedure still fails contact Technical Support Bio Plex Manager has Incorrect DD gate values used View histogram in Run Protocol window detected a problem and make sure DD peak falls within the with bead gate range If necessary adjust gates aggregation Clumped beads present Vortex plate at 900 rpm for 11 minutes Sheath reservoir is empty Refill sheath reservoir then perform Start Up Waste reservoir is overfilled Empty waste and reconnect Incompatible suspension buffer Check hardware manual for buffer used compatibility 202 Check Link in status Array reader or microplate Turn on array reader and microplate bar of software platform is not turned on platform Software is not communicating Cl
149. eated Select Yes then complete the protocol settings for each assay 51 Bio Plex Manager 6 0 Software User Manual Preparing Protocols Protocol Window The Protocol window steps you through a series of windows Pi Protocol1 Describe Protocol Protocol Settings Author MEE cv 1 Describe Protocol Description i 2 Select Analptes 3 Format Plate 4 Enter Standards Info 1 F 5 Enter Controls Info E 1 B Enter Sample Info 7 Run Protocol Figure 40 Protocol window The buttons are numbered to guide you through defining your protocol 1 Describe Protocol optional see page 53 amp e 2 Select Analytes o see page 53 3 Format Plate ZF see page 62 4 Enter Standards Info Us see page 72 5 Enter Controls Info fic see page 90 6 Enter Samples Info optional Ux see page 92 7 Run Protocol JB see page 102 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 52 Protocol Window 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 If you are using the Standard Edition of the software the Author field contains your computer login name You can enter a new name in the field
150. ed concentration should be within 70 to 13096 of the expected concentration The actual recovery rate for each standard and control is shown in the Obs Exp 100 column of the Report table see page 134 Unknown concentrations that fall in regions of the curve where standards fall outside the selected range will be reported as OOR or OOR in the Conc in Range column of the Report table See Report Table Error Codes on page 226 89 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 90 Step 5 Enter Controls Info UC 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 This is sometimes called the spiked recovery method because you are spiking your assay with known quantities of analytes In this step you enter the concentration values of your controls First you must select the analytes you will be analyzing in the reading as described on page 53 and identify the plate well s containing your controls as described on page 6 7 NOTE Only formatted wells are read by the array reader Be sure to define all your control wells before running a Protocol Click the Enter Controls Info tc button in the Protocol window to enter information about your controls P3 hum
151. ee eee 137 EXPO FOMal carre Soon ied Sloe jas B oai e Mun 3c OEC qid tr one ene Sa lan 138 vi Bio Plex Manager 6 0 Software User Manual Export a Subset of the Data 0 0 ccc ee 139 Export Preferences 000 cece eee eee 140 Use Multiple Worksheets 0 0000 141 Exclude Standard Curves llli llle 141 Exclude Column Headers FooterS 00 0c cee eee eee 141 Exclude Table Error Codes 0 00 cee eee 141 Standard GU VG 19 45 92 2 05 84 Sud Wed et los Wat he Pea ewe Se eee tate 142 Regression Methods eee ees 144 Linear vs Logistic Regression Methods 147 Copying the Standard Curve 0 000 cece ee eee 147 Exporting the Standard Curve 0000 eee ee ees 147 Printing the Standard Curve 0 00 cece eee 148 Gaping FUNCION Aisi tacit E E book cod ow RUE eee ed 149 Choosing Graph Options 2 000 eee eee 150 Viewing Information within Graphs 0000 00 eee 153 Adding New Graphs 0000 cee eee eee ees 155 Editing arm Existnd Gran a4 s s resit naa o NP EIER OE CCS 156 Deleting a Gap us erae oon eee IG dea a ob reote didici aiu 157 Exporting Graphs to Other Applications 157 Adjusting Your Graphs llle 158 Printing Graph Serent tats Ead obo dI ah ee to Argus ub rar 158 Document Export Options 0 000 ce ees 159 DIO lt FICX AMIVEXDOM 4 244 uou E ACIE ee EPOD
152. eeded or failed You also receive an alert which informs you how many days have elapsed since you last ran a Validation procedure If the calibration process failed check the MCV Plate IV to verify that the correct beads were added to the wells Then perform a fluidics Wash and repeat the above steps For more information consult the Troubleshooting chapter Logging the Calibration The Calibration Log provides a list of past calibration dates and times results instrument settings and other data obtained by Bio Plex Manager These data are stored in a log file called bioplexdata mdb By default the file is saved in the main Bio Plex Manager application folder on your computer NOTE This database is not compatible with versions of Bio Plex Manager earlier than 4 0 If you have an earlier version of Bio Plex Manager installing version 4 0 or later copies data from your existing database bioplex mdb into the new database A copy of the old database remains in the application folder 30 Calibration To open the Calibration Log go to the View menu and select Calibration Log The Calibration Log viewer opens Calibration Log Viewer File View Help amp IQ Calibration Log CONTROL NUMBER Date Time 25 Sep 2007 04 47 PM 25 Sep 2007 04 31 PM 14 Sep 2007 01 07 PM 14 Sep 2007 12 50 PM 14 Sep 2007 12 37 PM 29 Aug 2007 04 10 PM 29 Aug 2007 03 09 PM 29 Aug 2007 02 57 PM 29 Aug 2007 02 53 PM 29 Aug 2007 02
153. em The software features a standard Windows interface with pulldown menus toolbars and keyboard shortcuts Bio Plex Manager comes in two editions Standard Edition and Security Edition and with three available licenses Instrument Control Desktop and Network described below The computer included with the Bio Plex suspension array system comes preinstalled with a compatible operating system and a Bio Plex Manager Standard Edition Instrument Control license Software Editions Bio Plex Manager software comes in two editions e Standard Edition gives all users equal access to all features of the software with no restrictions and no electronic audit trail e Security Edition provides different levels of user access to various features and creates a complete electronic audit trail of all data generation and analysis The Security Edition can be run in Secure Mode with all the security features enabled or Standard Mode which behaves like the Standard Edition of the software NOTE Bio Plex Manager Security Edition must be installed on the Windows XP Professional operating system for full security and functionality Bio Plex Manager 6 0 Software User Manual Software Licenses Software Licenses Bio Plex Manager software is available with three different licenses e Instrument Control previously called Workstation license enables the software to control the array reader and microplate platform and to collect analyze and outp
154. er 6 0 Software User Manual Analyzing the Results 134 Obs Exp 100 Column Because the standard curve see page 142 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 90 to determine the overall accuracy of an assay The Obs Exp 100 column shows the recovery rate for the observed vs expected concentration of each standard This number should fall within the recovery percentage range selected in the Enter Standards Info window For example if you selected a recovery range within 80 to 120 the observed back calculated concentration of a standard should be within 80 to 120 of the expected concentration Values outside this range are flagged in the Conc in Range column Click Report Table Options ra and select the Obs Exp 100 checkbox to display this column Concentration in Range Column The concentration in range column Conc in Range provides a method for determining which unknown concentrations are reliable based on the recovery range of the standards see the Obs Exp 100 section above This column shows only observed concentration values that fall within the range of valid standards values that do not fall in the range are displayed as OOR lt or OOR gt The concentration in range values of the back calculated standards are also shown for comparison purpo
155. er 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 Plate Formatting Plate Groupings 1 2 3 4 8 6 7 8 9 COGOCOL JL JL JL IE OL L8 N N NM Replicate Group L E Set Sample Description Set Sample Group Figure 67 Right click context menu DEFINING THE REFERENCE To change the Reference member of the group click the Reference io button and click the desired Reference member in the group For replicates all replicate wells of the same number within the group are automatically designated as the same Reference You can also use the Select tool R to first select the Reference well within a group and then use the Set Sample as Reference Member command on the right click context menu to set the Reference PRINTING THE PLATE FORMAT To print the plate format for your records select Print from the File menu or pE click the Print button amp on the main toolbar For a preview of the printed format select Print Preview from the File menu 71 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 12 Step 4 Enter Standards Info Us 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
156. es 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 This can be useful for viewing the distribution of the fluorescence signal of a particular analyte e The Classification 1 channel measures fluorescence from the first classification dye embedded in each bead as it passes through the red laser e The Classification 2 channel measures fluorescence from the second classification dye embedded in each bead as it passes through the red laser NOTE Changing the channel type changes the channel numbers and the histogram display SCALING THE HISTOGRAM DATA If the histogram is too big or too small to fit comfortably within the display range click the Auto Scale button I on the histogram toolbar The height of the y axis changes to better fit the data You can also select this command from the right click context menu Select Set Scale from the right click histogram context menu to manually set the range of the y axis In the pop up box enter the maximum number of events to display on the y axis between 20 and 100 000 The default view of the data is log scale From the toolbar click the Log Linear button to change the display from logarithmic to linear Click again to return to log scale MAGNIFYING AND RESIZING THE HISTOGRAM Click the Zoom button Pg on the histogram toolbar to magnify the display With Zoom selected th
157. et up on your computer or computer network as described in the Bio Plex Manager 6 0 Upgrade and Configuration Guide part number 10018402 before you can use the application in Secure Mode This guide is provided on your software CD Users Passwords and User Levels In Secure Mode Bio Plex Manager Security Edition requires that users log in with a user name and password to run the software User names and passwords are set up by your Windows system administrator as described in the Bio Plex Manager 6 0 Upgrade and Configuration Manual NOTES e Contact your Windows system administrator about issues regarding your user name and password e Each user is assigned to a particular user level There are six user levels in Bio Plex Manager Security Edition and each level gives the user access to specific features and functions of the software A table listing the specific features and functions accessible at each user level is provided on page 211 in the Appendix The following is a general summary of each user level e Administrator Users at this level can enable or disable Secure Mode Administrator level users also can view log files Access to all other features and functions of the software is restricted 169 Bio Plex Manager Software User Manual Users Passwords and User Levels 170 e Supervisor Users at this level have full access to all features and functions of the software except that they cannot enable or disable
158. ex Manager installing version 4 0 or later will copy the data from your existing database biop ex mdb into the new database A copy of your old database will remain in the application folder To view this log go to the View menu and select Validation Log Validation Log Viewer File View Report Help fe i B e G9 d Ck GP Date Time Fluidics Validation Results Fons ink MESE Kit Control Z Expiration Date Carryover Result User Access Level 26 Jun 2007 10 56 AM 18 Apr 2006 04 50 PM 13 Apr 2006 08 41 AM 04 Apr 2006 09 48 AM 04 Apr 2006 09 17 AM 30 Mar 2006 03 09 PM 09 Mar 2006 11 41 AM 09 Mar 2006 11 27 AM 01 Mar 2006 03 25 PM 01 Mar 2006 03 07 PM 27 Jan 2006 02 56 PM 21 Oct 2005 01 49 PM 4 Aat ANNE NAAN MMA o Figure 30 Validation Log Viewer SI Lx200 TestC2 SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay SI LX200 John Pocekay Clinician 2 Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted Unrestricted I nem mil 300000489 Low 310000167 Low 300001055 Low 300001055 Low 300001055 Low 300001055 Low 300001055 High 300001055 High 300001055 High 300001055 High 300001055 High Test101205b High
159. ex Manager 6 0 Software User Manual Appendix This Appendix contains e Security Edition User Access by Function below e Normalization Formulas see page 215 e Basic Concepts of the Bio Plex suspension array system see page 219 e Microsphere Handling see page 221 e Software Warranty see page 224 e Reporting Problems to Bio Rad see page 225 e Report Table Error Codes see page 226 Security Edition User Access by Function The following table lists the functions and user levels in Bio Plex Manager 6 0 Security Edition software An X indicates that the user level has access to the function Functions that Apply to Whole Application Function View All Screens No Modify L RD o quen 43 3 3 X x x x x x Electronically Sign Document Do X X X X Functions that Apply to Instrument Control M L LOI Sa Remove Bubbles Wash Between Plates Run Calibration Enter Calibration Lot Exp Date Run Validation ENENE GERM a E tr kak t x a X X X lt x x x x 211 Bio Plex Manager 6 0 Software User Guide Appendix Enter Validation Lot Exp Date O CX XJ X OLX Instrument Reconnect ho TO xX XxX cX XxX Author Field Description Field Functions that Apply to Protocol File Select Analytes View Add Delete Rename Edt Panel x x Functions that Apply to Protocol File Formal Plate View Format Wells Change Number of Unknown Samples Functions that App
160. file If this option is selected table cells with error codes are exported as empty cells 141 Bio Plex Manager 6 0 Software User Manual Analyzing the Results Standard Curve i 142 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 72 Click the Standard Curve button LZ to open the Standard Curve window Ri Gene Expression 2 Read Only Standard Curve CER 4 is UC x APO Labels NONE w EmorBars NONE v Analyte gapdh 26 Regression Type Logistic 5PL Axis Transformation Loa x Linear y v Logistic Weighting Same regression type for all analytes C Swap XY Axes Show Conc Range Lines C Show Unknown Samples C Show Control Samples Curve Fit Fluorescence Intensity F1 LLOQ 0 026 C Apply across all analytes Show report after optimization 1 00 10 00 Concentration attomoles E Standard O Partial Outlier E Outlier Std Curve Fl 4 34013 35208 3 4 34013 1 Cone 58 4895 1 1863 0 905447 FitProb 0 5581 ResVar 0 5831 Figure 138 Standard Curve In the graph standards defined on the current plate are displayed as blue squares while external standards are displayed as
161. following columns are also displayed e Total Total bead count in the well e Region Number of beads that fell within defined regions e Gate Number of beads that passed the DD gate see page 100 e 96 Agg Beads Percentage of aggregated beads in the well calculated as 100 Gate Total 100 Region Gate Total Agg Beads 2058 2128 2385 11 Figure 109 Well data NOTE The number in the Region column is flagged by an asterisk if that well did not acquire the number of beads per region specified for the reading Sampling Error Codes During a reading the flow of beads bead count bead regions and platform temperature are continuously monitored The Sampling Errors column reports errors in any of these areas for each analyte These errors are reported in the table even if you chose not to pause the run due to an error condition see page 99 The sampling error codes in the column are listed in the following table Error Code Indication 1 Low bead number detected in the well 2 Aggregated beads detected in the well 113 Bio Plex Manager 6 0 Software User Manual Running Protocols 114 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 Optio
162. g displayed also appears in the title bar Uses for Multi Assay Protocols This functionality allows you to test the same or different samples with two or more separate assays simultaneously You can also run the same assay under different conditions for example when you are in the optimization phase of assay development 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 Enter the protocol settings for Assay 1 then save 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 Enter the protocol settings for the second assay into the right hand pane then Save NOTE Results of multi assay protocols are always stored in separate results files Each assay results file is stored under its own descriptive name such as Bio Plex Pro Human Group Il rbx NOTE Choosing the New Results from Protocol command from the File menu will generate results only for the assay that is currently open Changing a Protocol from Single to Multi Assay A Multi Assay Protocol can also be created from a single assay protocol 1 Open the single assay protocol and right click on the Protocol Settings tab in the left pane of the Protocol window Select Add Assay A message will open telling you that a new Multi Assay Protocol must be cr
163. g the performance of the reporter channel of the array reader The five primary parameters that verify accurate performance of the reporter channel include linearity dynamic range sensitivity accuracy and slope of the response A statistical measure of a good fit This is the weighted sum of the squared errors between the observed response and the response predicted by the fitted curve residual SSE divided by the number of degrees of freedom A small residual variance indicates a good curve fit A file containing the results of a reading including the raw data the settings information from the Protocol and tools for analyzing and exporting the data tables a standard curve export functions etc Each reading generates a new Results file In Bio Plex Manager Security Edition Results files may be Secure files in which case they have the file extension srbx A solution of analyte standards or unknowns that when added to an assay will bind to the microspheres in that assay and be identified and quantitated by the array reader A measure of the degree to which a sample differs from the entire population Immunoassay for the quantitation of a specific analyte through the use of monoclonal antibodies Ab A sandwich is formed when an analyte of interest binds to a capture Ab and then a second antibody detection Ab binds to a different epitope of the protein A Protocol or Results file that has been electronically signed by
164. gs view First select the ratio you want to calculate for each group Reference Member or Member Reference using the Ratio field pulldown list 69 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 70 DEFINING A GROUP To define an assay group click the Group button 9 and drag the cursor across the wells you want to group NOTES e Wells must be defined as Unknown samples Standards Controls or Blanks before they can be grouped e You can group across well types for example a single group can include Standards Unknown samples Controls and or Blanks e Replicate wells are automatically placed in the same group even if you drag over only one well in the replicate set Plate Formatting Plate Groupings 1 J 3 4 D a CEN ii i c JL IL Ife Figure 66 Defining a group of wells The grouped wells change color to indicate their grouping and the first member of the group is highlighted to indicate that it is defined as the Reference well Each group you create will be designated with a different color To remove individual members of a group or the entire group click the Ungroup Samples button x and click or drag over the members group Note that you can also use the Select tool X to first select a group of wells and then use the Set Sample Group command on the Format Options menu or context menu to select the group Protocol Window CONTEXT MENU Right click and drag ov
165. he wells in the template Use the Autofill buttons as described on the following page for defining multiple unknown wells and or replicate groups of unknowns Plate Formatting Plate Groupings 1 2 3 4 a Nisi ERR e L E TR Figure 55 Defining unknown sample wells 63 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 64 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 For more information about the Security Edition see the Appendix on page 211 WELL NUMBERING AND REPLICATE GROUPS For unknowns standards and controls wells with different numbers contain different samples Wells with the same number contain the same sample and are defined as a replicate group For each replicate group a mean value is calculated in the Report table along with the standard deviation and coefficient of variation AUTOFILLING WELL NUMBERS To define multiple single wells as different samples you can click them 123 1 2 individually or select Autofill Across or Autofill Down 24 and drag the cursor over the wells to number them sequentially Plate Formatting Plate Groupings 1 2
166. hod for adjusting the height of the sample needle using the MOV plate Iv See hardware manual or Bia Plex Manager user manual For procedure for adjusting sample needle Select the type of the plate for which you want to adjust the needle height and use MEY Plate Iv the Up Down button ta move the needle Select the Save button after adjusting the needle for the desired plate Standard Plate tl Down PCR Plate Figure 10 Adjust Needle Dialog 5 6 10 11 Click Eject Retract to eject the plate holder Place the MCV plate on the microplate platform with the black arrow facing toward the array reader Click on the Eject Retract button to retract the plate Tape the access door of the microplate platform open It will be necessary to be able to see inside the access door 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 plates In the Adjust Needle window click on the Up Down button The needle will move to the down position With the needle in the down position loosen the needle height adjustment thumbscrew at the top of the needle so that the needle housing can move up and down freely 19 Bio Plex Manager 6 0 Software User Manual Sample Needle Adjustment 20 NOTE All adjustments to the needle height must be made when the needle
167. hosen Edit Graph Graph Name Sample 1 Samples Analytes Analyte Sample 1 A3 A4 A5 Mo IL 1a 53 Sample 2 B3 B4 B5 Mo IL 1b 19 Sample 3 C3 C4 C5 3 Mo IL 2 36 Sample 4 D3 D4 D5 Mo IL 3 18 Sample 5 E3 E4 E5 Mo IL 4 32 Graph Options Sample 6 F3FAF5 Mo IL 5 52 Sample 7 G3 G4 G5 Mo IL 6 38 Sample 8 H3 H4 H5 Mo IL 9 33 Standard 1 Mo IL 10 56 Standard 2 Mo IL 12 p40 75 Standard 3 Mo IL 12 p70 58 Standard 4 Mo IL 13 37 Standard 5 Mo IL 17 72 Standard 6 Mo Eotaxin 74 Standard 7 Mo G CSF 54 Standard 8 Mo GM CSF 73 Mo IFN g 34 1 Mo KC 57 oo 0 000000000HKAK AB B5 C5 D6 Figure 158 Edit Graph dialog You can edit any graph by choosing it from your Saved Graph List Add delete or change elements by checking or unchecking the sample or analyte checkboxes Graphing Function 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 Click the Export to Excel button in the toolbar or choose Export under the Graphs menu The data open in an Excel spreadsheet Ri New Sample 23 Plex Standard PMT Graphs DER g Us Uc Ux Graphs All Unknowns amp Controls Ha ta A
168. hout connecting to the instrument To re enable automatic connection at startup select Reconnect from the Instrument menu see next section Disconnecting and Reconnecting Communication between the computer and the array reader and platform is closed when you exit Bio Plex Manager If you need to disconnect from the array reader and platform without shutting down Bio Plex Manager software for example to troubleshoot the instrument go to the Instrument menu and select Disconnect To reconnect to the array reader and platform while Bio Plex Manager is running select Reconnect from the Instrument menu This dalog also enables automatic connection each time you start the software if it has been disabled see previous section Menu and Toolbars 16 Bio Plex Manager includes a menu bar and main toolbar at the top of the application window The pulldown menus contain all the major functions of the software Note that these menus change depending on whether you are displaying a Protocol window a Results window or neither You can move quickly between any number of open Bio Plex Manager windows Hold down the Cntl key then press Tab The main toolbar includes the major instrument controls including Start Up Shut Down Wash Unclog etc The various Protocol and Results windows also contain their own toolbars with commands specific to those windows These are described in greater detail in the following chapters Status Bar
169. ic Mean Factorhk1 and multiple Factorhk2 Factorhk3 etc housekeeping genes 199 Bio Plex Manager Software User Manual Normalization Settings 200 Bio Plex Manager 6 0 Software User Manual Troubleshooting Follow these suggestions to troubleshoot your Bio Plex system Message Problem Bio Plex Manager 6 0 software has detected a problem with low bead number Bio Plex Manager has detected a problem with selection of regions More Likely Too few beads in the assay Check bead number calculations in 2 500 per region recommended assay Plate not shaken for 30 seconds Remove plate from array reader and before analysis shake for 30 seconds Buffer volume in wells is too low Resuspend wells in 125 ul Perform 125 pl is recommended Remove Bubbles Microbubble in cuvette Perform Remove Bubbles Perform Unclog to verify fluidics integrity Low no sheath fluid Refill sheath fluid check sheath connections Perform Start Up Possible clog Perform Unclog and rerun If unsuccessful repeat If still unsuccessful contact Technical Support Less Likely Incorrect needle height Adjust needle height Incompatible plate type used Replace with flat bottom or filter plate and adjust needle height Vacuum system not calibrated Calibrate vacuum system see hardware manual Red laser failure Contact Technical Support Filter plate not flat Check filter plate flatness see hardware man
170. if desired and a description of the assay reading If you are using the Security Edition the field contains your user name which cannot be changed Enter a description of the assay reading in the Description field n Step 2 Select Analytes In this step you select the analytes that you want reported in the reading 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 After a reading you can go back and correct your selections in the Protocol window and or the Results file and the detected analytes will appear in the tables Click the Select Analytes button to view the analyte selection window Pi Protocol Select Analytes EY Add Panel S Edit Panel Protocol Settings Panel Mo Cytokine 8 Plex A Available Selected Region Analyte Region Analyte Mo IL 2 Mo IL 1b Mo IL 10 Mo IL 4 5 Mo IFN g Mo IL 5 Mo TNF a Mo GM CSF Add All gt gt lt lt Remove All Figure 41 Select Analytes window o3 Bio Plex Manager 6 0 Software User Manual Preparing Protocols o4 Bio Plex Manager groups analytes by panels Preconfigured panels of analytes are built into the software these panels correspond to off the shelf Bio Plex assays and include human mouse and rat cytokines and phosphoproteins plus the newer human
171. in toolbar or select Save from the File menu After you have performed a reading see Running the Protocol on page 102 the data from that reading are also saved with the Protocol file These data are overwritten in the Protocol file whenever you rerun the Protocol To preserve the data from multiple readings you must save separate Results files See page 118 Use Save As on the File menu to save a Protocol under a different name You can create a new Protocol by opening an existing Protocol modifying the settings selecting Save As and specifying a new name for the modified Protocol Reducing the File Size of Protocols Protocol files that include all the data from a reading can be very large gt 5 MB Much of this file size is due to the raw bead event data from the reading which is used to generate the bead map and histogram displays If you require a smaller file size for instance to send the Protocol file via email you can save a copy of the file without this raw bead event data but with the other numerical data from the reading intact 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 Protocol Files In the Save As dialog box select the Compressed mode checkbox to save the Protocol file without the
172. in o ica Be a M redi ed 7 Types or FES AT 8 Key Software Feauires ivs deo de reins Pl E e raa Boise ea a a pd c per ded 8 Chapter 3 Software Installation 9 System RequirementS 0 0 0 een 9 Required Screen Resolution nnan nnna aana 10 Installing the Software issant pna ea usd AE ee bia ade 11 MiCroSO NET ux en adiit a a eek O Ss 11 Bioplexdata mdb File 222 4 usada em Rm CUR ee d 11 Luminex LXR Directory and Files llle 12 Hardware Protection Key HASP Key llle 12 UAIRSIN REDE CT CEN 12 Bio Plex Manager 6 0 Software User Manual Table of Contents Chapter 4 Starting the System 13 Starting Bio Plex Manager 0 00 eee eren 13 Connecting with the Array Reader and Microplate Platform 14 Communication between the Array Reader and Microplate Platform 15 Disconnecting and Reconnecting 00 cee eee ees 16 Menu and ToolbarS esse uev dereud SEG E RR Diei SER Pee ee eee E 16 UCL St luc ms teat Seater petits tob ease rae dedi aw tears i ipud urat ertt den 17 aUe CUIR EET TTE TERT TR TUTO TT TOT TIT 17 Sample Needle Adjustment lille 18 Chapter 5 Controlling the System 21 Bio Plex MCV Plate IM ava daa Rx eR REOR ROG Y mE A ee ERI ERES 22 lcge dM 23 Optics Warm Up and Shut Down 0 0 0 cee ees 24 AUS AON PRIM ETT 25 Opening the Calibration Dialog Box 00 00 cee ees 26 Se
173. ing Variance Model OK Weighting Coefficients Reset Defaults Power Law Variance Coefficient a Auto calculate coefficient a Coelficentb 1 8 Figure 190 Logistic weighting Under default settings data points that cannot be measured or calculated according to pre defined specifications display as error codes Figure 191 tabulates the most commonly encountered error codes and their corresponding causes The non numeric code symbols in the Report table OOR lt gt or before concentration values may interfere with macros in some types of spreadsheets including Excel 191 Bio Plex Manager Software User Manual Determining Outliers 192 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 T 7 Report Table Display O Report Scheme Select columns to display Layout table by Display _ Norm Ratio Std Err Single Analyte E MormRatio CV 2 Multiple Analyte E si Norm Fl I Norm FI Std Dev Organize samples by E MormFiStd Err None E MermFi cv Conc in Range BE Typs Obs Conc Group E Obs Conc Std Dev d ObsCencStd Err C Expand Replicate Info Fe Cone arii Exclude table error codes Ob
174. ion creates an electronic audit trail of user activity that cannot be altered or deleted Please Log In d Bio Plex Manager is in secure mode and requires a valid login to the domain IPD DOMAIN Username BP Supervisor Figure 171 Log In dialog 171 Bio Plex Manager Software User Manual Electronic Records Users are required to enter their user name and password when they e Start the application e Start a reading e Unlock the application e Sign a document such as a Protocol or Results file Different users may authenticate operations For example a Clinician 1 level user may be logged into Bio Plex Manager but a Supervisor level user may enter his user name and password to authenticate a particular reading Note that in this case the Clinician 1 level user will remain logged onto the application Your organization must establish its own guidelines for user authentication of specific operations Electronic Records 172 Bio Plex Manager Security Edition supports the creation and control of secure electronic records as defined by 21 CFR Part 11 In Bio Plex Manager the following are electronic records e Protocol files e Results files e Calibration Log included in the bioplexdata mab file e Validation Log included in the bioplexdata mdb file e Instrument Operations Log included in the bioplexdata mdb file These records are accessible only through Bio Plex Manager and its associated utilities
175. is used to calculate your initial results However these settings do not affect the reading and you can change these settings and recalculate your results after a reading is complete Finally if you want to use different regression methods for different analytes deselect the Same Regression Type for All Analytes checkbox Then when you select a different analyte from the pulldown list you can select a new regression method as well 87 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 88 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 Logistic Weighting Variance Model Power Law Variance Weighting Coefficients Reset Defaults Coefficient a Auto calculate coefficient a Coefficient b 1 8 Figure 86 Logistic Weighting dialog There are eight models for calculating the variance in the logistic weighting algorithm e Linear Variance e Logarithmic Variance e Exponential Variance e Power Law Variance default e Linear CV e Logarithmic CV e Exponential CV e Power Law CV COEFFICIENT A Weighting coefficient a will be calculated automatically using the following equation a 002 pow m_YMax m YMin 2 blank 2 Alternatively you can enter your own value by deselecting the Auto calc
176. istogram and Bead Map 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 r Bead Mage 100 region eji nl Au e i Each cluster of dots E Ti that falls within a HE white region 2 represents a unique o f NEN bead set analyte E within an assay ff z 1000 Classification 1 Figure 104 Bead map with pop up window indicating the analyte region under the cursor If one or more clusters do not fall within the white areas there may be a problem with the beads such as photobleaching or the analyte selections in the Protocol may be incorrect If all the data clusters have shifted out of the white regions there may be microbubbles in the cuvette See the Troubleshooting chapter on page 201 for troubleshooting suggestions In this way the map can be used to confirm that the bead sets are correctly selected measured and identified CHANGING THE X AND Y AXES OF THE MAP You can change the channel types of the axes Right click the bead map and select one channel from the x axis submenu and the other channel from the y axis submenu The available channels are e Doublet Discriminator This channel measures light scatter from the beads as they pass through the red laser Light scatter correlates to particle size e Re
177. late e Results files See page 117 contain the data from each reading and tools and reports for analyzing that data The application database file bioplexdata mab is installed on your hard drive or file server when you install the software This database contains calibration validation and instrument operation information See page 11 for more detail Key Software Features Bio Plex Manager 6 0 is a single integrated software package that has Comprehensive instrument functions e Data collection e Maintenance e Validation e Automatic error detection and logging Comprehensive data display e Raw data presentation including tables bead maps and histograms e Customizable tabular presentations e Customizable standard curve and bar graph displays Comprehensive Calculations e Data normalization internal controls for gene expression analysis e Auto calculation and display of usable assay range e Multiple standard curve fitting options including Brendan Scientific StatLIA Logistic 5PL Comprehensive and versatile automated export options e Excel now including optional standard curve graphs e Comprehensive xml file e New Luminex CSV comma separated values output e New customizable reports through style sheets e New custom report for export to custom macros CFR 21 Part 11 compliance e User access management e Comprehensive audit trail and instrument logs e Secure documents results files and protocols Bio Plex Manager 6 0
178. lation Do not cancel the installation during this period The Bio Plex Manager Installation Program opens displaying a navigation screen for performing the installation Microsoft NET Microsoft NET is automatically installed on your computer when you install Bio Plex Manager It is required to support the logistic curve fitting features of Bio Plex Manager Bioplexdata mdb File During installation you will be prompted to save the application database file bioplexdata mdb to a location on your hard drive or a file server This database file contains logs of calibration validation and instrument operations activity for your instrument e For more information about the Calibration Log see page 30 e For more information about the Validation Log see page 39 e For more information about the Instrument Operations Log see page 45 You can save the bioplexdata mdb file to any folder on your computer The default location is the Bio Plex Manager application folder If your computer is connected to multiple instruments each instrument must have a separate bioplexdata mdb file saved in a different folder For more information about the calibration validation and instrument operations logs in Bio Plex Manager 6 0 Security Edition see the chapter on Controlling the System page 21 11 Bio Plex Manager 6 0 Software User Manual Software Installation NOTE The bioplexdata mdb file is not compatible with versions of Bio Plex Ma
179. le NOTE You cannot generate a new unsigned uncontrolled Results file in Secure Mode all Results files generated in Secure Mode must be signed Signed Protocol and Results Files Protocol and Results files become secure files when they are electronically signed using a user name and password Once signed these files become read only you can make changes to the file but they must be saved under a different file name An audit trail of changes is built into each Secure Protocol and Results file Secure Edition Protocol files have the file extension spbx Secure Edition Results files have the file extension srbx To sign a Protocol or Results file go to the File menu and select Sign Document The Electronic Signature dialog will open This dialog opens automatically when a Results file is generated from a Protocol file Electronic Signature d Please enter the username and password of a valid account on the domain IPD DOMAIN Username Password Please enter your reason For signing the document Figure 172 Electronic Signature dialog NOTE In the dialog the signing party enters his or her user name and password in the appropriate fields This may be someone other than the current user The reason for signing the document must be entered in the text field Typical reasons may include review approval responsibility or authorship Calibration Validation and Instrument Operations Logs When you cli
180. le 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 Reason For Change You have performed an auditable action 1 of 1 Date Time Action User Access Level Version 04 Oct 2007 12 28 PM Format Wells BP SupervisorMame Supervisor 5 0 0 528 1 0 0 6 Is Details 48 Undefined What is your reason for this change Figure 179 Reason for Change dialog used to generate the audit trail The dialog notes the date and time of each auditable action and describes the change edit made You must enter a reason for each change in the bottom field If you have made more than one change use the Next and Back buttons to scroll through the changes When you have entered a reason for each change click the Finished button 180 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 Mouse_002 Read Only Audit Trail signed by BP SupervisorName gt E amp B Time Access Level Action Description Reason Version 04 Oct 2007 12 34 PM BP SupervisorName Supervisor Sign File DETAILS Sign Before Run 5 0 0 528 1 0 0 6
181. lecting Entering Calibration Control Numbers 26 Setting Up the Calibration 0 0 0 0 ees 29 Performing the Calibration 0 0 00 cee ee 30 Logging the Calibration 0 0c cc eee 30 Wash Between Plates 0 00 ce eee 32 Remove Air BUDDIES vs dy Bh ae bU OC ee x xd du o ate 33 ee eS it eee ees eI REM UE UU M pu EE M E 34 validation C RETRSTELLUTEITTST PPP 35 VAA ONIE TUE RN ES C E OT TT TE I P EI OR TIT T 35 Validation Kit Control Number 0 000 eee eee eee 35 Setting Up a Validation Run 2 0 0 0 cc ees 36 Control Number Selection 0 0 0 eee ees 36 Validation Type Selection 0 0 0 cc eee 37 Performing a Validation RUN 0002s 38 Validation RESURS x2 9133 ud oen tad tween te ee eee bow hee 38 MANGAL OO Gis Dn 39 Platform Bleater 2 22 acd 9 de Dee EN eoe a RR Grads at pod ed Gb enge 42 Instrument Information 0 0 0 0 0 ce rr 43 Elect Betraet PIale o 2a debts le Rep gute dur act Anio ie XE ROV VES RU uos 43 Additional Instrument Functions llle 43 arce Operator Soiree ait asa pds acr Sie we ae hace eds 45 SNA OIA user i Gait apt asses Beet Fan warp Searhces pee Aa ese Wh eee nee Ga ee ake 45 Instrument Operations LOG 0c ce eee eee 45 Bio Plex Manager 6 0 Software User Manual Chapter 6 Preparing Protocols 47 moet MINCE 47 Creating Opening Protocol Files 00 0 cece eee eens 47 S
182. licate groups For each replicate group a mean value is calculated along with the standard deviation standard error and coefficient of variation 65 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 66 CHANGING A REPLICATE GROUP To change the replicate group of a well or group of wells 1 Click the Select tool k 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 The selection is changed to the specified group number Plate Formatting Plate Groupings Plate Formatting Plate Groupings 1 2 3 4 5 6 7 8 9 10 s T mammam 20 D LOL ILI IL LI NNMNN OOL O00 Figure 60 Selecting a different replicate group RESEQUENCING WELL NUMBERS If your well numbering is out of sequence and you want to correct it select Resequence Well Names from the Format Options menu All the wells in the plate are renumbered sequentially based on whether you have selected Autofill Across or Autofill Down This is a cosmetic change and does not affect the analysis in any way DEFINING STANDARD WELLS Standard wells contain analytes of known concentration which are used to generate a standard curve of fluorescence intensity versus analyte concentration The regression equation for the
183. lly recalculate all the data in the Report Table If you are unsure whether your data have been recalculated select Refresh Calculations from the View menu Use the Axis Transformation pulldown list to select the standard curve axis scale Four combinations of linear and logarithmic axis scales are available NOTE Changing the axis transformation will only change the display of the curve not the calculated data You can use a different regression model for each analyte Deselect the Same Hegression Type for All Analytes checkbox select a different analyte from the pulldown list and select the regression type to use for that analyte Select the Swap XY Axes checkbox to change the x and y axes of the curve Select the Show Conc Range Lines checkbox to display this range as dotted lines on your graph Select the Show Unknown Samples checkbox to display your Unknown samples as green triangles in your graph Select the Show Control Samples checkbox to display your Control Samples as yellow triangles in your graph The Curve Fit area contains automated optimization controls discussed in greater detail in Chapter 10 Standard Curve Optimizer on page 185 You can optimize individual or all analytes undo the optimization and Clear Outliers from your display To display all the data for each standard sample point in the curve position your cursor over the point A pop up box will list the data Figure 139 Pop up data for a standa
184. ly to Protocol File Enter Standards Info View Enter Standards Info Description X X amp Concentration Enter Std Curve Regression X X Method Select External Standards E MWO X eee Functions that Apply to Protocol File Enter Sample Info View Enter Sample Info Sample Description Enter Sample Info Sample Dilution Functions that Apply to Protocol File Enter Controls Info View Functions that Apply to Protocol File Run Protocol View Security Edition User Access by Function Select Bead Count Method Per Well Per Analyte Set Sample Timeout P Advanced Settings EE Change Raw Data Display Options Print Histogram amp Bead Map E dul ALL gt lt E x x x Histogram Restore Default Gates Histogram Adjust DD Gates Histogram Autoscale Histogram Log Linear View tr tr Pas m XX m gt x Histogram Zoom Histogram Max Restore Bead Map Log Lin Bead Map Zoom EE BeadMap Max Restore X X X X Functions that Apply to Results File General New Protocol from Results Pf X X Change DD Gates X Refresh Calculations En rere 3 i x Functions that Apply to Results File Describe Protocol View Author Field Description Field u u X I X XJ X X X Xx x lt x lt x x lt x lt x x Functions that Apply to Results File Select Analytes View Select Remove Analytes p x qx oo dL Add
185. main toolbar or select Open 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 See Using the Security Edition page 167 for more Security Edition information NOTES e Secure Edition Results files can be opened only as read only files using the Standard Edition of Bio Plex Manager e Results files created or modified by the current version of Bio Plex Manager cannot be opened by earlier versions of the software Saving a Results File To save a Results file with the file open click the Save button on the main toolbar or select Save or Save As from the File menu Reducing the Size of a Results File Results files that include all the data from a reading can be very large gt 5 MB Much of this file size is due to the raw bead event data from the reading which is used to generate the bead map and histogram displays If you need a smaller file size for example to send via email you can save a copy of the file without this raw bead event data but with the other numerical data from the reading intact NOTE We strongly recommend keeping a copy of the original Results file with the raw bead event data intact These data are necessary for changing the DD gate range in the Results file See page 123 and so that data can be exported in XML file format Results Files In th
186. mand line Figure 162 Document Export Properties dialog box Here is a brief description of each option e Bio Plex XML This is a comprehensive export of individual results files in an xml format This format is useful for import into LIMS Laboratory Information System or other database applications e Output CSV format This mimics the output of third party software such as IS 2 3 or xPONENT with common options e Custom Export using style sheets Customer developed stylesheets can extract any of the data exported to the Bio Plex XML file See page 164 for more information Using stylesheets allows you to format the data in any way desired provided you have experience developing stylesheets Some default stylesheets are included for guidance e Command Line Export Custom export by passing a command line to external applications See page 165 for more information 159 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 160 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 Advanced Run Settings Bead Map Save phons v Auto gave after run e je Auto ML export after run DD Gates Sampling Engis C Ovemde gates 1 Pause run enor occurs C1 Low bea
187. map Click the Maximize button E to maximize the bead map portion of the window within the larger Protocol window Click the Restore button rH to return the bead map to its default state Bead Map Display Options To access additional Bead Map display settings right click the bead map and select Options Histogram and Bead Map 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 DotPlot Options Density Filtering Filter Levels Display Above Level 2 gt Level Multiple 2 C Zoom Logarithmic Linear Density Decaying Logarithmic Display Density DotPlot Linear Display Decaying DotPlot Figure 106 Bead map display options NOTE These settings only change the bead map display and do not affect the underlying data in any way First enter a number in the Level Multiplier field This number is used exponentially to determine the number of events required before a data point changes color For the default value of 2 the color level changes at 2 4 8 16 etc events If you want to filter lower numbers of events from the display select the Filter Levels checkbox and use the arrow keys
188. mat 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 Select the Scientific Notation checkbox to display the number in scientific notation Select the Same Number Format for All Columns checkbox to use the selected number format for all the columns NOTE Changing the number format changes only how the numbers are displayed and printed and does not affect calculations in any way Click OK to make the changes or Reset Defaults to return all columns to their default number formats Other Table Formatting To display the description of each sample in the table click the Show Hide Description Column button amp above the table The Description column displays To automatically size the columns to fit the width of the text click the Autofit button on the toolbar Copying the Raw Data 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 values you copy can be pasted into other Windows applications 115 Bio Plex Manager 6 0 Software User Manual Running Protocols Rerun Recovery Mode 116 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
189. may want to review the individual FI values and select the appropriate member of the replicate group as an outlier Exp Conc Expected concentration of the analyte standard or control as specified in the Enter Standards Info window or Enter Controls Info window respectively Obs Exp 100 Recovery rate for the observed versus expected concentrations of the standard or control See page 134 Group Defined group of the selected well see page 66 Ratio A calculation performed when you have grouped samples in the plate formatting section This calculation is the ratio of the member to reference measured values or vice versa For more information see Ratio Simple and the Grouping Function on page 218 of the Appendix Dilution Specified dilution factor of the analyte Bead Count Number of beads counted in region for the analyte Bead Mean Mean fluorescent intensity of the beads counted in region for the analyte Bead Std Dev Standard deviation of the bead mean Bead Std Err Standard error of the bead mean Bead CV Coefficient of variation of the bead mean Trimmed Mean Bead mean excluding the top 596 and bottom 596 of fluorescent intensities Trimmed Std Dev Standard deviation of the trimmed mean Trimmed Std Err Standard error of the trimmed mean Trimmed 96 CV Coefficient of variation of the trimmed mean Sampling Errors Error codes for a reading See page 99 133 Bio Plex Manag
190. nager earlier than 4 0 If you have an earlier version of the software installing version 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 Luminex LXR Directory and Files A directory called Luminex is automatically created in the Program Files folder on your computer during installation Inside this folder is a folder called LXR that contains various applications for monitoring and communicating with the array reader and platform A Windows service called LXService is also installed and started This service enables automatic communication with the instrument when you open Bio Plex Manager Hardware Protection Key HASP Key A hardware protection key also known as a HASP key is required to run Bio Plex Manager The HASP key determines the license Instrument Control Desktop or Network and the edition Standard or Security of your Bio Plex Manager A complete list of HASP key part numbers and the software versions they enable is in the Bio Plex Manager 6 0 Software Upgrade and Configuration Guide part 10018402 Instrument Control or Desktop HASP keys must be attached to a USB port on the computer running the software Network HASP keys must be attached to a USB port on the network file server computer The HASP key has a driver that is automatically installed when you install Bio Plex Manager Uninstalling 1
191. nd made read only the audit trail was continued in the new file alfred Nobel io Plex Supervisor Supervizor o Plex Supervisor Bio Plex Supervisor io Plex Supervisor Supervisar io Plex Supervisor Supervisar Alfred Mabel supervisar Bioa Plex Supervisor Supervisor o Plex Supervisor Alfred Nobel ign File Figure 182 Example of a Protocol audit trail with signatures To display the complete contents of the Description and Reason cells in the table hold your cursor over a table cell and the information for that cell is displayed in a context pop up box Or you can click the Expand Audit Entries En button in the Audit Trail window toolbar to expand all the cells Modify Controls Info Sri peras DET A Concentration Format Yells vies All analytes C1 0 0 Serie pera AR Concentration All analvyEes C1 10 0 Figure 183 Example of a pop up information box in the audit trail To copy the contents of the audit trail to the Windows clipboard click the Copy to Clipboard button in the window toolbar To print the audit trail click the Print button Protected Directories Your Windows system administrator may set up protected directories on your computer or computer network for the secure storage of Protocol and Results files These protected directories must be set up using Windows tools and are keyed to your Windows user name and password which may be different than your Bio Plex Manager user name a
192. nd password Consult your system administrator for information about protected directories 182 Locking Bio Plex Manager Security Edition Locking Bio Plex Manager Security Edition 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 To lock the application go to the Tools menu and select Lock Application You will be notified that the application is locked and the Log In dialog will be displayed To unlock the application enter your name and password and click OK Logging Off When you close Bio Plex Manager Security Edition you will be automatically logged off You can also log off without closing the application Note that you cannot log off while the array reader is in operation Logging off will close all open documents You will be prompted to save any changes before logging off To log off go to the Tools menu and select Log Off Application You will be notified that you have successfully logged off and the Log In dialog will be displayed If a Supervisor level user unlocks the application that Supervisor level user will then be logged in as the current user 183 Bio Plex Manager Software User
193. ng the Reporter Validation Kit accuracy is defined as the percent difference between the measured regression curve and the expected or actual data points Microspheres that have associated Two microspheres that have associated are called a doublet The substance being measured identified quantitated or otherwise analyzed in an experiment In the Bio Plex system analytes are molecules that are being quantitated Immunological protein capable of binding a unique antigen via a specific binding site or epitope Used in immunological assays to quantitate the amount of a specific analyte present The bead and reagent mixture that when combined with sample is loaded into microplate wells and read by the array reader Fluorescent intensity measured by the reporter channel that is due to non specific binding of a fluorochrome and the electronic noise of the array reader If you include blank wells in your microplates you can calculate the average background intensity and subtract it from the intensity readings of your standards and unknowns A microsphere A process that uses microspheres calibrators with a stable fluorescent intensity to adjust the gain settings in the array reader s detectors for optimal and consistent microsphere classifications and reporter readings over time and across different array readers Microspheres with stable fluorescent intensities used to standardize the signal output of the array reader An antibo
194. ng the System control basic system functions such as instrument start up warm up calibration validation and shut down NOTE The Start Up 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 WARNINGS The sheath fluid container and the waste fluid container should be closely monitored The sheath fluid bottle must be placed at the same level as the array reader unless you are using the Bio Plex High Throughput Fluidics HTF see Warning below or Luminex Sheath Delivery System SDS The fluid level should be below the air inlet connection and above the sheath outlet connection Always check the sheath fluid level before starting a run or procedure If you are using the Bio Plex HTF or Luminex SDS it should sit on the counter next to the array reader while the sheath fluid cube should be placed about 3 4 feet below the array reader for example on the floor The waste fluid container receives waste from the system Do not allow the waste container to overflow Empty the waste bottle each time the sheath fluid bottle is filled The waste container should be placed on the bench next to the instrument Never place this container on top of the instrument All waste containers should have vented caps 21 Bio Plex Manager 6 0 Software User Manual Controlling the System Bio Plex MCV Plate IV 22 Bio Plex Manager 5 0 and later re
195. normalize your data See Data Normalization on page 193 The Export button allows you to export your data to Excel or a word processing application See Exporting the Report Table on page 137 The Report Table Display Options button accesses the remainder of the toolbar functions See the Display Options section starts on page 128 The Show Hide Outliers button allows you to view or hide outlier data See Showing Hiding Outliers on page 135 The Organize by Type or Group button is discussed on page 130 The Single or Multiple Analytes button is discussed on page 129 The Show Replicates button is discussed on page 130 The Sort button allows you to sort the table data alohanumerically See Sorting Report Table Data on page 137 127 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 128 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 Ell Report Table Display Options Report Scheme Select columns to display Display Column 4 Type Vell Outlier Description Fl FI Bkgd Std Dev Std Err WEY Morm Ratio Morm Ratio Std Dev Morm Ratio Std Err Morm Ratio cy Morm FI Morm Fl Std Dev Morm FI Std Err Morm Fl CY Obs Conc Obs Conc Std Dev Obs Conc Std Err Obs Conc WEW Exp Conc Obs Exp
196. ns button or select the command from the Table Options menu Furor Esta Display Options iet Type eecnptiori Show Bani Fagon Counts magan Show Hitogam and Bead Hap Toa T Age Bidi Tangir Errors Karn but Des ce Error Fine E Regat Sassi end RFA Target Plater Heales Target Fisioa Temp C Bened Map Bees Count aF Sand Humba Fo DOB E L E e E O m L Ono 7 Select Al CI Save retrot stdin trewaca O Figure 110 Raw Data Display Options dialog In the Raw Data Display Options dialog select the columns to display using the checkboxes You can also choose between predefined report settings by selecting Standard or Diagnostic in the Report Scheme field these selections display different sets of columns Select the Show Bead Region Counts and Show Histogram and Bead Map checkboxes to set these display options as the default for the Raw Data table Select the Save Settings as Default for New Protocol checkbox to use the selections in this dialog as the default for new Protocol files Raw Data Table 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 Set Column Number Format Column Y Number Format Cancel Decimal places 1 Reset Defaults C Scientific Notation Sample 1234 0 C Same number format for all columns Figure 111 Set Column Number For
197. nt Norm FI is calculated as follows when housekeeping genes internal controls are chosen Avg ALHEG FI arre Norm Fl wel Fl Gene of Interest erus Eus F where Avg All HKG FI ait wells the average FI for all housekeeping genes in every well of the experiment This number serves as a mean factor around which all others will fall and converts a unitless ratio back into FI The next calculation averages per well data to arrive at the per sample normalized fluorescence value Norm FI ample Horm FI menyt Norm FI gt Norm FI jy Norm FI sample A When control is chosen Norm FI is calculated as follows Norm FI sample x Norm ratio sample x Average FI control sample Average Fl control sample Serves as a mean factor around which all others will fall and converts a unitless ratio back into FI 217 Bio Plex Manager 6 0 Software User Guide Appendix 218 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 Outside the group these numbers are arbitrary and meaningless unless the reference sample is another instance of the exact same biological sample in every group Since data outside of groups cannot be compared the grouping function in the plate form
198. nternal control divided by the Norm Ratio multiple control as defined in Case B for the control Norm Ratio Norm Ratio multiple internal Norm Ratio multiple E None Single Ratio of Fl Bkgd for the analyte and the control Norm Ratio Fl Bkgd FI control Bkgdcontrol NOTES e Replicates are done at the plate level See the section on plates to define replicates e When using multiple replicates the Norm Ratio for each replicate is calculated as described in Case A B C D and E For each sample type the replicates average is the Norm Ratio e For information about the formulas Bio Plex Manager uses to calculate normalization values see Normalization Formulas in the Appendix Normalization Settings Normalization Factor Calculations Normalization factors are determined in the following ways A Control sample Average of Fl Bkgd for the external control for present for all analytes normalization Factor Average Flcontrol Bkgdcontrol analyte1 Flcontrol Bkgdcontrol analyte2 Flcontrol Bkgdcontrol analytes etc B No control sample Average of Flhk Bkgdhk for all wells with this and single housekeeping gene Data are taken from raw housekeeping gene data table average replicate values for each sample type from report table are not included Factor Average Flhk Bkgdhk well1 FlIhk Bkgdhk well2 FIhk Bkgdhk well3 etc C No control sample Factor Geometr
199. nts RESULTS This column presents the optimization event results reported as three different outcomes e Successful The algorithm was successful It was able to improve the range of the assay or no changes were implemented e Not Optimized No changes were identified which could improve the range of the assay It is possible that manual optimization could improve the fit e Check Data There were significant challenges associated with this data file and it is recommended that the data be reviewed Review the included comments in any case Possible comments are described below 188 Determining Outliers COMMENTS Comments are included to point out particular issues that may be of interest e Low Bead Counts Detected Low Bead This message will be presented if fewer than 20 beads are detected This suggests one of the following problems Incorrect gates are set Incorrect region is set Insufficient agitation of plate before reading Perforated well if using a filter plate Some other contributor to bead loss e Standard Concentrations are not set Stand Conc Standards have either no value of 0 value entered Correct this by applying the appropriate concentration to your standards or renaming your O standard as a blank it is inappropriate to have a standard of 0 value e One or more standard has a CV higher than 20 High CV If one or more standard has a CV higher than 20 you may want to
200. ogram Files Bio Rad Bio Plex Manager 6 0 Custom Reportsl Report Template Gene Manager New Analysis xlt Use Browse button if you store templates in various locations Pull down menu contains available reports Generate Report Figure 116 Custom Reports Dialog Bio Rad plans to release custom report templates for specialized applications periodically via the web Bio Plex Manager 6 0 includes the Gene Manager an Excel based analysis application designed for genotype including SNP and Present vs Absent analysis It contains its own online help documentation and is launched from the Custom Report dialog box Sample results files with genotype data are present in the Bio Plex Manager example file directory The default directory is C Program Files Bio Rad Bio Plex Manager Example Files You can save different versions of Gene Manager with your own analysis preferences which show up in the list of available reports This is particularly useful if you are continually rerunning the same assay 121 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 122 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 Res
201. ol sample s calculated value When internal controls are not chosen Awerage FI pampk m Norm Ratio sample Agerage FI protol sample When internal controls are chosen Horm Ratio temple xi Norm Ratio ample x Norm Ratio tton mplei Norm Ratio When internal controls are chosen and no control samples are identified FI Gane of terest Norm Ratio wer E RT HEGI HEGa D To figure Norm Ratio per sample use the average normalized value of each sample i Norm Ratio myt Norm Ratio weng t Norm Ratio ey Norm Ratio sample E Norm Ratio mn eom n orm Fatio Norm Ratio Calculating Norm Ratio allows the researcher to express all data relative to the control so that data across experiments using the same control samples can be compared When control sample and housekeeping genes internal controls are chosen Horm Ratio templ xi Norm Ratio O sample x Horm Ratio tContml amp mple Normalization Formulas Normalized Fluorescence Values Norm FI This calculation requires internal controls to be chosen Normalized Fluorescence Values Norm Fl 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 It is left to the researcher to decide the appropriateness of this calculation for a particular experime
202. ols shown in the red rectangle to access your standard lots To save a Standard Lot for reuse name it and enter an expiration date if required The calendar under the Expiration Date pulldown menu enables you to set a specific date Save places your new Standard Concentration Lot into the Load Standard Lot window so it can be reused fi Load Standard Lot Select a Standard Lot to load Lok Mame Expiration Date 5005432 Mouse Group I Cytokines Mone Figure 81 Load Standard Lot window Load brings up the Load Standard Lot window so that you can choose between previously saved standard lots Manage Standard Lots brings up the Manage Standard Lots window that allows you to import or export your standard lots You can also select any number of Standard Lots for deletion 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 If you are using external standards the concentrations are automatically entered and you can skip to the next section on selecting concentration units page 87 To enter concentrations make sure the Standards Info tab is selected ENTERING CONCENTRAT
203. omization 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 If you are not familiar with stylesheets there are contractors who specialize in stylesheet design IT professionals who are familiar with XML can often develop custom stylesheets based on the examples given Once a style sheet is identified for document export it will be used to define the export file parameters and the resulting file will be placed in the location indicated in the destination line discussed below FILE DESTINATION OPTIONS Use the default file name and location or click Choose Folder to specify a new file name and location The exported file can be placed on your system or in a shared file Destination File Use Folder Choose Folder C Documents and Settings csetine Desktop Command line See Figure 166 Destination field of Document Export Properties Document Export Options Command Line Export This is an advanced option for customers who use custom designed applications to analyze their data You need knowledge of the following to use this option e he term command argument e Writing computer code e Implementing custom soft
204. on 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 It is not advisable to construct an assay without defining the concentration of the subject coupled microsphere preparation The total surface area total number of microspheres represented in an assay is a critical variable so the optimization and control of this variable begins with manipulation of known microsphere numbers Microspheres can be counted with a hemocytometer Please follow counting and calculation methods outlined in the instructions for your hemocytometer Microsphere Separation Methods Bio Plex assay development often requires separation of microspheres from an aqueous matrix This can be done using either centrifugation or vacuum methods CENTRIFUGATION Microsphere coupling protocols require separation of microspheres from reaction mixtures or wash buffers by means of centrifugation Generally a tabletop microcentrifuge that can spin 1 5 ml microcentrifuge tubes can pellet microspheres in 1 2 minutes The g forces vary in these instruments from approximately 6 000 to 13 000 X g VACUUM A vacuum separation method can be used for routine high throughput washing required for washed assay procedure
205. ontains the data from a reading the Protocol parameters used to collect that data and analysis tools for interpreting the data It includes all the information necessary to analyze raw data including a description of the assays well types concentrations dilutions and the regression method used for calculating the concentrations of unknowns from standards It also includes information about the instrument and the run conditions at each well at the time of data acquisition In the Results file you can change many of the parameters copied from the original Protocol file including the plate format analyte selection standards info etc and recalculate the results However you can change only the parameters of wells that have already been read and you can calculate only results based on the analytes that were present in the sample NOTE If you selected Auto Save After Run in the Protocol window the Results file is automatically saved after a reading Otherwise you must save your Results file as a separate step To generate a Results file from an incomplete reading or a previous reading open the Protocol file that contains the raw data for that reading and select gt New Results from Protocol from the Run Protocol toolbar or the File menu 117 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 118 Opening a Results File a To open an existing Results file click the Open button on the Quick Guide or
206. ormalized fluorescent intensity See Data Normalization on page 193 for more information on normalizing data Norm FI Std Dev Standard deviation of the Norm FI values for all the wells within a sample s replicate group Norm FI Std Err The normalized standard deviation Norm FI Std Dev divided by the square root of the number of replicate wells within the sample s replicate group Norm FI CV The normalized standard deviation Norm Fl Std Dev divided by the normalized Fl of the sample s replicate group 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 134 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 Note that the observed concentrations of standards are back calculated from their fluorescent intensities and the standard curve Obs Conc Std Dev Observed concentration of the analyte with the standard deviation of the replicate group of wells Obs Conc Std Err Observed concentration of the analyte with the standard deviation of a replicate group of wells divided by the square root of the number of replicates Obs Conc CV Observed concentration of the analyte with coefficient of variation of a replicate group of wells If this value is high you
207. ose and restart Bio Plex Manager with array reader Cables between computer and Check cables for proper connections array reader or microplate platform See System cable connections on are loose not connected page 14 Pressurizing in Leak in sheath bottle or cap Tighten sheath cap software status bar Platform heater not at Insufficient warm up time Allow at least 15 minutes for platform target temperature allowed heater to reach target temperature If heater does not reach target in 30 minutes contact Technical Support Platform heater not at Platform heater not functioning Contact Technical Support ambient temperature properly Insufficient cool down time Open platform door to facilitate cooling allowed An ice pack can also be placed on the platform plate holder to facilitate cooling No Assay Signal More likely Detected Sheath reservoir cap not on Tighten sheath cap Message should securely disappear within two minutes Sheath bottle lines are not Make sure that all hoses are connected connected properly to the appropriate ports and that they have clicked into place Less likely Sheath fluid level above the AIR Adjust sheath fluid level so that sheath port on the sheath container fluid is below the AIR port of sheath bottle Sheath bottle has a leak Try new sheath bottle Call Technical Support for further assistance High throughput fluidics HTF Turn on HTF and check to
208. osoft Corporation Pentium is a trademark of Intel Corporation No rights or licenses under any of Luminex Corporation s patents are granted by or shall be implied from the sale or acquisition of this Bio Plex system containing Luminex technology the System to you the end user By using this System you agree that i the System is sold only for use with fluorescently labeled microsphere beads authorized by Luminex Beads and ii you obtain rights under Luminex s patents to use this System by registering this System with Bio Rad in accordance with the instructions accompanying this System and purchasing a kit containing Beads Copyright 2001 2010 by Bio Rad Laboratories All rights reserved Bio Plex Manager 6 0 Software User Manual Table of Contents Chapter 1 Bio Plex Suspension Array System Overview 1 GOMDOMCIINS serae pa EUM es Pee eS M qoM ak VA i ela ae 2 AOValitades sc ux ve gd urbi ed CS eases a dr vat cw he e d d aou ues 3 For More Information s uides hic tmo Hate M re E cerco ek iu Sca d M ce E ane a 4 Bio Rad Technical Support 000 eene 4 Chapter 2 Bio Plex Manager Software Overview 5 SOItWare BQUONS sirara teu E oer aes aad eae eee EUR Da SUL hae ae es 5 Software LICENSeS uou x a a Ode PES E bare ae Pe Ed ete 6 Compatibility with Luminex Software 0 0 000 cee eee 6 General WOFKHlOW 5 s ck aoa Ete eU CR C oe E Je RM e ae DR 7 QUICK GUIE ease a ESAE Ps wate oid aise e a
209. p and histogram Alternatively hide the histogram bead map display by clicking on the Show Hide Histogram Bead Map button Lil to maximize the table Well Type Human IL 2 38 HumaniL i 52 Human IL 6 tia Human IL 8 HumaniL fU 56 Hu AT 1 AUG S 014 rS S 241 ZA 20 0115 SS SSS C1 Bi Sa z1558 OCIS ZUR OC z389 0 1171 z957210 125 3771 0 115 Ci 53 30065 0 113 25495 D 120 18454 0 107 3496 0 155 Sat OS 1123 Di 54 12722 5 1123 26195 114 2578 0 103 1613210 131 Sear 5 119 Ei 55 7210 Stra 165 0 1353 215 0 ts OES 0630 1356 0 1330 F1 35 1658 S 0230 1035 154 54502 VES iar 655 0 157 GH Sf 143 0 126 1045 122 55 5 104 13200 113 E orna Hi B 1T130 117 104 0 101 420 103 855 130 EIB5 119 A 51 3i T8 0 807 23785 D 127 306 0 101 301 68 0 1300 31875 5 100 E zr E dx 1055 Zonro 5 1123 23775 0 116 Sos 5 5 CE danti LOCI L 53 AO 33 OO ZAB34 D TXZ 1738950 1301 z2555 0 115 2200588 10 01253 Ci Sa 11556 D 135 2526 5 150 2273 0 110 15478 5 110 695 0 105 E2 55 T330 115 1680 124 230 0 112 1065 125 1414 0 1107 F2 55 Au t 104 5 110 TA D 114 137 0 111 716 0 126 io SF Tz DA B4 Der amp 8 5 112 V25 0 1584 y 2515 H B 115 5 140 B5 1440 8500 121 21 5 140 5345 100 Ls x1 zB595 D 115 P4950 5 125 25251 0 130 2618 5 159 2006 0 1013 Bi Ko 28025 5 1001 251425 127 244000 135 255554 D i 2702780 0107 3 Ka 30572 D 117 142355 5 125 9452 0
210. plate yielding up to 9 600 data points in about 30 minutes Instantly customize your experiments by mixing Bio Plex assays or creating your own assays Analyze results prepare reports and print and or export data immediately after each reading Provide a complete electronic audit trail of data generation and analysis in a secure digital environment with multilevel account access that is compliant with the Code of Federal Regulations Title 21 Part 11 Electronic Records Electronic Signatures Bio Plex Manager 6 0 Software User Manual 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 219 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 Phone 800 424 6723 option 2 Fax 510 741 5802 E mail LSG TechServ US Bio Rad com U S LSG TechServ IntlI Bio Rad com International Web _http www consult bio rad com Bio Plex Manager 6 0 Software User Manual 2 Bio Plex Manager Software Overview Bio Plex Manager 6 0 software runs on a computer installed with the Windows XP operating system and requires a hardware protection key also known as a HASP key installed on either the computer itself or the computer network syst
211. porter 1 This channel measures the reporter molecules bound to the analytes on each bead e Classification 1 This channel measures the first classification dye embedded in each bead e Classification 2 This channel measures the second classification dye embedded in each bead 109 Bio Plex Manager 6 0 Software User Manual Running Protocols 110 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 Bead Hap 100 region E e nj JA wa 1 a xi 1 g Hi ae ehana Lii X Axis Doublet Descriminator E b zu t g axis F Reporter 1 wr Fi E wv Classification 1 i Lii e Options i i o easton Auriliary 1 Tub 10000 100 Classification 1 Figure 105 Changing the channel displayed on the x axis SCALING THE BEAD MAP DATA The default view of the data is log scale From the toolbar click the Log Linear button e to change the display from log to linear Click again to return to log scale MAGNIFYING AND RESIZING THE BEAD MAP Click the Zoom button e in the bead map toolbar to magnify the display or With Zoom selected the cursor changes to a hand symbol v when you position it over the map Drag the hand cursor to view different magnified regions of the bead
212. ptions dialog The report table displays columns for data normalization The column titles associated with the normalization calculations begin with Norm Ratio or Norm FI Normalization Settings Predefined Normalization Report Schemes Select Norm Qualitative in the Report Scheme field The Norm Qualitative column is designed to be used with gene expression When selected in Report Table Display Options the following data normalization columns appear in the Report Table R Human Cytokine 17 Plex Standard PMT Read Only Report Table 4 is UC Ux A DW rire LC E FR Analyte Hu IL 1b 22 vi Type Wel Horm Ratio Horm Ratio Std Dev Horm Ratio Std Err Horm Ratio CV Horm Fl Horm FI Std Dev Horm FI Std Err Figure 196 Data Normalization Columns in the Report Table Horm FI CV Normalization Definitions The following columns and their associated calculations are provided when performing data normalization Here are definitions for these columns e Norm Ratio Normalized ratio See Normalization Formulas page 215 in the Appendix for details about the formulas used for this calculation e Norm Ratio Std Dev Standard deviation of the Norm Ratio values for all the wells within a sample s replicate group e Norm Ratio Std Err The normalized standard deviation Norm Ratio Std Dev divided by the square root of the number of replicate wells within the sample s replicate group e Norm Ratio
213. quires 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 0 with its Reporter and Classify bead sets It also includes two open needle wells for adjusting needle heights for microplates or PCR plates NOTE Bio Plex Manager 5 0 and later require the use of the Bio Plex MCV Plate IV the Bio Plex Validation Kit 4 0 and the Bio Plex Calibration Kit N CN eve QUSS _ pret e Yo s orn A pomt AK vac fez Figure 12 Bio Plex MCV Plate IV When a particular procedure such as calibration requires you to add solutions to the Bio Plex MCV Plate IV the dialog box describing the procedure includes a diagram of the MCV Plate IV with the wells to be loaded highlighted in blinking yellow CAL1 amp CAL2 Calibration This operation performs CAL1 and CAL2 calibration 1 Vortex CAL1 and CAL2 beads for 30 seconds 2 Load 5 drops of CAL1 and CAL2 beads 3 Fill DI H20 reservoir MCV Plate IV 4 Click Eject then load MCV plate 5 Press OK to start 4 Eiect Retract Plate Figure 13 Highlighted wells in diagram indicate wells to be filled Start Up Start Up Start Up is a series of fluidic functions that prepares the array reader to acquire
214. r Standards Info e 5 Enter Controls Info B Enter Sample Info 7 Run Protocol 78 Analyte Standard Lot Lot Hu IL 2 38 Std Regression Curve Regression Type Logistic 5PL Axis Transformation Loa x Linear y Same regression type for all analytes Same units for all analytes Units don t impact calculations Same recovery range for all analytes Concentration Units sd Expiration Date O Assign Standards Information Acceptable Recovery Range 70 130 v Calculate Concentrations Most Concentrated Standard 1 Apply dilution to all analytes v Apply same concentrations to all analytes Oss Calculate Clear All Concentrations Protocol Window 3 Enter the dilution factor and press calculate Pi Protocol2 Enter Standards Info PISIS Standards Info Extemal Standards Info Select Extemal Standards Standard Lot Lot Expiration Date Analyte 1 Describe Protocol Hu IL 2 38 Std Regression Curve Regression Type Assign Standards Information Std Description HuiL 2 9 Hum 4 5 Hut 5 53 HuiL 0 56 HulL t2 p70 75 HuIL t3 51 SL 2 Select Analytes Logistic SPL Axis Transformation Log x Linear v v Same regression type for all analytes Concentration Units 1 v Same units for all analytes Units don t impact calculations 1 Accept
215. range after a reading in the Results file and reanalyze the data Plate Loading Guidelines Before starting the run note the following plate handling guidelines and warnings 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 Sample may wick or leak from the bottom of the well causing problems when reading the plate Make sure that you have added at least 125 ul of sample to all the wells specified in your plate template before starting a reading If the array reader attempts to draw sample from an empty well air is sucked into the sample loop and injected into the flow chamber As a result bubbles form in the cuvette and interfere with the analysis If this happens perform a Remove Air Bubbles procedure page 33 and rerun the Protocol Shake the microplate on a plate shaker for 30 seconds prior to performing a reading 101 Bio Plex Manager 6 0 Software User Manual Running Protocols Running the Protocol 1 When you have selected and reviewed all your Protocol options and prepared your microplate click the Start button The Run Protocol dialog opens Protocol Run Protocol Lo m Js Ma 20e mi Beads 100 per region Run at High RP1 Target Sample Timeout sec none
216. ration calculations Note that if external standards and current standards are defined for the same analyte the external standards will always be used to calculate the concentrations of that analyte Standard Lots You can create reuse and import standard lots to ensure repeatable results between experiments and or researchers in a workgroup This function eliminates the need for you to enter or re enter this data manually Click the Enter Standards Info button in the Protocol Settings pane Choose the Standards Info tab 13 Bio Plex Manager 6 0 Software User Manual Preparing Protocols The Standard Concentration Lot window is at the top of the screen Standard Lot we ion ate E Assign Standards Information Stal Description Hu IL 5 33 Hu G CSF 5T Hu MIP 1b 18 51 0 00 0 00 0 00 E 0 00 0 00 0 00 53 0 00 0 00 0 00 54 0 00 0 00 0 00 55 0 00 0 00 0 00 zB 0 00 0 00 0 00 Calculate Concentrations Most Concentrated Standard 9 51 S6 Concentration of 51 Hu IL 5 33 Apply dilution to all analytes Dilution Factor o A Calculate Apply same concentrations to all analytes 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 Standard Lot nz
217. rd sample point in the curve Obs E xpi 100 100 143 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 144 Regression Methods There are seven regression types for calculating the standard curve e Logistic 5PL e Logistic 4PL e Linear e Cubic spline e Point to point e Linear Semi log e Point to point semi log The minimum number of nonzero standards required for each regression method type is shown in the following table Method Minimum number of standards Logistic 5PL 6 Logistic 4PL 5 Cubic spline 4 Linear linear and semi log 2 Point to point linear and semi log 2 Analyte Regression Type Logistic 5PL Axis Transformation Loats Linear y Adv Settings v Same regression type for all analytes Swap XY Axes iL gt 9 D E m o D o a t S5 t E 100 00 1000 00 10000 00 Concentration pg mL Bi Standards O Partial Outliers D Outliers Extemal Standards Partial Outliers Outliers Std Curve Fl 64 2831 30050 64 2831 1 Conc 1437 12 1 7272 0 53741 FitProb 0 3217 ResVar 1 1642 Ext Std Curve Fl 12 744 27388 9 12 744 1 Cone 1293 89 2 37881 0 389901 FitProb 0 9949 ResVar 0 0244 Figure 140 Example of two Logistic 5PL curves for standards on the current plate and external standards If you do not have the minimum number of standards for the selecte
218. re compliance with 21 CFR Part 11 Standard Mode vs Secure Mode Bio Plex Manager Security Edition can run in Standard Mode in which all the security and audit trail features are disabled so that software functions like the standard version of Bio Plex Manager or it can run in Secure Mode with the security functions enabled This chapter assumes you are running the software in Secure Mode unless otherwise noted When you are in Standard Mode the status bar displays an unlocked m symbol When you are in Secure Mode the status bar display a locked symbol 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 operating system for full Secure Mode functionality Installing and Starting Bio Plex Manager Security Edition Security Edition is installed and starts up just like Standard Edition After installation the software will run in Standard Mode until an administrator level user see Security Edition User Access by Function on page 211 in the Appendix enables Secure Mode When you start Bio Plex Manager in Secure Mode you are prompted to log in with your user name and password NOTE User names and passwords must be s
219. re finished the Selected External Standards list should include all the analytes that you want to use as external standards and their source file s L human_cyokines_001 Enter Standards Info P Setti n rotocol Settings Standards Info External Standards Info Select External Standards X 1 Describe Protocol Available External Standards Selected External Standards Analyte Ext Std Source Add Analyte Ext Std Source amp Select Anabtes 18 Hu MIP 1b Human Cytokine 17 Plex Hii 13 HulL 5 Human Cytokine 17 Plex High PMT 21 HulFN g Human Cytokine 17 Plex Hi Remove 32 Hull 1b Human Cytokine 17 Plex High PMT 3 Format Plate 33 HulL 5 Human Cytokine 17 Plex Hi 52 HulL 4 Human Cytokine 17 Plex High PMT 34 Hu GM CSF Human Cytokine 17 Plex Hi 4 Enter Standards Info Hu TNF a Human Cytokine 17 Plex Hii Hu IL 2 Human Cytokine 17 Plex Hi Hu IL 13 Human Cytokine 17 Plex Hit 5 Enter Controls Info Hu MCP 1 MCAF Human Cytokine 17 Plex Hi a Hu IL 8 Human Cytokine 17 Plex Hii 5 B Enter Sample Info B IL 10 Human Cytokine 17 Plex Hi UU u G CSF Human Cytokine 17 Plex Hii J 074 Uell 7 Lien m Cobak ina 17 Dla Ll 7 Run Protocol pS k lt Remove Al Fill Available List Clear Available List Figure 78 Selected external standards Click the External Standards Info tab to enter additional information about your external standards 82 Protocol Window NOTE The Stan
220. re than 2 C during the course of the day If the temperature changes by more than 2 C a message box prompts you to recalibrate e Before data acquisition if you switch between Bio Plex Manager and Luminex software installed on the same computer See page 6 NOTE Before calibrating make sure that optics warm up is complete The Bio Plex Calibration Kit contains calibration microspheres CAL1 and CAL2 beads with stable fluorescent intensities in the RP1 CL1 and CL2 wavelength ranges The calibration process uses these microspheres to adjust voltage settings for optimal and consistent microsphere classification and reporter readings over time and across different instruments Current calibrated settings are automatically applied to any new session Calibration using Bio Plex Manager requires the Bio Plex MCV Plate IV CAL1 beads and CAL2 beads from the Bio Plex Calibration Kit and distilled or deionized water The CAL1 beads calibrate the array reader s doublet discriminator and classification channels while the CAL2 beads calibrate the array reader s reporter channel for reporter fluorescence detection 25 Bio Plex Manager 6 0 Software User Manual Controlling the System Opening the Calibration Dialog Box To begin calibration click the Calibrate button a on the Quick Guide or main toolbar or select Calibrate from the Instrument menu The Calibrate dialog box opens Calibrate Enter user name Last calibration IPD DOMAINSBP
221. rformed simultaneously in the same reaction container The process of determining that an instrument is fit for its intended use The Bio Plex Validation Kit is a tool for operational qualification The process for verifying the alignment of the optics assembly of the array reader A well or replicate group of wells whose measured value is outside the normal range You can exclude outliers from statistical calculations A group of related bead sets For example the Human Cytokine panel includes bead sets for Human IL 2 Human IL 4 Human IFN gamma and other human cytokines You can select multiple bead sets from the same panel or sets from different panels in the same multiplex assay 207 Bio Plex Manager 6 0 Software User Manual Glossary Term PE Phosphoprotein Photobleaching Photomultiplier tube Phycoerythrin PE PMT Precision Protocol file pbx or spbx Quality control Recovery percentage Recovery range Reference Region 208 Definition See phycoerythrin An enzyme that functions to phosphorylate specific proteins Phosphoproteins are responsible for the activation of a variety of proteins through the process of phosphorylation A chemical reaction caused by exposure to light in which a fluorochrome is converted into a differently fluorescent or non fluorescent compound A light detector typically used in fluorescence detection systems designed to convert a fluorescent signal into
222. rol Numbers Available List Selected List Control Number Control Number 9999901 310000167 Test101205b 999999 310000003 300001055 lt lt Remoyve All 300000489 Figure 26 Add Remove Control Numbers dialog 36 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 gt gt 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 gt gt button Use the lt lt Remove and lt lt Remove All buttons to unselect them At least one Control Number must remain in the Selected List When you click OK the Control Numbers in the Selected List are added to the pulldown list in the Validate dialog To view the specifications for a Control Number select the number from the pulldown list and click the Show Specifications button The Control Number Specifications dialog box lists the validation specifications Specification for Control number 9999991 Optics Reporter Classification Fluidics High Low Values DD Median 4774 6593 CLICV 2 00 7 00 CL1 Median 3134 4067 CL2 CV95 3 00 8 00 CL2 Median 3190 4142 RP1CV 4 00 10 00 RP1 Median 14833 19251 Figure 27 Control Num
223. rotocol window of a Protocol file They also can be displayed after a reading in the Raw Data window of a Results file The histogram and bead map share window space with the Raw Data table If the histogram and bead map are not visible click the Show Hide Histogram Bead Map button ul to display them Bead Hap 100 region posa ge o ele of 1 J 8 L 1 J ix F 58 4 ii q J 5 4 i a un a F F j 1 1 1 4439 8535 12630 18726 333510000 Doublet Discriminator i Well Type MolL 1beta 19 MolL 2 36 MolL 4 32 Mo IL 5 52 Mo IL 18 56 Mo GM CSF 73 Mo IFM gamma 1 ar 81 2304 5 30071 0 E377 0 2331 0 26726 0 15422 0 14522 0 52 250750 232580 B240 3708 0 24058 0 3353 0 114430 Figure 99 Histogram and bead map You can see some rows of the Raw Data table below the histogram and bead map display if you maximize the window During a run the well number being read is displayed in blue in the upper left corner of the histogram After the run select the wells to display by selecting rows in the table below the graphs Histogram and Bead Map When viewing the histogram and bead map you can choose to display all analytes each analyte separately or all beads that pass through the discrimi nator gate Make your selection from the pulldown menu above the histogram Histogram Al Gated All Gated af Welt CI 2
224. rt function For example if you select Type organization any sorting will be within each type of sample EXPAND REPLICATE INFO If you have defined multiple wells as a replicate group in the Format Plate window the wells will be listed together in the Well column and mean data values for those wells will be reported in the relevant columns for the selected analyte unless the wells have been flagged as outliers see below Click the Expand Replicate Info checkbox in the Report Table Display Options dialog to review the data for each well in a replicate group Or click the Show Replicates button EP in the toolbar to expand the data for each well in a replicate group The mean values as well as the individual well values and are reported for each replicate group Type wen m Bj ABBECSDe 273 1 13384 3 S2 15129 85 FI Bkagual 27 3 9367 0 15102 5 AB BB c5 DE 133B7 n 197455 18388 8 15102 5 15351 3 14853 8 Figure 125 Hidden vs expanded data for replicate wells EXCLUDING DiSPLAYING TABLE ERROR CODES Error codes such as OOR or appear in the Report Table if data cannot be measured or calculated or if a sampling error occurs To hide error codes in the table select the Exclude Table Error Codes checkbox Report Table Click the Display Help for Table Legends button on the toolbar to get definitions for any error conditions SETTING NUMBER FORMATS Cli
225. s In this case the microsphere reactions are performed in microtiter filter bottom 96 well plates A vacuum is applied to the plate allowing the liquid to filter through while retaining the microspheres on the filter Resuspension is accomplished by adding adequate fluid to each well and by repetitive vigorous pipetting 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 Microsphere Stability and Storage xMAP microspheres are light sensitive and should be protected from light during all stages of storage and usage In addition freezing conditions and organic solvents should be avoided 223 Bio Plex Manager 6 0 Software User Guide Appendix Software Warranty 224 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 The Purchaser must notify Bio Rad in writing within 30 days of
226. s 224 Reporting Problems to Bio Rad 0 00 cee eee 225 Report Table Error Godes 2x edu Sake tee AC ace OR Os 226 ICI X 2 5 Ri dotasaa E ata pai a cU Sed E UB EUN HAC UR CONDI ER pfe si A 2217 Bio Plex Manager 6 0 Software User Manual Table of Contents 1 Bio Plex Manager 6 0 Software User Manual 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 The system detects and measures molecules bound to the surfaces of fluorescent microspheres providing highly accurate real time digital analysis of serum or culture media samples as small as 50 ul This allows quantitative analysis of a wide variety of cell biology assays including immunoassays complex genetic assays and enzymatic assays The Bio Plex suspension array system consists of a fully integrated array reader and microplate platform with an optional HTF high throughput fluidics sheath delivery module Figure 1 Bio Plex array reader and microplate platform with optional HTF Bio Plex Manager 6 0 Software User Manual 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 0 software New angiogenesis acute phase diabetes and iso
227. s 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 Open the Display control panel 3 Inthe Display Properties dialog select the Settings tab see Figure 3 4 Drag the Screen Area slider to the right toward More until you have selected 1024 x 768 pixels Click OK to accept the settings Display Properties Themes Desktop Screen Saver Appearance Settings Drag the monitor icons to match the physical arangement of your monitors Arrange Icons By gt Refresh Paste Color quality Graphics Properties ur qom Graphics Options gt B i Highest 32 bit New b IR CREENEN Figure 4 Changing the screen resolution settings 10 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 e Before reinstalling Bio Plex Manager 6 0 we recommend that you first uninstall any existing version of Bio Plex Manager on your computer e We recommend that you turn off any antivirus protection software before installation Such software if active can greatly slow the progress of the installation If you are unable to turn off your antivirus protection software allow 15 minutes for complete instal
228. s may resolve the problem If not please contact technical support for assistance d Do not connect at startup Reconnect Close Figure 6 Connection Error dialog Make sure the instrument is turned on check the cable connections and then click Reconnect in the dialog If you are still unable to connect try restarting the computer array reader and platform You can also choose the Run Diagnostic option found under the Instrument menu This produces a report which can help Bio Rad Technical Support determine the cause of the problem Bio Plex Manager The Flash best has passed Ox4FFODO78 The RAM best has passed Ox4FFO1079 The NVR AIM test has passed Ox4FFDT0TA The DSP ORC best has passed Oe4FF0 L078 The DSP run best has passed xdFF LDZC The high wolbage best has passed OxsFFOTOTDO The background test has passed x4FF 107E The pressurize best has passed Ox4FFO107F The actuator test has passed Ces FFOTOBD The syringe test has passed red FFOTOS T The debubbler wake best has passed Oct FFOT0EZ The backflush valve best has passed x4FF TOS3 Figure 7 Run Diagnostics Report 15 Bio Plex Manager 6 0 Software User Manual Menu and Toolbars 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 wit
229. se A copy of your old database will remain in the application folder For more information about the Calibration Log see page 30 For more information about the Validation Log see page 39 For more information about the Instrument Operations Log see page 45 Secure Protocol and Results Files Bio Plex Manager Security Edition supports the creation of Secure Protocol and Results files Secure files are documents that have been electronically signed by a user Once signed these files are controlled documents that cannot be overwritten by Bio Plex Manager and that preserve a built in audit trail of all saved changes Unsigned Protocol and Results Files When you create a new Protocol file in Bio Plex Manager Security Edition it is initially unsigned and is not a secure file Protocol or Results files created with previous versions of the software or created in Standard Mode are also unsigned uncontrolled documents and you are notified that these files are not secure when you open them in Secure Mode You can save and overwrite these files without restrictions and no audit trail will be generated for any changes 173 Bio Plex Manager Software User Manual Calibration Validation and Instrument 174 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 179 will be preserved in the new file and the file will remain a secure fi
230. selected number of lots to collaborators by pressing the Export button When you export the lots they are saved in an XML format which can be sent to other users of Bio Plex Manager for import Deleting Standard Lots Unneeded standard lots are deleted by pressing the Delete button 19 Bio Plex Manager 6 0 Software User Manual 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 Kits fall into two categories those which have various starting concentrations and those which have the same starting concentration for each analyte When starting concentrations vary across all analytes There are a number of ways to edit values and apply dilutions The following outlines the most efficient approach 1 Enter the starting concentration of each analyte directly in the spreadsheet within the interface Enter each analyte before proceeding to the next step Make sure the Apply same concentration to all analytes box is unchecked default PH Protocol2 Enter Standards Info Protocol Settings Peace aetna Standards Info External Standards Info Select External Standards Standard Lot Analyte r Lot Expiration Date Hu IL 5 33 v Std Regression Curve Load Save Manage Stand
231. ses Obs Exp 100 Back calculated standard EI 23818 08 23919 08 concentrations within a specified orc NE SS 1325 95 1325 95 recovery percentage range of ret eer 80 120 101 89 101 89 JOR 15 29 JOR JOR JOR 16775 83 16775 53 Concentrations of unknowns 4514 B7 451467 within range 1155 94 1155 84 E 343 57 343 57 m OOR 89 56 Concentrations of unknowns OOR OOR lt outside range ES OOR OOR Figure 127 Concentration in Range column Report Table For example if only standard concentrations from 1 9 to 8 100 pg 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 185 for information about how the Conc in Range column can help you optimize your Standard Curve fit Showing Hiding Outliers In some cases you may want to exclude a well or replicate group of wells from statistical calculations for example when the 96CV of a set of replicates is high or the observed concentration of an analyte is out of range The Report Table Outlier column allows you to flag a well or replicate group of wells as an outlier dl Click the Show Hide Outliers Column button g in the toolbar to show or hide the column Each row in the column contains a checkbox When you check the checkbox the data for the selected analyte is excluded and the table is automati
232. show the number of curves that can be printed on a page If you are printing the curve for a single analyte that curve will be sized based on the layout you select for example the cH layout will size the curve at one quarter the full size of the page The regression type will be printed below each curve Print Standard Curve Select graph layout CO EH EH BB Cu Mate The size af the printed standard curve will depend on the above selected Figure 143 Print Standard Curve dialog Select a layout and click OK to display the standard Windows Print dialog Graphing Function Graphing Function p Click the Graphs button to display your Results data as graphs You can choose one of two default graphs from the Graphs dropdown menu All Unknowns amp 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 dropdown menu R New Sample 23 Plex Standard PMT Graphs es P us Uc Ux Graphs All Unknowns amp Controls H3 ta e Result Y axis Fl v Analyte Mo IL 1a 53 v Zoom out Results Mo IL 1a 53 at Raw Data Hii Report T able A Standard Curve n Graphs Figure 144 Unknowns and Controls graph with FI y axis Ri New Sample 23 Plex Standard PMT Graphs SEE g Bs Bc Ux Graphs All Samples Ra ta A r a Result Y axis
233. siExp 100 Group Ratio E Dihtion DE Bead count DE Bead ean Bead sta Dev E BeadStdErr j Bead cv 7 Trimmed Mean E Trimmed Std Dev E Trimmed Std Err EH Trimmed cv Sampling Errors Lj O Select All O Save settings as default for new protocol Figure 191 Excluding error codes Bio Plex Manager Software User Manual 11 Data Normalization There are two main reasons to use data normalization e You may need to account for differences in the amount of material loaded in each sample This is accomplished using analytes that will not vary from sample to sample for example housekeeping genes reference genes and internal controls e You may want to express all other samples measured values relative to an assigned control sample The normalized control sample has a value of 1 and other samples have measured values relative to the control for example 2 fold or 5 57 fold higher than mRNA or protein level compared to control Normalization Settings ix Normalization is off by default Click the Normalization Settings I button on the toolbar or choose View gt Normalization Settings to enable data normalization from any view of the experiment results The dialog allows you to specify how you want to normalize data The Normalization Settings dialog box provides the following options e Internal Control Housekeeping Genes None Single and Multiple
234. sl U E z xm I 555 A B C D E F G H z File Name C Program Files Bio Rad Bio Plex Manager 5 0 Example Files Mouse Cytokine 23 Plex Star Acquisition Date 09 Jun 2005 04 34 PM Reader Serial Number LX10004167313 RP1 PMT Volts 647 13 RP1 Target 3832 Plate ID Well Type Mo IL 1a 53 Mo IL 1b 19 Mo IL 2 36 Mo IL 3 18 Mo IL 4 32 Mo IL 5 52 Mo IL 6 38 N 19961 13415 223035 197725 15599 20205 8545 5 18710 5 7561 162055 15378 5 16538 21662 6389 12875 3432 10588 9734 14599 15366 5 3697 5615 970 4838 4855 7821 7358 5 1191 5 1819 235 1657 1941 5 4349 5 1748 335 609 5 72 443 5 653 1766 471 95 A7 AIDA PUE Lat dak ras or ATM AP f el ran ann M 4 Mouse Cytokine 23 Plex Standard Ready Figure 120 Example of an Excel spreadsheet with exported data You can also export the raw data to a text file With your raw data displayed go to the File menu and select Export Table to File This command opens a Save As dialog box in which you can specify a file name and location for the text file NOTE You can copy any or all values in the table to the Windows clipboard Select the entire table or the desired rows columns or entries and select Copy from the Edit menu The values you copy can be pasted into other Windows applications 125 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 126 Raw Data Table Error Codes If data cannot be measured or a sampling error occurs in a well it
235. sly monitors its internal temperature voltage pressure and other systems for diagnostic purposes You can access this information by clicking the Information button on the main toolbar or Value Lasers status Warmed up Lasers shutoff time 2 hrs 44 min DD Temp Celsius 23 96 High Voltage volts 10 02 Air Pressure psi 0 01 Sheath Pressure psi 4 08 Calibration Pressure psi 5 90 Manufacturing Pressure psi 5 90 Figure 34 Instrument Information dialog Click the tabs in the dialog to access information about the instrument See the hardware manual for detailed instrument specifications Eject Retract Plate The Eject Retract Plate command gt on the main toolbar and Instrument menu ejects and retracts the plate carrier in the microplate platform Note that every function that requires insertion of a plate into the platform for example Start Up Wash Calibrate etc includes an Eject Retract button in its dialog box Additional Instrument Functions The Additional Functions submenu under the Instrument menu contains secondary instrument fluidics functions Selecting one of these commands opens a dialog box to guide you through the procedure Many of these functions require the use of the Bio Plex MCV Plate IV 43 Bio Plex Manager 6 0 Software User Manual Controlling the System NOTE Major fluidics functions such as Wash Between Plates Remove Bubbles and Unclog consist of combinations of the following
236. ss of business or profits special or indirect damages of any nature whatsoever No amendment waiver or other alteration of the warranties in this agreement may be made except by mutual agreement in writing Purchaser agrees that Bio Rad s liability arising out of contract negligence strict liability in tort or warranty shall not exceed the amount of software license fees paid by Purchaser This manual and the software computer program described in it are copyright Bio Rad Laboratories Inc with all rights reserved worldwide Under the copyright laws this manual and the software program contained herein may not be copied in whole or in part without the prior written consent of Bio Rad except in the normal use of the software or to make a backup copy This exception does not allow copies to be made for others whether or not sold but all of the materials purchased with all backup copies may be sold given or loaned to another person Under the law copying includes translating into another language or format A multiuse license may be purchased to allow the software to be used on more than one computer owned by the Purchaser including a shared disk system 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
237. ssay Handbook David Wild Editor Nature Publishing Group New York NY 2nd Edition 2001 The GraphPad Guide to Nonlinear Regression Harvey Moltusky PhD GraphPad Software San Diego CA 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 For more information see Principles of Curve Fitting from the website www bio rad com or from Technical Support Nevertheless in certain cases you may want to use linear regression to analyze the data For most cytokines the linear portion of the curve is well within the biological range of concentrations expected for patient samples Thus the linear regression may be useful for serum The main advantage of a linear regression is that fewer points as few as 2 can be used Copying the Standard Curve To copy a bitmap image of the curve to the Windows clipboard right click in the Standard Curve window and select Copy Graph to Clipboard You can then paste this image into your spreadsheets or other documents using the Paste command in your other applications Ra IL 1a 21 Copy Graph to Clipboard E me gt Ez D E D x pu o e D
238. t After Run checkbox With this checkbox selected Bio Plex Manager automatically generates an XML file and saves it as specified in the XML Export Properties dialog Sampling Errors During a reading the system continuously monitors the flow of beads the bead count the bead regions and the platform temperature If the system detects a problem in any of these areas it can stop the reading and trigger a warning This feature is enabled if any or all of the checkboxes under Sampling Errors are selected 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 Sampling Errors C Pause run if enor occurs 1 Low bead number 12 Aggregated beads 3 Classify efficiency C 4 Region selection 5 Platform temperature Figure 96 Sampling Errors checkboxes in Advanced Settings window NOTE All errors are logged in the Raw Data and Report tables whether or not the checkboxes are selected see page 113 99 Bio Plex Manager 6 0 Software User Manual Running Protocols 100 The reading errors and their possible causes are listed in the following table Error Possible Cause 1 Low bead number Too few beads in the assay buffer volume in well is too low plate was not shaken properly before analysis microbubbles in the cuvette
239. ta file for CFR 21 Part 11 compliance Document Export Options 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_4 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 This schema file can be used to automatically validate the XML file 161 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 162 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 Document Export Properties Content C2 Bio Plex XML 9 Output CSV Format Export Options Analyte Labels Name Region Mi Well Labels Both 1641 2 61 30C1Y w Analyte Sort Order Priza He eet e nra m ch alae Figure 164 CSV Content options in the Document Export window ANALYTE LABELS Various software packages require different analyte naming conventions such as IL 2 which appear in the column heading within the CSV files Refer to the
240. tandard 5 Software Licenses Desktop 6 Instrument Control 6 Network 6 Software Warranty 224 Solobug Reporting Problems 225 Sorting Report Table Data 137 Standard Concentration Lots 73 Standard Curve 142 Copying 147 Linear vs Logistic Regression Methods 147 Printing 148 Regression Methods 144 Standard Curve Optimizer 185 Starting Bio Plex Manager 13 System cable connections 14 System Controls Eject Retract Plate 43 Instrument Information 43 Optics Warm Up and Shut Down 24 Platform Heater 42 Remove Air Bubbles 33 Start Up 23 Unclog 34 Validation 35 Wash Between Plates 32 Uninstalling Bio Plex Manager 12 Using the Security Edition 167 V Validation Setting Up a Validation Run 36 Validation Kit 35 Validation Log 39 Validation Type Selection 37 W Wash 44 Well Numbering and Replicate Groups 64 231 Life Science Group 10018879 Rev A Bio Rad Laboratories Inc Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 20 8732 2339 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 8840 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38
241. te 21 H IFN gamma from the list by selecting it and clicking the Remove button or you can remove all analytes by sj clicking the Remove All button To edit an analyte in the list select it then click the Edit button A dialog box opens in which you can change the analyte region and name Remove When you are finished creating the panel click OK to save your changes and return to the Protocol window IMPORTING A PANEL OF ANALYTES Follow these steps to import a new panel 1 Open Excel and create a Regions and Analytes list as shown You may choose to include the Panel name as shown or omit it 2 Save this as a CSV file by choosing Save As from the File menu Choose CSV MS DOS from the Excel Save as type dropdown list amp Microsoft Excel New Panel csv iBj Ele Edit view Insert Format Tools Data Window Help Adobe PDF ELI 3c EH 9 5828 EE New Panel Name 1 Analyte 1 2 Analyte 2 3 Analyte 3 4 Analyte 4 E Name 3 Desktop 3 AIA X Gy EH Toos Size Type 6 Analyte 6 7 Analyte 7 8 Analyte 8 9 Analyte 8 17 Analyte 17 18 Analyte 18 19 Analyte 19 20 Analyte 20 21 Analyte 21 5 Analyte 5 4 My Recent Documents My Network Places Lamy Documents Y My Computer my Network Places Bio rad CDC Atlanta Bio Plex by Product files clean up 9 22 09 Desktop Clean up 11 24 09 desktop cleanup 7 23 09 Directories Gene Manager
242. te Layout above select which columns you want to export you can select one column or all visible columns by choosing from this pull down list You can export only the selected analyte or export them all 139 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 140 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 137 for an example C Program Files Bio Rad Bio Plex Manager Examples Human Cytokine 16 plex rbx FI Bkad Hu TNF alpha 35 1 2 3 4 5 6 7 8 g 10 11 12 A 31 00 31880 00 364 00 27041 00 32043 00 3221450 3204550 32735 00 32666 00 32735 00 32735 00 32735 00 B 33 00 2921350 380 00 2620500 31977 50 31616 00 31204 00 32735 00 30125 00 29382 00 27725 00 30507 50 C 28 00 29646 00 46 00 20294 00 20248 00 15367 00 23669 00 30275 00 4824 50 3129 00 3249 00 3843 00 D 28781 00 45 00 1370 50 1441 50 2077 00 1830 00 3462 50 268 50 206 50 205 50 257 00 E 30630 00 6489 00 47 00 113 00 87 00 119 00 140 00 267 50 38 50 31 00 31 00 37 00 F 31636 50 6372 00 20 00 11 00 13 00 24 00 14 00 38 00 18 00 18 00 15 00 10 00 G 31561 50 6606 00 20 00 4 00 1 00 3 00 5 50 19 00 4 00 4 00 0 50 1 00 H 31243 00 372 00 20 00 7 00 7 00 8 00 5 50 0 00 32735 00 32735 00 32735 00 32735 00 Hu IL 4 52 1
243. te that this selection exports only a single column of data at a time _1 Filename C Program Files Bio Rad Bio Plex Manager 5 0 Example Files Human Cytokine 17 Plex High PMT rbx 2 Data Type FI Bkgd _3 Analyte Hu IL 2 38 _4 Plate ID 2 Bog 6 123456 7 8 9 10 11 12 7 A 08 133 37 48 8 B 15 3 3 3 40 42 3 c 393 383 10 D 167 3 163 3 11E 7003 7813 12 F 3579 3 3693 3 43 53 13 G 13669 3 13316 3 163 8 3 14 H 26568 3 25882 3 103 3 120 3 i 15 v M 4 M Hu IL 2 38 Hu IL 4 52 Hu IL 6 19 Hu IL 8 54 Hu IL iC Figure 135 Example of an Export worksheet 96 well layout Click the Advanced Export Options button to access additional options Table Export Options Export Format oK Single Analyte Layout 9 Multiple Analyte Layout SB well plate Help Cancel Columns to Export In this section Report table column to export choose a subset of data lt All Visible Columns to export from the Report Table Export Source Selected Analyte All Anales Export Destination 5 Excel Workbook These are Preference Use Multiple Worksheets Settings governing Text File Tab Delimited where and how your Report Table Exclude Standard Curve is exported Exclude Column Fiow Headers and Page Headers Footers Exclude table error cades Figure 136 Report Table Advanced Export Options Export a Subset of the Data If you selected 96 Well Plate or Multiple Analy
244. ted Single Analyte This choice generates a workbook with one worksheet for each analyte Multiple Analyte If you check the Use Multiple Worksheets checkbox in the Advanced Export options dialog you will generate a workbook with one worksheet for each column Fl Fl Bkgd etc with data for all analytes If you leave the Use Multiple Worksheets checkbox unchecked all of the data appears on one large worksheet which can be viewed by scrolling with the elevator bar 96 well If you check the Use Multiple Worksheets checkbox in the Advanced Export options dialog you will generate a workbook with one worksheet for each analyte If you leave the Use Multiple Worksheets checkbox unchecked all of the data appears on a single worksheet Exclude Standard Curves Standard curve images are automatically exported to Excel by default You can exclude them from the export by checking the box next to Exclude Standard Curve Exclude Column Headers Footers Select the Exclude Column Headers Footers and Page Headers Footers checkbox to exclude this information from the exported file Exclude Table Error Codes See the Appendix on page 226 for a table of possible Report Table Error Codes The error code symbols in the Report Table OOR lt gt before concentration values can interfere with macros in some types of spreadsheets including Excel spreadsheets Select this checkbox to exclude these symbols from the exported
245. ted list to move it back Hold down the Shift or Ctrl key and click multiple analytes in the Available list to select them as a group Then click the Add gt gt button to move them over to the Selected list Use the Remove button to return multiple selected analytes back to the Available list Protocol Window Use Add All gt gt to move all the available analytes into the Selected list and lt lt Remove All to move them back 1 Select the panel of analytes F Prslocoli Selc M V Add Panel 5E Pane pu Cyan DH A Ay peli Ben Anaye 2 Select the analytes in x Mail 55 Ma IL 0 M M3 Fg i the Avanabye column Ma THF 3 Use the buttons to move your selections ta the Seect d column Figure 42 Selecting the analytes to be reported NOTE Some analytes share the same region Only one analyte per region can be added to the Selected list CUSTOMIZING ANALYTES AND PANELS Bio Plex Manager comes preconfigured with a selection of panels and analytes that Bio Rad provides as Bio Plex assays You can edit these and or create new ones NOTE If you have assay kits for analytes that are not included in Bio Plex Manager you must add these analytes using the commands described in this section 55 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 56 CREATING A NEW PANEL OF ANALYTES ae Click the Add Panel button iss in the Select Analytes toolbar to create a new panel of analytes
246. tem pressurization 5 to 20 seconds the time remaining in the operation displays in the Bio Plex Manager status bar 34 Validation Validation 9 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 Start Up and Calibration have been performed Validation is dependent on successful calibration for accuracy You should perform a validation reading e Oncea month e Each time you move the array reader or e To diagnose any problems with the array reader that cannot be solved by other procedures calibrating washing unclogging etc Validation Kit Bio Plex Manager requires the use of Bio Plex Validation Kit 4 0 to perform validation It must be used in conjunction with the Bio Plex MCV Plate IV Each Bio Plex Validation Kit consists of reagents and procedures to evaluate e Optical alignment e Reporter channel performance e Efficiency of multiplexing e Integrity of fluidics The Bio Plex Validation Kit validates the operation of all of the primary components of the array system and can also be used to discriminate between assay and instrumentation problems Validation Kit Control Number Each Bio Plex Validation Kit has a control number linked to the specifications for the kit You must use the mini CD included in the kit to install the control number on your compu
247. ter After installation you can select the control number in Bio Plex Manager as described in the following section See the Bio Plex Validation Kit 4 0 Instruction Manual part 171 203001 for more information 35 Bio Plex Manager 6 0 Software User Manual Controlling the System Setting Up a Validation Run To set up a validation reading click the Validation button a on the main toolbar or select the command from the Instrument menu The Validation dialog box opens Validation User Name Last CAL2 Calibration C Use High RP1 Target Control Number 3333331 bl Add Remove Expiration Date 15 Oct 2008 tS Show Specifications Note The Control Number is located on the Validation Kit box Validation Type All Optics Fluidics Reporter Classify Optics Fluidics Reporter Classify 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 Control Number Selection In the Validation dialog box select the Control Number for your specific Bio Plex Validation Kit from the Control Number pulldown list The expiration date for the control number is listed below the field If the control number is not available in the list or the list is too long and you want to limit it to the control numbers you are using click the Add Remove button Add Remove Cont
248. th the Control Number for that run in the lower window Then click Create Report EK 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 EJ Microsoft Excel BPValidation3_Reportt L EM i43 DR s lw i AT E Bio Plex suspension Array System Validation Report Test Performed by chloe Date Time 14 Oct 2003 11 17 PM Validation Kit Cantral 96290 High Reader Serial LXTODOO273003 RP1 Target value 17677 RP1 PMT Voltage 03 54 I Optics Validation Rasy leted Sp H Parameter CL2CV 400 800 523 Pass RPI Wedian 15205 18583 17258 Pass RPI Cv 5 00 10 00 IM Reporter Validation Resul Completed A Calculated Values B Raw Values PassFail Bead Median FI Blank 8 10 Pass 0 0690 Sensitivit 200 MESF 16 j 4 70 L5 16235 IV Classify Validation H 4 r M i Report ippw L Aytoshapest gt w 162 5d s Result Completed Figure 31 Validation results spreadsheet Microsoft Excel 41 Bio Plex Manager 6 0 Software User Manual Controlling the System Platform Heater 5 42 The microplate platform has a heater for warming samples The heater has an operating range of 35 C to 60 C 95 F to 140 F and can be controlled through Bio Plex M
249. the Cut Copy and Paste commands on the Edit menu to copy your descriptions and or dilutions from and to other applications such as Microsoft Word or Excel Click a cell row or column in the table to copy and paste Bio Plex Manager 6 0 Software User Manual 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 ee in the Protocol window to select the final settings and run the Protocol WARNINGS When performing the following operations the sheath fluid container and the waste fluid container should be closely monitored The sheath fluid bottle must be placed at the same level as the array reader unless you are using the HTF High Throughput Fluidics see the next Warning The fluid level should be below the air inlet connection and above the sheath outlet connection For more information refer to the Bio Plex 200 System Hardware Instruction Manual Always check the sheath fluid level before starting a run or procedure If you are using the HTF it should sit on the counter next to the array reader while the sheath fluid cube should be placed 3 4 feet below the reader for example on the floor The waste fluid container receives waste from the system Do not allow the waste container to overflow Empty the waste bottle each time the sheath fluid bottle is filled Do not place it on the instrument All waste containers should
250. to Apply same concentrations to all analytes Standards Info External Standards Info Select External Standards Standard Lot Lot Expiration Date Hu IL 5 33 Std Regression Curve Regression Type Analyte Assign Standards Information Logistic 5PL Std Description Hu IL 5 33 Hu G CSF 57 Hu MIP 1b 18 Axis Transformation 0 00 A 0 00 Loa s Linear glz v am TAE mmm 0 00 Logistic Weighting Logistic weighing T Same regression type for all analytes 0 00 Concentration Units Same units for all analytes Units don t impact calculations EE EHE REA Acceptable Recovery Range 70 1307 v Most Concentrated Standard 1 Os6 Concentration of 1 Hu IL 5 33 Same recovery range for all 0 140 Apply dilution to all analytes Dilution Factor lx 70 130 ESS 80 z 120 C Apply same concentrations to all analytes CisaiAl eancantakons Figure 186 Selecting the Recovery Percentage Range 186 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 lt OOR or OOR gt Fi Bkad Cons in Range ObsrExp 100
251. to the application when each calibration was performed The Result column notes the result of each calibration including whether it passed or failed NOTE If you see a drastic change in a detector s voltage from one calibration to the next it could indicate a problem with the instrument A steadily increasing detector voltage may indicate that the laser is decreasing in intensity On the toolbar click Calibration Detail Bh to display additional information for each calibration including system temperature and pressure and the e reader software and firmware versions Click Calibration Log to return to the default view For CAL1 only calibrations the RP1 column displays an entry of NA not applicable For CAL2 only calibrations the DD CL1 and CL2 columns display entries of NA 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 31 Bio Plex Manager 6 0 Software User Manual Controlling the System Wash Between Plates 32 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 pro
252. toggle between the views using the tabs above the plate diagram As you toggle the plate template remains the same but the commands toggle between formatting controls and grouping controls e Plate formatting tools are used to define the types of wells ina plate sample control standard blank etc e Plate groupings tools are used to organize the well types into groups with one member of each group defined as the Reference or Primary member as in a Western blot kinase assay The ratio of each member s fluorescent intensity to the fluorescent intensity of the Reference can then be calculated NOTE Plate formatting is required to perform a reading because the array reader will read only formatted wells whereas plate groupings are optional and can be defined later 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 The different well types you can select are x Unknown sample S Standard C Control Blank aR Undefined Remember that all undefined wells are not read by the array reader DEFINING UNKNOWN SAMPLE WELLS To define wells containing unknown samples first click the Unknown Sample button x then click or drag t
253. tokines and Human Cytokines II without going to the trouble of creating custom panels 1 Select the panels individually from the pulldown list 2 Use the Add All gt gt command to move the analytes from each panel into the Selected list 3 Save the entire Protocol as you would normally Your analyte selections are saved when you save the Protocol 61 Bio Plex Manager 6 0 Software User Manual Preparing Protocols 62 Step 3 Format Plate amp 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 NOTE Only formatted wells will be read by the array reader Be careful to format all the wells that you want to read before running a Protocol Click the Format Plate button in the Protocol window to display the plate template and the formatting controls Pi human cyokines 001 Format Plate ie Sil vik eO Ok 3 4 5 6 7 8 9 10 11 12 LILI s WWIII c JL JL JL IL IL ILILIL o IL IL TL TEIL IL IL JL JL IL IL JLJL ILICE JL IL JL JL JL UL FL WWIII e JL Ld Figure 53 The plate template and formatting toolbar Plate Formatting view The Format Plate window has two views the Plate Formatting view and the Plate Groupings view You can
254. ton Ux in the Results toolbar to review or change the sample information as described on page 92 Results Files Changing the Doublet Discriminator Gate Range You can change the doublet discriminator DD gate range See page 100 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 The Change DD Gates dialog opens displaying the histogram and bead map with controls for changing the DD gate range See page 104 for a complete description of other histogram and bead map controls Change DD Gates Histogram Bead Map 100 region wel Ay Mcaed w it i ej e ol Ej ej a 21 10000 Jed ee CUM i e a pd zl oy aa prs J la n PSIE F Ss pP 1000 d aun Pb ff of 100 Classitication 2 1 40 i00 1000 10000 100 1000 10000 623 26741 Doublet Discriminator Classification 1 Select Well O00 Low DO High Al 623 26741 Update Gates Same DD gates for all Formatted wells Figure 117 Change DD Gates dialog In the Change DD Gates dialog first select the well you want to change or select the Same DD Gates for All Formatted Wells che
255. ts can include enzyme substrates receptors antigens and antibodies A conjugated bead reactant mixture can be mixed with a sample to create for example a capture sandwich immunoassay NOTE To ensure the stability of the spectral address it is essential to protect the microspheres from light Do not subject microspheres to prolonged high temperatures and protect them from freezing and thawing Reporter Fluorochromes When you mix your samples with the microspheres the target analytes bind to the reactants on the surface of the beads The amount of analyte that binds to the beads is then quantitated through the use of a fluorochrome R phycoerythrin The intensity of this fluorochrome as detected by the array reader is directly proportional to the amount of analyte present The excitation wavelength of the array reader reporter channel is 532 nm the emission wavelength is 575 12 nm Specific information on R phycoerythrin is shown in the following table R Phycoerythrin Specifications Formula weight Daltons 240 000 Absorbance max nm 480 546 565 Extinction max M 1cm 1 1 960 000 Emission max nm 578 Quantum yield 0 82 219 Bio Plex Manager 6 0 Software User Guide Appendix 220 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 dri
256. tween 3000 and 4000 The High RP1 target value remains on the bottle label because it can be used with earlier versions of Bio Plex Manager Assays that require calibration using the High RP1 target value can be run by selecting the checkbox labeled Run at High RP1 Target in the Run Protocol window When you are done click Add to close the dialog box and save your changes Your new control numbers are added to the selection lists in the main Calibrate dialog To delete a particular control number first select it in the Calibrate dialog box and then click the Delete button 28 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 amp 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 CAL1 amp CAL 2 Calibration This operation performs CAL1 and CAL2 calibration 1 Vortex CAL1 and CAL2 beads for 30 seconds 2 Load 5 drops of CAL1 and CAL2 beads 3 Fill DI H20 reservoir MCV Plate IV 4 Click Eject then load MC plate 5 Press OK to start 4 Eiect Retract Plate Figure 19 Preparing the Bio Plex MCV Plate IV for CAL1 amp CAL2 calibration NOTES When preparing the MCV Plate IV do the following e Important Before vortexing remove the calibration beads from 2 to 8 C 36 to
257. typing assays have been introduced with this version of the software Check www bio rad com bio plex x plex for newly available assays and reagents The assays contain sets of microscopic color coded beads each of which is conjugated with a different reactant Reactants can include e DNA e Enzyme substrates e Receptors e Antigens e Antibodies These can be used to create for example a capture sandwich immunoassay To perform a multiplex reading samples are mixed with conjugated microsphere and reactant mixtures next fluorescent reporter molecules are added The assays are loaded into the wells of a 96 well microtiter plate and the plate is inserted into the microplate platform The platform and array reader are controlled by a computer running Bio Plex Manager software The detection system in the array reader uses two lasers to analyze the microspheres in a flow stream The first laser identifies each microsphere and its associated analyte based on the fluorescent signature of the microsphere and the second measures the amount of analyte using the reporter molecules attached to the analytes When the reading is complete Bio Plex Manager displays the raw data and generates detailed summary reports Components A certified Bio Rad service engineer will install the complete Bio Plex suspension array system at your site including the array reader microplate platform and computer The system setup procedure is described in
258. uJfe e of Events 150 75 dy g181 1803 4335 7390 Doublet Discriminator Figure 100 Selecting the analyte s to display To print the histogram and or bead map click the Print Histogram Bead Map j This opens a small dialog in which you can select the histogram bead map or both to print Histogram The histogram plots the number of events per channel number for the selected well analyte s and channel type An event is generated when particles such as a bead or aggregated beads pass through the path of the lasers Each event generates signals in different channels e The fluorochromes embedded in each bead generate signals in the Classification 1 and Classification 2 channels e The fluorescent molecules bound to the analytes generate a signal in the Reporter 1 channel e The light scatter of each bead generates a signal in the Doublet Discriminator channel The amount of light scatter is directly proportional to bead size The signals are reported as channel numbers In the histogram the y axis represents the events and the x axis represents the channel numbers from 1 to 32 766 for the selected channel type 105 Bio Plex Manager 6 0 Software User Manual Running Protocols 106 DOUBLET DISCRIMINATOR GATE RANGE The Doublet Discriminator DD range specified under the Run Protocol Advanced Settings See page 100 is shown on the histogram as two red lines perpendicular to the x axis In the Protocol window
259. ual Incompatible suspension buffer Check buffer compatibility see used hardware manual Incorrect bead regions were Compare bead regions in the assay selected in the Protocol with those selected in the Protocol Incorrect regions were selected Verify regions chosen during assay when preparing the assay preparation Too few beads in the assay in Verify regions chosen during assay one or more regions preparation Too few beads in the assay in Verify that the correct number of beads one or more regions was used by referring to the specific assay preparation procedure 201 Bio Plex Manager 6 0 Software User Manual Troubleshooting Bio Plex Manager has More likely detected a possible Calibration was performed Perform 30 minute warm up and problem due to before the array reader was recalibrate microbubbles in the warmed up cuvette or Calibrated with incorrect CL1 or Check that the target values of the CAL CL2 target values beads match values entered in the software If different recalibrate Array reader was calibrated with Clean MCV Plate IV and recalibrate a dirty MCV Plate IV Misalignment of optics Perform Optics Validation Contact Technical Support if values are not within range photobleached beads Less likely Microbubbles present in cuvette Perform Remove Bubbles Calibration beads are Recalibrate with new CAL1 beads photobleached do not expose to light for mor
260. ulate coefficient a checkbox and entering a value in the Coefficient a field COEFFICIENT B Weighting coefficient b has a default value of 1 8 You can enter a different value in the Coefficient b field Protocol Window RECOVERY PERCENTAGE RANGE Because the standard curve page 142 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 185 for how Bio Plex Manager incorporates Recovery Range data automatically For each analyte standard an observed concentration is back calculated from the standard curve and the fluorescence intensity This is divided by the expected concentration as entered in the Enter Standards Info window and multiplied by 100 to give the recovery percentage Recovery percentage Observed conc Expected conc 100 Recovery percentages are also used with controls page 90 to determine the overall accuracy of an assay In the Enter Standards Info window use the Acceptable Recovery Percentage Range pulldown menu to select an acceptable recovery range Same units for all analytes Units don t impact calculations Acceptable Recover Range 70 130 Same recovery range for all 60 140 HENE eu 1205 X 30 110 Figure 87 Selecting the recovery percentage range For example if you select a range of 70 to 13096 the observ
261. ulate 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 Assign Standards Infomation aoao 1101 12 42 12 13 s se0 00 e 9 sg swo oo 900 oo s 2000 00 oo 900 99 s s0000 90 900 99 s o 99 900 99 s 325 99 900 99 Calculate Concentrations Most Concentrated Standard 51 56 Concentration of 51 32000 10 10 v Apply dilution to all analytes Dilution Factor 4 Calculate E Apply same concentrations to all analytes Clear All Concentrations Figure 83 Entering standard concentrations automatically 2 Enter the concentration of the most concentrated standard in the Concentration of S1 field and the dilution factor for the remaining standards in the Dilution Factor field NOTE Calculations are made by dividing by the dilution factor so do not enter fractional values for the dilution factor If each standard is half the concentration of the previous standard enter 2 for the dilution factor If each standard concentration is one fourth the previous enter 4 and so on 3 Click the Calculate button when all the information is entered The concentrations in the table are automatically updated If the Apply same concentrations to all analytes checkbox below the concentrations table is selected the concentration values
262. ults window NOTE Any changes you make in the Results file will not change the original Protocol file or the data from the reading They will change only how these data are displayed in the Results file tables Click the Describe Protocol button in the Results toolbar to review or change the description of the Protocol as described on page 53 d e Click the Select Analytes button e in the Results toolbar to review the analyte selection or select different analytes as described on page 53 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 not appear in the final reports and tables After a reading you can go back and change your analyte selections and any detected analytes will appear in the tables Click the Format Plate button ew in the Results toolbar to review or change the formatting of the plate Plate formatting is described on page 62 NOTE Only formatted wells are read by the array reader You must define all the wells you want to read before running a Protocol However you can change the formatting of a defined well for example from unknown to standard after a reading and the analysis of that well will change Click the Enter Controls Info button Us in the Results toolbar to review or change the concentrations of the controls as described on page 90 Click the Enter Sample Info but
263. ut data e Desktop license enables the software to analyze data files but not control the array reader and platform The instrument communication and control functions are not available with this license e Network license provides Desktop licenses to multiple users over a computer network The Desktop license enables the software to analyze data files but not control the array reader and platform This user guide assumes that you are using the Standard Edition with an Instrument Control license unless otherwise noted The Security Edition user levels and restrictions are described in detail in the Appendix on page 211 Compatibility with Luminex Software Bio Plex Manager 6 0 is compatible with Luminex xPONENT software Because the Luminex LXR library has been updated older Luminex IS 2 3 software will not function once you upgrade to Bio Plex Manager 6 0 You must upgrade your Luminex IS 2 3 software to the current version in order to run both applications on the same machine If you install Bio Plex Manager 5 0 or later on a computer with Luminex xPONENT software note the following e To avoid communication conflicts with the array reader and platform do not run Bio Plex Manager and Luminex software at the same time e If you switch between the two software applications you must recalibrate the array reader before acquiring data ANALYZING LUMINEX DATA IN Bio PLEX MANAGER Bio Plex Results Generator 3 0 converts CSV Output files
264. ven system transports a user specified volume of sample from a microplate well to the cuvette The sample is injected into the cuvette at a steady rate and the microspheres in the sample are aligned in a single file through the path of the two lasers Following analysis the first sample path is automatically purged with sheath buffer fluid via the second fluidics path This process effectively removes residual sample in the tubing valves and sample needle Excitation There are two lasers in the array reader one for classifying each microsphere and its associated analyte and the other for quantitating the amount of analyte bound to each microsphere The first laser the classification or red laser excites the dyes embedded in each microsphere the fluorescent signal is discriminated with selective emission filters and converted into intensity units by fluorescence detectors and a digital signal processor and the microsphere is classified The second laser the reporter or green laser excites the fluorochromes bound to the target analytes on the surface of the microspheres again the fluorescent signal is discriminated with selective emission filters and converted into intensity units by fluorescence detectors and a digital signal processor and the amount of analyte is quantitated Microsphere Handling Microsphere Handling Microsphere Dispersion xMAP microspheres will settle and aggregate if left undisturbed You should always
265. ware applications Select Command Line and click Select Application to locate the software application you want to automatically launch when the file is generated This application must accept a command line filename argument After you locate and select the application the application pathname will appear in the Command Line field enclosed in quotation marks and the string amp amp XML amp amp will be automatically appended to it as shown in Figure 167 NOTES e If you manually enter the application pathname it must be enclosed in quotation marks e The amp amp XML amp amp token must also be enclosed in quotation marks If the application can be found by using the system executable search PATH only the application name needs to be entered Command line Select Application The command line must contain the XML Filename token amp exmL eg Figure 167 Example of a command line 165 Bio Plex Manager 6 0 Software User Manual Analyzing the Results 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 Document Export Content C Bio Plex XML Output CSV Format Export Options Analyte Labels Region Well
266. will be flagged in the Raw Data table The possible error conditions in the table are listed below Column Error Code Cause F1 No data present Low bead number detected in the well Aggregated beads detected in the well z Classify efficiency problem detected in the well Region selection problem detected in the well Platform temperature problem detected in the well Printing the Raw Data You can print the Raw Data table using the Print command Cj on the main toolbar or File menu Print Preview on the File menu displays the table as it will appear in the printout Report Table Report Table __ The Report Table provides detailed information about the analytes in your samples Click the Report Table button HE 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 R Phosphoprotein 6 Plex Read Only Report Table gs F us Bc Ux oy J Pi E um ka Analyte PhosphoJNK 34 v Display Show Options Organize Single Replicates Show by or Multiple Hide Type or Analytes Export Outliers Group Sort Norm Plate Formatting Tools Options Figure 121 Report Table Toolbar The first six tools allow you to adjust any plate formatting or protocol settings See Viewing Changing the Protocol Settings on page 122 The Normalization Settings button allows you to
267. y reader including those that do not pass the internal discriminator gate Manually Stopping a Reading While the sample is being read the Start button changes to a Stop button in the Protocol window You can click Stop to cancel the reading An alert box notifies you that you can generate a report with the incomplete results or rerun the Protocol as described on page 116 Generating Results from a Protocol The reading stops when you click Stop when an error stops the reading or when all samples have been analyzed At the end of the reading the sample needle lowers and the syringe pump purges the sample loop and needle with sheath fluid sending a fraction of sheath fluid approximately 160 ul back into each well 103 Bio Plex Manager 6 0 Software User Manual Running Protocols At the end of a complete reading the Results are automatically calculated and displayed Note that the Results file automatically saved if you selected Auto Save After Run page 99 in the Protocol Otherwise you must save your Results as a separate step see the next chapter starting on page 117 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 ne 104 selected Histony Well number 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 P

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