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User Manual - Hitachi Solutions America

1. es Rm 105 5 038 Mouse IL 10 alb MA 106054 Mouse IL 2 Wen c EMAN 2352073 Mouse IFN gamma MEME EPI 400 077 Mouse IL 4 300 ah nu M 200 ee Select All 15047777 aa i M EN Un select all 100 7 zi 50 Export chart 034 Mouse IL 5 054 Mouse IL 2 077 Mouse selected Analytes Mouse Tip Styles d e x Labels Y Values Multi Well Chart Selected Parent Plate IL5 Project mlx Figure 7 19 Multi well chart Right click a bar to display a pop up menu of options Modifying Chart Properties You can modify the appearance of a chart e Select Chart gt Chart Properties from the menu bar The Chart Properties dialog box appears and displays a list of the charts in the Data Chart window Figure 7 20 MasterPlex QT www miraibio com 119 CHA PTER7 DATA CHARTS Chart Properties Charts 3D Legends Walls Seres Panel Well D2 Chart Fi Senes for D2 Modify Series Ue Exit Figure 7 20 Chart Properties dialog box NOTE The types of chart property options available in each tab depends on the type of chart that is open Charts Tab Alternative formats are available for the multi well chart 1 To view the available multi well chart formats click Modify Series Figure 7 20 The Chart Gallery appears Figure 7 21 NOTE The Modify Series button is only available is a multi well chart is open in the Data Chart window MasterPlex QT www
2. IL5 Project mlx lt IL5 csv gt Sl LP LZ Ge za Uk Gel fag bg The Cel H E EB ee OX 1 2 3 4 5 6 Analytez A 034 Mouse IL 5 amp 038 Mouse IL 10 BE ga Select Entire Group ssign Model Equation Mark Wells Che Unmark wells Cl Group Wells B Ungroup Wells Copy mt 034 Mouse IL 5 Number of Wells 96 Ctrl HC Copy Horizontal List Copy Vertical List 2 NN Auto Group Figure 5 1 Standard wells selected in the well grid 2 Right click a standard well and select Auto Fill from the pop up menu that appears Alternatively click the Auto Fill button ES The Auto Fill dialog box appears Figure 5 2 MasterPlex QT www miraibio com 61 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION Auto Fill Dilution parameters Dilution Factor Fill in for all bead Figure 5 2 Auto Fill dialog box 3 Make a selection from the Analyte drop down list 4 Enter the starting concentration for the standard data set 5 Enter the dilution factor 6 Make a selection from the concentration unit drop down list 7 To select a dilution direction for the standard data set click a dilution direction arrow gt The gradient map shows the location and direction of the dilution gradient s Figure 5 3 MasterPlex QT www miraibio com 62 CHA PTER5 STANDARD CURVES amp AN
3. C ET Equation E 2 Model Equations A D 7 Click the Calculate button HE Polynomials F x D cem 1 399 regression radio button b qp Set LIII A ES Four Parameter Logistics then select ES Five Parameter Logistics Jii amp Competitive Binding Mode Ei Assign Model el Set As Default Gi Exit Dana LERTE geen Fl Pasa es H PED Dy EH bower l 3 m JaME 004 01 La IE gt id Hoe EFI AAA ARA RNE 8 To view the Dose Response curve click the Standard Curves button fra Figure 6 10 Steps to generate a Dose Response curve MasterPlex QT www miraibio com 92 CHA PTERG VIRTUAL PLATES 1 Create a virtual plate and use the virtual pipette to add the data for the Dose Response curve to the virtual plate See Creating a Virtual Plate on page 6 2 NOTE After you finish creating the virtual plate click the f button to turn off the virtual pipette 2 Select the wells that you want to include in the regression analysis data set The regression analysis data set includes the wells that contain the response data to different dosages of the agent 3 Right click the selection and choose Mark Wells Regression from the pop up menu that appears Alternatively click the Mark regression wells button or select Template gt Mark Wells gt Mark Regression Wells from the menu bar 4 Right click the regression data set and select Group Wells from the pop up menu
4. Paint Index Format Point Labels E Te Header AML C3 HTML Table Delimiter 3 Excel Figure 7 14 Export Chart dialog box Data tab 4 Make a selection from the Series drop down list To choose the data from all the wells in the chart select All 5 Choose a file format Text option saves the data is plain ASCII text format Figure 7 16 XML option saves the data in extensible markup language HTML Table option saves the data to a table in an HTML file Excel option saves the data in xls file format Copying to the system clipboard is not available for this option 6 If you are exporting data to a text file make a selection from the Delimiter drop down list The delimiter specifies how data values are separated 7 If desired choose the Point Index Point Labels or Header options Point Index Numbers the members of the data series Point Labels Displays the analyte name for each series Header Displays a header for each column of the data series MasterPlex QT www miraibio com 115 CHA PTER7 DATA CHARTS 8 To copy the data series to the system clipboard click Copy 9 To save the data to a file a Click Save gt The Save As dialog box appears Figure 7 15 b Enter a name for the file c Select a file format from the Save as type drop down list d Click Save Save As Save n opm File name 038 Mouse IL 10 1056 054 Mouse IL 2 1723 5 073 Mouse IFN gamma 1674 077 Mouse IL 4 2721 5 Point lab
5. concentration Unknown Wells that contains analytes of unknown Quantification gt Unknown Mark Wells gt Absolute concentration Control Wells that contain analytes that function as Quantification gt controls for a particular assay design Control Regression Mark Wells gt Absolute Wells of a virtual plate that are members of Quantification gt a regression data set See Generating a Regression Dose Response Curve on page 6 11 for more information on regression analysis data sets Assay Background Mark Wells gt Relative Wells that contains analytes of assay Quantification gt background concentration Assay background Mark Wells gt Relative Quantification gt Treatment Sample Wells that contain analytes that function as treatment patient sample Treatment Sample Mark Wells gt Relative Quantification gt Control Sample Wells that contain analytes that function as controls known as reference sample Control Sample e NOTE To show or hide the color coded well types click the Show well type MasterPlex QT www miraibio com 33 CHA PTER4 DEFINING A PLATE button 3 To unmark a well right click the well and select Un Mark Wells from the pop up menu Alternatively select the well s and click the Unmark wells button OX or select Template gt Unmark well from the menu bar cF If a well belongs to a group unmarking the well also removes the well from the group 4 Repeat
6. og A eee me en mds vs sall eso xa E 201 00 AT E Figure 4 7 Well groups 4 Once you are satisfied with the selection just release the mouse button The software will automatically finish the grouping Figure 4 8 Figure 4 8 Well groups NOTE When starting drag vou can move the cursor either downwards or rightwards which results in different ways to select wells To switch between the two modes just drag the cursor back into the pattern group and then drag out in either direction So it is determined by your first move direction when you are dragging the cursor out of the pattern group MasterPlex QT www miraibio com 38 CHA PTER4 DEFINING A PLATE 1 2 3 a s A 177 000 266 00 ie 609 50 11 00 B 93 00 3083 00 190 00 60 00 1 50 C EE n aab ol 188 00 203 50 f DD D 212 0 E INL FID ol 206 50 440 00 26 00 E 356 50 e 199 50 4631 00 DU 50 07 00 E 24 00 NT G DA ol za ol SE on 190 00 341 00 apr so eel ol 320 o 01 00 344 00 1 E A d 2 a 177 00 2966 00 162 50 609 50 11 00 as 93 00 3083 00 190 00 00 00 12 50 247 e ee pl 188 00 203 50 f DD EA l2 00 10478 00 06 50 440 00 6 00 PE 356 50 16523 00 199 58 4631 00 DU 50 oF Joe DO 13902 00 07 00 VM IZ 24 00 E G 545 00 202 00 74 00 190 00 341 00 H 04 50 21 00 320 00 01 00 344 00 Figure 4 9 Well groups Dragging downwards as the first move ab
7. Chart window 1 To minimize a multi well click the Ed arrow Figure 7 2 2 To restore a chart click the Ed arrow Alternatively click the button to minimize all charts Figure 7 3 Click the button again to restore all charts 3 To normalize the data for all open multi well charts click the Normalize button P gt The well data are plotted using the y axis scale from the chart with the MasterPlex QT www miraibio com 102 CHA DIER DATA CHARTS largest y axis value NOTE You can choose different colors for the chart bars in the Color tab of the Plate window see Plate Window Components on page 3 9 Select the data type for the chart MFI 034 Mouse IL 5 6 038 Mouse IL 10 M054 Mouse IL 2 2 Ahora Mouse IFN gamma 5077 Mouse IL 4 ENNN 52034 Mouse IL 5 MA 105 5 038 Mouse IL 10 ENNN 106054 Mouse IL 2 ENNN 292073 Mouse IFN gamma Place a check 400 077 Mouse IL 4 mark next to the analytes to include in the chart I 034 Mouse IL 5 054 Mouse IL 2 077 Mouse IL 4 selected Analytes Multi Well Chart Selected Parent Plate ILS Project mlx Figure 7 2 Multi well chart for C2 Multi well charts for A2 and well B2 are minimized Click a bar to display a tool tip analyte name or y axis data Right click a bar for tool tip options MasterPlex QT www miraibio com 103 CHA PTER7 DATA CHARTS a a Ss Ex M034 Mouse ILS 038 Mouse IL 10 j 88 054 Mouse IL 2 a P A0
8. D4 76 3000 731 2116 palm E4 17 0000 214 0734 Gem F4 14 0000 283 3351 palm A 3 0000 145 8435 Gem H4 7 0000 202 2244 pom Mean 120 1887 797 4510 Std Dew 169 2687 819 1842 CMS 140 8616 102 7253 ocr Paga oz DUE ria mix Figure 8 10 Report per unknown replicates MasterPlex QT www miraibio com 147 MasterPlex QT Report Per Control Replicates Report Date 2 8 2007 Report Time 12 32 30 AM Data File ILS csv Plate Name ILS Project mix MasterPlex OT Version 3 0 1 166 CHA PT ERG Hun Date 3 20 01 Run Time 12 45 59 PM Hardware Serial No LX1000028000 Operator AC Analyst MASTERPLEX QT REPORTS 034 Mouse IL 5 Background 20 00 Well Sample Name MFI Concentration Unit AZ Ind mouse serum 347 0000 1935 7353 Gem BZ 10 405 OD 21538 3012 Gem C2 62 0000 551 5248 Gem D2 GR 5000 702 5327 palm EZ 17 5000 319 0262 Gem F2 12 0000 261 7078 Gem Gl 2 000 44 0515 pa mL H2 1 0000 71 1064 Gem Mean 114 1250 763 2751 Std Dev 165 8243 825 3844 CWE 145 3012 107 4237 Well Sample Name MFI Concentration unit A 50 10 buffer 485 2000 2400 0634 palm B3 450 500 ooo 4645 palm ca 68 0000 555 4455 pa mL D3 Bo 0000 551 5248 palm E3 18 0000 A231 93127 palm F3 12 0000 251 7078 pom Gi 2 DX 44 0515 pon H3 2 2000 Kee pa mL Mean 137 8123 BAR 2655 Std Dew Z08 2244 S80 12522 CWE 131 6512 11S 478 Br Daza cT LE rci mis Figure 8 11 Report per control replicates MasterPlex QT www miraibio com 148 CHA PT ER 8 M
9. I L 1 1 I 1 1 1 I E I 1 LI 1 1 1 L 1 1 I 1 1 1 1 E 1 I I 1 1 TAT T ze en es zm zm db sn sn zs zm zm zm jm Ab e zs zm ss ke zs zm zm e zm zm zl e zm zs zs zm zs e bk ss zs zm zm zm em zs en zs zm zm sl e en zs rs zm zm em ke zs zs rm em zm zs ECKER A 8 8 8 s CL cc IT T zs zm zm zm em zm ke re zm zm zs en zm zl e e zs zs zm zm zm bk zs zm rs rm em zm zi T TGG 1 000 2 000 3 000 4 000 5 000 6 000 T 000 8 000 5 000 10 000 ira Figure 8 14 Standard curves report Shows the standard curve s for each analyte MasterPlex QT www miraibio com 151 CHA PTER8 MASTERPLEX QT REPORTS MasterPlex QT Tabular MFI Report Report Date 2 8 2007 Run Date 3 20 01 Repon Time 12 38 42 AM Run Time 12 45 39 PM Data File ILS csw Hardware Serial Mo LX10000280002 Plate Name ILS Project mix Operator AC MasterPlex OT Version 3 0 1 166 Analyst Well Sample Mame 034 Mouse IL 3 033 Mouse 034 Mouse IL 2 073 Mouse IFN 077 Mouse IL 4 A1 curve 0 00 au 0 00 au 0 00 Bi 2 1248 00 SARA 530 3427 00 3039 50 7724 00 CG A 1064 50 1229 00 5878 50 2413 00 3704 00 Di i 08 00 1319 00 2417 00 1905 50 2312 00 El 5 181 00 383 060 308 00 459 00 1408 50 Fl a 48 50 Ka 23 00 7 00 516 00 Gi 7 19 00 23 00 6 00 2350 137 00 H1 A 6 50 300 5 00 as 35 00 A2 Ind mause serum 387 00 97 30 1522 X0 1083 00 1331 50 Bi id 408 00 725530 1343 50 1235 0
10. change a color for an analyte right click the color swatch and choose or define a color in the color palette that appears Untitled Plate 2 lt 1L5 csv gt 5 Tt li Rd Be Tie i oe oe Bead Set Properties Standards 2000 Enter 1 2 3 4 5 6 4 im Local Standards A 034 Mouse IL 5 A 038 Mouse IL 10 A 054 Mouse IL 2 F A 073 Mouse IEN gamma La 077 Mouse IL 4 341 00 Importe Number of Wells Oe Figure 3 11 Plate window and Standards tab Click a tab at the bottom of the Standards tab to display a list of the local or imported standards for the plate For more information about standards see Chapter 5 on page 5 1 MasterPlex QT www miraibio com 20 CHA PTER3 GETTING STARTED 3 6 Thresholds You can enter an MFI count or concentration threshold for a plate The software can identify wells that contain data less than the user specified threshold To set a threshold s 1 Click the Preferences button 23 gt The Preferences dialog box appears Figure 3 12 Preferences Application Plate Regression Calculations Background Plate Info Plate Name IL5 Project Analyst Name Thresholds Subtract Background When ta subtract All calculations C3 Regressions only Background Type Concentration Average Beete C3 Peak value 000 C2 Lowest value Bead Count Background
11. e111 054 Mouse IL 2 ENNN 232072 Mouse IFN gamma 309 5 077 Mouse IL 4 034 Mouse IL 5 054 Mouse IL 2 077 Mouse IL 4 selected amp nalytes Legend Figure 7 24 Well chart Settings in the Legends tab determine the location and appearance of a chart legend User Modifiable Legend Settings Gradient Visible Choose this option to display the legend with a gradient background No Middle Direction Make a selection from the drop down list to set the direction of the gradient displayed in the legend Start Mid End Click to open a color palette Make a selection from the color palette to set the background color for the start middle or end portion of the gradient Positions Choose an option to display the legend to the left right top or bottom of the chart Shadows Color Click to open a color palette Make a selection from the color palette to set the color for the shadow at the legend perimeter Size select a number to set the thickness of the shadow at the perimeter of the legend Text style Make a selection from the drop down list to select among MasterPlex QT www miraibio com 124 CHA PTER7 DATA CHARTS display options that include order of items placement of data hide or unhide data display percentage values and display the x axis value in the legend Show Legends Choose this option to display a legend in a data chart Walls Tab 1 To view the chart wall properties click the Walls tab in the Chart Propert
12. standard deviation and CV for each control group Includes the mean standard deviation and CV for the background wells Displays a table of MFI data per analyte column and well row Displays a table of Fold Change data per analyte column and well row A layout of the plate that shows the well type the group number for each well and the links between standards and unknown groups 4 To apply a log scale to the x axis of standard curves in the Standard curves report choose the Log on X axis option 5 Click Generate to display the report The report window opens and displays the report The report name is added to the plate navigator under the report node for the plate 6 To display a log scale x axis in the Standard curves report choose the Log on X axis option 7 To print the Standard curves report on a monochromatic printer choose the Gray Scale option MasterPlex QT www miraibio com 139 CHA PT ER 8 MASTERPLEX QT REPORTS 8 2 Working with Reports If the report window 1s open the report toolbar is available Figure 8 2 You can print report or save a report 2414 0000 54 Figure 8 2 Report window toolbar Table 8 2 Report window toolbar button functions Toolbar Click to Button Open files ab Exit report window Change the magnification in the Report window Make a selection from the drop down list of view options Display the first page in the report 1 Displav the
13. standard deviation and CV for each standard group Figure 8 11 on page 8 12 Standard data with expected and calculated values residuals and per cent recovery Figure 8 12 on page 8 13 Local standard curves generated on the current plate Figure 8 14 on page 8 15 Displays a table of MFI data per analyte column and well row Displays a table of concentration data per analyte column and well row A layout of the plate that shows the well type the group number for each well and the links between standards and unknown groups MasterPlex QT www miraibio com 138 CHA PT ER 8 MASTERPLEX QT REPORTS Table 8 2 MasterPlex QT reports for Relative Quantification analysis Report Name Report per analytes Report per sample well Plate format report Report per unknown replicates Report per control replicates Report per background replicates Tabular MFI report Tabular Fold Change report Well map report Contents MFI bead count and concentration data organized by analyte name Figure 8 7 on page 8 8 MFI bead count and concentration data organized by sample well Figure 8 8 on page 8 9 Organized by data types MFI bead count or concentration that are displayed in the well grid format Figure 8 9 on page 8 10 Considers each group of unknown wells a replicate Includes the mean standard deviation and CV for each unknown group Considers each group of control wells a replicate Includes the mean
14. 00 1288 00 409 00 460 50 372 00 1064 50 62 00 66 00 58 00 505 00 68 50 62 00 76 50 181 00 17 50 18 00 17 00 46 50 12 00 12 00 14 00 1900 200 200 3 00 6 50 1 00 2 50 7 00 Figure 5 15 Print Preview dialog box Preview the well grid contents 3 To print the well grid click Print 9 9 Working With Standard Curves After the standard curves are generated you can view the curves residual plots the standard data and information about the standard data in the Standard Curves window Figure 5 16 Opening the Standard Curves Window 1 Click the Open Standard Curve button Fra Alternatively select Plate gt View Curves from the menu bar gt The Standard Curves window opens and displays the Standard Curve Chart tab Figure 5 16 The window displays a tree of local standard curves imported standard curves and regression curves for the active plate MasterPlex QT www miraibio com 75 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION Standard Curves Ema lx E GI Local Curves Standard Curve Chart Residual Plot Standard Data da EE All Selected Standard Curves Std Curvea 1 AA 034 Mouse IL 5 o Std Curve 1 A 038 Mouse IL 10 Std Curve 1 A 054 Mouse IL 2 Std Curve 1 A O73 Mouse IFN gamma Std Curve 1 A 077 Mouse IL 4 ie qoem Bi Regrfssions Click the standard curve s that vou want to displav o E VW get E el Lr E m H ER 2 Les LL E a T
15. 3 1 lists the types of data available for display in the well grid 3 To view background subtracted data click the Subtract background button S gt The Plate window displays background subtracted data For more information on background calculation options see Background Type on page A 4 Data type drop down list Edit box MasterPlex QT www miraibio com 16 CHA PTER3 GETTING STARTED PAE Ea lx UN M gig Su i Cri fad mma GB Oy SS I Bead Set Properties Standards 2000 E Local Standards d 034 Mouse IL 5 038 Mouse IL 10 d 054 Mouse IL 2 073 Mouse IEN gamma e 077 Mouse IL 4 Local Importe Number bf Wells 96 034 Mouse IL 5 Bead set tab Well grid Figure 3 9 Plate window and Bead Set tab Bead Set tab shows the analytes in the plate Plate Window Components Well grid microtiter plate that displays the well contents for the analyte selected from the Bead Set panel and data type selected from the data drop down list Shows the types of data available for display in the Data type well grid Make a selection from this drop down list drop down list to choose the data type displayed in the well grid Click the dropdown arrow to view the list and select a data type Alternatively click the E arrows to scroll forward or backward through the list See Table 3 1 for a description of the data types Edit box A representation of a MasterPlex QT www miraibio com 17 Bead set
16. A Fl a m t E x 1 d 3 4 ki e a 02 ep 1288 00 405 00 50 372 00 1064 50 62 00 65 00 55 00 D 505 00 68 50 62 00 76 50 E 181 00 17 50 15 00 17 00 F 45 50 12 00 12 00 14 00 G 19 00 NI NI 3 00 H 6 50 1 00 7 00 mm Number of Wells 96 034 Mouse IL 5 Figure A 3 Plate window Red border identifies wells with a MFI value less than the user specified threshold for the selected analyte A 3 Calculations Tab The Calculations preferences Figure A 4 specify how to fit the standard curve when there are replicate standard data sets display concentrations for diluted unknowns that were diluted the diluted concentration or the original undiluted concentration MasterPlex QT www miraibio com 164 APPENDIXA PREFERENCES Preferences Application Plate Regression Calculations Standard Points O Average standards Individual points Check this option to include dilution factor in the Calculation Type concentration calculation e Absolute Quantification Fold Change Normalization Calculate Dilution Factor C Regression Outlier Option I Automatic Outer D etected Outlier Values utomatic Outlier Detecte G MFI iu Error Range x olde Concentration Default ay Ok ae Cancel c Apply Figure A 4 Preferences dialog box Calculations tab Standard Points If the plate contains replicate standard data sets there are two ways to fit a standard curve Average The replica
17. File gt Preferences from the menu bar gt The Preferences dialog box appears Figure 5 10 MasterPlex QT www miraibio com 70 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION Preferences Application Plate Regression Calculations Standard Points OAveragestandards SE Individual points Check this option to include dilution factor in the Calculation Type concentration calculation 5 Absolute Quantification 3 Fold Change 3 Normalization Calculate Dilution Factor 3 Regression Outlier O ptior Automatic Outlier Detected Outlier Values uromatic OUIET L ELECE MFI H A Sieh etes ion Concentration Default a Ok 3X Cancel Apply Figure 5 10 Preferences dialog box Calculations tab 2 Click the Calculations tab 3 Choose a Standard points option click Apply and click OK 4 To return all preference settings to the factory set defaults click Default and click OK Generating Standard Curves amp Viewing Analyte Concentrations 1 To generate the standard curves and compute interpolate or extrapolate the analyte concentrations click the Calculate button El Alternatively select Calculations gt Calculate STD Curves from the menu bar gt A message box confirms the calculations are completed Figure 5 11 MasterPlex QT www miraibio com 71 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION SUCCESS Protein Quantification All calculations
18. Generates a Dose Response curve for the selected regression analysis data set using the selected model and determines LogEC50 Calculate Calculate STD Curves Fit Regression Curves MasterPlex QT www miraibio com 170 APPENDIXB MASTERPLEX QT TOOLBARS B 5 Chart Toolbar The chart toolbar is available when a Data Chart or Standard Curves window 1s open To show or hide the chart toolbar select Show gt Show Hide gt Chart Tools from the menu bar M 99 gt Figure B 5 Chart toolbar Table B 5 Chart toolbar buttons and functions Menu Bar Function Command Toolbar Button Charts gt Select MS Displays a drop down list of chart types Charts Charts gt Chart 39 Toggles the display of a chart between a 3D two or three dimensional view Charts Enables you to rotate a chart using a Rotate Chart click and drag operation Charts gt Zoom Enables you to magnify a user selected area Chart in a chart using a click and drag operation Displays or hides the analyte panel in the Data Chart window Displays or hides the scale bar in the Data Chart window Opens the Chart Print Preview dialog box Charts Print Chart MasterPlex QT www miraibio com 171 APPENDIXB MASTERPLEX QT TOOLBARS B 6 Report Toolbar The report toolbar is available when the Report window 1s open A ICI AT e A A OOUD va Figure B 6 Report toolbar Table B 6 Report toolbar buttons and functions Menu Bar Main Function Co
19. IL 8 m e E kd m 2 GM CSF m E E m LG n Figure 7 6 Intensity map Click the map to display a tool tip analyte name or y axis data Right click the map for tool tip options MasterPlex QT 107 CHA PTER7 DATA CHARTS The intensity map is color coded Figure 7 6 shows the default colors Red peak intensity Green mid range intensity Blue low intensity The default colors may be changed See Intensity Map Settings on page 7 35 Each row in the map represents the relative intensities of the analytes in one well This row represents the third well selected in the Plate window LL m C Re E Bes KA E i A m oh a z E This box represents This box represents This box represents the intensity of the intensity of IFN the intensity of l 1Beta relative to the gamma relative to the GMCSF relative to the intensity of the other intensity of the intensity of the other analytes in the third other analytes in analytes in the third well the third well well Figure 7 7 Intensity map In this example map Il 1Beta has the highest intensity and GM CSF has the lowest intensity in the third well that was selected in the Plate window MasterPlex QT www miraibio com 108 CHA PTER7 DATA CHARTS Single Analyte Chart This chart shows the data MFI count or concentration for a single analyte across all user selected wells Figure 7 8 Each bar represents the selected analyte data from one w
20. Please enter the path and name of the data file FML tal LS CSW to be opened rou can also click on the folder button ta the right of the edit box to browse the data file This data file contains all data acquired by Lumines system during the run Plate wizard will extract all these data into easy to read plate window Figure 3 3 Plate Wizard Import File tab 4 Enter the file path for the csv xls or Ixd that you want to import Alternatively click tz Browse button The Open dialog box appears Figure 3 4 Look in 9 6 P m Beh AverageStd csv Bah H 10Plex csv Pl CG Files of type All Files f mb kd xls csv Open as read only Figure 3 4 Open dialog box 5 Navigate to the directory of the csv xls or Ixd that you want to import 6 Select one or more csv xls or Ixd files and click Open MasterPlex QT www miraibio com 12 CHA PTER3 GETTING STARTED To select adjacent files press and hold the Shift key while you click the first and last file in the selection To select nonadjacent files press and hold the Ctrl key while you click the files of interest 7 Click Finish in the Plate Wizard The Plate window opens and displays the results data Figure 3 5 MasterPlex QT 3 0 Untitled Plate 1 lt IL5 csv gt Ed File Edit Plate Calculations Show Window Help AH o m zez ar Y um gm vo i 3 a EE Work Space i s m Na Wu LE CU Ba T C SS Sa OX E E Untitled Plate 1 Bead Set P
21. Select Plate Type tab appears Figure 6 3 MasterPlex QT www miraibio com 82 CHA PT ER6 VIRTUAL PLATES Plate Wizard Select Plate Type e F l LU S z pe G Ji E Amb dh Please select a plate type by clicking an one of the radio button below il a E i wv wi Y Y B a M a Plate Type Open a plate 9 A virtual plate ty Einigh Figure 6 3 Plate Wizard Select Plate Type tab 4 Choose the Virtual Plate option and click Next gt The Plate Dimensions tab appears Figure 6 4 Plate Wizard Plate Dimensions Please enter dimensions for the virtual plate below Virtual Plate Dimensions The row and column defaults can be edited in the Preferences dialog box click the d button Figure 6 4 Plate Wizard Plate Dimensions tab MasterPlex QT www miraibio com 83 CHA PT ER6 VIRTUAL PLATES 5 Enter the number of rows and columns for the virtual plate Click Finish gt A Plate window opens and displays the empty well grid of the virtual plate Figure 6 5 Untitled Plate 2 Virtual Plate gt Bead Set Properties Analytes Figure 6 5 Virtual plate Selecting Data from a Source Plate The virtual pipette copies aspirates data from user selected wells in a source plate and pastes dispenses the data into a virtual plate The virtual pipette aspirates all of the analyte data in a well including the computed analyte concentrations It remains load
22. actual curve model The nonlinear least square algorithm accomplishes this task The nonlinear or linear least square algorithm assumes that all points have the same variability so all points influence the curve fit equally However data that exhibit non constant variability violate this assumption As a result data points with greater variability assert more pull on the curve Data points at higher concentrations have more variability then those at lower concentrations and have greater influence on the curve fit than the points at a lower concentration As a result accuracy or sensitivity at the lower concentrations decreases C 4 Weighted Nonlinear Least Square The weighted nonlinear least square method of curve fitting 1s one way to correct for non constant variability In this method weights are assigned to each point so that all points have equal influence on the curve Instead of minimizing the residuals the method minimizes residuals based on the weight at each point In mathematical terms the non constant variability 1s made constant again by these additional weighting factors If the weight for a point 1s higher it will influence the curve fit more The weight is the inverse of the variance so points with low variability have more influence during curve fitting which seems logical As a result the curve fit represents the data better and the sensitivity often increases at lower concentrations Weighting By 1 Y We
23. at a time Bead Set Properties E Analytes 2 034 Mouse IL 5 T Figure 4 26 Well grid Red border identifies wells with concentration less than user specified threshold for the selected analyte Calculations Preferences The Calculations preferences Figure 4 27 specify how to fit the standard curve when there are replicate standard data sets display concentrations for unknowns that were diluted prior to the assay the diluted concentration or the original undiluted concentration can be displayed MasterPlex QT www miraibio com 56 CHA PTER4 DEFINING A PLATE Preferences Application Plate Regression Calculations Standard Points WEE Individual points Check this option to include dilution factor in the Calculation Type concentration calculation e Absolute Quantification Fold Change Normalization Calculate Dilution Factor Regression Lutlier Option Outher Values e MFI Error Range s 3tdev Concentration Automatic Outlier Detected Default ay Ok ae Cancel Poly Figure 4 27 Preferences dialog box Calculations tab Standard Points If a plate contains replicate standard data sets there are two ways to fit a standard curve Average The replicate standard data points are averaged and the standards standard curve is fitted to the single set of averaged data Choose this option if the experimental errors are depen
24. copies of the Software itself LICENSE You may a Use the Software on a single machine at any given time b Obtain limited numbers of Copy Protection Devices Additional Copy Protection Devices are provided only as a convenience of running the software c In no manner engineer or reverse engineer the copy protection hardware or whole or part of the software d Copy the software only for backup provided that you reproduce all copyright and other proprietary notices that are on the original copy of the Software provided to you Certain Software however may include mechanisms to limit or inhibit copying Such Software 1s marked copy protected e Transfer of the Software and all rights under this Agreement to another party together with a copy of this Agreement if the other party agrees to accept the terms and conditions of this Agreement If you transfer the Software you must at the same time either transfer all copies MasterPlex QT www miraibio com 1 LICENSE AGREEMENT whether in printed or machine readable form to the same party or destroy and copies not transferred RESTRICTIONS You may not use copy modify or transfer the Software or any copy in whole or in part except as expressly provided for in this Agreement Any attempt to transfer any of the rights duties or obligations hereunder except as expressly provided for in this Agreement is void YOU MAY NOT RENT LEASE LOAN RESELL FOR PROFIT OR DISTRIBUTE T
25. data for the selected wells in the new format Toggle the chart display between a two and three dimensional view Put the chart in rotate mode so that you can rotate the chart around the X Or y axis Put a chart in zoom mode so that you can magnify a user selected area of the chart Opens the Chart Print Preview window so a chart may be printed Normalize the data for the open multi well charts Plots the selected well data using the y axis scale from the chart with the largest y axis value Note This function is only available for the multi well chart select all analytes in the analyte panel for display in the chart Removes all analytes from the chart Minimize or restore all well charts in the Data Chart window Click to Hide or show the analyte panel Hide or show the scale bar To change the y axis scale move the scale slider up or down the scale bar MasterPlex QT www miraibio com 112 CHA PTER7 DATA CHARTS Copying Saving or Sending a Chart You can copy a chart to the system clipboard or save a chart 1 Open the chart of interest 2 Select Edit gt Export Chart from the menu bar gt The Export Chart dialog box appears Figure 7 11 Picture Native Data Format Options Size as Metafile Enhanced as Bitmap O as WML HTM Figure 7 11 Export Chart dialog box 3 In the Picture tab choose a file format option Metafile saves the chart in a scalable wmf format Choose the Enhanced
26. dialog box appears Figure 4 21 Look in E examples eg Ez Files of type Master Plex Template mxt Cancel Figure 4 21 Open dialog box MasterPlex QT www miraibio com 49 CHA PTER4 DEFINING A PLATE 2 Choose the directory with the template that you want to import 3 Select the template and click Open The template name is added to the Template Manager Deleting a Template You can delete a template mxt from the system 1 In the Template Manager click the template that you want to delete 2 Click Delete button y gt A confirmation box appears Figure 4 22 Confirm 4 Are you sure you want to delete MSPlexReplicates template qw OK 2 Cancel Figure 4 22 Confirmation box 3 Click OK to delete the template gt The template is removed from the Template Manager WARNING This permanently removes the template from the system WARNING 4 3 Setting Preferences Preferences are user modifiable software settings They are displayed in the Preferences dialog box e To open the Preferences dialog box Figure 4 23 click the Preferences button dh Alternatively select File gt Preferences from the menu bar MasterPlex QT www miraibio com 50 Preferences CHA PTER4 DEFINING A PLATE Application Plate Regression Calculations Application Preferences Set Preferences Default b rutal Plate Dimensions Rows 1207 Columns 12 Automatic Well Grouping
27. how the _ Other E names are affected in the preview below C Use last settings Or Br Om vor Figure 4 10 Specifving delimiters 3 Step 2 specifv sample tvpe replicate group Users choose which segmented column represents sample tvpe e g standard unknown background and which column represents replicate group Figure 4 11 MasterPlex QT www miraibio com 40 CHA PTER4 DEFINING A PLATE Auto Group Sample Type Replicate Group Please set a column which has sample pe information IF wou have replicates please alza indicate a column that has replicate group information Figure 4 11 Specifying sample types 4 Step 3 specify group type Based on users choice on sample type in step 2 they need to choose the segmented string for each group type Figure 4 12 Auto Group Specify Group Type Analysis Type e Absolute Quantification C5 Relative Quantification EE std ha Sp Treatment Unknown Un Dackoroud bkg Control Control ctrl Regression Assay Bkg oe Finish Figure 4 12 Specifying group types MasterPlex QT www miraibio com 41 CHA PTER4 DEFINING A PLATE 5 Finish l A and 1 Fa 3 4 5 b T C Figure 4 13 Autogrouping result a NOTE User s choices in the wizard will be saved into a file each time Next time user can simply choose Use the last settings to further speed u
28. images user has to use some tool or utility to do the conversion MasterPlex QT www miraibio com 158 APPENDIXA PREFERENCES APPENDIX A Some of the software settings can be modified This appendix explains the preferences user modifiable software settings that are available to you These settings are displayed in the Preferences dialog box To open the Preferences dialog box Figure A 1 click the Preferences button ge Alternatively select File gt Preferences from the menu bar Preferences Application Plate Regression Calculations Application Preig ences Set Preferences Default Virutal Plate Dimensions Use this dialog box Rows ta sel various pre ferences related to Columns 12 MasterPles GT Automatic Well Grouping Enable plate wizard at start up Enable Relative Quantiication Gene Expression Default ay Ok a Cancel Apply Figure A 1 Preferences dialog box Application tab default settings A 1 Application Tab The application preferences Figure A 1 specify MasterPlex QT www miraibio com 159 APPENDIXA PREFERENCES the number of rows and columns for a virtual plate displayed in displayed in the plate wizard whether the plate wizard appears when the MasterPlex QT software Starts 1 To change the row and column dimensions of the well grid enter the number of rows and columns for the well grid 2 If you do not want to display the plate wizard at program start u
29. meoriajjnm UA ze mme e KA El Work 5 me Work space Plate Wizard Plate Welcome to MasterPlex OT plate wizard z This wizard will guide you through creating Navigator and configuring a new plate window Please click Nest to begin creating and configuring a new plate Figure 3 1 MasterPlex QT user interface MasterPlex QT www miraibio com 10 CHA PTER3 GETTING STARTED 3 3 Importing Luminex Results To begin a MasterPlex QT analysis import a csv xls or Ixd file from the Luminex 100 200 system using the Plate Wizard toolbar or menu bar commands NOTE The Luminex default directory is named Output Importing Luminex Results Using the Plate Wizard 1 If the Plate Wizard is not open click the Plate Wizard button g The Plate Wizard appears Figure 3 1 2 In the Welcome tab of the Plate Wizard click Next gt The Select Plate Type tab appears Figure 3 2 Plate Wizard Select Plate Type Please select a plate type by clicking on ane of the radio button below ATAR E em SE g me E ae Zen un s M A d E a 3 Plate Type e Open a plate CO A virtual plate Qc Qs Next ww Fini Figure 3 2 Plate Wizard Select Plate Type tab 3 Choose the Import a new plate option and click Next The Import File tab appears Figure 3 3 MasterPlex QT www miraibio com 11 CHA PTER3 GETTING STARTED Plate Wizard Open File
30. miraibio com 120 CHA PTER7 DATA CHARTS Chart Gallery Horiz Bar Bar Join Histogram Line Point Click the down arrow to display a sub menu of bar chart formats Fast Line Bezier Pyramid LL Chart Gallerv Horiz Bar Bar Join Histogram Line Point Lolors Pyramid Elipse Inv Pyramid Stack Stack 100 Sides Sub menu of bar Side All chart formats Figure 7 21 Chart gallery of available formats for the multi well chart MasterPlex QT www miraibio com 121 CHA PTER7 DATA CHARTS 2 To change the chart format click the format of interest 3 To view a sub menu of styles for the selected format click the down arrow Figure 7 21 4 If you want to display the chart format in 3 dimensions select the Show 3D option 5 Click OK gt The selected format is applied to the multi well chart 3D Tab To view 3D chart properties click the 3D tab in the Chart Properties dialog box Figure 7 22 Chart Properties Charts 3D Legends Wals Dees Panel Rotation Elevation Perspective Orthogonal Horiz Offset Vert Offset Figure 7 22 Chart Properties dialog box 3D tab User Modifiable 3D Chart Properties Enable 3D Choose this option to display a three dimensional view of the chart 3D Increases or decreases the three dimensional thickness of the graph bars Orthogonal Choose this option to display a front view of the chart no rotation around the vertical axis MasterPlex QT
31. option to save the charts in enhanced metafile format emf Bitmap saves the chart in bmp format Depending on the chart dimensions the bmp file size can become large VML HTM saves the chart as an HTML document htm that can be viewed using a web browser 4 To specify a size option for the chart a Click the Size tab b Enter the width and height in pixels for the chart c Choose the Keep aspect ratio option to maintain the same dimension ratio at the new chart size 5 To copy the chart click Copy 6 To save the chart click Save gt The Save As dialog box appears Figure 7 12 MasterPlex QT www miraibio com 113 CHA PTER7 DATA CHARTS Save As Save n O 8 l File name Save Figure 7 12 Save As dialog box 7 To e mail the chart a Click Send c The Choose Profile dialog box appears Figure 7 13 Choose Profile Profile Mame Figure 7 13 Choose Profile dialog box b Select a recipient for the e mail and click OK El NOTE This dialog is only functional with Microsoft Outlook E MasterPlex QT www miraibio com 114 CHA PTER7 DATA CHARTS Copying or Saving Data The software can export data from a chart to other applications The data may be copied to the system clipboard or saved in different file formats 1 Open the chart of interest 2 Select Edit gt Export Chart from the menu bar gt The Export Chart dialog box appears Figure 7 11 4 Click the Data tab Include L
32. predicts the data perfectly The steps to create a standard curve include 1 Mark the standard wells 2 Group the wells in a standard data set 3 Link the standard data set to the unknown well group s of interest The analyte concentrations are interpolated from the standard curve that 1s linked to the unknown well group 4 Enter the standard concentrations 5 Select a model equation for the standard data set 6 Calculate the standard curves NOTE A plate can have more than one standard data set The standard data sets may have different concentrations or model equations Entering Standard Concentrations Enter the standard concentrations after you mark the standard wells group them into a standard data set and link the standard data set to a group s of unknown MasterPlex QT www miraibio com 60 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION wells For more information on setting well types and groups see Designating Well Type and Group on page 4 1 There are two ways to enter standard concentrations Use the autofill feature to automatically enter the analyte concentrations Manually enter the analyte concentrations Using the Autofill Feature The autofill feature enters the analyte concentrations for selected standard wells based on the user specified starting concentration and dilution factor 1 Open the results of interest csv xls Ixd or mlx and select the standard data set Figure 5 1
33. sat D Report per analytes MI 8 034 Mouse IL 5 038 Mouse IL 10 e INN 034 Mouse IL 5 V 42 054 Mouse IL 2 pet MI 038 Mouse IL 10 V 4 073 Mouse IFN aamma l 3 I 054 Mouse IL 2 V 077 Mouse IL 4 ji em Rm 073 Mouse IFN gamma il i i i EN 077 Mouse IL 4 Figure 3 15 Plate Navigator window If multiple windows are open click an item in the Plate Navigator to view it in the main displav area EE Work Space ci ES IL5 Project U Reports Go Report per analytes bir Bar Graph Lo Standard Curves EE Untitled Plate 3 Click this to change the view to Untitled Plate 3 in the 2l n Reports main display area Qa Report per sample well Lo Standard Curves FEE Human 10 plex project Open windows that are associated with Untitled Plate 3 Click an item to display it in the main display area Click to expand or collapse the plate node Figure 3 16 Plate Navigator Shows the windows that are open in the main display area MasterPlex QT www miraibio com 24 CHA PTER3 GETTING STARTED Table 3 2 shows the types of windows available in the software Table 3 2 MasterPlex QT windows Window Name Displays See Plate a CSV Xls Ixd or mlx file Figure 3 17 otandard Curves the Standard Curves window for the Figure 3 18 current Plate window Data Chart the current Data Chart window for the Figure 3 19 selected plate Report a report that was g
34. seamless 1 Start the plate merge wizard by clicking the toolbar button aq Merge Plates 2 You will see a list of plate names which are currently opened Figure 6 14 If you want to add more plates to merge you can click Open button to load more plates Then you need to check the plates to be merged from the list and go to the next page Merge Plates Select Plates To Merge Flatelz opened vl 44 Untitled Plate 1 FF Untitled Plate 2 FFA Untitled Plate 3 This wizard will guide you through merging multiple plates into a virtual plate The left list shows the plates currently opened If you want to open mare plates please use the Open button above Then check the plates from the list which you want to merge Then click Next to continue Figure 6 14 Selecting plates MasterPlex QT www miraibio com 97 CHA PT ER6 VIRTUAL PLATES 3 Now you will see a list of bead analyte names which appear in all selected plates Figure 6 15 If you confirm they are the common house keeping genes and want to exclude them from the virtual plate you may choose Automatically merge then finish wizard Otherwise you may choose to Manually select house keepers then go to the next step Merge Plates House Keeper Selection Common Bead Name 2 DEZ SsRINA The left list shows the bead names which appear in all selected plates Please c
35. standard curve fits the observed data o Recovery Observed Concentration Expected Concentration 100 The closer Recovery is to 100 the better the curve fit at that point If Recovery 1s less than 100 the point is below the curve If it 1s greater than 100 the point 1s above the curve Figure C 5 and Figure C 6 show the Recovery for two different standard curve MasterPlex QT www miraibio com 180 APPENDIXC MODEL EQUATIONS fits The standard data in Figure C 6 is from a four parameter logistic equation with no weighting Note that the Recovery for H1 is poor This means the sensitivity deteriorates significantly at lower concentrations The data in Figure C 6 1s from the same equation with 1 Y weighting After we apply weighting the Recovery for G1 and H1 improves significantly Standard Curve Chart Residual Plot Standard Data Sample Name Outliers MFI Calculated Expected Residuals ve Recovery Background a n 65 00 ale 9 085 26 96 7 30 66 00 7 E 197 00 84 81 87 24 2 04 97 21 66 00 e F 516 00 273 63 261 73 11 90 104 55 68 00 5 E 1406 50 Dad Gi 785 19 79 70 110 15 66 00 4 D 212 00 199 04 2300 56 156 52 93 36 68 00 3 KE 3104 00 75 33 7D56 67 70 06 103 95 68 00 d E FRE UO 0793 06 100 00 06 14 95 06 66 00 Figure C 5 Example standard curve data Four parameter logistics equation fitted to standard data with no weighting Standard Curve Chat Residual Plot Standard Data Well Samp
36. step 1 and step 2 to mark and group other well s Designating Well Groups After you have defined the wells the wells must be organized into groups so that the software can identify Replicate unknowns A standard data set A regression analysis data set for a virtual plate only For more information on virtual plates see Chapter 6 on page 6 1 MasterPlex QT automatically places all background wells into one group You can define one or more groups of control wells per plate e NOTE A group can include nonadjacent wells A plate can have more than one group of standards or unknowns A virtual plate can have more than one set of regression data To show or hide the group borders click the Show well grouping button E NOTE Grouping does not set replicate unknowns Standard unknowns control and background groups can be treated as replicates to obtain statistics such as mean standard deviation and CV in the final report Automatic Well Grouping If the Automatic Well Grouping option is chosen in the Preferences dialog MasterPlex QT www miraibio com 34 CHA PTER4 DEFINING A PLATE box Figure 4 4 the software automatically groups wells when you set the well type The automatic well grouping is the factory set default Preferences Application Plate Regression Calculations Application Preferences Get Preferences Default Virutal Plate Dimensions Use this dialog box Rows to set various pre ferences re
37. the Government is subject to restrictions as set forth in subparagraph c 1 11 of the rights in Technical Data and computer software clause of DFARS 52 227 7013 Hitachi Software Engineering America Ltd 601 Gateway Boulevard Suite 100 South San Francisco CA 94080 EXPORT LAW ASSURANCES You acknowledge and agree that the Software 1s subject to restrictions and controls imposed by the United States Export Administration Act The Act and the regulations thereunder You agree and certify that neither the Software nor any direct product thereof is being or will be acquired shipped transferred or reexported directly or indirectly into any country prohibited by the Act and the regulations thereunder or will be used for any purpose prohibited by the same GENERAL This agreement will be governed by the laws of the State of California except for that body of law dealing with conflicts of law Future updates of the Software will be available for purchase by licensees for a fee provided a registration card has been received by Hitachi Software Engineering America Ltd Should you have any questions concerning this Agreement you may contact HISAL at http www miraibio com You acknowledge that you have read this Agreement understand it and agree to be bound by its terms and conditions You further agree that it is the complete and exclusive statement of MasterPlex QT www miraibio com IN LICENSE AGREEMENT the agreement
38. were completed successfully Figure 5 11 Message box 2 To view analyte concentrations in the Plate window select Concentration from the data type drop down list and select an analyte in the Bead Set tab Figure 5 12 Select an analyte EE EEN from the eae down list n a ptes 48 034 Mouse ILB O39 Mouse IL 10 Figure 5 12 Well grid displays concentration data for the selected analyte If the standard curve uses a sigmoidal model for example the four parameter logistics equation Figure 5 13 the software interpolates the analyte concentration when Highest standard MFI x MFI x Lowest standard data point The software extrapolates the analyte concentration when A MFI Lowest standard MFI Or Highest standard MFI MFI D where A is the bottom asymptote and D is the top asymptote of the sigmoidal MasterPlex QT www miraibio com 72 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION curve Figure 5 13 A MFI value less than A or greater than D is beyond the range of the standard curve model and the concentration value cannot be extrapolated If MFI lt A the well grid displays the lowest standard MFI preceded by lt Figure 5 12 If MFI 2 D the well grid displays the highest standard MFI preceded by gt Four Parameter Logistics R Square 0 9991 a 16 335 b 2 392 c 306586 d 1373 175 o em o sm e k Median Fluroscense I
39. you can perform a Dose Response analysis The Dose Response analysis provides information on the relationship between the agent and changes in the level of cytokines Dosages To conduct a Dose Response analysis different dosages of the chemical biological or other agent are prepared For example eight serial dilutions of an agent mg ml could be prepared and given to eight different mice A blood sample taken from each mouse is then used to perform a cytokine multiplex assay on the Luminex 100 200 Responses Using MasterPlex QT you can interpolate the cytokine concentrations of the blood samples from the standard curves of the reagent kit Now you have both dose and quantified response data mg ml Regression Analysis The software uses regression analysis to fit a dose response model to the data points x dose and y response After the model is fitted the software provides the LogEC50 Take the antilog of the logEC50 to determine the EC50 the agent dosage that elicits 50 of the response If 50 of the test subjects die MasterPlex QT www miraibio com 182 APPENDIXD DOSE RESPONSE ANALYSIS at the EC50 value the EC50 value is the LD50 value In this way you can determine the effectiveness or the toxicity of an agent MasterPlex QT www miraibio com 183 APPENDIXE RELATIVE QUANTIFICATION ANALYSIS APPENDIX E This appendix provides an introduction to Relative Quantification Relative Quantification Assav Researche
40. 0 1354 00 C2 2 00 22 50 78 00 190 00 332 00 De e x 37 00 323 00 130 00 121 50 E2 17 50 13 50 14 50 39 00 30 00 F2 12 00 17 00 2200 39 00 35 00 2 z 00 0 00 15 00 71 00 6 50 H2 71 00 71 00 14 00 200 200 A3 30 10 butter 286 50 1073 00 101 50 157200 203 30 53 450 50 1015 00 1853 00 121200 2308 00 Ca 69 00 13200 32 00 191 50 317 00 Di 200 13 00 87 00 198 00 731 00 el 14 00 27 00 10 00 22 00 191 530 Fa 12 0d 27 00 1250 20 50 204 00 GA 2 00 71 00 400 3 00 1 00 H3 2 50 1 00 3 00 150 71 00 Al Sigma poded 121 00 330 00 1195 00 745 00 1478 00 372 00 388 00 120 00 206 00 1904 00 zz 33 00 111 00 2200 70 50 Kelt oe 76 50 113 00 46 00 35 00 332 00 zl 17 00 20 00 17 50 39 00 30 00 Fi 14 00 2200 13 00 33 00 33 00 iB 30 71 00 11 00 2300 71 00 Hi 3 00 1 00 7a 14 00 300 Page 7 Ta Tec om Figure 8 15 Tabular MFI report MasterPlex QT www miraibio com 152 CHA PTER8 MASTERPLEX QT REPORTS MasterPlex QT Tabular Concentration Report Run Date 3 20 01 Rum Time 12 45 35 PM Hardware Serial No Operator AC Analyst Report Date 2 8 2007 Repon Time 12 39 22 AM Data File ILS5 csv Plate Mame ILS Project mix MasterPlex OT Version 3 0 1 166 Well Sample Name 034 Mouse IL 3 038 Mouse 054 Mouse IL 2 073 Mouse IFN 077 Mouse IL 4 A1 curve 158 2300 156 2300 158 2300 1312 1908 51 2 SEKR 3688 100083 370 108635 325 Su eres C1 A o0 0818 49913516 2535 0517 Ren DA 2233 7311 23039 5060 Z0 56257 2255 5258 El 3 1277 3756 1251 30
41. 00 486 50 321 00 JAF 1288 00 409 00 460 50 372 00 1064 50 62 00 68 00 58 00 n E Aspirate 62 00 76 50 18 18 00 17 00 4 i 12 00 14 00 p 6 50 1 00 2 50 7 00 Figure 6 6 Well grid Right click selected wells to display the pop up menu 4 To clear the data from the virtual pipette right click and select Clear from the pop up menu Adding Data to a Virtual Plate After the virtual pipette aspirates data from the source plate it 1s ready to dispense the data into the virtual plate MasterPlex QT www miraibio com 85 CHA PT ER 6 VIRTUAL PLATES 1 Position the virtual pipette over the virtual plate 2 Click the first well to which the data will be added 3 Right click the well and select Dispense from the pop up menu that appears gt The data are added to the virtual plate in the same configuration as in the source plate Figure 6 7 NOTE If the number or names of the analytes in the virtual pipette is different from that in the virtual plate the virtual analyte filter automatically appears For more information on using the filter see Selecting and Renaming Analytes on page 6 9 dR NOTE Data in a virtual plate cannot be removed but can be overwritten 4 After you finish adding data to the virtual plate click the Virtual Pipette button to turn off the virtual pipette MasterPlex QT www miraibio com 86 CHA PT ER6 VIRTUAL PLATES 1 Open a mlx Or CSV Click the IL5 Project mix xIL5 csv button t
42. 00 software If you want to edit the plate name enter a new name Analyst Name Displays the analyst name entered in the Luminex 100 200 software If you want to edit the analyst name enter a new name Thresholds MasterPlex QT www miraibio com 162 APPENDIXA PREFERENCES You can enter an MFI count or concentration threshold for a plate The software can identify wells that contain data less than the user specified threshold To set a threshold s 1 Enter the MFI count or concentration threshold in the Preferences dialog box Figure A 2 2 Click Apply when you are finished 3 To return the plate preferences to the factory set defaults click Default To identify the wells in the Plate window that contain data less than threshold 1 Make a selection from the analyte panel 2 In the Plate window select the data type MFI count or concentration from the data type drop down list 3 To identify wells with MFI data less than threshold click the button A red border is placed around the well Figure A 3 To identify wells with bead count data less than threshold click the FLI button A red border is placed around the well To identify wells with concentrations less than threshold click the 7 button A red border is placed around the well MasterPlex QT www miraibio com 163 APPENDIXA PREFERENCES IL5 Project mlx lt IL5 csv A 7 ai A3 E j el l Misi C Ulum sekli Bead Set Tepes sa G gan
43. 000 pom 213136 102 235 Di 506 0000 Zo Een 1 93778 C1 d 10645000 50000000 pom 27725 100 055 Bi a 1288 0000 S385 3756 10000 0000 pom 13 124 39 869 Well Sample Heme NEI Calculzte Expected Unit Residuals Recorery Hi 3 3 0000 1582500 pom GI 7 28 00 312 3000 pam AN R33 F1 vd 52 0000 425 0000 pom 5080 El 3 33 0000 1250 00 pom 1 3083 Di 13190000 2300 00 pom 123683 c d 3223 000 SOLO pom 0 0878 Bi 2 553 5000 1000 00 pom Dii Well Sample Heme MFI Calculzte Expected Unit Residuals SRecorery Hi 3 5 0000 19582300 pom NA NA GI 7 4 0000 312 5000 pom NA NA Fi tal 23 0000 ex Oy pom 1183 43 118372 El 3 SCHO 12384083 1250 00 pom 11 5203 28 073 Di 2170000 pom 3 5060 100 140 Lac Taga xz ILA Project mix Figure 8 13 Report per standard data MasterPlex QT www miraibio com 150 CHA PT ERG MASTERPLEX QT REPORTS asterPlex QT 3 0 Standard Curve Report Hun Date 3720 01 Report D ate 2 8 2007 Hun Time 12 45 55 PM Report Time 12 38 02 AM Data File Hardware Serial No LX 100007800 Operator AC ILS cs Plate Name ILS Project mix Analyst MasterPlex OT Version 3 0 1 166 Five Parameter Logistics 3 660 c 0 434 a 2 878 b 4403 824 d 1318 477 e 0 9999 Y Intercept A Square eE se ee 1 100 1 050 I 1 1 I 1 1 L 1 1 1 1 1 1 I r 1 1 1 1 I 1 L I 1 1 1 I 1 1 L I 1 1 1 1 I I r 1 LI 1 1
44. 1651 00 123500 BIO E Toe n0 CLICA pinin n 2445 00 111 00 IEEE T4 Ui BT En oci an ar 4700 24 on 4300 xz am on Analyte 073 Mouse IFN gamma 1155 00 1674 00 ety 3141 50 1337 00 1516 00 BIZ o 251500 ZZ DI ze E 17250 ML ce Pag 2 Figure 8 9 Plate format report Each data type is displayed in a well grid format MasterPlex QT Background 0 00 zu oc 26700 Ec ED 24 1 00 Background 0 00 Tz oC Background 0 00 ze o0 Background 0 00 TORO LS Prepa min www miraibio com 146 CHA PT ER 8 MASTERPLEX QT REPORTS MasterPlex QT Report Per Unknown Replicates Report Date 2 8 2007 Run Date 3 20 01 Sun Time 12 45 53 PM Report Time 12 30 33 AM Data File ILS5 csv Plate Name ILI Project m MasterPlex OT Version 3 Ix 0 1 165 034 Mouse IL 5 Hardware Serial No LX 1000078 Operator AC Analyst Background 20 00 Well Sample Name MFI Concentration Unit AZ Ind mouse serum 347 0000 1235 1303 pom B2 10 209 00000 2138 O12 palm C2 62 00000 661 248 Gem De GH 5000 JU2 e pom E 17 3000 319 0262 pom F2 12 000 261 7078 palm G2 2 XX 44 0515 palm H2 1 0000 71 1064 palm A 30 10 buffer ABE 5000 24 30 0534 Gem B3 ABU 000 2113 1545 Gem C3 68 00000 Ga 4453 palm D3 62 00000 551 5248 Gem E3 18 00 A i7 palm F3 12 0000 261 7078 palm G3 ONI 44 0513 Gem H3 2 XX 23 90075 Gem Ad Sigma pooled 321 0000 1833 8552 pa mL 24 372 00000 2026 4183 palm CA 28 000000 619 244 palm
45. 17250 LL ene wm Cer miie KR I Figure 3 20 Report viewer Plate format report MasterPlex QT www miraibio com 21 CHA PTER3 GETTING STARTED 3 8 Saving Plate Data After you import a Luminex results file csv xls or Ixd the data can be saved to a MasterPlex QT file format mlx The mlx file includes all data associated with a plate such as well definitions and computed interpolated or extrapolated concentrations To save results data csv xls or Ixd to a MasterPlex file mlx 1 Click the Save button i Alternatively select File gt Save from the main menu gt The Save As dialog box appears Figure 3 21 Save As A IL5 Project mlx File name Figure 3 21 Save As dialog box 2 Confirm the default directory where the file will be saved or choose another directory 3 Enter a file name and click Save Opening a MasterPlex File mlx 1 Click the Open button EA Alternatively select File gt Open Plate from the main menu The Open dialog box appears Figure 3 21 MasterPlex QT www miraibio com 28 CHA PTER3 GETTING STARTED Lock in 9 d amp m IL5 Project mlx Bil AverageStd csv E H10Plex csv EN IL5 csv File name pen Files of type All Files mix kd ads ce Figure 3 22 Open dialog box 2 Confirm the default directory or choose another directory 3 Select a file name mlx and click Open A Plate window opens and displays the results data Fig
46. 55 T2457 1208 10602 1235 3 012 F1 33 3375 MEM 22335723 805901 23733581 al 7 3116136 TMIN 156 2500 2332183 d E38 H1 3 135 7183 122 3821 158 2300 x 158 2300 181 2227 A2 ind mause serum 1335735 1108917 2133 28253 18 6 7282 1238 1352 10 21585012 17737768 AE 3 533 A 7133 14133113 c2 601 348 813 5828 315 2481 877 1313 413 8751 D2 MASES EZA OE 2700294 362 6805 470 5550 E2 SIE ei 253 35325 817 0083 2233872 212 0205 F2 281 178 ITI ax 371 2233872 216 4227 2 20513 156 2500 fi EBU 1562500 138 0313 H2 31 1088 x 158 2300 aa i15 158 2300 133 38 2 Ag 3010 butter LEAD AL 2137 23530 2113 3512 ZELLE 2251 7300 Es 33 5525 21227271 2053 20 2306 2835 21875778 c3 Ver 382 0156 300 120 873 33 5 TATH D3 601 348 1139113 887 1300 885 728 7320852 E3 21331272 X 8574 S308 1933 35 3788 300 1457 Fa 261 178 d 8372 391 038 212 5085 310 8352 a3 220515 158 2300 158 2300 158 2300 133 0901 H3 23 3035 158 2300 207 7250 21562500 1312308 AI Sigma poded 1833 8562 2004 2695 1523287 150050 112 526 zl 212 4183 2090 7112 1885 3135 1233 2382 1222 3322 zz 835227 830 7417 805 5333 33 0877 255 1800 W 1312116 466131 421 005 Gah 2052 225 180 EI 3120754 ore 2278 a350345 3233872 212 0205 l 283 3381 Z3 24100 80 4287 233016 2145700 is 125 8235 x 158 2300 2232385 DES 131 2308 Ai EA 156 2500 156 2500 352 1322 1333772 Pag e ILE Project mix Figure 8 16 Tabular concentration report MasterPlex QT www miraibio com 153 CHA PT ER 8 MASTERPLEX QT REPO
47. 5745073 Mouse IFN gamma eege AR ee ears e 2721 5 077 Mouse IL 4 2 5004 Place a check zoe puse mark next to m the analytes E that you wantto ui A 1 000 displav in the Ie well chart e soo 1 034 Mouse IL 5 054 Mouse IL 2 077 Mouse IL 4 Selected Analytes Parent Plate IL5 Project mlx Scale bar and slider Figure 7 1 Multi well chart MFI data for the selected analytes in well B1 MasterPlex QT www miraibio com 100 CHA PTER7 DATA CHARTS 3 To display another graph format for the selected wells click the button a and make a selection from the drop down list gt The well data are displayed in the selected format 4 To change the data type in the chart MFI count or concentration make a selection from the data drop down list Figure 7 1 5 To remove an analyte from a chart remove the check mark next to the analyte name in the analyte panel 6 To view other well data in the current chart format a Return focus to the Plate window b Select the well s of interest c Click the Data Chart window The Data Chart window is updated and displays the new well data in the selected format 7 To change the y axis scale move the slider up or down the scale bar 7 2 Data Chart Types MasterPlex QT provides seven different chart types Table 7 1 To choose a chart format for the selected well data click the iu button and make a selection from the drop down list of data charts bk NOTE The
48. 7 To enter other standard concentration values for the same analyte repeat step 4 and step 5 8 To enter standard concentration values for a different analyte repeat step 2 through step 5 1 Click the Well Editor button to put the Plate ndok iredi d 3 Select l l window in edit mode Standard Indepe 5 Displays the concentration for ndent Values the selected well Edit the p an value and click Enter IR Aw mao el E 1 r Wo Space E D ILS Project mls EE 11 Reports Analptes ROMS Bug Sed Unk Cri ge E e ES Le Well grid Bead Set panel 034 Mouse IL 5 Figure 5 6 Plate window 4 Click the well that you want to edit Steps to manually enter a standard concentration MasterPlex QT www miraibio com 65 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION Selecting a Model Equation for the Standard Data Set 1 Select an analyte from the Bead Set panel Figure 5 6 2 In the well grid select a standard data set 3 Right click the standard data set and select Assign Model Equation from the pop up menu that appears Alternatively click the Select Model button dn or select Calculations Model Equations from the menu bar The Model Equations dialog box appears Figure 5 7 NOTE The Model Equations dialog box only appears if you selected a standard data EJ se Model Equations Available
49. 73 Mouse IFN aamma 2 d 077 Mouse IL 4 I Multi Well Chart Selected Parent Plate IL5 Project mlx Figure 7 3 Multi well charts are minimized TIP You can conveniently reorder the multi well charts in the Data Chart window by dragging a chart to the desired position in the list MasterPlex QT www miraibio com 104 CHA PTER7 DATA CHARTS 3D Depth Chart The 3D depth chart is a convenient way to compare the MFI count or concentration data in one well and across different wells Figure 7 4 Each bar represents an analyte The analytes of a particular well are identified by color and row Select the data type for the chart Color coded legend identifies the wells in the chart A Oe e m EX L IER SO 3D Depth Chart d 034 Mouse IL 5 MI 8008 Mouse IL 10 T Am 4 Mouse IL 2 selected analytes vs all selected wells v Mora Mouse IEN aamma SI aM O77 Mouse IL 4 Place a check mark next to the analytes to include in the chart 3D Depth Chart selected Parent Plate IL5 Project mlx Figure 7 4 3D depth chart compare analyte data in the same well or between different wells Each bar represents an analyte Each row of color coded bars represents the analyte data from one well Click a bar to display a tool tip analyte name or y axis data Right click a bar for tool tip options MasterPlex QT www miraibio com 105 CHA PTER7 DATA CHARTS Inverted 3D Dep
50. 77 Mouse IL 4 034 Mouse IL 5 054 Mouze IL 2 077 Mouse IL 4 Selected Analytes Multi Well Chart selected Parent Plate IL5 Project Figure 3 19 Data Chart window MasterPlex QT www miraibio com 26 CHA PTER3 GETTING STARTED Report Date 4 27 2007 Run Date 3 20 01 Report Time 9 41 02 AM Hun Time 12 45 22 PM Data File ILS csv Hardware Serial Mo LX 10000280002 Plate Name Untitled Plate 2 Operstor AC MasterPlex OT Version 3 0 1 166 Analyst Analyte 034 Mouse IL 5 Background 0 00 ES E EE a aj 20 00 ES 367 00 505 50 341 00 RS 1308 00 425 00 450 50 35200 E d 1084 50 S200 SS 00 72 00 MET sso DEN EZ 00 se zo ESI 201 00 37 50 SE 37 00 La 5550 3200 34 00 e 3800 18 00 18 00 2300 Rn 26 50 18 00 17 50 27 00 Analyte 038 Mouse IL 10 Background 0 00 ER Ee EA A O EI RE ee a E E E 13 00 710 50 1o OO Ex DO 547650 HE 1063 00 1001 00 X242 00 105 50 185 00 124 00 1332 00 110 00 161 00 126 00 407 00 32 50 40 00 33 00 107 00 30 00 40 00 35 00 41 00 13 00 12 00 12 00 71 00 12 00 12 00 14 00 Analyte 054 Mouse IL 2 Background 0 00 ae ETE EES TS PO E O EE O ee eee eee ZS 1550 ED 1723 50 123 00 7355 00 1617 50 1651001 5705 50 105 00 TO MD 2345 00 111 00 5500 335 00 4250 ZS O0 34 00 43 00 3200 35 00 45 00 37 00 35 50 Analyte 073 Mouse IFN gamma Background 0 00 e A O pn O ee ae eee 10200 115500 167400 84700 3141 50 1357 00 1516 00 03 00 2315 00 ADO E ED
51. 8 5 Open dialog box 3 Double click the report that you want to open The plate viewer displays the report MasterPlex QT www miraibio com 142 CHA PT ER 8 MASTERPLEX QT REPORTS 4 To close the report viewer click the Close button Hl Printing a Report 1 Double click the report that you want to print 2 Click the Print Report button e The Print dialog box appears Figure 8 6 Printer Mame G Wsfdo01 SF_MAIN_HPLJ5si v Page range Copies 8 Al Number of copies Curent O Pages Collate Enter page e and or page ranges separated by commas For example 1 3 5 12 Print All pages hal Figure 8 6 Print dialog box 3 Choose the print options of interest and click OK MasterPlex QT www miraibio com 143 CHA PTER8 MASTERPLEX QT REPORTS MasterPlex QT Report Per Analytes Report Date 5 8 2007 Run Date 3 20 01 Report Time 12 09 14 AM Run Time 12 45 55 PM Data File IL5 csw Hardware Serial No LX10000280002 Plate Name IL5 Project mix Operator AC MasterPlex QT Version 3 0 1 166 Analyst Analyte Name 034 Mouse IL 5 Background 0 00 Well Sample Name MFI Concentration Unit Count Al curve 20 9 30 pg mL 63 B1 2 1308 993 69 pg mL 95 C1 3 1084 5 500 28 pg mL 72 D1 4 525 240 45 pg mL 79 El 5 201 127 80 pg mL 81 FI 6 66 5 55 70 pg mL 80 EU 7 39 33 36 pg mL 78 H1 8 26 5 19 57 pg mL 63 A Ind mouse serum 367 19357 pg mL 6 B2 10 429 215 85 pg mL 66 C2 82 66 15 pgimL 79 D2
52. 88 5 70 26 pg mL 66 E 37 5 31 90 pg mL 82 F2 32 26 17 palmL 79 G2 18 4 41 pg mL 81 H2 19 7 11 pg mL 69 A3 90 10 buffer 506 5 243 01 pg mL 76 B3 480 5 233 95 pg mL 78 Ca 88 69 94 palmL 75 D3 HA 66 15 pg mL 76 ES 38 32 39 pg mL SCH F3 32 26 17 pg mL 56 a3 18 441 pg mL B8 H3 17 5 2 59 pg mL 80 Ad Sigma pooled 341 183 99 pg mL 80 B4 392 202 64 pg mL 79 GA 78 63 55 pg mL 70 D4 96 5 75 12 pg mL 74 E4 af 31 41 pg mL 96 F4 34 28 33 pg mL TA G4 23 14 58 pg mL 69 H4 2 20 22 pg mL 42 Figure 8 7 Report by analyte Data are organized by analyte name MasterPlex QT www miraibio com 144 CHA PT ERG MASTERPLEX QT REPORTS ort Per Sample Well Hun Date 3 20 01 Report Date 3 8 2007 Report Time 12 26 05 AM Data File ILS csv Plate Mame ILS Project mix MasterPlex OT Version 3 0 1 166 Hun Time 12 45 55 PM Hardware Serial No LX10000280002 Operator AC Analyst Analyte Name Sample Name MFI Concentration BKG Unit Count 034 Mouse IL 5 curve 20 3 30 0 00 pgimL 63 038 Mouse IL 10 curve 13 15 62 0 00 pa mL 138 054 Mouse IL Z curve 2B 15 62 0 00 pgimL 238 073 Mouse IFN Le curve 102 lt 15 62 0 00 Pom 103 077 Mouse IL 4 curve 63 13 22 0 00 pa mL 112 Analyte Name Sample Name MFl Concentration BKG Unit Count 034 Mouse IL 5 2 1308 308 69 0 00 Deum 55 038 Mouse IL 10 2475 5 1000 00 0 00 pa mL 120 054 Mouse IL 2 2 7355 1000 78 0 00 pone 52 073 Mouse IFN gamma 2 3141 5 1052 74 0 00 pam 128 O77 Mouse IL 4 d TIE 300 7
53. 9 ATTE Bead Set Properties gt D MFI v E Wir Bead Set Properties Analytes E mr v E 000 1 2 Analytes Siz n en Lem L re L JB Untitled Plate 7 Virtual Piste Virtual plate NE Bead Set tere E i bails Anales ids o e e m Sg A men EE NEUE ee eee Figure 6 1 Add ser selected data from actual plates csv xls pp or r mix to a virtual plate In a virtual plate you can perform a regression analysis on user selected data to generate a Dose Response curve and determine the LogEC50 value MasterPlex QT www miraibio com 81 CHA PTER6 VIRTUAL PLATES 6 1 Creating a Virtual Plate 1 Open the Luminex results files csv xls or Ixd or MasterPlex QT files mlx that are the data sources for the virtual plate 2 If the Plate Wizard is not open click the Plate Wizard button g The Plate Wizard appears Figure 6 2 NOTE If the Display wizard at start up option is chosen the Plate Wizard is displayed when MasterPlex QT starts Plate Wizard Welcome Welcome ta MasterPlex OT plate wizard This wizard wall quide you through creating and configuring a new plate Please click Next to begin creating and configuring a new plate Display wizard at start up ES cance Figure 6 2 Plate Wizard Welcome tab 3 Click Next gt The
54. 9 0 00 pg ml CR Analyte Name Sample Name MEI Concentration BKG Unit Count 024 Mouse IL 3 3 1084 5 500 25 0 00 pa mL D O33 Mouse IL 10 3 A242 200 01 0 00 Poin 123 054 Mouse IL 2 3 3706 5 499 82 0 00 pa mL 60 073 Mouse IFN gamma 3 2515 470 38 0 00 palm 115 077 Mouse IL 4 3 2 72 300 20 0 00 pa mL 128 Analyte Name Sample Name MFI Concentration BKG Unit Count 034 Mouse IL 5 4 ve 243 43 0 00 Deum 79 O38 Mouse IL 10 4 132 249 97 0 00 pg mil 123 054 Mouse IL 2 2445 230 11 0 00 pone Ti 073 Mouse IFN gamma 4 1707 5 239 85 0 00 py mL 136 077 Mouse IL 4 4 2380 248 55 0 00 pa mL 83 cre EIERE EE Figure 8 8 Report per sample well Data are organized by well location MasterPlex QT www miraibio com 145 CHA PT ERG MASTERPLEX QT REPORTS Report Date 3 8 2007 Run Date 3 20 01 Report Time 12 28 27 AM Run Time 12 45 39 PM Data File ILS3 csw Hardware Serial Mo LX10000280002 Plate Name IL5 Project mix Operator AC MasterPlex OT Version 3 0 1 166 Analyst Analyte 034 Mouse IL 5 Tx OU Ces 450 50 SEO 10564 sn Eon SS DO Tec EA OD EE vu B EE ED zu on ATE zem Oi 3700 EE En xz on az on 00 KZ 15 001 IS CO za on 26 ED 1810001 17 50 2700 Analyte 038 Mouse IL 10 T1050 105 010 Ec on EXT ED Tee Pai UK HK u 161 060 ES DU 105 mn 185 00 124 001 1332 OU 110 001 161 00 126 001 107001 zn oc 40 00 zE DO 4 00 13 00 12 00 1200 2100 1200 1200 14 001 Analyte 054 Mouse IL 2 1550050 1712350 L Tess n 1617 50
55. ALYTE CONCENTRATION Details Dilution direction Click an arrow to choose a dilution direction This gradient map specifies a separate dilution gradient in each column of the standard data set The starting concentration is at the top of a column Details Dilution direction This gradient map specifies one dilution gradient per standard data set The starting concentration is at the upper left well and the end concentration is at the lower right well Click an arrow to choose a dilution direction Figure 5 3 Example dilution gradient maps Click a dilution direction arrow to choose the dilution gradient configuration for a standard data set 8 If you want to specify the same starting concentration dilution factor and concentration units for all analytes in the standard data set choose the Fill in for all bead sets option If you want to specify a different starting concentration dilution factor or concentration unit for a different analyte repeat step 2 through step 4 9 Click Fill when finished entering the concentration dilution and dilution direction for all analytes in the standard data set MasterPlex QT www miraibio com 63 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION A confirmation message appears Figure 5 4 SUCCESS Automatically filled standard values for all analytes l Figure 5 4 Message box confirming autofill 10 Select Standard Independent Values from the data type drop d
56. ASTERPLEX QT REPORTS MasterPlex QT Report Per Standard Replicates Report Date 2 8 2007 Run Date 3 20 01 Report Time 12 33 33 AM Run Time 12 45 35 PM Data File ILS cs Hardware Serial No LX 10000280002 Plate Name ILS Project mix Operator AC MasterPlex OT Version 3 0 1 166 Analyst Well Sample Name MFI Concentration Unit Bi 2 1258 Ab RI ma mil Mean HA HA Std Dew HA HA CN NA HA Replicates Set 1 Well Sample Mame MFI Concentration Unit Ci 3 1054 3002 7725 pa mL Mean HA HA Std Dew HA HA CMS ALA NA Replicates Set 2 Well Sample Name MFI Concentration Unit Di 4 205 2454 4558 Gem Mezan 205 0000 2454 4558 Std Dew 505 0000 2454 4558 CNW 100 0000 100 0000 Replicates Set 3 Well Sample Name MFI Concentration Unit ce Taga fi Ia Perez ox Figure 8 12 Report per standard replicates This partial view of the report shows data for the first three IL 10 standard replicates MasterPlex QT www miraibio com 149 CHA PTER8 MASTERPLEX QT REPORTS MasterPlex QT Report Per Standard Data Report Date 2 8 2007 Run Date 3 20 01 Report Time 12 37 16 AM Run Time 12 45 52 PM Date File ILS csw Hardware Serial No LX10000 Plate Name ILS Project mix Operator AC MasterPlex OT Version 3 0 1 166 Analyst Well Sample Hame MFI Calculzte Expected Unit Residuals SRecorery Hi 3 i 195 7183 pom E 123 280 ai 7 13 000 3 3125000 pom 21 1156 108 757 F1 a 6 5000 625 0000 pom 53 00024 33 120 El u 181 0000 1230 0
57. Al IL 6 Click here to assign d Al IL 8 Click here to assign d A TNF alpha Click here to assign 1 d 3 4 KI 6 Fi 8 9 Int a Add all as new Save this assignment Use last saved assignment SC Check All Se Apply Template age DE 3 amp Cancel Figure 6 8 Virtual analyte filter shows the analytes in the virtual pipette Selecting and Renaming Analytes If the virtual analyte filter appears you must select and 1f necessary rename the analytes to match the number and names of the analytes in the virtual plate 1 In the virtual analyte filter Figure 6 9 place a check mark next to each analyte that you want to add to the virtual plate To select all analytes for the virtual plate click Check All 2 To rename an analyte so that it is consistent with the nomenclature in the virtual plate a Click here to assign next to the analyte that you want to rename A drop down list shows the names of the analytes in the virtual plate Figure 6 9 b Select a name from the drop down list gt The virtual analyte filter displays the new name for the analyte MasterPlex QT www miraibio com 89 CHA PTER6 VIRTUAL PLATES Virtual Analyte Filter Click on the link for a pull dawn menu No New Analytes Assign Aliases 1 ILl AkEM CSF Click here to assign i 2 B IFN gamma Click here to assign List of analytes in the virtual i 3 ER Al il 1 Beta Click here to assign pipette i 4 H L t Clic
58. Alternatively click the Group button Gi or select Template gt Group Wells from the menu bar 5 If the agent was serially diluted follow these steps to enter the agent concentration using the auto fill function a Choose Standard Independent Values from the data type dropdown list b Right click the regression data set and select Auto Fill from the pop up menu that appears Alternatively click the Auto Fill button EH The Auto Fill dialog box appears Figure 6 11 MasterPlex QT www miraibio com 93 CHA PT ER6 VIRTUAL PLATES Auto Fill Dilution parameters Starting Dilution Factor Fill in for all bead Figure 6 11 Auto Fill dialog box Use the auto fill function to set concentrations for a serially diluted agent c Enter the starting concentration for the analyte displayed in the Analyte drop down list d Enter the dilution factor e If the starting concentration and dilution factor are the same for all analytes choose the Fill in for all bead sets option If not make another selection from the Analyte drop down list and repeat step b and step c f Click Fill after the starting concentration and dilution factor are entered for all analytes 6 If the agent was not serially diluted follow these steps to manually enter the agent concentration for each analyte a Click the Edit mode button b Choose Standard Independent from the data type drop down list c Select an analyte in the Analyte panel d Cl
59. CH DAMAGES OR FOR ANY CLAIM BY ANY OTHER PARTY SOME STATES DO NOT ALLOW THE LIMITATION OR EXCLUSION OR LIABILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES TO THE ABOVE LIMITATION OR EXCLUSION MAY NOT APPLY TO YOU GOVERNMENT LICENSEE If you are acquiring the Software on behalf of any unit or agency of the United States Government the following provisions apply The Government acknowledges HISAL s representation that the Software and its documentation were developed at private expense and no part of them is in the public domain The Government acknowledges HISAL s representation that the Software is Restricted Computer Software as that term is defined in Clause 52 227 19 of the Federal Acquisition MasterPlex QT www miraibio com 111 LICENSE AGREEMENT Regulations FAR and is commercial Computer Software as that term is defined in Subpart 227 401 of the Department of Defense Federal Acquisition Regulations supplement DFARS The Government agrees that If the Software is supplied to the Department of Defense DOD the Software is classified as Commercial Computer Software and the Government is acquiring only restricted rights in the Software and its documentation will be as defined in Clause 52 227 19 c 2 of the FAR If the Software is supplied to any unit or agency of the United States Government other than DOD the Governments rights in Software and its documentation RESTRICTED RIGHTS LEGEND Use duplication or disclosure by
60. Displays a well grid using colors to denote the different well types Blue standard well green unknown well gray background well orange control well In the well grid displays a border around the wells in a group Displays a red border around wells that contain MFI data less than the user specified threshold Displays a red border around wells that contain MFI data less than the user specified concentration threshold Displays a red border around wells that contain count data less than the user specified count threshold Displays background subtracted data in the Plate window Displays the Print Settings dialog box Displays a print preview of the well grid for the selected analyte in the active plate window MasterPlex QT www miraibio com 169 APPENDIXB MASTERPLEX QT TOOLBARS B 4 Calculation Toolbar The calculation toolbar is available when a Plate window is open To show or hide the calculation toolbar select Show gt Show Hide gt Calculation Tools from the menu bar Figure B 4 Calculation toolbar Table B 4 Calculation toolbar buttons and functions Menu Bar Main Function Command Toolbar Button Calculation Model Equations Displays the Model Equations dialog box so that you can select a model for a standard or regression analysis data set Generates the standard curves for the selected standard data set using the selected model and interpolates analyte concentrations from the standard curves
61. ERM This Agreement is effective until terminated You may terminate it at any time by destroying the Software together with all copies in any form This Agreement will immediately and automatically terminate without notice if you fail to comply with any term or condition of this Agreement You agree upon termination to promptly destroy the Software together with all copies in any form LIMITED WARRANTY HISAL warrants for the period of ninety 90 days from the date of delivery of the Software to you as evidenced by a copy of your receipt that 1 The Software unless modified by you will perform the function described in the documentation provided by HISAL Your sole remedy under the warranty is that HISAL will undertake to correct within a reasonable period of time any marked Software Error failure of the Software to perform the functions described in the documentation HISAL does not warrant that the Software will meet your requirements that operation of the Software will be uninterrupted or error free or that all Software Errors will be corrected 2 The media on which the Software is furnished will be free from defects in materials and workmanship under normal use HISAL will at its option replace or refund the purchase price of the media at no charge to you provided you return the faulty media with proof of purchase to HISAL HISAL will not have any responsibility to replace or refund the purchase price of the media damaged by a
62. Id P 037 mir 213 Ol Z 043 mir 27a O F A 045 mir 324 5p O FA O47 mir 3 Vd O67 SsRNA Figure 6 16 Manual analyte selection NOTE If there are duplicated analyte names in the final virtual plate the tool will automatically rename them by adding n at the end of the name n indicates the number of duplicates Finally a report of the renaming process will be presented ina popup window MasterPlex QT www miraibio com 99 CHA PTER7 DATA CHARTS CHAPTER H MasterPlex GT displays MFI count or concentration data in several graph formats in the Data Chart window 7 1 Viewing a Data Chart 1 In the Plate window select one or more wells of interest To select adjacent wells press and hold the mouse button while you drag the mouse pointer to select the wells of interest To select nonadjacent wells press and hold the Ctrl key while you click the wells of interest 2 Click the Open Data Charts button fiij Alternatively select Plate gt Data Charts from the menu bar The Data Chart window opens and displays the well chart s default for the selected well s Figure 7 1 Select a data type for the chart from the drop down list n PESA 7 A Mouse IL 5 Ee v i zi 038 Mouse IL 10 EE v B D54 Mouse IL 2 E o Selected analytes in well A3 v d 73 Mouse IFN gamma i EN 505 5034 Mouse IL 5 WI M077 Mouse IL 4 ENNN 1 086 038 Mouse IL 10 30001 Se SS Rm 1 729 5 054 Mouse IL 2 MIA 16
63. Load button F gt The template is applied and the well grid shows the new well attributes well type well group and links to standard data sets Overwriting a Template You can overwrite an existing template with the current plate definition 1 In the Template Manager select the template that you want to overwrite 2 Click the Overwrite button A confirmation box appears Figure 4 19 4 Are you sure you want to overwrite selected template Y OK x Cancel Figure 4 19 Confirmation box 3 Click OK to overwrite the selected template with the current plate definition Exporting a Template You can export a template to a user specified location 1 In the Template Manager click the template that you want to export 2 Click the Export button 3 gt The Save As dialog box appears Figure 4 20 MasterPlex QT www miraibio com 48 CHA PTER4 DEFINING A PLATE Save in E examples Save as type Master Plex QT Template mxt Cancel Figure 4 20 Save As dialog box 3 Choose the directory for the template that you want to export 4 Enter a name for the template mxt NOTE A template must have a mxt file extension Changing the extension will render the exported template unusable Importing a Template You can import a template mxt from a user specified location Templates from MasterPlex QT 1 0 can also be imported 1 In the Template Manager click Import button The Open
64. Models Model Options 1 Boo ptirrizatiari Bnalutez O34 Mouse IL 5 qg C Use Weighting Select this model for all analytes FIFised lower asymptote at zero Equations U IT pression E gt Model Equation E d Polwnomials be Quadratic m Linear B e Sigmaldal fed Four Parameter Logistics CH Log Logit Rem Dose SSIES EOE Mens slop C Con Competitive Binding Mode E Assign Model Set As Default H Exit Figure 5 7 Model Equations dialog box Model equations available for regression analysis of a standard data set MasterPlex QT www miraibio com 66 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION 4 Select a model equation 5 To apply the selected model to all analytes choose the Select this model for all analytes option 6 To fix the bottom asymptote of the four parameter logistics curve or the five parameter logistics curve to zero sets A 0 choose the Fix bottom at zero option 7 To apply weighting during curve fitting choose the Use Weighting option and select a weighting method from the drop down list 8 Click Assign Model 9 To choose a model equation for another analyte repeat step 1 to step 3 and click Assign Model NOTE For more information about model equations and weighting methods see Appendix C 5 2 Working With Diluted Unknowns If you need to dilute a sample prior to an assay you can specify a dilution factor in the well g
65. RPLEX QT REPORTS 3 To generate report for absolute quantification analysis choose a report category and the Absolute Quantification option To generate a report for relative quantification analysis choose the report categories of interest and the Relative Quantification option Table 8 1 and Table 8 2 lists the available types of reports Table 8 1 MasterPlex QT reports for Absolute Quantification analysis Report Name Report per analytes Report per sample well Plate format report Report per unknown replicates Report per control replicates Report per background replicates Report per standard replicates Report per standard data Standard curves report Tabular MFI report Tabular concentration report Well map report Contents MFI bead count and concentration data organized by analyte name Figure 8 7 on page 8 8 MFI bead count and concentration data organized by sample well Figure 8 8 on page 8 9 Organized by data types MFI bead count or concentration that are displayed in the well grid format Figure 8 9 on page 8 10 Considers each group of unknown wells a replicate Includes the mean standard deviation and CV for each unknown group Considers each group of control wells a replicate Includes the mean standard deviation and CV for each control group Includes the mean standard deviation and CV for the background wells Considers each group of standard wells a replicate Includes the mean
66. RTS MasterPlex QT Well Map Report Report Date 2 8 2007 Run Date 3 20 01 Report Time 12 40 31 AM Run Time 12 45 39 PM Data File ILS csw Hardware Serial Mo LX10000280002 Plate Name ILS Project mix Operator AC MasterPlex OT Version 3 0 1 166 Analwst ESE EEN Uunk 1 Unk 1 Urit 1 Sidi Uni 1 Link 1 Unk 1 Sati Une 1 Uni 1 Unk 1 Sid Une 1 Une 1 Une 1 Sidi Link 1 Limk 1 Unk 1 Sidi Uni 1 uni 1 Unk 1 Sati Une 1 Link 1 Unk 1 Sidi Uni 1 Link 1 Une 1 Daer Paga 1 z 8 ra m s Figure 8 17 Well map report MasterPlex QT www miraibio com 154 CHA PTER8 MASTERPLEX QT REPORTS 8 3 Custom Report Custom report is a powerful and flexible tool for presenting and exporting your data Compared to regular report custom report has great flexibility on what and how to present data As QT stores its analysis results in the format of XML document it is possible for users to present their data in whatever format they want The only thing users need to do is to define their presenting formats in XSL files Extensible Stylesheet Language QT will apply the stylesheets to transform and present data with certain format 1 Start Report Generator Choose Custom report as report type 2 Click View button to bring out Style Sheet Manager Figure 8 18 Custom Report 1 Lal View T d Please Select View from the toolbar to generate a custom report Figure 8 18 Custom report viewer 3 Click Import t
67. USER MANUAL Version 3 0 for Microsoft Windows MasterPlex QT Multiplex Data Analysis Software MiraiBio A Group of Hitachi Software For Research Use Only 601 Gateway Blvd Suite 100 South San Francisco CA 94080 TELEPHONE 1 888 615 9600 toll free 1 650 615 7600 FACSIMILE 1 650 615 7639 Part no P 33020 10200 TRADEMARKS MicroSoft is a registered trademark of Microsoft Corporation COPYRIGHT O 2001 2007 Hitachi Software Engineering America Ltd All Rights Reserved MasterPlex QT www miraibio com LICENSE AGREEMENT LICENSE AGREEMENT BEFORE OPENING THIS PACKAGE YOU SHOULD CAREFULLY READ THE FOLLOWING TERMS AND CONDITIONS BY OPENING THIS PACKAGE YOU AGREE TO BECOME BOUND BY THE TERMS AND CONDITIONS OF THIS AGREEMENT WHICH INCLUDES THE SOFTWARE LICENSE AND LIMITED WARRANTY IF YOU DO NOT AGREE WITH THESE TERMS AND CONDITIONS YOU SHOULD PROMPTLY RETURN THE PACKAGE UNOPENED TO HITACHI SOFTWARE ENGINEERING AMERICA LTD HISAL or HISAL Distributor AND YOUR MONEY WILL BE REFUNDED The enclosed software is licensed not sold to you for use only upon the terms of this Agreement and HISAL reserves any rights not expressly granted to you You are responsible for the selection of the Software to achieve your intended results and for the installation use and results obtained from the Software You own the media on which the Software is originally or subsequently recorded or fixed but HISAL retains ownership of all
68. Use this dialog bos ta set various pre ferences related to MasterPlex HT Enable plate wizard at start up Enable Relative Euantification Gene Expression Default ay Ok a Cancel Apply Figure 4 23 Preferences dialog box Application tab default settings Application Preferences The application preferences Figure 4 23 include Default Virtual Plate Dimensions Automatic Well Grouping Enable plate wizard at startup The default number of rows and columns for a virtual plate displayed by the plate wizard during virtual plate setup Choose this option to automatically group wells after they are defined Choose this option to display the plate wizard at startup MasterPlex QT www miraibio com 51 CHA PTER4 DEFINING A PLATE 1 To set the row and column dimensions of the well grid for a virtual plate displayed by the plate wizard enter the number of rows and columns 2 If you do not want to automatically group wells after they are defined remove the check mark from the Automatic Well Grouping option 3 If you do not want to display the plate wizard at program start up remove the check mark from the Enable plate wizard at start up option 4 Click Apply when you are finished 5 To return the application preferences to the factory set defaults click Default Plate Tab The Plate preferences Figure 4 24 specity how to compute background and when to subtract background threshold values fo
69. a a 400 600 600 1000 1 200 Concentrations pg mL Parent IL5 Project mix Figure 5 16 Standard curves window Standard Curve Chart tab 2 To display a standard curve click the curve s of interest in the data tree To select multiple curves press and hold the Ctrl key while you the click the curves E NOTE If the Standard Curves window shows data points only you need to generate the standard curves click the Calculate button a Viewing the Residual Plot The residual plot helps you identify outliers and detect non constant variability in a standard curve In a residual plot each point represents the difference between the expected standard concentration and the calculated standard concentration for a selected analyte Residual Observed or calculated concentration Expected concentration The observed calculated concentration is interpolated or extrapolated from the MasterPlex QT www miraibio com 76 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION standard curve The expected concentration is the standard data value that you enter using the autofill function or manually If the residual is positive we have detected a concentration that 1s greater than the expected value If the residual is negative the detected concentration 1s less than the expected value 1 To view the residual plot click the Residual Plot tab in the Standard Curves window 2 Click the standard curve of interest gt The
70. a well and select Mouse Tip Styles gt Y Values from the pop up menu that appears Figure 7 19 gt The tool tip displays the MFI concentration bead count value for the well depending on the data type selected in the analyte panel 2 To change the tool tip display right click a well and select Mouse Tip Styles gt Y Values from the pop up menu that appears Figure 7 19 gt The tool tip displays the MFI concentration bead count value for the well depending on the data type selected coer 034 Mouse IL 5 M038 Mouse IL 10 88 054 Mouse IL 2 E M D73 Mouse IFN damma 17 or Mouse IL 4 JE Selected analytes in well C2 WO GC 034 Mouse IL 5 ee id ENNN 105 5 038 Mouse IL 10 a nt T E i ie ENNN 106 054 Mouse IL 2 i i MIA 292073 Mouse IFN gamma 400 077 Mouse IL 4 ed ee zm zm vs db e e e rm ps rr rs A wl zm e zs e rm e zm zm e e e zm hemm 077 Mouse IL 4 034 Mouse IL 5 054 Mouse IL 2 077 Mouse IL 4 Selected Analytes Parent Plate H D Project mix Figure 7 18 Multi well chart Click a bar to display a tool tip MasterPlex QT www miraibio com 118 CHA PTER7 DATA CHARTS Select the data type for the chart y axis MFI bead count or concentration d r 98 2 E M034 Mouse IL 5 jii ET 488 038 Mouse IL 10 a E Me054 Mouse IL 2 NE A073 Mouse IFN aamma M077 Mouse IL 4 D Kaf ENNN 520234 Mouse IL 5
71. acts background MFI from unknown MFI then interpolates or extrapolates the unknown analyte concentration from the standard curve This method of computing background subtracted analyte concentrations subtracts the background MFI from each member of the standard data set then fits the standard curve The method does not subtract the background MFI from the unknown MFI before interpolating or extrapolating the unknown analyte concentration Eje NOTE The All Calculations method is recommended The Regressions Only 1 0 2 0 method provides backward compatibility with data generated in MasterPlex QT MasterPlex QT www miraibio com 53 CHA PTER4 DEFINING A PLATE Background Type If there are three or more background wells in the assay choose one of the following methods for computing background MFI Average Background Bkg MFI Bkg MFI Bkg MFI Bkg MFT n Peak Value where n the number of background wells in the plate Highest background MFI value Lowest Value Lowest background MFI value Plate Info Plate Name Displays the name assigned to the result file in the Luminex 100 200 software To edit the plate name enter a new name Analyst Name Displays the analyst name entered in the Luminex 100 200 software To edit the analyst name enter a new name Thresholds You can enter an MFI count or concentration threshold for a plate The software automatically marks wells that contain data less than th
72. alette Choose a color for the Background display area behind the chart Color Log scale on X axis Choose this option to display a log scale on the x axis for a standard curve only NOTE The Gradient option overrides the Background option MasterPlex QT www miraibio com 136 CHA PTER8 MASTERPLEX QT REPORTS CHAPTER 8 This chapter explains the types of reports that are available and how to use the report generator You can preview print or save a report 8 1 Generating a Report 1 Open the plate csv xls Ixd or mlx for the report oF NOTE The report generator is only available if a plate window is open and active Mm 2 Click the Report Generator button gt The report generator appears Figure 8 1 Report Generator Report Type Absolute Quantification Reports Absolute Quantification U 2 Report per analytes C2 Relative Quantification trs ERU C5 Report per sample well 5 Plate format report Report per unknown replicates Report per control replicates Vertical C5 Report per background replicates C2 Horizontal C Report per standard replicates Reports CO Report per standard data Report per analytes organizes Standard curves report plate data by analytes Well data is grouped under each Tabular MFI report analytes C5 Tabular Concentration report CO Well map report Generate Figure 8 1 Report generator MasterPlex QT www miraibio com 137 CHA PTER8 MASTE
73. almost never know what the weight should be at each point so it must be estimated One of the most widely used estimation methods 1s based on a working hypothesis that variance variability depends on the square of the mean the y value This implies that the standard deviation of a detected MFI value for a particular concentration 1s directly proportional to the expected MasterPlex QT www miraibio com 178 APPENDIXC MODEL EQUATIONS concentration In other words standard deviation is proportional to the mean G u o Some Constant u o u Some Constant where o standard deviation and u mean Since standard deviation mean 1s the coefficient of variation CV we can now expect CV to be constant for all points In this scheme weight W can be estimated using the following expressions W 1 Variance W 1 0 where weight is the inverse of the variance and variance standard deviation Since we assume O Lt W 1 Y Note that u Mean F x Y where W is the estimated weight and Y F x and F x is the mean function which is represented by the equation of the model In this scheme we can think of minimizing the residuals based on their relative MFI values This method is appropriate when higher MFI values have larger residuals than lower MFI values The wedge shape pattern of data on a residual plot usually confirms this Weighting by 1 Y Another weighting scheme is 1 Y This method is useful when the MFI
74. ange of the four parameter logistics model It is not possible to mathematically extrapolate values that are equal to or beyond the asymptote values For example imagine the function F x Log x The vertical asymptote for this function is the line x 0 This means the function can never reach the line where x 0 Try calculating Log 0 to see what happens In Figure C 1 B is the slope at the inflection point It is the speed of the function as it moves away from the inflection point C is the most interesting parameter since it corresponds to the x value that is associated with the y value at the midpoint between the minimum and maximum limits of the function In a biochemical assay C corresponds to the concentration since it 1s on the x axis that produces a 50 response In drug discovery terms it is also known as ED50 the 50 effective dosage It can also be interpreted as LD50 if this dosage kills 50 of the sample population C 2 Five Parameter Logistic Curve Five Parameter Logistics Std Curwe 1 R Square 0 9999 A 034 house IL 5 a 2 878 b 2 308 c 4333 921 d 1318 477 2 0 434 1 300 1 200 1 100 l E a y E l B 1 wes em em rm rm rm mm rm rm rs e e rm rm em rm rm re em e rm rm rm rm vm rem zm mm vm an Andes AS drum c N EERE E rri el E Mam ET o CEA E I trm fa Zt ns 2 S bi velt rm fa WALL wf ISA D tem Mi KIM si Median Fluroscense Intensity 100 1 000 10 000 Concentrations
75. ays a list of the standard curves imported from the source plate Figure 5 19 MasterPlex QT www miraibio com 79 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION Destination plate mm g moa mol n b X Click the Local tab to display a list of standards that were defined in the plate Click the Imports tab to display a list of standards that were imported to the plate Figure 5 19 Standard curves can be exported Local and imported standard curves are listed in the Standards tab of the destination Plate window NOTE To view and conveniently compare standard curves open the Standard Curves window click the Ee d button For more information see Opening the Standard Curves Window on page 5 16 MasterPlex QT www miraibio com 80 CHA PT ER6 VIRTUAL PLATES CHAPTER 6 A virtual plate is a software simulation of an emptv microtiter plate User selected unknown and standard data from one or more actual plates csv e Ixd or mlx can be added to a virtual plate In a virtual plate you can Compare and analyze samples from user selected actual plates csv xls Ixd or mlx Specify custom row and column dimensions to increase analysis throughput Generate a Dose Response curve for user selected data Actual plates csv xls Ixd or mlx Untitled Plate 2 lt IL5 csv gt Sele Untitled Plate 3 lt H10Plex csv gt EIE 77 Big Sui Usb Gri fum Ba Tu Cr mmm ZC 79
76. between us which supersedes any proposal or prior agreement oral or written and any other communications between us in relation to the subject matter of this Agreement MasterPlex QT www miraibio com V MuiraiBio MasterPlex QT 3 0 Analysis software for multiplex data from the Luminex 100 200 system CONTENTS CHAPTER 1 PAGE Welcome About This Manual sssseessesnsnnenazennenzzznnnnnzznnnnnnznz 1 Technical Support HH 9 CHAPTER 2 Installing MasterPlex QT Requirements BEE 3 Installing MasterPlex OT HH 4 Installing a License mA 8 CHAPTER 3 Getting Started Overview of MasterPlex QT Analysis si 9 Starting MasterPlex QT e 10 Importing Luminex Results HA 11 Using Windows Explorer to Import csv xls Ixd or Open D E NETTES DIST 15 Viewing Data in the Plate Window se 16 Re e E 21 The Plate Navigator EE 23 Saving Plate Data sas ee 28 CHAPTER 4 Defining a Plate Designating Well Type and Group sen 30 MasterPlex QT www miraibio com Working With Templates A ies A5 Setting Prei rejfi e ica 50 Saving a Plate eene 58 CHAPTER 5 Standard Curves amp Analyte Concentration Specifying Standard Data MM 60 Working With Diluted Unknowns sen 67 Generating Standard Curves amp Computing Analyte C ONCENITAHONS nani 70 Printing the Well Grid eee 74 Working With Standard Curves A 75 Importing Standard Curves HM 79 CHAPTER 6 Virtual Plates Creating a Virtual Plate sisas NI Working With the Virtual Ana
77. cal axis labels to the left right or left and right of the chart Show in Legend Choose this option to display the series name in the chart legend Color Each Point Choose this option to display each bar in the series in a different color Sort Make a selection from the drop down list to choose a descending or ascending sort order for the series Note After a series is sorted it cannot be unsorted To display the series prior to sorting simply close the chart then regenerate it Bar Settings MasterPlex QT www miraibio com 130 CHA PTER7 DATA CHARTS Chart Properties Charts 3D Legends Walls Series Panel Series for 42 Ww Apply to all series Marks General Bars Multi Bars Appearance 9 None Bar Style Rectangle w Side O Stacked 100 Side All Bar Dark 30 E Exit Figure 7 30 Chart Properties dialog box Series tab Bar settings Multi Bars None Choose this option to display the bars in the default orientation For example Figure 7 31 shows the default orientation of the 3D depth chart Side Choose this option to display the bars side by side along the X axis Figure 7 31 Stacked Choose this option to combine the separate bars of a series into one vertical column Figure 7 32 Stacked 100 Choose this option to display the percentage data for a series in one vertical bar Each segment of the bar represents the percent a particular series value contributes to the total bar
78. ccident abuse or misapplication THE ABOVE WARRANTIES ARE EXCLUSIVE AND IN LIEU OF ALL OTHER WARRANTIES WHETHER EXPRESS OR IMPLIED INCLUDING THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR MasterPlex QT www miraibio com 11 LICENSE AGREEMENT PURPOSE NO ORAL OR WRITTEN INFORMATION OR ADVICE GIVEN BY HISAL ITS EMPLOYEES DISTRIBUTORS OR AGENTS SHALL INCREASE THE SCOPE OF THE ABOVE WARRANTIES OR CREATE ANY NEW WARRANTIES SOME STATES DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES SO THE ABOVE EXCLUSION MAY NOT APPLY TO YOU IN THAT EVENT ANY IMPLIED WARRANTIES ARE LIMITED IN DURATION TO NINETY 90 DAYS FROM THE DATE OF DELIVERY OF THE SOFTWARE THIS WARRANTY GIVES YOU SPECIFIC LEGAL RIGHTS YOU MAY HAVE OTHER RIGHTS WHICH VARY FROM STATE TO STATE LIMITATIONS OF REMEDIES HISAL s entire liability to you and your exclusive remedy shall be the replacement of the Software media or the refund of your purchase price as set forth above If HISAL or the HISAL s distributors are unable to deliver replacement media which is free of defects in materials and workmanship you may terminate this Agreement by returning the Software and your money will be refunded REGARDLESS OF WHETHER ANY REMEDY SET FORTH HEREIN FAILS ITS ESSENTIAL PURPOSE IN NO EVENT WILL HISAL BE LIABLE TO YOU FOR ANY DAMAGES INCLUDING ANY LOST PROFITS LOST DATA OR OTHER INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OR INABILITY OF SU
79. cked option left Stacked 100 option right MasterPlex QT www miraibio com 132 CHA PTER7 DATA CHARTS nee P PS pum z l aa Tr mi mm m l owas amp t l l A m mm Pm um mm Go wen UU um um DL Mouse IL 57 r 077 Morse IL 4 Aral ez Figure 7 33 3D depth chart Side All option Intensity Map Settings MasterPlex QT www miraibio com 133 CHA PTER7 DATA CHARTS If an intensity map 1s open the Series tab shows Intensity settings Figure 7 34 Chart Properties Charts 3D Legends Walls Series for 2D Intensity C e Apply ta all series Marks General Intensity Map Color Scheme Peak Color Peak IN Mid Range Color Mid BEN Low Color Low MN Exit Figure 7 34 Chart Properties dialog box Series tab If an intensity map is open in the data chart window the intensity map color scheme settings are available To change a color peak mid range or low color in the intensity map 1 Click the button for the color that you want to change for example _ Pesk BIL gt The color palette appears Figure 7 35 l l Custom color field Luminosity scale Es ES P Custom colors Web Red 255 i l Sat Green Define Custom Colors gt gt Coloris
80. ckground value for the plate 3 To specify an outlier place a check mark next to the standard data point of interest Figure 5 18 MasterPlex QT www miraibio com 78 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION Alternatively you can select outliers in the Standard Curve Chart tab a Click the Standard Curve Chart tab b Click the Chart Selection Tool button then click a point in the standard curve that you want to make an outlier The Standard Data tab highlights the selected point c Place a check mark in the highlighted outlier check box 4 Recalculate the standard curve and analyte concentrations Click the Calculate button Mor select Calculations gt Calculate STD Curves from the menu bar 5 6 Importing Standard Curves You can import standard curves from another plate so that you can easily compare standards from different plates 1 Open the plate that contains the standard curves for export source plate 2 Open the plate that the standard curves will be imported to destination plate 3 Select the E Import standards menu from plate menu The plate windows are in import export mode NOTE The plate windows must be in import export mode to export standard curves from the destination plate to the source plate 4 Use the click and drag method to move the standard data set of interest from the source plate to the well grid of the destination plate gt The Standard Curves window displ
81. ction from the drop down list to set the direction of the gradient Start Mid End Click to open a color palette Choose a color for the start middle or end of the gradient Note If the No Middle option is chosen the Mid button is not displayed Series Tab A series is a group of related bars in a chart For example in the 3D depth chart the bars that represent the analytes in a particular well comprise a series Figure 7 27 In the series tab you can modify the display of a series 1 To view the series properties for an open chart s click the Series tab in the Chart Properties dialog box Figure 7 28 2 To modify the settings for a series make a selection from the Series drop down list If you want to apply the settings to all series in the graph choose the Apply to all series option MasterPlex QT www miraibio com 127 CHA DIER DATA CHARTS Series for well B2 Figure 7 27 3D depth chart In the 3D depth chart the bars that represent the analytes in a well are an example of a series Chart Properties Charte 3D Legends Walls Series Panel series dropdown list i C Apply ta all series Ghada Ma Middle visible Make Ande Figure 7 28 Chart Properties dialog box Series tab Marks settings MasterPlex QT www miraibio com 128 CHA PTER7 DATA CHARTS User Modifiable Series Settings Marks Settings These settings determine the display of the name labels marks 1n a data chart Figur
82. d Outlier Option Outlier Values AE e MFI Error Range s 3tdev CO Concentration Automatic Outlier Detected Default ay Ok ae Cancel Apply Figure 5 8 Preferences dialog box Calculations tab Editing a Dilution Factor 1 Click the Well Editor button Fi Alternatively select Plate gt Edit Wells from the menu bar c The Plate window is in edit mode ek NOTE You can only edit a dilution factor when the Plate window is in edit mode MasterPlex QT www miraibio com 68 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION 2 Select Dilution from the data type drop down list 3 In the well grid click the dilution factor s that you want to edit To select all of the dilution factors for a group right click a dilution factor and choose Select Entire Group from the pop up menu that appears 4 To edit a single selected dilution factor use the 7 arrows Alternatively double click the dilution factor in the well edit the value in the edit box and click Enter 5 To edit the dilution factor for several wells simultaneously a Click the Selection Tool button Y to put the plate in selection mode b Click the wells that you want to edit c Click the edit box and enter the dilution factor 1 Click Well Editor 2 Select Dilution 3 Click the dilution 4 Enter a new dilution factor in button to put the Plate from the data type factor s that you the edit box and click Enter Or window in edi
83. data chart toolbar is available if a Data Chart window is open MasterPlex QT www miraibio com 101 CHA PTER7 DATA CHARTS Table 7 1 MasterPlex QT data charts Displays See Page Separate bar graphs of analyte data for each 7 4 user selected well Bar graph of analyte data that includes all Data Chart Multi Well Chart 3D Depth Chart Inverted 3D Depth Chart Intensitv Map Single Analvte Chart Stacked Analvte Chart Stacked Well Chart Multi Well Chart selected wells Analytes from a particular well are organized by row Bar graph of analyte data that includes all selected wells Analytes from a particular well are organized by column A qualitative map of the intensity data for the analytes in one well The map colors indicate the relative intensity of an analyte compared to the other analytes in the same well Bar graph of a single analyte across all user 7 10 selected wells Bar graph of user selected wells Each color coded segment of a bar represents the contribution of an analyte to the total data value for the well Bar graph of analytes Each color coded segment of a bar represents the contribution from a well to the total data value for the analyte The multi well chart displays a bar graph of analyte data MFI count or concentration for a user selected well s Each bar represents an analyte The multi well chart is the default format that 1s displayed when you open the Data
84. dent on each another MasterPlex QT www miraibio com 57 CHA PTER4 DEFINING A PLATE Individual The replicate standard data points are not averaged and the points standard curve is fitted to all of the data points For example if there are three replicates of eight standard wells the standard curve is fitted using all 24 data points Choose this option if the experimental errors are independent of each other This option is set by default In most cases if you re not sure about the nature of the distribution of the experimental errors try this option first Dilution for Unknowns Samples can be diluted prior to the assay and analysis After MasterPlex QT interpolates the diluted unknown analyte concentrations from the standard curve it can compute and display the original undiluted concentration in the Plate window Original concentration Diluted concentration Dilution Factor The dilutions factors are manually entered in the Plate window For more information see Working With Diluted Unknowns on page 5 8 4 Click Apply when you are finished To return the plate preferences to the factory set defaults click Default 4 4 Saving a Plate The software saves the imported results csv xls or Ixd in MasterPlex QT native file format mlx The mlx file also includes the well and group definitions or the associated template well and bead set analyte color data calculated for the plate for example analyte conce
85. e 7 27 Gradient Visible Click to display a gradient background in the name label Direction Make a selection from the drop down list to set the direction of the gradient Start Mid End Click to open a color palette Choose a color for the start middle or end of the gradient No Middle Choose this option to display a two color gradient If this option 1s not chosen the gradient has three colors start middle and end color Visible Choose an option to display the name labels for the selected series Marks Angle Click the up or down arrows to set the display angle for the name labels Shadow Color Click to open a color palette Choose a color for the shadow of the name label Size Click the up or down arrows to set the thickness of the name label shadow General Settings MasterPlex QT www miraibio com 129 CHA PTER7 DATA CHARTS Chart Properties Charts 30 Legends Walle Seres Panel Series for z v Apply to all series Marks General Bars Series Color Show In Legend Horizontal Axis Color Each Point LE Exit Figure 7 29 Chart Properties dialog box Series tab General settings Series Color Click to open a color palette Choose a color for the selected series Horizontal Axis Make a selection from the drop down list to display the horizontal axis labels at the bottom top or bottom and top of the chart Vertical Axis Make a selection from the drop down list to display the verti
86. e a Dose Response curve Figure 6 10 For more information on Dose Response curves see Appendix D Dose Response Analysis on page D 1 The software determines the LogEC50 value from the Dose Response curve The logEC50 value is the log of the agent concentration for example a drug or other chemical that produces a response half way between the baseline and maximum response 1 Select concentration from the data type drop down list MasterPlex QT www miraibio com 91 CHA PT ER6 VIRTUAL PLATES Untitled Plate 2 lt IL5 csv gt L e gui Uk Cr ka Ba Tw Cl Gu d OY SS OX Bead Set Properties 34700 Analytes 1 amp 034 Mouse IL 5 Sele Untitled Plate 9 lt Virtual Plate gt LZ II e Sed Gk Cri ad Ba Ta Cui SE GE Bead Set Properties Standard Independent Values Y E 500000 Analytes 034MowelL 5 Pomo 2 Create a virtual plate 3 Transfer response data to the virtual plate using the virtual pipette 4 Mark and group wells into a regression data set 5 Select Standard Independent values from the data type drop down list and enter the agent concentrations use the Autofill function or enter manually Model Equations Available Madels mi 6 To select a Dose Response model right click the Arabes Uso weighing regression data set and select Assign Model Select this model for all analytes Fixed lower asymptote at zero
87. e user specified threshold with a red border Figure 4 18 To set a threshold s 1 Enter the MFI count or concentration threshold in the Preferences dialog box Figure 4 25 MasterPlex QT www miraibio com 54 CHA PTER4 DEFINING A PLATE Preferences Application Plate Regression Calculations Background Plate Info Plate Name IL5 Project mls Analyst Name pO Thresholds Subtract Background When to subtract Background Type S ds Concentration MFI 20 00 Bead Count GENN Sa TER Ie SKI before calculation Assay Ek k Prompt before recalculation Default ay Ok ae Cancel Apply Figure 4 25 Preferences dialog box Plate tab 2 Click Apply when you are finished 3 To return the plate preferences to the factory set defaults click Default To identify the wells that contain data less than threshold 1 Choose an analyte in the Bead Set panel 2 Select the data type MFI count or concentration from the datatype drop down list 3 To identify wells with a MEI less than threshold click the button b Bead count data less than threshold click the Li button c Concentration less than threshold click the button A red border marks wells that contain data less than threshold for the selected MasterPlex QT www miraibio com 55 CHA PTER4 DEFINING A PLATE analyte Figure 4 26 NOTE The threshold buttons Ca Flor are mutually exclusive Only one is active
88. ection This gradient map specifies one dilution gradient per standard data set The starting concentration is at the upper left well and the end concentration is at the lower right well Click an arrow to choose a dilution direction Figure 4 15 Example dilution gradient maps Click a dilution direction arrow to choose the dilution gradient configuration for a standard data set 7 To specify the same starting concentration dilution factor and concentration units for all analytes in the standard data set choose the Fill in for all bead sets option To specify a different starting concentration dilution factor or concentration unit for a different analyte repeat step 2 through step 4 8 Click Fill when finished entering the concentration dilution and dilution direction for all analytes in the standard data set gt A confirmation message appears Figure 4 16 MasterPlex QT www miraibio com 44 CHA PTER4 DEFINING A PLATE Success Automaticalh filled standard values for all analytes Figure 4 16 Message box confirming autofill Linking a Standard Data Set Background control and unknown wells must be associated with or linked to the standard data set that will be used to calculate concentrations By default the first standard that you define will be linked to the background control and unknown well groups If there 1s more than one standard on a plate you can link a user selected standard to a user selected
89. ed i Parent Plate IL5 Project mlx Figure 7 9 Stacked analytes chart Click a bar to display a tool tip analyte name or y axis data Right click a bar for tool tip options MasterPlex QT www miraibio com 110 CHA PTER7 DATA CHARTS Stacked Well Chart Each bar in the stacked well chart represents the sum total of the MFI count or concentration values of an analyte across the user selected wells The color coded segments of a bar show the contribution of each well to the total value Select the data type for the chart a de A IE WI di 0134 Mouse IL 5 ii X Stacked wells for all selected analvtes V 098 Mouse IL 10 ie in 7 LOSA Mouse IL 2 E Fr Mouse IEN aamma La M077 Mouse IL 4 A D3 Place a check mark next to the analytes to include in the chart Stacked MF I 034 Mouse IL 5 054 Mouse IL 2 077 Mouse IL 4 Selected Analytes Stacked Well Chart selected Parent Plate IL5 Project mlx Figure 7 10 Stacked well chart Click a bar to display a tool tip analyte name or y axis data Right click a bar for tool tip options MasterPlex QT www miraibio com 111 7 3 CHA PTER7 DATA CHARTS Working With Data Charts Table 7 2 explains the toolbar button functions in the Data Chart window The available buttons depend on the type of data chart that 1s open Table 7 2 Toolbar buttons in the Data Chart window Button Click to Choose another data chart format The software plots the
90. ed until you dispense or clear the pipette NOTE The data source plates must contain the same type and number of analytes otherwise concentrations cannot be calculated If the source plates contain the same number of analytes but they are named differently use the virtual analyte filter to rename analytes so that the nomenclature Is consistent See Selecting and Renaming Analytes on page 6 9 1 Click the Virtual Pipette button A gt The virtual pipette appears when the mouse cursor is positioned over a plate window MasterPlex QT www miraibio com 84 CHA PT ER 6 VIRTUAL PLATES 2 In the source plate select the wells of interest To select adjacent wells press and hold the mouse button while vou drag the mouse pointer to select the wells of interest a NOTE Selecting non adjacent wells is not recommended 3 Right click the selected wells and select Aspirate from the pop up menu that appears Figure 6 6 gt The data for the analytes in the selected wells are added to the virtual pipette and is ready to dispense into a virtual plate e NOTE If the background is subtracted in the source plate the virtual pipette aspirates and transfers background subtracted values If you do not want to aspirate background subtracted values make sure the background subtraction is turned off before you aspirate data into the virtual pipette Click the S button to turn background subtraction on or off 1 2 3 4 A 0 00 347
91. el Figure 7 16 Example of data series for well A3 saved to a text file MasterPlex QT www miraibio com 116 CHA PTER7 DATA CHARTS Printing a Chart mum I 1 Click the Print current chart button iz The Chart Print Preview window opens Figure 7 17 Chart Print Preview Printer XsfdcOTXSF MAIN HPLJEW Orientation Portrait 2 Landscape Marginz Y ia mo S ms o e Reset Ma Mew Marginz Detail More Hor mal J Proportional Figure 7 17 Chart Print Preview window 3 To print the chart click Print Rotating or Magnifying a Chart 1 To rotate the chart around the x or y axis a Click the Chart rotate tool button gf b Press and hold the mouse button while you move the mouse pointer to rotate the graph 2 To zoom in on a chart area a Click the Chart zoom button 7 MasterPlex QT www miraibio com 117 CHA PTER7 DATA CHARTS b Press and hold the mouse button while you draw a rectangle from the upper left to lower right corner over the graph area that you want to magnify 3 To zoom out and display a chart without magnification press and hold the mouse button while you draw a rectangle from the lower right to upper left corner over the graph Displaying Chart Tool Tips 1 To show a tool tip that displays information in a bar chart a Click the bar of interest gt A tool tip displays the analyte name b To change the tool tip display right click
92. el 1 650 615 7600 Toll Free 1 888 615 9600 Fax 1 650 615 7639 E mail support miraibio com www miraibio com MasterPlex QT www miraibio com 2 CHAPTER2 INSTALLING MASTERPLEX QT CHAPTER 2 This chapter explains the minimum hardware and software requirements needed to install and use MasterPlex QT 3 0 It provides installation instructions for a computer connected to the Luminex 100 200 system 2 1 Requirements For optimum performance MasterPlex QT requires hardware and software that meet or exceed the following specifications It is also strongly recommended that you use the Luminex XY platform Minimum Hardware Requirements Platform PC CPU Intel Pentium A 1 5 GHz or equivalent Intel Pentium4 2 GHz or better recommended Memory RAM 512MB or higher for Windows 98 ME 2000 XP Storage space HDD 30 MB available space for the installation Input devices Keyboard and mouse or any other pointing device Video RAM 16MB or higher Monitor resolution XGA 1024x768 pixels or higher 1280 x1024 recommended Monitor color 16 bit color high color or higher CD ROM drive Required for CD media version Not applicable for download version Software Requirements Operating system Microsoft Windows 98 98SE Me NT4 0 SP6 2000 XP Windows 2000 or XP recommended MasterPlex QT www miraibio com 3 CHAPTER2 INSTALLING MASTERPLEX QT 2 2 Installing MasterPlex QT 1 Insert the MasterPlex QT CD ROM in the workstation c
93. ell Select the data type for the chart vi OA Mouse IL 5 M038 Mouse IL 10 Al 054 Mouse IL 2 073 Mouse IFA aamma 077 Mouse IL 4 Place a check mark next to the analytes to include in the chart CH D3 Selected Wells Single Analyte Chart selected Parent Plate IL5 Project mlx Figure 7 8 Single analyte chart Each bar represents data for a single analyte from one well Click a bar to display a tool tip analyte name or y axis data Right click a bar for tool tip options MasterPlex QT www miraibio com 109 CHA PTER7 DATA CHARTS Stacked Analyte Chart Each bar represents a single well The color coded segments of a bar represent the contribution of each analyte to the total MFI count or concentration value in the well Figure 7 9 To include all analytes in the chart click the Select All Analytes button ES s E H BES 7 M034 Mouse IL 5 O36 Mouse IL 10 Select the data type for the chart Stacked Analytes in all selected wells 054 Mouse IL 2 NAS Mouse IEN aamma M Boor Mouse IL 4 MN 034 Mouse IL 5 EN 038 Mouse IL 10 EN 054 Mouse IL 2 EN 073 Mouse IFN gamma 077 Mouse IL 4 IVANA LI CORR RARA To include user specified analytes in the chart place a check mark next to the analytes of interest Stacked MF I ER B3 C3 D3 EA F3 selected Wells uii nui nmi rnm n nm nnn nmn A nenna Stacked Analyte Chart select
94. enerated for the Figure 3 20 current plate Untitled Plate 2 IL 5 csv Bu Sed Uk CH fug B r Cii EE YR OK Bead Set Properties Standards 2000 Enter 1 2 3 4 5 NEM amp jy Local Standards A 034 Mouse IL 5 A 038 Mouse IL 10 e 054 Mouse IL 2 He OF Mouse IEN gamma La 077 Mouse IL 4 Imports Number of Wells 96 034 Mouse IL 5 Figure 3 17 Plate window MasterPlex QT www miraibio com 25 CHA PTER3 GETTING STARTED Standard Curves e SE Local Curves standard Curve Chart Residual P Plot Standard Data E A 034 queue Pa ions Five Parameter Logistica Gtd Curve 1 R Square 0 9999 A 034 Mouse IL 5 ER Std Curve 1 ARR 7 a 2 878 b 3 660 c 4403 824 d 1318 477 8 0 434 B A 054 Mouse IL 2 B std Curve 1 5 de 073 Mouse IFN gamma GA sta curve 1 B de 077 Mouse IL 4 GA std Curve 1 pr Imported Curves d oF Regressions dl 034 Mouse IL 5 e 038 Mouse IL 10 e 054 Mouse IL 2 P F A 073 Mouse IFN gamma e 077 Mouse IL 4 Median Fluroscense Intensity a zs wn Se ka zs wn Se zs vn w el ze zm wa by ze zm wm ze db wn mm mcm ven wl pn wm em 2 000 4 000 6 000 6 000 Concentrations pg ml Data Chart MFI wi Mouse IL 5 Lal 038 Mouse IL 10 t Bosa Mouse He Selected analytes in well 42 zy ue Mouse IEN gamma Rm 367034 Mouse IL 5 Wi MOF Mouse IL 4 mm 710 5 038 Mouse IL 10 MIA 1550 5 054 Mouse IL 2 M1185 073 Mouse IFN gamma MO 1 388 5 0
95. es of analytes in the pipette and the virtual plate If the number of analytes in the pipette 1s greater than that in the destination plate the virtual analyte filter automatically appears Figure 6 9 The virtual analyte filter displays a list of the analytes that are present in the virtual pipette It enables you to choose the analytes that you want to add to the virtual plate and if necessary rename them to be consistent with the number and name of analytes in the virtual plate If you add data to a virtual plate from source wells that contain different analyte names or a different number of analytes data holes are created As a result a well in the virtual plate appears blank if the analyte selected in the analyte panel is not present in the well If a plate csv zs Ixd mlx or virtual contains data holes the concentrations cannot be calculated _ da NOTE In order to prevent data holes if the number of analytes in the virtual pipette is less than the number of analytes in the destination plate the data cannot be added to the virtual plate MasterPlex QT www miraibio com 88 CHA PT ER6 VIRTUAL PLATES Virtual Analvte Filter Click on the link For a pull down menu New Analytes Assign Aliases LI dM GM CSF Click here to assign LI A IFN gamma Click here to assign d Aa Beta Click here to assign Al IL 10 Click here to assign d An Click here to assign d Al IL 4 Click here to assign d e 1 5 Click here to assign
96. ex QT www miraibio com 14 CHA PTER3 GETTING STARTED MasterPlex QT 3 0 Untitled Plate 1 lt IL5 csv gt Ed File Edit Plate Calculations Show Window Help P ue m zx e a aa ze um gm vo la 3 EE Work Space x i sug Sul Unk Sel ug Dame 3 des i Si 0x E ES Untitled Plate 1 Bead Set Properties SUS b a 5 6 11 Reports A 034 Mouse LS e 038 Mouse IL 10 ji 054 Mouse IL 2 Le 073 Mouse IFN gamma e 077 Mouse IL 4 Navigator Plate Window Window Figure 3 7 Navigator window Plate window More than one Plate window can be open at the same time 4 To import additional Luminex results files repeat step 1 to step 3 Each set of results data 1s displayed in a separate Plate window 3 4 Using Windows Explorer to Import csv xls or Open Ixd mlx Files 1 Open Windows Explorer and adjust the window size so that you can view both the MasterPlex QT and Windows Explorer application windows 2 Use Windows Explorer to navigate to the csv xls Ixd or mlx file s that you want to open 3 Select the file s of interest then click and hold the mouse button while you drag the selected file s to the MasterPlex QT application window Figure 3 8 To select adjacent files press and hold the Shift key while you click the first and last file in the selection To select nonadjacent files press and hold the Ctrl key while you click the files of interest 4 Release the mouse button The file s open in Maste
97. gt Open Displays the Open dialog box so that a Luminex results file csv Ixd BioPlex file xls or MasterPlex QT file mlx may be opened File Report Displays the report viewer Viewer File Close Closes the current opened window File Save Displays the Save As dialog box so that a results file csv Ixd or xls may be saved in MasterPlex QT file format mlx File Save as Displays the Save As dialog box so that a results file csv Ixd or xls may be saved in MasterPlex QT file format mlx File Preferences Displays the Preferences dialog box Edit Copy Copies the data in the active window to the system clipboard Edit Copy Copies the data horizontally in the active window je mis Horizontal List to the system clipboard MasterPlex QT www miraibio com 167 APPENDIXB MASTERPLEX QT TOOLBARS Edit 7 Copy Vertical List Edit 7 Export Chart Charts 7 Chart Copies the data vertically in the active window to the system clipboard Displays the Export Chart dialog box Displays the Chart Properties dialog box Properties Displays the Plate Wizard Plate 7 Template Opens the Template Manager Manager EN EN EN o Show 7 Show Hide 7 Navigator Shows or hides the Plate Navigator Displays the Plate Wizard Displays the Plate Merge Wizard Displays the Auto Grouping Wizard B 2 Plate Toolbar The Plate toolbar is available when a Plate window is open To show o
98. iability Heterogeneous variability or heteroscedasticity occurs in most types of observed data This is especially true for biochemical assays where concentration or dose is the predictor and response is often based on count Therefore we can expect that measurement error varies with respect to the mean In the Luminex 100 200 system MFI median fluorescence intensity values are based on bead counts and vary with the concentration In this case we expect the error in detecting MFI values to increase as concentration increases This is best seen in Figure C 3 a residual plot from a Luminex 100 200 cytokines assay Residual Plot IL 10 Standard 0 4 572 4 572 4 572 4 572 4 572 4 572 13 347 13 717 13 47 1 13 717 13 717 13 717 41 152 41 152 41 152 41 4152 2 41 152 41 152 123 457 123 457 123 457 123457 2 123 457 y 123 457 370 37 370 37 31 Residuals 370 37 11 370 37 370 37 3 370 37 39 1411411 14141411 1411411 1411111 1 111411 1411111 48 3 333 333 195 452 3 333 333 267 781 3333333 33057 9900 069 0 000099 009099099909900000009 02929 H1 G1 F1 E1 D1 C1 B1 A1 Predicted Concentration Figure C 3 Residual plot MasterPlex QT www miraibio com 176 APPENDIXC MODEL EQUATIONS A residual plot is a graphical representation of how far away an observed concentration 1s from its expected value It plots residuals against observed concentrations In Fig
99. ick a well in the regression data set and enter the agent concentration in the edit box e Click Enter The value is displayed in the well grid f Repeat step b to step e for the remaining analytes MasterPlex QT www miraibio com 94 CHA PT ER6 VIRTUAL PLATES 7 Right click the regression data set and select Assign Model Equation from the pop up menu that appears Alternativelv click the regression data set and click the Select Model button E 3 The Model Equations dialog box appears Figure 6 12 Model Equations Available Models Model Options 1 Optimizations Analtes O34 Mouse IL 5 wt Use Weighting 7 Select this model for all analytes Fixed lower asymptote at zero Function Expres sion H E Model Equations ER Polynomials ES Quadratic Competitive Binding Made E Assign Model Set As Default H Exit Figure 6 12 Model Equations dialog box Select a model for the Dose Response curve 8 Select the Dose Response variable slope model equation and choose the Select this model for all analytes option 9 Click Assign Model 10 Click the Calculate button then select the regression The software computes the Dose Response curve and displays a confirmation message when the calculation is completed MasterPlex QT www miraibio com 95 CHA PT ER6 VIRTUAL PLATES 11 To view the Dose Response curve click the Standard Curves button EE The Dose Response curve shows the agent dose indepe
100. ick the Ungroup wells button EEE sor select Template gt Ungroup wells from the menu bar gt The grouping is removed Grouping Wells by Pattern The purpose of pattern grouping is to provide users another way to easily and quickly group wells Pattern here means two things the group type e g MasterPlex QT www miraibio com 36 CHA PTER4 DEFINING A PLATE standard unknown and the dimensions of the group 1 e rows and columns This function acts similarly to the Resizing feature of Microsoft Excel It is especially useful when the plate has many groups replicates which follow similar group patterns 1 Define the group pattern by selecting a group of wells and marking and grouping them together We will group other wells into this pattern 2 Select all wells of the pattern group Figure 4 6 d 3 4 5 6 367 00 506 50 341 00 429 00 480 50 392 00 82 00 5 00 78 00 88 50 2 00 96 50 37 50 38 00 37 00 F 65 50 32 00 232 00 34 00 G 39 00 18 00 18 00 23 00 H 26 50 19 00 17 50 27 00 Figure 4 6 Well groups 2 Point mouse cursor to the bottom right corner of the selection When you see the cursor turns into a black cross push down the left button of mouse and start to drag During dragging you will see in real time that new wells are selected and grouped into the pattern as indicated by a red line border Figure 4 7 MasterPlex QT www miraibio com 37 CHA PTER4 DEFINING A PLATE 1 a 3 4 5 1 a 3 4 5 Ca a al eee
101. identifies wells with a MFI value less than threshold for the selected analyte active at a time NOTE The threshold menus Ha Fl or m are mutually exclusive Only one is MasterPlex QT www miraibio com 22 CHA PTER3 GETTING STARTED 3 7 The Plate Navigator At startup MasterPlex QT displays the Plate Navigator Figure 3 14 The plate navigator workspace shows a tree of the windows that are open during a session The Plate Navigator organizes the open windows that are associated with a plate It provides a convenient way to change the view in the main display area e To view a particular window click the item in the Plate Navigator window Figure 3 15 e To show or hide the Plate Navigator select the EN avigator menu from Show gt show hide gt Navigator MasterPlex OT 3 0 File Plate Calculations Show Window Help Ines zesin eOr S au IA 2Vvmevloj a re EE Work Space if e em j d l ad i i E jg j Ln pa jil Plate Navigator Main Display Area window Figure 3 14 Plate Navigator window at start up If no windows are open in the main display area the Plate Navigator window is empty MasterPlex QT www miraibio com 23 CHA PTER3 GETTING STARTED MasterPlex OT 3 0 SE File Edit Charts Show Window Help A Bl2 aa k amp o gt lge Ba ze mas 0 SA EE Work Space IL5 Project lt IL5 csv gt ES IL5 Project 115 Reports Report per an Data Chart fii Bar Graph
102. ies dialog box Figure 7 25 These settings determine the appearance of the left back and bottom walls in a three dimensional chart Figure 7 25 2 To change a setting click a wall Left Back or Bottom and modify the settings Chart Properties Charts 3D Legends Walls senes Panel Left Right Bottom Back Appearance Gradient visible No Middle 7 Visible Direction Top Bottom Thickness v Dark 3D Transparent LE Exit Figure 7 25 Chart Properties dialog box Walls tab MasterPlex QT www miraibio com 125 CHA PTER7 DATA CHARTS Left wall Back wall E E Figure 7 26 3D depth chart Bottom wall User Modifiable Wall Settings Appearance Color Click to open a color palette Choose a color for the wall from the color palette Visible Choose this option to display the wall color Thickness Change this value to increase or decrease the wall thickness Dark 3D Choose this option to apply a darker color to the inside surface of the wall Transparent Choose an option to display the a transparent wall without color MasterPlex QT www miraibio com 126 CHA PTER7 DATA CHARTS Gradient Visible Choose this option to display a gradient wall color No Middle Choose this option if you do not want to display a gradient with only two colors start and end color If this option 1s not chosen the gradient has three colors start middle and end color Direction Make a sele
103. ight click the selection and choose Mark Wells and a well type from the pop up menu that appears Figure 4 3 You can also use toolbar buttons or menu bar commands to define the wells Table 4 1 gt The well type is applied to the selected well s MasterPlex QT www miraibio com 31 CHA PTER4 DEFINING A PLATE NOTE If the Automatic Well Grouping option is selected in the Preferences dialog box the wells are automatically grouped when you set the well type For more information on this option see Application Tab on page A 1 1 2 3 4 5 6 Fi A 0 00 347 00 486 50 321 00 1288 00 409 00 460 50 172 00 TA Assign Model Equation f Select Entire Group Mark Wells d Absolute Quantification KE 19 De Unmark wells Relative Quantification Bil Standard 6 E Group Wells Ul Unknown St Control lut EB Ungroup Wells Jse IL 5 88 Regression Copy Ctril HC Copy Horizontal List Copy Vertical List EB Auto Fill E Auto Group Figure 4 3 Well grid pop up menu Right click a well to display the pop up menu Table 4 1 Button and context menu to define wells MasterPlex QT www miraibio com 32 CHA PTER4 DEFINING A PLATE Well Type Context menu on the plate window Background Mark Wells gt Absolute Wells that contain no analytes BI Quantification gt Background Standard Mark Wells gt Absolute Wells that contains analyte of known Quantification gt Standard Mark Wells gt Absolute
104. iple wells To select adjacent wells Figure 4 1 press and hold the mouse key while you drag the mouse cursor over the wells that you want to select Click and release the mouse button to select the highlighted wells To select adjacent wells press and hold the Shift key while you click the first and last well in the selection To select nonadjacent wells Figure 4 2 press and hold the Ctrl while you click the wells MasterPlex QT www miraibio com 30 CHA PTER4 DEFINING A PLATE 1 2 3 A A 0 00 347 00 485 50 321 00 B 1758 00 408 00 450 50 372 00 C 1064 50 62 00 65 00 58 00 D 505 00 68 50 62 00 76 50 E 181 00 17 50 18 00 17 00 F 46 50 12 00 12 00 14 00 G 19 00 2 00 2 00 3 00 H 6 50 1 00 2 50 rA Figure 4 1 Well grid To select adjacent wells press and hold the Shift key while you click the first and last well in the selection Alternatively press and hold the mouse key while you drag the mouse over the wells of interest 1 d 3 4 A 0 00 347 00 486 50 321 00 B 1288 00 409 00 450 50 372 00 C 1064 50 62 00 65 00 58 00 D 505 00 68 50 62 00 76 50 E 181 00 17 50 18 00 17 00 F 46 50 12 00 12 00 14 00 G 19 00 2 00 2 00 3 00 H 6 50 1 00 2 50 7 00 Figure 4 2 Well grid To select nonadjacent wells press and hold the Ctrl key while you click the wells of interest Designating Well Type Table 4 1 shows the types of wells that are available 1 Select the well s that you want to define 2 To define or mark the well s r
105. k here to assign i 5 E Al IL 2 Click here to assign i 6 H Af Click here to assign m 11 5 miar ss Ser Click to display a dass a pud 9 LI Al Il 8 034 eeneg IL 5 drop down list of dee 38 Mouse IL Ivte names in TNF a 038 Mouse IL 10 analyte analyte to add i 10 LN 054 Mouse IL 2 the virtual plate to the virtual SE Ae SE Select a name from plate the list to rename the analyte from the source plate Add all az new Save this assignment Use last saved assignment Check All Se Apply Template ag OF A Lancel Figure 6 9 Virtual analyte filter 3 To save the renaming assignments for use again with the same source plate csv xls or Ixd or mlx during the current session choose the Save this assignment option If you want to aspirate other data from the same source plate choose the Use last saved assignments option in the virtual analyte filter to automatically rename all of the analytes in the filter 4 Click OK gt The data are added to the virtual plate and the virtual analyte filter closes 6 3 MasterPlex QT www miraibio com 90 CHA PT ER 6 VIRTUAL PLATES Generating a Dose Response Curve A Dose Response curve plots the dose of an agent independent variable on the x axis and the response computed concentration on the v axis After vou have created a virtual plate that contains the concentration data of interest VOu can run a regression analvsis on the data in the virtual plate to generat
106. lated to Columns MasterFlex OT Choose this option to Automatic ell Grouping automatically group wells when you set the well type Enable plate wizard at start up Enable Relative Quantitication Bene Expression Default ay Ok ae Cancel Apply Figure 4 4 Preferences dialog box To display the Preference dialog box click the button Gor select File gt Preferences from the menu bar Manually Grouping Wells You can only place the same type of wells in a group 1 Select the wells that you want to group NOTE The grouping function is only available if you have selected wells that have been defined 2 Right click the selection and select Group Wells from the pop up menu that appears Alternatively click the selection and click the Group Wells MasterPlex QT www miraibio com 35 CHA PTER4 DEFINING A PLATE button GC or select Template gt Group Wells from the menu bar C A border appears around the grouped wells Figure 4 5 3 To display the well grouping Figure 4 5 click a well Bead set Properties Analutes A O34 Mouse IL 5 Group of Group of standard data Unknowns A2 to H4 B1 to H1 Figure 4 5 Well groups A red border identifies a group in the well grid Ungrouping Wells 1 Right click the group NOTE The ungrouping function is only available if you have selected grouped wells E 2 Select Ungroup wells from the pop up menu that appears Alternatively cl
107. le Mame Outliers MFI Calculated Expecbed Residuals amp Recovery Background Hi Tial a 55 00 27 92 29 08 1 16 96 00 66 00 e Gi ai T 197 00 92 56 87 24 5 32 106 10 68 00 s Fi e T 516 00 256 56 261 73 4 87 98 14 68 00 4 El si TI 1406 50 822 09 785 19 36 90 104 70 68 00 3 Di 4 l 2212 00 2192 21 ECK 163 35 93 07 66 00 sul e ai T 5704 00 7625 68 7066 67 559 01 107 91 68 00 e ai E 7724 00 20322 65 21200 00 877 35 05 86 66 00 1 Bt Figure C 6 Example standard curve data Fitting the four parameter logistics equation to standard data using 1 Y weighting improves the Recovery at lower concentrations MasterPlex QT www miraibio com 181 APPENDIXD DOSE RESPONSE ANALYSIS APPENDIX D This appendix provides an introduction to Dose Response analysis Dose Response Analysis MFI values are raw response values measured by the Luminex 100 200 Based on these measured responses standard curves are used to approximate the actual analyte concentration in a sample Analyte concentrations interpolated from a standard curve are quantified responses Quantified responses can be used to study dependent variables inherent to a biological or chemical process For example suppose a chemical agent that is given to mice causes an increase in the level of certain cytokines The chemical agent dosage is the independent variable and the cytokine concentration is the dependent variable To understand the potency of the agent
108. les csv xls or 1xd from the Luminex system designate standard unknown control and background wells generate standard curves compute analyte concentrations generate data charts and reports What s New in MasterPlex QT 3 0 MasterPlex QT 3 0 offers new features including the ability to Merge plates using virtual plate feature so that 1t can analyzes beyond 100 panels at one time Make a sample marking and groups easily and quickly using Auto grouping feature or dragging grouping feature Calculate a fold change especially for being used relative gene analysis Normalize the data so that 1t can analyze between difference plates generate a custom reports using style sheet Conventions Used in This Manual This manual describes the steps required to perform the various tasks associated with the MasterPlex QT software The manual uses a step format to explain the various tasks associated with MasterPlex QT The symbol may follow a step instruction It indicates the software response to the action performed by the user MasterPlex QT www miraibio com 1 CHA PT E R 1 WELCOME Screen Captures Screen captures may accompany the step instructions for further illustration The screen captures in this manual may not exactly match those displayed on your screen 1 2 Technical Support You can contact MiraiBio Technical support at Hitachi Software Engineering America Ltd 601 Gateway Boulevard Suite 100 South San Francisco CA 94080 USA T
109. lyte Filter e 88 Generating a Dose Response Curve sen 91 Auto Plate Merging A USE uM SUE US 96 CHAPTER 7 Data Charts Viewing a Data Chart eee 100 Data Chart Tvpes SEENEN 101 Working With Data CHATS eses 112 CHAPTER 8 MasterPlex QT Reports Generating a Report A ETO l 137 Working With Reports eee 140 Custom Report Iudae ue iac bU Ei DX EN Oan EM US 155 MasterPlex QT www miraibio com II APPENDIX A Preferences Application Tab HH eee eee 159 Poe Tah SE 160 Calculations Tab E 164 APPENDIX B MasterPlex QT Toolbars Man Toolbar seisoi is eein ies 167 Plate Toolbar urna ip ice 168 Calculation Toolbar essenenenensenezzennsnnenzenzznnenza 170 Chant Keel EE 171 Report Toolbar eH eee 172 APPENDIX C Model Equations Four Parameter Logistic Case eean 173 Five Parameter Logistic Gilia e insna 174 Heteroscedasticity ni eras 176 Weighted Nonlinear Least Square e 178 Results of Weighting HMM 180 APPENDIX D Dose Response Analysis Dose Response Analysis HMM 182 APPENDIX E Relative Quantification Assay Relative Quantification Assay M 184 MasterPlex QT www miraibio com 111 CHA DIER WELCOME MiraiBio MasterPlex M QT 3 0 CHAPTER 1 Welcome to the MiraiBio MasterPlex QT User Manual MasterPlex OT software analyzes results files csv xls or lxd from the Luminex amp 100 200 system 1 1 About This Manual This manual explains how to use the MasterPlex QT 3 0 software to import results fi
110. mmand Toolbar u Open saved reports Close current report window Displays a drop down list of magnification options for viewing the report in the Report window Displays the first page in the report g Displays the previous page in the report Displays the next page in the report Displays the last page in the report Report gt Save Displays the Save As dialog box so that you Report may save a report frp Report gt Print Displays the Print dialog box so that you Report may print a report L 2 a NM m L r MasterPlex QT www miraibio com 172 APPENDIXC MODEL EQUATIONS APPENDIX C This appendix provides background on the four and five parameter logistic curves It also explains how weighting methods can improve the fit of nonlinear models to data with non constant variabilitv heteroscedasticitv C 1 Four Parameter Logistic Curve D Upper Asymptote Mid way between A and Dor D A 2 A Lower Asymptote C X value corresponding to the Y value that is half way between A and D Figure C 1 Four parameter logistics curve In Figure C 1 the asymptotes D and A are the upper and lower limits of the model An asymptote is a value that the function never reaches Therefore the value of a function at or beyond an asymptote cannot be MasterPlex QT www miraibio com 173 APPENDIXC MODEL EQUATIONS predicted If MFI lt A or MFI lt D the MFI value is out of the calculable r
111. nager button es Alternatively select Plate gt Template Manager from the menu bar The Template Manager appears Figure 4 17 2 Click a template in the Available Templates list to view information about the template MasterPlex QT www miraibio com 46 CHA PTER4 DEFINING A PLATE Template Available Term plates Fa MsPlexSingleStd NS MasterPlex 21 Template zs Murine IL Project Template Mame Murine IL Project Generated by version 2 Created modified in 712003 xg H10PlexDuelstd Created modified at2 31 12 PM Template Management Aa Load LI Dave B Export fa Import Overwrite fa Delete Figure 4 17 Template Manager shows available templates Click a template to view information about the template Saving a Template Y ou can save the current plate definition to a template 1 After you have finished defining a plate open the Template Manager and click the Save button LR The Template Name box appears Figure 4 18 Template Name Please enter NEW template name li OH x Cancel Figure 4 18 Template Name box 2 Enter a name for the template and click OK gt The new template is added to the Available Template list MasterPlex QT www miraibio com 47 CHA PTER4 DEFINING A PLATE Loading a Template You can apply or load a saved template to the current plate 1 In the Template Manager select the template that you want to apply to the plate 2 Click the
112. ndent variable on the x axis and the response interpolated concentration on the y axis Figure 6 13 It displays the parameters of the model equation and the LogEC50 value 12 To compute the EC50 value take the antilog of the LogEC50 Bese Aesponse Hamabi shpel Btj Curva 1 F Square 8887 A house L A ge 210139 b 11414 L0pECS 0 3 3135 d 1425 83D mm pe rm eee m me om mm um m mm om cum m cum he ee mm Pe m pe em Pe e Median Flurostense nternaty Concentrations pg Ant Figure 6 13 Dose Response curve Dose independent variable on the x axis and response computed concentration dependent variable on the y axis 6 4 Auto Plate Merging The purpose of auto plate merging is to quickly merge multiple plates at analytes dimension into a virtual plate That 1s assuming all plates have different analytes we want to merge plates together by adding all their analytes as new So if a plate with 5 analytes 1s merged with another plate with 7 MasterPlex QT www miraibio com 96 CHA PT ER6 VIRTUAL PLATES analytes we will get a virtual plate with 12 analytes This tool is especially useful for miRNA analysis where assay results of the same sample pool are usually distributed in several plates It will be extremely useful if we can put these results into one place and do analysis together Compared to use virtual pipette to manually add wells this tool makes the merging task much faster and
113. nformation available in the Standard Data tab GE Local Curves Standard Curve Chart Residual Plot Standard Data il 034 Mouse IL 5 Well Sample Name Outliers MFI Calculated Expected Residuals Recovery Background Hi 8 6 50 15 62 15 62 NA MA 20 00 19 00 22 85 31 25 8 40 73 11 20 00 46 50 63 54 62 50 1 04 101 66 20 00 G 038 Mouse IL 10 Std Curve 1 Sd 054 Mouse IL 2 Std Curve 1 GZ 073 Mouse IFN gamma Std Curve 1 GA 077 Mouse IL 4 5td Curve 1 505 00 243 00 250 00 7 00 97 20 20 00 1064 50 315 08 500 00 15 08 103 02 20 00 Fi 6 3 4 151 00 135 07 125 00 10 07 108 06 20 00 z 2 1 1288 00 956 55 1000 00 43 45 95 65 20 00 al Imported Curves E Regressions Place a check mark in a check box to make a data point an outlier Figure 5 18 Standard Data tab Table 5 1 Standard Data Item Description Outliers To make a standard data point an outlier place a check mark in the check box next to the standard data point The software ignores outliers during the calculation of the standard curve MFI Mean fluorescence intensity of the data point in the selected analyte standard Expected The standard concentration for the selected analyte that was entered using the autofill function or manually Calculated The standard concentration for the selected analyte that was interpolated or extrapolated from the standard curve Residuals Calculated Expected Recovery Calculated Expected x 100 Background MFI ba
114. ntensity TA mm um mm mm mm em mm mm em em mm mm rm em rm em rm em L em rm em rm rm rm em rm mm rm et Figure 5 13 Four parameter logistics model equation x axis log scale A 16 335 bottom asymptote D 1373 175 top asymptote MFI values less than A or greater than D are beyond the range of the model equation Right click the graph to select log scale for the x axis 9 4 MasterPlex QT www miraibio com 73 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION Printing the Well Grid You can print the contents of the well grid l 1 To set print preferences click the Plate Print Settings button 4 gt The Print Settings dialog box appears Figure 5 14 Print Settings General Headers Footers Margins Preferences Borders Border Border Style General Repeat Fixed Columns Autosize Columns Repeat Fixed Rows AukoSize Rows Print graphics Center on Page Fonts 4 Table Font 4 Fised Font Use display font 4 Header Font 4 Footer Font mee Figure 5 14 Print Settings dialog box 2 To display a print preview of the well grid click the Print button The Print Preview dialog box displays the currently selected well contents data type and analyte MasterPlex QT www miraibio com 74 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION Print Preview IL5 Project mlx IL5 csv O34 Mouse IL 5 1 2 3 4 3 6 7 8 9 10 11 12 0 00 347 00 486 50 321
115. ntration Yes associated with a well that is a member of a regression data set A Dose Response curve is generated from a regression data set User specified name for the well Yes A check mark indicates the well is outlier and Yes the well data are not included in the calculation of concentrations or a Dose Response curve The color that represents each analyte in the Yes bar chart The group number of the well Wells that belong to the same group have the same group number User specified replicate type name for the Yes well Shows the standard number that is linked to Yes each well or well group shows the average within the group No Shows the std within the group No Shows the CV within the group No shows the Normalized data within the group No MasterPlex QT www miraibio com 19 CHA PTER3 GETTING STARTED Untitled Plate 2 lt IL5 csv gt HE Big Sod Gk Cel Rag fe The Cel Gh HD rn SES OX Bead Set tag d 034 Mouse IL 5 1 038 Mouse IL 10 3 4 5 6 F 20 00 357 00 341 00 054 Mouse IL 2 1306 00 429 00 073 Mouse IFN gamma 077 Mouse IL 4 1084 50 82 00 525 00 88 50 201 00 37 50 66 50 32 00 18 00 19 00 Number of Wells 96 034 Mouse IL 5 Figure 3 10 Plate window and Color tab The Color tab shows the color that reoresents each analyte in the multi well chart To
116. ntrations standard data curves or regression analysis curves for virtual plates only data manually entered in the plate for example dilution factors or independent data values plate preferences MasterPlex QT www miraibio com 58 CHA PTER4 DEFINING A PLATE 1 To save a plate click the Save button il c The Save As dialog box appears Figure 4 28 Save As Save n O 8 em IL5 Project mlx is Save as type MasterPlex QT Plate Files mi Figure 4 28 Save As dialog box Save the current plate data in native mlx format 2 Enter a name for the file 3 Click Save MasterPlex QT www miraibio com 59 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION CHAPTER o This chapter explains how to generate standard curves and compute interpolate or extrapolate analyte concentrations from the standard curves 5 1 Specifying Standard Data Each well in a standard data set represents an x y data point The MFI value is plotted on the y axis and the concentration is plotted on the x axis MasterPlex QT uses regression analysis to fit a user specified model equation to the standard data set and generate a standard curve E NOTE The standard curve may not pass through each point in the standard data set The software computes the R value 0 lt R lt 1 for the model equation R measures the goodness of fit of the model equation to the standard data set where R 1 is the probability that the model
117. o load a stylesheet into manager Figure 8 19 MasterPlex QT www miraibio com 155 CHA PT ER 8 MASTERPLEX QT REPORTS Style Sheet Manager Stile Sheet Mame OL File Details Determination Es Determination_nevw En Determination_nevw En Unknowns Only en GL HTML ez Bea HTML Dutpu es CSV file es Cross Tab Include standard curve images es Al HTML es Copy All Style Sheet Management en Control HTML Em Determination EE ipee BB Export a Delete Figure 8 19 Import custom report stylesheet 4 Select a stylesheet from the manager window The XSL file details window will display the information of the selected stylesheet including name created time and output type e g text html xml Figure 8 20 LaReport _per_Unknown_Replicates html xsl Style Sheet Manager Files of type Style Sheets xsl Style Sheet Mame Determination en Determination new dm Determination_new dm Unknowns Orly en All HTML en Beadl HTML en CSV file en Cross Tab es AILHTML en Copy Al en Control HTML dm Determination ABL File Details MasterPlex OT Style Sheet Style Sheet Name Report per Unknown Aeplicates_ html Created modified ir 5 5 2007 Created modified at 12 47 10 AM Output Method HTML Dutpu l C Include standard curve images Stile Sheet Management fe Import fa Export a Delete Figure 8 20 Stylesheet information window Ma
118. o turn on the virtual pipette Analytez de 034 Mouse IL 5 2 Select the wells of interest in the source plate csv or mlx Right click the selected wells and choose aspirate from the pop up menu Dispense Number of Viele Oe 034 Mouse IL 5 3 In the virtual plate select the first well where you want to dispense the data Analytez A B C D E F G H 4 Right click the well and select Dispense from the pop up menu The data are added to the virtual plate starting at the Analptes selected well in the same configuration as in the source plate M ji Aspirate Dispense Clear I lnm o AD ml Number of Wells 96 Figure 6 7 Adding data to a virtual plate Open a source plate mlx or csv xls or Ixd and create a virtual plate click the G button to start the virtual plate wizard MasterPlex QT www miraibio com 87 CHA PT ER 6 VIRTUAL PLATES 6 2 Working With the Virtual Analvte Filter In a multiplex assay all of the plate wells must contain the same tvpes of analvtes bead sets with the same nomenclature the same number of analvtes This is true for virtual plates as well When vou add data to a virtual plate MasterPlex QT compares the name and number of the analytes in the virtual pipette to those in the virtual plate The virtual pipette will not dispense if there are discrepancies between the number or nam
119. of the three ways gt Normalize by the geometric mean of several house keeper genes gt Normalize by a single house keeper gene gt Normalize by a constant value MasterPlex QT www miraibio com 184 APPENDIXE RELATIVE QUANTIFICATION ANALYSIS 5 For this demo we will choose Fold Change with Normalization based on our previously selected house keeper gene 6 Click Ok and a popup window will display the calculation status 7 To view the normalized data select Normalized data from the Data Type pull down menu To view the final Fold Change results select Concentration Fold Change from the drop down menu To view results for a different bead region just click its name from the bead set window MasterPlex QT www miraibio com 185
120. olid Blue Figure 7 35 Color palette 2 To select a predefined color click one of the basic colors MasterPlex QT www miraibio com 134 CHA PTER7 DATA CHARTS 3 To define a custom color a Click Define Custom Colors then use the click and drag method to move the cross hairs in the custom color field b Adjust the color brightness using the luminosity scale to the right c When finished click Add to Custom Colors to apply the color 4 Click OK to close the color palette Panel Tab The panel settings determine the appearance of the display area behind the chart Chart Properties Charts 3D Legends Walls Series Panel Gradient Visible Direction Bottom Top No Middle Background Chart Panel Background Log scale on X axis Figure 7 36 Chart properties dialog box Panel tab Gradient Visible Choose this option to display a gradient in the area behind the chart No Middle Choose this option to display a two color gradient start and end color in the area behind the chart If you do not choose this option you can specify three colors for the gradient start middle and end Direction Make a selection from the drop down list to set the direction MasterPlex QT www miraibio com 135 CHA PTER7 DATA CHARTS of the gradient Start Mid End Click to open a color palette Choose a color for the start middle or end of the gradient Background Chart Panel Click to open the color p
121. omputer and double click MasterPlex QT exe gt The installation begins and the InstallShield Wizard appears Figure 2 1 MasterPlex QT 3 0 InstallShield Wizard ES Welcome to the InstallShield Wizard for MasterPlex OT 3 0 The InstallShieldR wizard will install MasterPlex BT 3 0 on your computer To continue click Next naai Figure 2 1 InstallShield Wizard Welcome screen 2 To continue the installation click Next The Choose Destination Location window appears Figure 2 2 MasterPlex QT www miraibio com CHAPTER2 INSTALLING MASTERPLEX QT MasterPlex QT 3 0 InstallShield Wizard E Choose Destination Location Select folder where setup will install files Setup will install MasterPles HT 3 0 in the following folder Ta install to this folder click Next To install to a different folder click Browse and select another folder r Destination Folder E Program FilessHitachiSoftNM asterPlex OT 3 04 Instalisie Figure 2 2 Install Shield Wizard Choose Destination Location window 3 To accept the default destination folder click Next To specify a different destination folder click Browse choose the folder and click Next c The Start Copying Files window appears Figure 2 3 MasterPlex QT CHAPTER2 INSTALLING MASTERPLEX QT MasterPlex QT 3 0 InstallShield Wizard Start Copying Files Review settings before copying files Setup has enough information
122. oncentrations Save the Luminex results file in MasterPlex QT file format mlx The mlx file includes information associated with the file for example well definitions and interpolated concentrations After the concentrations are calculated you can view the results in graphs or several different report formats create a virtual plate a simulated microtiter plate that contains data from user selected actual plates csv xls Ixd or mlx generate a Dose Response curve and determine the Log EC50 value for user selected data in a virtual plate MasterPlex QT www miraibio com CHA PTER3 GETTING STARTED 3 2 Starting MasterPlex QT e On the desk top double click the MasterPlex QT icon Alternatively you can click the Windows start menu button and select Programs gt MasterPlex QT 3 0 gt MasterPlex QT 3 0 The MasterPlex QT user interface appears and displays the Plate Wizard and Navigator window Figure 3 1 For more information about the Plate Navigator window see page 3 14 chosen in the Application preferences or the Display wizard at startup option is chosen in the Plate Wizard NOTE The Plate Wizard appears if the Enable plate wizard at start up option is The Plate Wizard guides you through the steps to import a Luminex results file csv xls or 1xd or create a virtual plate For more information on virtual plates see Chapter 6 on page 6 1 MasterPlex OT 3 0 File Show Window Help jem b sajjiu
123. onfirma if they are the only House Keepers IF pes you may click the Automatically radio button below The plates will be merged without the House Keepers Otherwise you may click the Manually radio button Then you can select the House Keepers for each plate at the nest page Merge Type C3 Automatically Then click Nest to continue e Manually select house keeper Figure 6 15 Merge method selection 3 At this step you can manually select analytes Figure 6 16 from each plate to be excluded from the virtual plate Then go the next page to finish wizard MasterPlex QT www miraibio com 98 Merge Plates Selected Plates E Untitled Plate 1 Merge Plates Select House Keeper Manually Selected Plates EE Untitled Plate 1 EE Untitled Plate 2 22 Untitled Plate 4 For each plate please check the bead sets which are house keepers Then click Next to continue Select House Keeper Manually ai Untitled Plate 2 FW Untitled Plate 4 CHAPTER6 VIRTUAL PLATES Merge Plates Select House Keeper Manually 1 de 017 let 7a amp 019 let 7d El A O21 let 7e SN Untitled Plate 1 77 g O17 let 7b AS O19 let 7f O de 021 let Fi 7 g 033 mir 130b 77 i 035 mir 196 ei P 017 mir 124a OI F A 013 mir 125b O FA 021 mir 128b L P 0133 mir 1 9b L P O35 mir 217
124. ove vs dragging rightwards as the first move below Grouping Wells by Sample Name The purpose of grouping wells by sample name is to provide users an alternative way to easily and quickly group wells based on wells sample names When using this tool users will be asked in three steps to provide information on what string segment of sample name are related to sample type and what segment is related to replicate group The segment of sample type provides information about well s group type e g standard unknown background the segment of replicate group provides information about replicates assuming wells in one replicate group have an identical string segment in their names Based on the information this tool will automatically group wells together into different types The auto grouping tool is especially useful when the data file is MasterPlex QT www miraibio com 39 CHA PTER4 DEFINING A PLATE generated strictly following the naming rules for its analytes 1 Start wizard by click toolbar button Cd or right click dropdown button Auto Group 2 Step 1 specify delimiters Users choose or set delimiters to segment sample names The segmented results are displayed at the same time Figure 4 10 Auto Group Specify Delimiterz Delimiters This wizard will quide you through grouping Tab Space wells automatically Dash Underzcore First please set the delimiters which well names contain ou can see
125. own list to view the analyte concentrations for the selected standards Figure 5 5 Select Standard Independent Values from the data type drop down list ILS Project mlx lt IL5 csv gt ETT Properties IE a Standard Independent Values Analtes 8 034 Mouse IL jib 038 Mouse IL 10 d i 054 Mouse IL 2 FA 073 Mouse IEN gamma de OF Mouse IL 4 Number of Wells 96 034 Mouse IL 5 Figure 5 5 Standard concentrations for the selected analyte The autofill feature entered the analyte concentrations for the selected standard set based on a user specified starting concentration of 10 000 and a dilution factor of 3 Entering Standard Concentrations Manually 1 Open the results of interest csv xls Ixd or mlx 2 Click the Well Editor button Fi Alternatively select Plate gt Edit Wells MasterPlex QT www miraibio com 64 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION from the menu bar c The Plate window is in edit mode NOTE You can only edit the standard concentrations when the plate window is in edit mode 3 Select an analyte from the Bead Set panel Figure 5 6 4 Select Standard Independent Values from the data type dropdown list 5 In the well grid click the well that you want to edit gt The concentration value for the selected well is displayed 6 Enter a concentration value and click Enter gt The well grid displays the new concentration value
126. p remove the check mark from the Enable plate wizard at start up option 3 Click Apply when you are finished 4 To return the application preferences to the factory set defaults click Default A 2 Plate Tab The Plate preferences specify how to compute background and when to subtract background threshold values for concentration MFI and bead count the plate and analyst name MasterPlex QT www miraibio com 160 APPENDIXA PREFERENCES Preferences Application Plate Regression Calculations Background Plate Info Plate Name Untitled Plate 3 Analyst Name IMI Thresholds Subtract Background hen to subtract e All calculations C Regressions only Background Type z as Concentration Average Peak value Dm Lowest value EM Background target Gene Prompt before calculation Assay Bk k Prompt before recalculation Default ay Ok ae Cancel Apply Figure A 2 Preferences dialog box Plate tab default settings a NOTE If a plate csv xls Ixd or mlx is not open the Preferences dialog box does notdisplay the Plate tab Background Options You can specify whether you want to consider background data in the calculation of analyte concentrations There are two methods of computing background subtracted analyte concentrations All Calculations or Regressions Only Subtract Choose this option if you want to compute Background background subt
127. p the grouping procedure Setting Standard Concentrations After you define and group the standard wells use the autofill feature to help you automatically enter the standard concentrations For more information on specifying standard data see Chapter 5 on page 5 1 1 Right click the standard data set and select Auto Fill from the popup menu Alternatively click the Auto Fill button Hor select Template gt Auto Fill from the menu bar gt The Auto Fill dialog box appears Figure 4 14 MasterPlex QT www miraibio com 42 CHA PTER4 DEFINING A PLATE Auto Fill Dilution parameters 10000 000 A T oricentratien Unit 034 Mouse IL 5 Dilution direction Figure 4 14 Auto Fill dialog box 2 Make a selection from the Analyte drop down list 3 Enter the starting concentration for the standard data set 4 Enter the dilution factor 5 Make a selection from the concentration unit drop down list 6 To select a dilution direction for the standard data set click a dilution direction arrow c The gradient map shows the location and direction of the dilution gradient s Figure 4 15 MasterPlex QT www miraibio com 43 CHA PTER4 DEFINING A PLATE Details Dilution direction Click an arrow to Y choose a dilution direction This gradient map specifies a separate dilution gradient in each column of the standard data set The starting concentration is at the top of a column Details Dilution dir
128. pg mL Figure C 2 Five parameter logistic curve MasterPlex QT www miraibio com 174 APPENDIXC MODEL EQUATIONS A five parameter logistic curve introduces an additional parameter to the four parameter logistic model to compensate for asymmetric data In Figure C 2 we can see that the curve is not symmetric because the bottom part of the curve behaves quite differently from the top portion The additional parameter E compensates for this behavior and corrects for asymmetry Fixing Parameter A to Zero If an assay contains background or blank wells readings from these wells can be considered noise in the detection By deducting this noise from the MFI values we can obtain more accurate responses If we subtract a reasonable background value then we can further assume that the lowest detectable value should now be zero Since parameter A represents the lowest detectable concentration value it can be set to zero E Before background After background subtraction subtraction Figure C 1 Five parameter logistics curve MasterPlex QT www miraibio com 175 APPENDIXC MODEL EQUATIONS Log Log Model The Log Log model transforms the data to log scale for the x and y values It applies linear regression to fit a straight line through these points This model is appropriate for data that are intrinsically linear C 3 Heteroscedasticity Fitting nonlinear models to observed data is often complicated by non constant or heterogeneous var
129. previous page Displav the next page Displav the last page Open the Save As dialog box soooes Open the Print dialog box Figure 8 6 1 MasterPlex QT www miraibio com 140 CHA PT ER 8 MASTERPLEX QT REPORTS Saving a Report 1 Click the Export Report button gt The Save As dialog box appears Figure 8 3 File name Save as type Report file frp kul Figure 8 3 Save As dialog box 2 Enter a file name 3 Choose a file type from the Save as type drop down list A report may be saved in frp csv txt emf wmf pdf bmp rtf or htm format 4 Click Save NOTE A report saved in the frp file format can only be opened in the MasterPlex QT application Viewing Reports To view a report generated in the current session click the report of interest in the Plate Navigator To view a report saved during a previous session use the report viewer 1 To open a saved report select File gt Report Viewer from the menu bar gt The Report Viewer appears Figure 8 4 MasterPlex QT www miraibio com 141 CHA PT ERG MASTERPLEX QT REPORTS Report Viewer ul OOOO va Please click open report button to view a report Figure 8 4 Report viewer 2 Click the Open button Y The Open dialog box appears Figure 8 5 Look in O P fil Well map report frp Tabular conc report frp IL 5 replicates frp File name Files of type Report file CG frp Cancel Figure
130. r concentration MFI and bead count the plate and analyst name Preferences Application Plate Regression Calculations Background Plate Infa Plate Name IL5 Project mis Analvst Name IT Thresholds Subtract Background When ta subtract Bead Count Be Background Type E al Concentration Back d target G i ackground target Gene v Prompt before calculation Assay Bk v Prompt before recalculation Default aye Ok ae Cancel ad Apply Figure 4 24 Preferences dialog box Plate tab default settings MasterPlex QT www miraibio com 52 CHA PTER4 DEFINING A PLATE NOTE If a plate csv xls Ixd or mlx is not open the Preferences dialog box does not displav the Plate tab Background Options You can specify whether you want to consider background data in the calculation of analyte concentrations There are two methods of computing background subtracted analyte concentrations All Calculations or Regressions Only Subtract Background All Calculations Regressions Only Choose this option if you want to compute background subtracted analyte concentrations If this option is not chosen the background MFI value is not considered during calculation of the analyte concentrations This method of computing background subtracted analyte concentrations subtracts the background MFI from each member of the standard data set then fits the standard curve The method subtr
131. r hide the plate toolbar select Show gt Show Hide gt Plate Tools from the menu bar W Y DD ga Figure B 2 Plate toolbar Table B 2 Plate toolbar buttons and functions Menu Bar Main Function Command Toolbar Button Plate Click this button to enable users to select wells in Selection Tool Plate 7 Edit E Click this button to enable users to edit Wells user modifiable data in the Plate window the Plate window using the mouse cursor MasterPlex QT www miraibio com 168 Plate gt Virtual Pipette Plate gt Analytes Filter Plate 7 Import Standards Plate 7 Data Charts Plate View Curves Show 7 Show Well Tvpes Show 7 Show Well Groups Plate gt Show MFI lt Limit Plate gt Show CON lt Limit Plate gt Show Count lt Limit Plate gt Subtract Background Plate 7 Print Plate Wa APPENDIXB MASTERPLEX QT TOOLBARS Enables the virtual pipette that is used to transfer analyte data from a source plate to a virtual plate Opens the virtual analyte filter Note This button is only available when the virtual pipette 1s turned on When toggled on this button enables the user to copy a standard data set from one plate to another using a drag and drop operation Opens a Data Chart window and displays the well chart of the selected data type for the selected well s Opens the Standard Curves window for the active Plate window Displays the Report Generator dialog box
132. rPlex QT MasterPlex QT www miraibio com 15 CHA PTER3 erPlex QT 3 0 DER File Plate Calculations Shov Window l Er E p FT i arju a GE ZY ma le ab Tm 3 FS Ix 4 mlo MESIE ji als ley EX Stay nlt Fie Edi Wee Feyies Tooke Hao G Qo d 5 E fici ES Cop roga Fe c HEachESa tatere OH E Dl estadales ze Ee Fries x Ke a Tess Data Had fieri CH Matatani GT vage csv JEN Merce Pere Conn Serer 2716 430 PIN J VH MaeberPle QT 24 HDP oxy XiED Mirari Era Corea Ze 1D 231200 27 0M SET Aus cr S4 ED Miromafi Errol Comma Sapa MECO 10 58 PA 1 donurenbahone Sink IR PX Pis TH 1000 re L3 eres A LI HTL hap W sho E eta Sham misis inns Ini H O hiie E L3 hiim Expl or H Lj krios B G Litr C heny bei E gt Figure 3 8 MasterPlex QT and Windows Explorer application windows Use a drag and drop operation to open a csv xls Ixd or mlx file s in the MasterPlex QT application window 3 9 Viewing Data in the Plate Window 1 If more than one Plate window is open select the Cascade ET ile Horizontally or Tile Vertically menu from the window menu bar to arrange the Plate windows for easier viewing 2 To change the data displayed in the well grid a Click an analyte in the Bead Set panel b Make a selection from the data type drop down list The well grid displays the data for the selected analyte Figure 3 9 shows the components of the Plate window Table
133. racted analyte concentrations If this option is not chosen the background MFI value is not considered during calculation of the analyte concentrations MasterPlex QT www miraibio com 161 APPENDIXA PREFERENCES All This method of computing background subtracted analyte Calculations concentrations subtracts the background MFI from each member of the standard data set then fits the standard curve The method subtracts background MFI from unknown MFI then interpolates the unknown analyte concentration from the standard curve Regressions This method of computing background subtracted analyte Only concentrations subtracts the background MFI from each member of the standard data set then fits the standard curve The method does not subtract the background MFI from the unknown MFI before interpolating the unknown analyte concentration ek NOTE The All Calculations method is recommended The Regressions Only method provides backward compatibility with data generated in MasterPlex QT 1 0 Background Type If there are three or more background wells in the assay choose one of the following methods for computing background MFI Average Background Bkg MFI Bkg MFI Bkg MFI Bkg MFI n where n the number of background wells in the plate Peak Value Highest background MFI value Lowest Value Lowest background MFI value Plate Info Plate Name Displays the name assigned to the result file in the Luminex 100 2
134. residual plot is displayed Figure 5 17 Click the standard curve of Difference between calculated and expected interest for the residual plot Br Ee Local Curves Standard Curve Chart Residual Plot Standard Data El de 034 Mousf IL 5 Residual Plot 034 Mouse IL 5 Standard 1 de 038 Mouse IL 10 bo gf Std Curve 1 a A 054 Mouse IL 2 E 5td Curve 1 B WI 073 Mouse IFN gamma beg Std Curvel MIT 077 Mouse IL 4 ut Std Curve 1 E Tec Curves de tires 034 Mause IL 5 038 Mouse IL 10 054 Mouse IL 2 de 073 Mouse IEN gamma 077 Mouse IL 4 Residuals Figure 5 17 Residual plot Each point represents the difference between the expected and calculated concentrations of the standard data for the selected analyte The residual plot helps you identify non constant variability heteroscedasticity in the data For more information on detecting heteroscedasticity using a residual plot see Appendix C Viewing Standard Data amp Specifying Outliers In the Standard Data tab you can view information about each standard and MasterPlex QT www miraibio com 77 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION specify outliers in a standard data set The software ignores outliers in the calculation of the standard curve 1 In the Standard Curves window click the Standard Data tab 2 Click the standard curve of interest gt The data for the selected standards are displayed Figure 5 18 Table 5 1 explains the i
135. rid MasterPlex QT can compute the diluted or original analyte concentration For a diluted unknown Original concentration Dilution factor Calculated concentration Setting the Concentration Calculation Preference When you work with diluted unknowns you must specify whether the software computes the diluted or original analyte concentration 1 Click the Preferences button 9 Alternatively select File gt Preferences from the menu bar gt The Preferences dialog box appears Figure 5 8 2 Click the Calculations tab 3 To display the diluted analyte concentration for a diluted unknown in Plate window confirm that the Calculate dilution factor option is not chosen MasterPlex QT www miraibio com 67 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION 4 To display the original undiluted analyte concentration for a diluted unknown in the Plate window choose the Calculate dilution factor option ek NOTE If you change the Undo dilution option you must recalculate the analyte concentrations Preferences Application Regression Calculations Standard Points O Average standards IG Individual points Check this option to include dilution Factor in the Calculation Type concentration calculation e Absolute Quantification Fold Change Normalization Calculate Dilution Factor C Regression Choose this option to display the original undiluted analyte concentration in the well gri
136. roperties SUS j 4 5 6 11 Reports malytes 3 4 88 034 Mouse LS 038 Mouse IL 10 2 054 Mouse IL 2 jib 073 Mouse IEN gamma A OF Mouse IL 4 Navigator Plate Window Window Figure 3 5 Plate window displaying results data More than one Plate window can be open at the same time 8 To import additional Luminex results files using the Plate Wizard click the Plate Wizard button G and repeat step 1 to step 6 Each set of results data 1s displaved in a separate Plate window Importing Luminex Results Using the Toolbar or Menu Bar Xou can import a Luminex results file using the toolbar or menu bar 1 To import a csv xls or Ixd file click the Open button or select File gt Open from the menu bar gt The Open dialog box appears Figure 3 6 MasterPlex QT www miraibio com 13 CHA PTER3 GETTING STARTED EN Averagestd csv E Hi0Plex csv EE File name Files of type All Files mix bd xls csv Figure 3 6 Open dialog box 2 Navigate to the directory of the csv xls or Ixd that you want to import NOTE The Luminex default directory is named Output 3 Select one or more csv xls or Ixd files and click Open To select adjacent files press and hold the Shift key while you click the first and last file in the selection To select nonadjacent files press and hold the Ctrl key while you click the files of interest The Plate window opens and displays the results data Figure 3 7 MasterPl
137. rs sometimes are interested in studying the quantified relationships between groups Instead of calculating the absolute concentration values they may want to know the response ratio Fold Change between Control and Treatment groups The following demo will guide your through setting up and analyzing a single QuantigenePlex plate 1 First we will begin by designating the bead regions House Keeper that you want to normalize the data with Right click on a bead region and select housekeeping from the drop down menu 2 Now we will start to group our wells according to their sample type There are basically three types of the groups in relative quantification Background Control and Treatment There is at most one background group while the other two types may have multiple groups Each treatment group is linked to one control group 3 Now we are ready to calculate the Fold Change Click Calculation button from the toolbar to open the calculation dialog On the Calculation dialog there are three options for relative quantification analysis 1 Fold Change without normalization this will only calculate the MFI ratio between the Control and Treatment groups without any data normalization 2 Fold Change with normalization this will perform a normalization step first and then calculate the Fold Change between groups based on the normalized data 3 Normalization only this will only perform normalization on plate in one
138. sterPlex QT www miraibio com 156 CHA PTER8 MASTERPLEX QT REPORTS 5 Click Apply to generate custom report which is then displayed in a report window Figure 8 21 P View b Save a Print Report Per Unknown Replicates Ind A2 mouse 1935 74 1710 69 2023 28 serum B2 10 2158 50 1775 78 2059 35 C2 661 52 613 58 915 25 D2 702 55 628 00 927 03 E2 319 03 269 99 617 01 F2 261 71 247 44 696 57 G2 44 05 NaN 624 29 H2 71 11 NaN 660 42 A3 a 2430 09 2197 29 2119 35 B3 2339 46 2124 73 2093 40 C3 699 45 782 05 900 32 Unknown 1 pz SE 797 45 169 27 140 86 9 987 88 389 71 148 89 57 33 1056 8 E3 323 93 326 66 504 19 F3 261 71 326 66 581 70 G3 44 05 NaN NaN H3 25 90 NaN 417 74 A4 ine 1839 86 2004 27 1843 45 B4 2026 42 2090 71 1885 33 C4 635 52 670 74 805 60 D4 75121 676 61 821 04 E4 314 08 274 23 655 09 F4 283 34 290 41 670 43 G4 145 85 NaN 543 50 H4 202 22 NaN NaN Figure 8 21 Opened custom report 6 From the report window user has options to save and print report as well as to load report previously saved Figure 8 22 MasterPlex QT www miraibio com 157 CHA PTER8 MASTERPLEX QT REPORTS La View Ld Dave Print Report Per Unknown Replicates Figure 8 22 Save and Print icon for custom report NOTE When creating custom report in html xml format user has an option to export Standard Curve images in base64 string into the report To convert the base64 string from the custom report back to
139. t License Obtain Product License CO Network Client License Mode Requires MasterPlex network license server License Server IP Address License Server Porti Server Name Description Password Remember Password License Expires Figure 2 5 License Information dialog box 2 To view instructions on how to obtain a license lic click Obtain Product Licenses 3 After you have obtained a license click Install New License The Open dialog box appears 4 Use the Open dialog box to locate the license lic and double click the file gt The license is installed MasterPlex QT www miraibio com 8 CHA PTER3 GETTING STARTED CHAPTER 3 This chapter provides a brief overview of data analysis using MasterPlex QT 3 0 It also explains how to start the software import a Luminex 100 200 results file csv xls or lxd and the user interface components 3 1 Overview of MasterPlex QT Analysis MasterPlex QT software analyzes results files csv xls or Ixd from the Luminex 100 200 system The analysis steps include Import a Luminex results file csv xls or 1xd Designate well types standard unknown background or control and well groups identifies members of a standard data set or replicate unknowns Define the standard data set enter standard concentrations and select a model equation for the standard curve Associate or link a standard data set to an unknown group s Compute the analyte c
140. t mode drop down list want to edit click the arrows to edit a single dilution factor ja 4 Y IE TECA T Be Ma cu Go MS Bead Set Properties Str dore vn 200 Cer A O34 Mouse IL 5 Figure 5 9 Steps to edit a dilution factor MasterPlex QT www miraibio com 69 CHA PTER5 STANDARD CURVES amp ANALYTE CONCENTRATION 5 3 Generating Standard Curves Computing Analvte Concentrations MasterPlex QT carries out a two step calculation sequence when it fits the standard curves The software fits a standard curve for all defined standard data sets interpolates or extrapolates analyte concentrations for the unknown groups that are linked to the standard data set Standard Curve Preferences If a standard data set includes replicates MasterPlex QT can fit a standard curve two different ways Individual points method Treats each point in the standard data set default individually for curve fitting For example if there are three replicates of eight standard wells the software fits a standard curve using all 24 data points If the experimental errors are independent choose this option Average standards method Computes the average of the replicate standard data points and fits the standard curve to the averaged points If the experiment errors are dependent on each another choose this option 1 To view or change the standard curve preference click the Preferences button Alternatively select
141. ta start copying the program files IF you want ta review or change any settings click Back If vou are satisfied with the settings click Next to begin copying files Current Settings Installation Path C Program FilessHitachiS oft MasterPlex QT 3 04 Installshield Figure 2 3 InstallShield Wizard Start Copying Files window 4 To copy the files to the selected directory click Next gt After the installation is completed the InstallShield Wizard Complete window appears Figure 2 4 MasterPlex QT www miraibio com 6 CHAPTER2 INSTALLING MASTERPLEX QT MasterPlex QT 3 0 In InstallShield Wizard Complete Setup has finished installing MasterPles QT 3 0 on pour computer BI View release notes InstallShield ps Figure 2 4 InstallShield Wizard Complete window 5 Choose the View release notes option and click Finish MasterPlex QT www miraibio com CHAPTER2 INSTALLING MASTERPLEX QT 2 3 Installing a License 1 Double click the MasterPlex QT icon EISE on the workstation desktop Al The License Information dialog box appears Figure 2 5 License Information Product Name MasterPlex OT 3 0 version 3 0 1 Build 166 User Name Institute Division Number of Licenses Date Issued Licensed Version Licensed Machine ID Expiration Date Machine ID af this PC 3D FS BABL BAD 7 e Stand alone License Mode Obtain Demo License Install License File Void Curren
142. tab Color tab Standard tab Displays the selected data type value for the CHA PTER3 GETTING STARTED active selected well Some data types can be edited see Table 3 1 Enter a new value in this box to edit well data Displays a list of the analytes bead sets in an assay Shows the color that represents each analyte in the multi well chart To change a color for an analyte right click the color swatch and choose or define a color in the color palette that appears Displays a list of the local or imported standard curves for the plate MasterPlex QT www miraibio com 18 CHA PTER3 GETTING STARTED Table 3 1 Data Types in the well grid Data Type Edit Data Median The median fluorescence intensity measured No Fluorescence Intensity MFI Count Concentration Dilution Standard Independent Value Sample Names Outlier selection Well Colors Group Numbers Replicate tvpe Standard Control Links Sample Average Standard Deviation CV Normalized Data by the Luminex 100 200 system for a bead set count The number of beads per bead set detected No by the Luminex 100 200 system specified by the user in the Luminex software The analyte concentration that is computed Yes interpolated or extrapolated from the user selected standard curve The dilution factor for the well Yes standard Value Analyte concentration fora Yes standard Independent Value The agent conce
143. target Gene Prompt before calculation A Bk v Prompt before recalculation Default ae Ok a Cancel ad Apply Figure 3 12 Preferences dialog box Plate tab 2 Click the Plate tab 3 Enter the concentration MFI or bead count threshold 4 Click Apply when you are finished 5 To return the plate preferences to the factory set defaults click Default To identify wells in the Plate window that contain data less than threshold 1 Make a selection from the Bead Set panel Figure 3 13 2 Select the data type MFI count or concentration from the data type MasterPlex QT www miraibio com 21 CHA PTER3 GETTING STARTED drop down list 3 To identify wells with MFI data less than threshold select the GB show lt MFI Limit from plate menu A red border is placed around the well Figure 3 13 To identify wells with bead count data less than threshold select the I show Count Limit menu from plate menu C A red border is placed around the well To identify wells with concentrations less than threshold select the m Show CON Limit menu from plate menu C A red border is placed around the well Click the analyte of interest Select MFl concentration or count Untitled Plate 2 iL 5 csv L Ge Bead Set Properties E Analutes amp 034 Mouse IL 5 201 00 65 50 T 2650 ES Number of Wells 56 034 Mouse IL 5 Figure 3 13 Well grid in the Plate window Red border
144. te standard data points are averaged and the standard standards curve is fitted to the single set of averaged data Choose this option if the experimental errors are not independent of each other Individual Replicate standard data points are not averaged and the standard points curve is fitted to all of the data points For example 1f there are three replicates of eight standard wells the standard curve 1s fitted using all 24 data points Choose this option if experimental errors are independent of each other MasterPlex QT www miraibio com 165 APPENDIXA PREFERENCES Diluted Unknowns Samples can be diluted prior to the assay and analysis After MasterPlex QT interpolates the diluted unknown analyte concentrations from the standard curve it can compute and display the original undiluted concentration in the Plate window Original concentration Diluted concentration Dilution Factor The dilutions factors are manually entered in the Plate window For more information see 4 Click Apply when you are finished To return the plate preferences to the factory set defaults click Default MasterPlex QT www miraibio com 166 APPENDIXB MASTERPLEX QT TOOLBARS APPENDIX B The toolbars that are available depend on the types of windows that are open in the main display area B 1 Main Toolbar Ah e z Figure B 1 Main toolbar Table B 1 Main toolbar buttons and functions Menu Bar Main Function Command Toolbar Button File
145. th Chart Each bar of the chart represents an analyte in a selected well The bars are organized into columns and each column represents the analytes in respective selected wells Figure 7 5 Select the data type for the chart a El Se pr E Wl Ana Mouse IL 5 v 038 Mouse IL 10 Inverted 3D Depth Chart A SC Selected wells vs all selected analytes 054 Mouse IL 2 7 v Mora Mouse IEN gamma 077 Mouse IL 4 MN 034 Mouse IL 5 MIA 038 Mouse IL 10 MA 054 Mouse IL 2 ENN 073 Mouse IFN gamma 077 Mouse IL 4 Place a check mark next to the analytes to include in the chart Inverted 3D Depth Chart selected Parent Plate IL5 Project mlx Each column of bars represents the analytes in one well Figure 7 5 Inverted 3D depth chart Click a bar to display a tool tip analyte name or y axis data Right click a bar for tool tip options MasterPlex QT www miraibio com 106 CHA PTER7 DATA CHARTS Intensity Map The intensity map is a qualitative picture of the intensity data in the wells selected in the Plate window The map provides a convenient way to look at a large number of samples and identify analytes that consistently have a high or low intensity compared to the other analytes in the same well FP NOTE The intensity map compares and ranks data in the same well It does not compare data across wells TTS 2 E 7 i EA ra l ie IL 2 IL 4 IL 5 IL 6
146. ure 3 23 IL5 Project mlx IL 5 csv EA Concentration Fold Change ze B 2617 Enter 7 4 5 6 2 034 Mouse IL 5 Be 054 Mouse IL 2 Number of Wells 96 034 Mouse IL Figure 3 23 Plate window MasterPlex QT www miraibio com 29 CHA PTER4 DEFINING A PLATE CHAPTER 4 After you import a Luminex results file csv xls lxd your analysis begins by defining a plate This chapter explains how to define and save a plate The steps to define a plate include Designate well type to identify the standard unknown background and control wells Designate well groups to identify replicate unknowns members of a standard data set or members of a regression data set Create a standard data set s by entering the concentration for each well in the standard data set and selecting a model equation for the standard curve A plate can have more than one standard data set Link each well group to a standard data set to specify the standard that is used to compute interpolate or extrapolate the analyte concentrations The plate definition can be saved as a template that can be applied to other plates The Template Manager helps you manage your templates For more information on templates see Working With Templates on page 4 9 4 1 Designating Well Type and Group Selecting Wells To select a well in the Plate window click the well in the well grid There are three ways to select mult
147. ure C 3 we can see that the deviation of the observed concentration from the expected value increases as concentration increases This means the variability is not constant Residual plots help you detect non constant variability as well as outliers If a residual plot exhibits data points in a wedge or funnel shaped pattern then we can expect the underlying data to have non constant variability Non constant variability complicates curve fitting because the regression process assumes the errors are constant across all data points When the data violate this assumption the resulting curve fit is less than optimal This is illustrated in Figure C 4 Predicted S concentration Residuals get larger as concentration increases Figure C 4 Data with non constant variability The residual increases as concentration increases When we fit a model to data the curve is applied to all of the data points as closely as possible so that the distances between the predicted and expected concentrations are minimized In Figure C 4 we can think of the lines that MasterPlex QT www miraibio com 177 APPENDIXC MODEL EQUATIONS represent the residuals as ropes and each data point as a wrench Curve fitting can be imagined as pulling the curve line so that it is as close to each point as possible without snapping the actual curve The best curve fit is reached when the curve is pulled as close as possible to each data point without breaking the
148. value Figure 7 32 Side All Choose this option to display all bars of the same type in a side by side orientation Appearance Bar Style Make a selection from the drop down list to choose a bar style Bar Width Click the up or down arrows to change the bar width Bar Dark 3D Choose this option to display three dimensional bar demarcations Border Click Border to open a dialog box of style options for borders of bars MasterPlex QT www miraibio com 131 CHA DIER DATA CHARTS 1500 T 1600 T GER 1500 1 400 y il 1301 10 100 12004 1244 77 NM M OO 11001 VAM i cm 1H 42 c7 o E 77r77 oo D DW D C D QC QN anbl MM WU O OM Tel A Ban d ml gn ml mm DO OAM wmr mlq STI GW M O wo wot soo so E UE eC t 034 Mouse IL 5 O34 Mowe ILS r 055 Morze IL 10 r 065 Moze IL 10 i i l ye AlaWes O73 Mo ze IEN gamma AlaWeES 073 Morse IFN gamma gt Figure 7 31 3D depth chart None option is the default display left Side option right 3 400 T u cT EE 0 32004 amni gt wl L al 2 00 2500 1 simili cas oh a 2904 L 2000 Bis eee vc MEE igmtH c B m B HH STRESS TE 054 Mouse ILS 7 7 D38 Morse IL 10 034 Morse ILS gt 035 Morse IL 10 pm i ies Batz 073 Mowe IEN gamma Ames 073 Mowe IEN gamma Figure 7 32 3D depth chart Sta
149. values follow a Poisson distribution In a Poisson distribution the standard error of the MFI values is equal to the square root of that value Since weights are inverses of variances we have W 1 Variance W 1 0 W 1 Y W 1 Y MasterPlex QT www miraibio com 179 APPENDIXC MODEL EQUATIONS where weight W is the inverse of variance variance standard deviation o VY Y MFI This scheme works well with most Luminex 100 200 assays It is a compromise between minimizing absolute residuals and minimizing residuals based on their relative intensity values If the 1 Y scheme is too strong for a particular data set this scheme should be tried even if the underlying data distribution is not understood Weighting by 1 X and 1 X These schemes are rarely used They give more weight to a data point that 1s closer to the left side of the graph A data point on the right side of the graph receives less weight Weighting by 1 Stdev If the distribution of MFI values for a particular assay type is understood we can derive standard errors for the values In this case the weights can be calculated exactly as 1 Stdev2 You can enter the standard deviations into the well grid under the Standard Deviation data type and MasterPlex QT will automatically calculate the weights C 5 Results of Weighting In addition to the R Square value MasterPlex QT computes 9o Recovery values that measure how well the calibration curve
150. well group s 1 To link a well group to a standard data set press and hold the Ctrl key while you click the group and the standard data set that you want to link NOTE A standard data set can be linked to multiple groups of the same well type but each group can have only one standard 2 Right click the selection and choose Link Standard from the popup menu that appears Alternatively click the Link button Link Standard from the menu bar dor Select Template gt 4 2 Working With Templates A plate definition includes well types and well groups standards including standard concentrations associated model equation and concentration units MasterPlex QT www miraibio com 45 CHA PTER4 DEFINING A PLATE links between the standard s and well groups data calculated for the plate for example analyte concentrations standard data curves or regression analysis curves for virtual plates only data manually entered in the plate for example dilution factors or independent data values plate preferences You can save the plate definition as a template You can apply a template to an active plate Templates may also be exported imported or deleted NOTE If a plate window is not open you can still delete import or export templates however you cannot load save or overwrite a template Opening the Template Manager The Template Manager is a tool that helps you manage your templates 1 Click the Template Ma
151. www miraibio com 122 CHA PTER7 DATA CHARTS Angle Enter a value to change the display angle for the graph bars when the orthogonal option is chosen Rotation Move the slider to rotate the chart around its vertical axis Elevation Move the slider to rotate the chart around its horizontal axis Perspective Move the slider to change the perspective angle of the chart The perspective effect is the dimensional appearance of the chart with respect to distance from the viewer If this option is set to zero the perspective option is disabled Note If the orthogonal option is chosen the chart perspective cannot be changed Horiz Offset Move the slider to adjust the horizontal position of the chart in the Data Chart window Vert Offset Move the slider to adjust the vertical position of the chart in the Data Chart window Legends Tab To view the chart legend properties click the Chart tab in the Chart Properties dialog box Figure 7 23 These settings determine the location and appearance of the legend in a data chart Figure 7 24 Chart Properties Charts 3D Legends Walle Series Panel Gradient Positions Visible Direction O el No Middle e Right Top O Baton Shadows Text Style ie Show Legends LE Exit Figure 7 23 Chart Properties dialog box Legends tab MasterPlex QT www miraibio com 123 CHA PTER7 DATA CHARTS selected analytes in well D2 ENNN 585034 Mouse IL 5 ENNN 110038 Mouse IL 10

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