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User Manual - Hitachi Solutions America

1. Ere bum Farm 1n Om EEEUIMLI C s sr r Y E Figure 7 14 Print Preview 2 To print click the Print amp icon from the menu bar Print setting dialog appears NOTE For more print options see appendix A section A 2 Print Preview menu MasterPlex ReaderFit www miraibio com 110 CHA 7 DATA CHARTS la Copying or Saving Chart Image The software can export the chart image to other applications The data may be copied to the system clipboard or saved in different file formats 1 Right click on the chart you want to copy or save 2 To copy the chart image in bitmap format click Copy The image is sent to the clipboard and you can paste the image data on other applications 3 To save the chart image in other formats click Export Image c The File Save dialog is opened Figure 7 15 4 Input the file name and select one of the file formats from the Save as type drop down list There are five formats available bitmap png html pdf and Excel oar Save in BD E My Recent L ocuments Desktop My Documents 33 My Computer gt mm Network Saveastype Figure 7 15 Chart Image Export
2. eee 5 9 Creating a Virtual Plate s 62 Working with the Virtual Analyte Filter 77707 67 Quality Control Manager mH 70 5 Standard Curve amp Concentration Fit Curves tab Go to Fit Curves tab M 75 Generating Standard Curve amp Computing Analyte Conc entrations eee 7 8 Reviewing Calculated Standard Data 7777007 80 Best Fit Calculation Option m 96 Statistics Toolbox aede 95 Printing and Exporting the Standard Data 7 7 89 CHAPTER 6 Reviewing Data View Results tab Add or Delete Column 02 Sort Of Filter the Column Data e8eee e 04 Exporting a Data em 06 Printing a Data 08 CHAPTER 7 Data Charts Create Graphs tab Viewing a Data Chart 09 Chart format eee eT 104 Analyte Selector eee Te RR CUR 105 Changing Color Palette Teer er eee E eC 107 Changing Chart Properties Tr 108 Printing a Chart 109 MasterPlex ReaderFit www miraibio com Copying or Saving Chart Image TU 111 amp Export Data Customized Report Manager tab Importing a User Defined Stylesheet veteres 113 Exporting a User Defined Stylesheet 114 Delete Stylesheet File from Style Sheet List 115 Including standard curve images 116 Transfo
3. Are obese the Finish boron do ext Aree raune Arpt 1 E t 1 Dim roam ed m omm iMm 5 8241 11 SL 13 76 ARE A5 m b IL IL 2 TL 3i x H0 42 0 2 L z 2 L z IL 1 Lia LE 08 12 TE IX ITE uii ELH XT ci TE Le Lhd EU IL zu ox za Lua LIB I 4140 A 41 50 454 m a 5 2 2 1 4 EE ALH 15 00 41430 s l E ILU E TE 5 FEL s 1250 124 51 334 Figure 3 20 Analyte Assign dialog MasterPlex ReaderFit www miraibio com 22 GETTING STARTED 3 9 Import csv txt xls or Open mlx Files by drag and drop 1 Open Windows Explorer and adjust the window size so that you can view both the MasterPlex ReaderFit and Windows Explorer application windows 2 Use Windows Explorer to navigate to the csv txt xIs or mxqs 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 application menu bar area Figure 3 21 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
4. Desktop examples 912 2008 1 L 6 CHher Documents Settings tslumizu Desktop exemples H1 Plex C Documents aid ettings tshimzu Desktop exemples H1 UPlex Documents ad 8 1272008 6 10 PME 1 FN gamma Lad 198 12 2008 8 19 PME 1 L 8 Cher 3 LES 19 12 2008 8 19 PME 1 TSF pha g1 5nng Eg T i FRE Dhar Figure 8 6 Transformed Data HTML format 3 Save to file or print from the menu button MasterPlex ReaderFit www miraibio com 117 APPENDIXA PREFERENCES APPENDIX A This appendix explains more details about Data Grid for Fit Curves tab and View Results tab and Print Preview A 1 Grid Customize Menu Grid customize menu allows you to customize your grid data viewing more flexibly and efficiently Grid Customize Menu 1s invoked by right clicking on the both grid column standard curve data grid and data table grid Figure A 1 T Wel Sample Name Group Nar Wel P a 3 dd l Sort Ascending 2 l Sort Ascending Analyt 12 a 1 Sort Descending a Bead 1 Sort Descending E nn A 7 x 5 S Group By This Column T ci amp Group By This Column Hide Group By Box 1 Hide Group By Box Remove This
5. n nn ee Click one of the Click same well again Input the value Click other well well you want to press character key or enter key or input the value press F2 to enter the ESC key to exit edit mode the edit mode Figure 4 16 Input manually using edit mode MasterPlex ReaderFit www miraibio com 47 CHA PTER4 DEFINING A PLATE 4 3 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 is more than one standard on the plate you can link a user selected standard to a user selected 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 Click the Link Standard button 3 To check the status select Standard Links data type from upper or lower drop down Figure 4 17 Simi 1 E F tt Sd f EST Em oo m 1 ded E Seeded 3oedadg 5 Hh Figure 4 17 Checking Linking Status MasterPlex ReaderFit
6. 63 67 76 80 1308 00 429 00 480 50 392 00 95 66 78 79 1084 50 82 00 88 00 78 00 72 79 75 70 525 00 88 50 82 00 96 50 D ee ee 79 66 76 74 201 00 37 50 38 00 37 00 E ee 81 82 85 46 66 50 32 00 32 00 34 00 NEN L LLL e 80 79 66 74 39 00 18 00 18 00 23 00 uud ee 2 78 81 68 69 Figure 4 1 Well grid selection 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 button while you drag the mouse over the wells of interest 1 2 3 4 20 00 387 00 506 50 341 00 A ee es 53 67 765 80 1308 00 422 00 480 50 392 00 95 66 78 9 1084 50 82 00 88 00 78 00 T2 79 75 70 525 00 88 50 82 00 96 50 D 79 66 Jb 14 201 00 37 50 38 00 37 00 81 82 85 96 66 50 32 00 32 00 34 00 F 7 7 7 _ m onnnnnnnnnnnnm 80 79 66 74 38 00 18 00 18 00 23 00 3 wee ee ee ee Figure 4 2 Well grid random selection To select nonadjacent wells press and hold the Ctrl key while you click the wells of interest MasterPlex ReaderFit www miraibio com 34 CHA PTER4 DEFINING A PLATE 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 click one of the icons located on the upper well
7. E Horror ite onion Lg Sree L 4 0 1 Gy ipm Vectra 15 o 80 Hron e 5 e ee 9 cendi us im ETEF a 2 Harmon is TE E xe eub 4 H ipeo 4 INE del TE 10 lL z 14 L i 6 La Analyte Figure 7 13 Multiple Well Selection MasterPlex ReaderFit www miraibio com 108 CHA 7 DATA CHARTS Table 7 4 Chart Properties Properties Icon Features Legends Customize the legend s properties Diagram Customize the diagram s properties Axes Customize X and Y axes of the diagram Note that you may select an axis to be modified on the chart preview Chart Titles Add chart titles to be displayed within a chart Point Labels Customize point label properties of the selected series Note that you may select a series to be modified on the chart preview Series Views Customize series view properties of the selected series Note that you may select a series to be modified no the chart preview bo Appearance Choose a palette to color series or their data points Also choose the style which specifies the chart s appearance depending on the current palette 7 6 Printing a Chart You can print your data with the Print button 1 Click the Print Preview button Print preview window appears Figure 7 14 MasterPlex ReaderFit www miraibio com 109 CHA 7 DATA CHARTS
8. B Im 1 i EIT 1 Calculating best fits Figure 5 18 Progress Window for BEST FIT Calculation 3 Once the best fit has been found for all analytes and all calculations have been done the Fit Curves tab is displayed Figure 5 17 gt amp Analyte IL 10 002 zm Five Bi 510001 El 10515 00 10023 06 10000 00 23 06 003 Lil Ci 510001 6152 00 493491 5000 00 65 09 F x ha D1 5001 E 3104 00 2582 15 2500 00 82 18 004 EX 514001 E 1040 00 1194 31 1250 00 55 59 J Selec Fi 510001 292 00 585 60 625 00 33 40 Information 3 eus C Fixec 2 All calculations were completed successfully E Indiv Aver 4 70687 IL 16 10000 8000 Figure 5 19 Notification Dialog of BEST FIT calculation 5 MasterPlex ReaderFit www miraibio com 86 5 STANDARD CURVES amp CONCENTRATION Best Fit method options Best Fit search is based on the index value selected in the preference dialog There are three searching index available in the preference dialog Root Mean Square Use RMSE index to choose the best curve fit Error RMSE combination R Square Use R square to choose the best combination Least deviation of Use LD of Recover to choose the best This index Recovery is good for the needs of lower concentration accuracy MasterPlex ReaderFit www miraibio com 87 5
9. Foundry NINIENN Grayscale In A Fog Median Metro 8 Mixed Ti T Module Figure 7 11 Color Palettes Nature Colors Northern Lights Office Opulent Oriel Origin Paper Pastel Kit Solstice Technic Terracotta Pie The Trees Trek Urban Verve EERE EN BN HAH NN LL BEBE ERE EU OEE TENE EETHEN MasterPlex ReaderFit www miraibio com 107 CHA 7 DATA CHARTS 7 9 Changing Chart Properties The Create Graphs tab has great flexibility in customizing the chart To enter the chart properties dialog click Chart Propertid itton You can choose to change the entire chart s properties or just one of them To change the entire chart s properties click the button To change one of the chart s properties click small drop down arrow right side of the button Figure 7 12 Figure 7 13 shows Chart Properties dialog and Table 7 4 explains the property categories Click small down arrow mark to show the each chart s property selection Chart for well A1 Chart for well B1 Chart for well C1 dh Hm ered p ood pa Iz kre Selected analytes in well C2 yr mba 1000 4 ioo x 86 a
10. 1051600 1051 xi 10023 16 152 00 49343 310400 15216 1 i 1184 31 Stoo 56 00 39 00 1003 50 mum 2951 50 990 50 48344 109400 1064 04 262 92 300 00 200 00 e006 e mm 225 45 Link Linkin Page 1242 EA Figure 6 12 Print Preview 2 To print click the Print amp icon from the menu bar Print setting dialog appears NOTE For more printing options see appendix section A 2 Print Preview menu MasterPlex ReaderFit www miraibio com 98 CHA 7 DATA CHARTS CHAPTER 7 Data Charts Create Graphs tab MasterPlex ReaderFit can display Response Value concentration or standard data in many graph formats in the create graphs tab Create Graphs tab ETE Pla Det Types Cert oe sil ID ae ii f TF Pe Vie 5 4 E e Response Vakjes vs selected wells W si Hc 21 a BH BE xx Hu Fz x Response Unies Fil wi Mx Wels in analis Figure 7 1 Create Graphs tab 7 1 Viewing a Data Chart 1 Click the Well Selector 2 button mini sized well plate is displayed under the button Figure 7 2 MasterPlex ReaderFit www miraibio com 99
11. IL 2 Sample name grouped by Analyte Chart for analyte groups 9 an 3 6 gt mu 00 C i sx c erpupod by Sample Name Figure 7 6 Group by Sample name chart MasterPlex ReaderFit www miraibio com 102 CHA 7 DATA CHARTS Replicate View Plotting group values is available by clicking the Replicate View button il Replicate View is available only on Bar style charts If you are on another type of chart when you click the Replicate View button 1t automatically re selects the Bar chart and displays the data with error bars Figure 7 7 shows an example of the replicate view chart Selected analytes in group Standard 1 Average Stdev am IFM gamma NE 1 1 Beta Average s Stdev E i 1L 6 1 Average alpha value 18801 5 12299 75 Respgnse Values 10275 25 0 GM CSF gamma 11 1 Beta 11 10 IL 2 IL 4 IL 5 IL 6 8 alpha Selected Analytes Figure 7 7 Replicate View chart MasterPlex ReaderFit www miraibio com 103 CHA 7 DATA CHARTS 7 2 Chart Format MasterPlex ReaderFit provides various chart formats Table 7 3 To change a chart format for the selected well data click the Chart Gallery and make a selection from the drop down list of Chart Gallery Table 7 3 Available chart format Icon Features Bar style
12. 7 DATA CHARTS Figure 7 2 Well Selector 2 Select the wells you want to display on the chart You can select multiple wells by pressing CTRL key Figure 7 3 Er ul Cate Pil Cams am Dark Date Geet te 1 A Reponse VUES oh Response Values selected wells E QE ap ES 124 iai M Wels analis Figure 7 4 Bar Chart for Selected Wells MasterPlex ReaderFit www miraibio com 100 CHA 7 DATA CHARTS 3 To display another data type for the selected wells click the Data Type drop down list and select one of the data type Table 7 1 Data Types Data Type Displays Response Value Response Value Concentration Calculated concentration 4 To change the data type click the Chart Type drop down list and select one of two chart types Table 7 2 Chart Types Chart Type Displays Well Group Analyte data for each user selected well Wells by Analyte Analyte data for each user selected well by analyte Wells per Analyte Analyte data for each user selected well per analyte Group by Analyte Plot the selected data type which has sample name across the analytes Figure 7 5 Group by Sample Name Plot the selected data type which has sample name across the sample name Figure 7 6 MasterPlex ReaderFit www miraibio com 101 CHA 7 DATA CHARTS i Response Values _ ER
13. STANDARD CURVES amp CONCENTRATION 9 9 Statistics Toolbox The Statistic Toolbox has a EC effective concentration or IC inhibition concentration value list Figure 5 20 MasterPlex ReaderFit calculates the EC or IC value whenever the fitting curve 1s drawn You can check the default EC o IC5 value from here or you can add any percentage value you want to check Statistic Toolbox EC or IC 4nything Analyte Standard Log 50 4 0943065 3 3685123 3 55678182 34762025 4 27256911 4 874758 2 962325 4 001921 3 9349929 37079918 Stdoo1 Stdo01 Stdo01 Stdo01 Stdo01 Stdoo1 Stdoo1 Stdo01 Stdo01 Stdo01 12225 297 5 23356 7904 45653 9125 24 2103 18736 514 12231 415 JO A52 10166 9853 agar 6222 8104 9544 Figure 5 20 or ICanything list Adding a percentage 1 Choose EC or IC you want to display by the radio button 2 Click the text box above Add button and then input desired value 3 Click Add button To calculate any EC or IC value other than ECSO TCSO a percentage value e g an in Ehe textbox below Click the Add button The new EC ar IC values will appear in the table an the left hand side ECxx Percentages EC 0 zx 100 Add Remove Print Export MasterPlex QT calculates the EC or IC value based on the inputted value and add the result in the
14. Welcome to the Installshield Wizard for MasterPlex The Installshield R Wizard will install MasterPlex on your 1 computer To continue click Next WARMING This program is protected by copyright law and international treaties Figure 2 1 InstallShield Wizard Welcome screen 2 To continue the installation click Next The Customer Information window appears Figure 2 2 MasterPlex ReaderFit www miraibio com 4 CHAP TER2 INSTALLING MASTERPLEX READERFIT 1 MasterPlex InstallShield Wizard Destination Folder Click Next to install to this folder or click Change to install to a different folder e Install MasterPlex to J C Program Files HitachiSoft MasterPlex Figure 2 2 Customer Information screen 3 Input both User Name and Company Name the click Next The Set Type window appears Figure 2 3 MasterPlex ReaderFit www miraibio com 5 CHAPTER2 INSTALLING MASTERPLEX READERFIT 1 MasterPlex InstallShield Wizard Custom Setup Select the program features you want installed Click on an icon in the list below to change how a feature is installed Feature Description EE EMI N pm The application platform E QT This feature requires 70MB on your hard drive It has 3of 3 subfeatures selected The subfeatures require 1160KB on your hard drive Install to Program Files Hitachisaft MasterPlex Y ecco zi L
15. drop down icon There are 8 file types and 7 image types to export the document See appendix A section A 2 Print Preview Menu for further details and task options NOTE You can also copy or export the Standard Curve chart by right clicking the mouse button on the chart It will Show Copy and Export Image in the pop up menu The Copy menu enables you to copy the chart image in bitmap format MasterPlex ReaderFit www miraibio com 90 5 STANDARD CURVES amp CONCENTRATION 6 Reviewing Data View Results tab View Results tab is to review the data across all analytes In this tab you can Add or delete the data column via column selector box Sort or filter the column data Change the column layout Make groups to categorize the data Print or export the data View Results tab RH Pee oe mean CLAD eer Peer Bl Tekh npo 1271 23 50 2 Ji EA 6225 5 47511 F Ed Alp po zm Dm rr Br 4 Ei 538901 LN L2LL BE Lea 63 Fl TEFEN amc Dp rrr Bop E pn m r 1 6 50 rii m p 11 5 1 Ex Lon EITI Pees Lg 53 5 003 6225 50 OD t aur m 2951 8 mar ad E 501 1054 0 TI WT pp nee p z 116 39 28 336 75 51 30 m2 Dp 155 Le DA i3 37 06 om
16. xls 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 Create a standard data set s by entering the concentration for each well in the standard data set 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 section 4 5 4 1 Designating Well Type and Group Selecting Wells To select a well in the Input Data tab click the well in the well grid There are three ways to select multiple wells To select adjacent wells Figure 4 1 press and hold the mouse button while you drag the pointer 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 ReaderFit www miraibio com 33 CHA PTER4 DEFINING A PLATE 1 2 3 4 20 00 367 00 506 50 341 00 A ee ee ee
17. 0 BI BE DD ax DC DG Cc DU e 38 00 04M m ca AL Di Oak D Dr p D E 45 00 4 F3 m pp DER T D Er D DE r 4 00 re D DE p Da i4 11200 04x 00 Figure 6 1 View Results Table MasterPlex ReaderFit www miraibio com 91 5 STANDARD CURVES amp CONCENTRATION 6 1 Add or Delete a column 1 Click Show Columns Selector 50 Columns Selector button Column selector appears Figure 6 2 Customization Background Calculated Calculated Mean Calculated SD Response oly Response Mean Response SD Response Values Background Figure 6 2 Column Selector box 2 To add a data column select one of the data types and drag amp drop it onto the data grid Figure 6 3 Response Values 1 Indepe Y Residual R Customization 10516 00 6152 00 3104 00 Cakulated CV Background 1040 00 Mean 292 00 Calculated 5D 108 50 Response 25 00 Response Mean Response SD Response Values Background Figure 6 3 Add data type to the data grid 3 To delete the column from the data grid select desired column and drag amp drop it away from the column Figure 6 4 MasterPlex ReaderFit www miraibio com 92 5 STANDARD CURVES amp CONCENTRATION Response Values Response Mean Response SD Independ 55 00 55 50 0 71 156 25 3 06 56 00 55 50 x 156 25 3 06 104 00 106 25 18 312 50 6 45 Figure 6 4
18. 00 6152 00 3104 00 1040 00 292 00 108 50 56 00 Calculated Expected Residuals Recovery 10023 06 10000 00 4934 91 5000 00 2582 15 2500 00 1194 31 1250 00 585 60 625 00 392 43 312 50 240 25 156 25 23 06 100 23 65 09 98 70 82 16 103 29 55 69 735 534 39 40 93 70 25 93 112 78 80 00 157 60 Relative Luminescence Units 12000 10000 8000 6000 4000 2000 All Selected Standard Curves 1000 Concentrations Figure 5 10 Multiple Standard Data jL 10 Standard 1 L 2 A Standard 2 JL Standard 3 TNF alpha Standard 4 jL t2 Standard 5 10000 2 In the standard data grid you add delete sort interchange the column position and set filtering for customized data viewing Figure 5 9 10 11 There are 15 data types available to review Table 5 1 2 Well Sample Name Ignored Response Values Indepe Residuals Background Calculated CV Calculated Mean Calculated SD Response Response Mean Response SD 6152 00 6299 00 3104 00 2990 50 1040 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 10000 00 10000 00 5000 00 5000 00 2500 00 2500 00 1250 00 10000 00 10000 00 5000 00 5000 00 2500 00 2500 00 1250 00 ii OE a nn Select one of the data types from the pop up menu and drag amp drop it onto the data grid where you would like to add the c
19. 1040 g 70 0 j Mark Wells EL aus 22 1 a 29 0 Figure 4 35 Aspirating Data Right click selected wells to display the pop up menu 3 To clear the data from the virtual pipette right click and select Clear from the pop up menu Figure 4 36 P a Dispense m i Delete Selected Wels Mark Wells b Figure 4 36 Clear Aspirated Data MasterPlex ReaderFit www miraibio com 64 CHA PTER4 DEFINING A PLATE 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 1 Position the mouse pointer 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 The data are added to the virtual plate in the same configuration as in the source plate Figure 4 37 oF 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 Working With the Virtual Analyte Filter on section 4 9 oF NOTE Data in a virtual plate cannot be removed but can be overwritten 1 Open a mlx or csv 10516 00 10034 50 66 00000 4911 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 Pr
20. 255 In front All Pages Behind Enter page numbers and or page ranges separated by commas For example 1 3 5 12 Clear All Figure A 12 Watermark dialog Table A 6 Text Watermark properties Property Function Text Input text word in the text box or select predefined words from the drop down list Predefined words ASAP CONFIDENTIAL COPY DO NOT COPY DRAFT EVALUATION ORIGINAL PERSONAL SAMPLE TOP SECRET URGENT Direction Select text direction from one of the followings Horizontal Vertical BackwardDiagonal ForwardDiagonal Color Open color picker and choose text font color Font Open font dialog and choose text font Size Specify text font size Transparency Set transparency for the insert text Position Select text position In front or Behind Page Range Set page range for the text water mark MasterPlex ReaderFit www miraibio com 129 APPENDIXA PREFERENCES Table A 7 Picture Watermark properties Property Load image Size mode Horizontal alignment Vertical alignment Tiling Transparency Position Page Range Function Open file open dialog and specify the image file to be inserted Select one of the size mode from followings Clip Insert the image as same size as original image Stretch Stretch the image to horizontal direction Zoom Zoom the image to the page Set horizontal alignment left center or right Set vertical alignment left center or right Fill up
21. 3 In the virtual plate select the first well where you want to dispense the data A MasterPlex ReaderFit www miraibio com 65 CHA PTER4 DEFINING A PLATE 4 Right click the well and select Dispense from the pop up menu A Clear Pipette Delete Selected Wells 1 2 3 5 The data are added to the virtual plate starting at the selected well in the same configuration as in the source plate 10516 00 5152 00 3104 00 1040 00 Figure 4 37 Adding data to a virtual plate Open a source plate mlx or csv txt or xls and create a virtual plate click the 2 button to generate the blank virtual plate MasterPlex ReaderFit www miraibio com 66 CHA PTER4 DEFINING A PLATE 4 8 Working With the Virtual Analyte Filter In a multiplex assay all of the plate wells must contain The same types of analytes with the same nomenclature The same number of analytes This is true for virtual plates as well When you add data to a virtual plate MasterPlex ReaderFit 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 names of analytes in the pipette and the virtual plate If the number of analytes in the pipette is greater than that of the destination plate the virtual analyte filter automatically appears Figure 4 38 The virtual analyte filter displays a list of the analyte
22. 617 8 9 10 11 12 13 14 15 16 17 18 19 20 1 2 P Pla 11 P E L RAF 1 1 Al 1 12 96 3 Te 2 45 6 7 9 4 25 5 SEESPRENFNENERENPRER 6 q 7 IRR 8 por 4 4 9 2444 DEN 11 o J ss Boe Bee Bevo Onn 12 13 14 Figure 3 11 Data selection by mouse dragging MasterPlex ReaderFit www miraibio com 17 3 GETTING STARTED 4 1 In case of List First select well address by mouse dragging then click Done button for well address column Figure 3 12 Column next to the well address 1s automatically selected Figure 3 13 ca VUH T OF PE SEL CENS and click Done when finished Column Well Address 4 few Figure 3 13 Auto Intensity Data Selection 4 2 In case of List Next select intensity data by mouse dragging then click done button for intensity column If auto selected column is the intensity data column just click done button for intensity column Figure 3 14 uoum Well Address Done 2 Intensity 3 Figure 3 14 Click Done Button for Intensity Column MasterPlex ReaderFit www miraibio com 18 GETTING STARTED 5 Click Next to proceed Figure 3 15 Figure 3 15 Next Button MasterPlex ReaderFit www miraibio com 19 GETTING STARTED Analyte Assignement Page Analyte
23. Delete data type from the data grid Table 6 1 Data Types in the Data Table Data Type Well Analyte Name Sample Name Replicate Group Name Outlier Response Values Response Values Background Response Mean Response SD Response CV Calculated Independent Values Calculated Mean Calculated SD Calculated CV Residuals Recovery EC50 IC50 Dilution Factor Description Well name Analyte name User specified name for the well The group number of the well Wells that belong to the same group have the same group number Well data are not included in the calculation The light intensity measured by the plate reader instrument Background subtracted value from Response Value Shows the Response Value average within the group Shows the Response Value standard deviation within the group Shows the Response Value CV within the group The concentration that is calculated interpolated or extrapolated from the user selected standard curve Standard data user inputted Shows the concentration average within the group Shows the concentration standard deviation within the group Shows the concentration CV within the group Residual Observed or calculated concentration Expected concentration Recovery Calculated Expected x 100 Half maximal effective concentration ECso half maximal inhibitory concentration ICao Dilution factor for Background Unknown and Control MasterPlex ReaderFi
24. Release the mouse button The file s open in MasterPlex ReaderFit Fle Wee Fpi To Hep Quue 29 27 seme res 35 EC AP gran Pie si TH Peres e zu v m Te Date Fidi fied H GT ines jr xxv Cones per Me HP J iC Masten QT SB Hopes ore SED Mince Comma Sepa JOG 270 C da AES cre Mirco sper ODIO 15 35 LJ donumentations 4200 MU Pie TEI 7 31 ari E L2 Hip oth pE Figure 3 21 MasterPlex ReaderFit and Windows Explorer application windows Use a drag and drop operation to open a csv txt xls or mxqs file s in the MasterPlex application menu bar area MasterPlex ReaderFit www miraibio com 23 GETTING STARTED 3 6 Tab categorized work flow ReaderFit application module consists of five tab pages Input Data Fit Curves View Results Create Graphs and Customized Report Manager Figure 3 22 designed to match the work flow in a typical multiplex data analysis session Plate Standard Curve Data Table Export Manager Figure 3 22 ReaderFit application module tabs bas er RS ae ead 34 Maps Mark sample as um coU cat on est eal Background Standard mm seb uut ami Unknown or Control A Fill out standard value Draw Standard Cur
25. Response Value concentration or error range threshold for a plate The software automatically marks wells that contain data less than the user specified threshold with a red border Figure 4 18 To set a threshold s 1 Check Show threshold marker box 2 Check one of the radio button in front of the data type you want to use as a threshold marker 3 Select equity equal symbol and input the value in the box 4 Chck Apply to reflect current setting to the plate or click OK to reflect and close the dialog box red border marks wells that contain data less than or greater than threshold for all analyte Figure 4 28 1 4 5 6 7 8 9 10 2 3 2 1022 00 126 00 1932 50 1964 00 787 00 271 00 105 04 11083 00 10831 00 587 00 5457 00 133 50 621 50 1465 00 472 50 128 00 9072 00 8899 00 5459 00 201 00 866 50 5011 50 a 7044 00 7087 00 4589 50 481 00 om 511 00 3186 00 4987 00 4440 00 575 00 2722 00 415 00 4701 00 2289 50 154 0 2824 00 2861 00 280 00 218 50 49 00 3372 00 1472 00 Bi 1484 00 1451 00 2190 00 157 50 63 00 4727 00 2160 50 831 00 310 50 59 0 Figure 4 31 Well grid Outlier Options Show threshold Show red rectangle indicator inside the grid if the Marker threshold conditions meet the criteria Response Value Use Response Value for threshold conditions MasterPlex ReaderFit www miraibio com 60 Concentration Error Range Automatic outliers CHA PTER
26. and displays the results data Figure 3 28 oo 7 BE b E Analyte Name 1 0 x a g BRENNEN zm tps pos jJ s Fle 42 00 39 00 37 00 112 00 92 00 2426 50 43 00 36 00 48 00 80 00 B A SIEGE Ei ucc laszsmsmsmsu ss It 10 1 10 1 10 IL 10 IL 10 IL 2 IL 2 IL 2 IL 2 IL 2 10516 00 10034 50 55 00 4911 00 105 50 185 00 41 00 38 00 45 50 55 00 1 10 1 10 10 1 10 1 IL 2 IL 2 IL 2 MEZ sz 6152 00 8289 00 39 00 5011 00 65 00 43 00 38 00 52 00 40 00 45 00 t sbeee be hee ee eee ee oe eee emcee eee eee 2 3104 00 2990 50 41 00 5591 00 3989 5 113 00 38 00 44 00 40 00 42 00 RSS ERE EA mto He 1 10 1 10 1 10 1 10 IL 10 1 2 IL 2 2 H IL 2 1040 00 1094 00 45 00 3070 00 1808 00 270 00 49 00 40 00 36 00 47 00 IL 10 1 10 1 10 1 10 1 10 1 2 1 2 1 2 1 2 1 2 Figure 3 28 Input Data tab MasterPlex ReaderFit www miraibio com 32 CHA PTER4 DEFINING A PLATE 4 Defining a Plate Input Data tab After you import a scanning results file csv txt or
27. appears Figure 4 11 Auto Fill Dilution Parameters Analyte Target Wells 610001 Highest Value 10000 000 Dilution Factor Unit Replicate Options Replicate Number Replicate Orientation Details Dilution Direction Fill amp Close Apply amp View Apply amp View 2 Close Figure 4 11 Auto Fill dialog box MasterPlex ReaderFit www miraibio com 42 CHA PTER4 DEFINING A PLATE Make a selection from the Analyte drop down list Select target standard group you want to fill out Enter the starting concentration for the standard data set Enter the dilution factor Make a selection from the concentration unit drop down list To select a dilution direction for the standard data set click a dilution direction arrow The gradient map shows the location and direction of the dilution gradient s Figure 4 12 tn 92 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 WERE 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 12 Example dilution gradient maps Click a dilution direction arrow to choose the dilut
28. automatically when the program 1s launched Figure 3 2 B De foes Lim EA jr zd ad Yos r Plain 1 ritual 1 Inpur Docs mm rr m a 1 m rr vix m Fapenes n i 1j i Figure 3 2 Default blank plate Paste copied data in the blank plate 1 Copy the result data from plate reader 2 Select the top left cell you want to paste the data 3 Press Ctrl V or right click and select Paste Data command Figure 3 3 Data type selection window appears Figure 3 4 1 z 4 v B Delete Selected Wells Aspirate T Figure 3 3 Paste Data command from right click menu MasterPlex ReaderFit www miraibio com 13 GETTING STARTED Choose Paste Data Type x Paste Response Values Paste Independent values Paste Sample Names Figure 3 4 Data type selection window 4 Choose appropriate data type you want to paste in 5 Copied data is pasted in the blank plate Figure 3 5 1 2 3 4 5 6 f 8 9 10 11 41 00000 39 00000 37 00000 112 0000 92 00000 2426 500 43 00000 36 00000 48 00000 80 00000 b 10516 00 10034 50 66 00000 4911 000 106 5000 186 0000 41 00000 38 00000 45 50000 55 00000 B 6152 000 6299 000 39 00000 5011 0
29. being installed Please wait while the InstallShield Wizard installs MasterPlex This may take several minutes status Figure 2 6 InstallShield Wizard Start Copying Files window 7 After the installation is completed the InstallShield Wizard Complete window appears Figure 2 7 MasterPlex ReaderFit www miraibio com 8 CHAPTER2 INSTALLING MASTERPLEX READERFIT i MasterPlex InstallShield Wizard InstallShield Wizard Completed The InstallShield Wizard has successfully installed MasterPlex Click Finish to exit the wizard Figure 2 7 InstallShield Wizard Complete window 5 Click Finish to finish the installation and close the window MasterPlex ReaderFit www miraibio com 9 CHAP TER2 INSTALLING MASTERPLEX READERFIT 2 3 Installing a License 1 Double click the MasterPlex icon on the workstation desktop The License Information dialog box appears Figure 2 5 License Information Product Hame HMasterPlex HeaderFit 2010 version 2 0 0 Build 1 Machine ID af this User Mame Institute Division Date Issued Licensed Version Expiration Date Licensed Machine ID Obtain Demo License Install License File Obtain Product License Please send an email with the saved License Request File attached to the following email address reqistercomiraibia com Network Client License Mode Requires M asterPles network
30. cannot always predict the weight values because of unknown error distribution But for many measured datasets it is often the case that the Response Values are more accurate at their lower levels so it may be reasonable to use the 1 Y weighting method to put more weight on the lower part of the curve 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 is 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 1 Y for when you expect the average distance of the points from the curve to be higher when Y is higher but the relative distance to be a constant e 1 Y for when it follows a Poisson distribution or there s a counting error e 1 X or 1 X for weighting the left part of the graph more than the right The only problem with weighting methods is
31. com 83 5 STANDARD CURVES amp CONCENTRATION or Highest standard Response Value lt Response Value lt D where A is the lower asymptote and D 1s the upper asymptote of the sigmoidal curve Figure 5 12 A Response 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 Response Value lt A the well grid displays the lowest standard Response Value preceded by lt Figure 5 12 If Response Value gt D the well grid displays the highest standard Response Value preceded by gt Equation parameters Five Parameter Logistics R Square 0 9996930 a 16 702 b 0 689 c 1246 323 d 28652 032 e 4 707 IL 10 Standard 1 10000 8000 6000 Chart area 4000 Relative Luminescence Units 2000 0 1000 10000 Concentrations Figure 5 14 Five Parameter Logistics model equation x axis log scale A 38 367 bottom asymptote D 22988 119 top asymptote Response Value values less than A or greater than D are beyond the range of the model equation MasterPlex ReaderFit www miraibio com 84 5 STANDARD CURVES amp CONCENTRATION Specifying Outliers for Standard Data Point 1 Click one of the data points on the chart you want to eliminate from the calculation Figure 5 13 Small data window pops up under the cursor and the corresponding data is highlighted in the data table F
32. 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 25 MasterPlex ReaderFit www miraibio com 53 CHA PTER4 DEFINING A PLATE Warning x Ay Are you sure you want to overwrite M5PlexSinglestd Cancel Figure 4 25 Confirmation box 1 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 you want to export 2 Click the Export button The Save As dialog box appears Figure 4 26 Save in _ examples 2 empty 52 data My Recent Documents Desktop My Documents 3 My Computer q a My Network Saveastype MasterPlex Template mda Figure 4 26 Save As dialog box 3 Choose the directory for the template that you want to export MasterPlex ReaderFit www miraibio com 54 CHA PTER4 DEFINING A PLATE 4 Enter a name for the template mxtq dr NOTE A template must have a mxtq file extension Changing the extension will render the exported template unusable Importing a Template You can import a template mxtq from a user specified location 1 In the Template Manag
33. dialog MasterPlex ReaderFit www miraibio com 111 CHAPTERS EXPORT DATA CHAPTER 8 Export Data customized Report Manager tab This chapter explains how to export MasterPlex ReaderFit data transformed by the user Custom report is a powerful and flexible tool for presenting and exporting your data Compared to regular report Custom report has greater flexibility on what and how to present data While ReaderFit stores its analysis results in an XML format 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 Extensive Stylesheet Language Figure 8 1 Customized Report Manager tab MasterPlex ReaderFit www miraibio com 112 CHAPTERS EXPORT DATA 8 1 Importing a User Defined Stylesheet 1 Click Import button m The file open dialog appears Figure 8 2 Look in 3 examples all_csv xsl norm xml xsl My Recent a jall xml xsl Documents first_analyte_html xsl 27 i conc unk xml xsl 2 html xsl L esktop data n My Documents My Computer File name My Network Files of type MasterPlex Template 7 Figure 8 2 Open File Dialog 2 Select the file you want to import to the Customized Report Manager Style sheet s name is displayed in the Style Sheet list and XSL I
34. for the selected wells 10 Cancel Figure 4 21 Dilution Factor Input Dialog 4 Edit value directory or change the value using spin button 5 Click OK when finish Dilution factor is updated on the well grid Figure 4 22 MasterPlex ReaderFit www miraibio com 50 CHA PTER4 DEFINING A PLATE 4 22 Inputted Dilution Factor Dilution for Unknowns Samples can be diluted prior to the assay and analysis After MasterPlex ReaderFit interpolates the diluted unknown analyte concentrations from the standard curve it can compute and display the original undiluted concentration in the well grid Original concentration Diluted concentration Dilution Factor MasterPlex ReaderFit www miraibio com 51 CHA PTER4 DEFINING A PLATE 4 5 Working With Templates A plate definition includes Well types and well groups Standards including standard concentrations associated model equation and concentration units Links between the standard s and well groups Data calculated for the plate for example analyte concentrations or standard data curves Data manually entered in the plate for example sample names or dilution factors 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 Opening the Template Manager The Template Manager 1s a tool that helps you manage your templates 1 Click the Template
35. grid Figure 4 3 You can also right click the selection and choose a well type from the pop up menu that appears Figure 4 4 Table 4 1 c The well type is applied to the selected well s Figure 4 3 Sample mark icons Paste Data Aspirate c D Mark Wells E Unmark F d Flate Dilution E 56 00 55 00 51 00 55528 0 Figure 4 4 Well grid pop up menu Right click a well to display the pop up menu MasterPlex ReaderFit www miraibio com 35 CHA PTER4 DEFINING A PLATE Table 4 1 Sample mark icon and context menu to define wells Well Type Button Context menu on the well erid Background Background Wells that contain no analytes Standard 3 Standard Wells that contains analyte of known concentration Unknown Unknown Wells that contains analytes of unknown 5 concentration Control Control Wells that contain analytes that function as controls for a particular assay design Unmark Unmark Clear the current marking oF If a well belongs to a group unmarking the well also removes the well from the group 3 Repeat step 1 and step 2 to mark and group other well s Designating Well Groups After you have defined the wells the wells are organized into groups automatically so that the software can identify Replicate unknowns A standard data set MasterPlex ReaderFit automatically places all background wells into one group You can d
36. grid display Well grid can be separated into upper and lower grid Each grid displays separate data type MasterPlex ReaderFit www miraibio com 30 GETTING STARTED 3 8 Saving Plate Data After you import a scanning results file csv txt or xls the data can be saved to a MasterPlex ReaderFit file format mxqs The mxqs file includes all data associated with a plate such as well definitions and computed interpolated or extrapolated concentrations To save results data csv txt or xIs to a MasterPlex file mxqs 1 Click the Save button Alternatively select File gt Save from the main menu c The Save As dialog box appears Figure 3 26 Figure 3 26 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 mxqs 1 Click the Open button Alternatively select File gt Open Plate from the main menu The Open dialog box appears Figure 3 27 MasterPlex ReaderFit www miraibio com 31 GETTING STARTED Lookin Project mlx AverageStd csv 10Plex csv E IL5 csv Femme 5 Files of type Al Files mix bed xls csv Figure 3 27 Open dialog box 2 Confirm the default directory or choose another directory 3 Select a file name mxqs and click Open c An application module window opens
37. is a fraction between 0 0 and 1 0 A high R value means the curve came close to the points When all calculations have been completed R is reported on the curve chart of the Standard tab Calculating SSyeg 15 the sum of the squares of the distances of the points from the best fit curve determined by nonlinear regression Figure C 6 A sigmoidal or S shaped curve SS 15 the sum of the square of the distances of the points from a horizontal line through the mean of all Y values MasterPlex ReaderFit www miraibio com 147 APPENDIXC MODEL EQUATIONS VA Figure C 8 A sigmoidal or S shaped curve R2 1s calculated using this equation SSrec R22 10 77 SStot Note that is not really the square of anything Also could be negative R will be negative when the best fit curve fits the data worse than a horizontal line at the mean Y value This could happen if you pick an inappropriate model or fix a parameter to an inappropriate constant value Weighting Methods 1 Y for when you expect the average distance of the points from the curve to be higher when Y is higher but the relative distance to be a constant e 1 Y for when it follows a Poisson distribution or there s a counting CITOT MasterPlex ReaderFit www miraibio com 148 APPENDIXC MODEL EQUATIONS 1 X or 1 X for weighting the left part of the graph more than the right The only problem with weighting methods is that the user
38. like the Four Parameter Logistics 4PL equation shown below A D E 1 2 Four Parameter Logistics Model F x D The assumption that the lower asymptote 1s zero 1s probably incorrect in some cases but it allows the user to reduce the number of parameters in the equation The asymptote sets a bound for the parameter In the 4PL model there are lower and upper asymptotes defined as the A and D parameters respectively See diagram below MasterPlex ReaderFit www miraibio com 140 APPENDIXC MODEL EQUATIONS D Upper Asymptote B Slope at inflection point 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 MasterPlex ReaderFit extrapolates the analyte concentration when A Response Value Lowest standard Response Value or Highest standard Response Value lt Response Value lt D where A is the bottom asymptote and D is the top asymptote of the sigmoidal curve An Response Value less than A or greater than D 1s beyond the range of the standard curve model and the concentration value cannot be extrapolated In the 2 dimensional representation of the curve shown above Response Value is the Y axis value Theoretically Response Value cannot be less than zero So setting the lower asymptote to zero 1s reasonable But there can be measured data that actually has some negative Respo
39. mode selection 2m SingleFlex Wizard Analyte Assignment Please inspect the plate at the bottom If incorrect cick the Back button to dd proper analyte made and follow the below instructions to assign analytes Single Analyte There is only one analyte in the plate Please click the Finish button to exit There is nothing need to be done Single Analyte Default 1 Multiple Analytes Analyte gt Analyte 1 Available Analytes list Remove analyte button Mini plate preview Figure 3 16 Analyte Assignment Page 1 Select wells for the analyte your are trying to specify 2 Click Assign button c Selected wells are registered as an analyte and listed the Available Analytes list Figure 3 17 3 Rename the analyte name or the color if you want default analyte name 15 Analte n color is randam Figure 3 18 4 Repeat analyte assignment Figure 3 19 5 When all wells were assigned click OK Analyte assignment dialog closes and plate is build on plate view Figure 3 20 MasterPlex ReaderFit www miraibio com 20 3 GETTING STARTED mii Are fuged Peme rogati piris brir AP ores Ew Mkh erg ey ee Pe ee rate cde ndi eee h BS arenes LIII Thee muitis To de fna prote creer dm rp Fe i mute fum cece Gran bution Yo
40. move above vs dragging rightwards as the first move below MasterPlex ReaderFit www miraibio com 40 CHA PTERA DEFINING A PLATE Select all wells within the group at one time 1 While hovering over a replicate group border the mouse pointer changes to a hand icon Figure 4 9 5 00 F 10034 50 66 00 2 00 f 6299 00 39 00 4 00 41 00 0 00 1094 00 45 00 2 00 300 00 39 00 4911 00 5011 00 5591 00 3070 00 1129 00 10 398 180 11 5 00 F 10034 50 2 00 6299 00 39 00 4 00 2990 50 41 00 0 00 1094 00 45 00 2 00 300 00 29 00 Figure 4 9 Mouse pointer changes to hand icon 2 Click the border while mouse pointer is hand icon Entire wells within the group are selected Figure 4 10 10034 50 4911 00 5011 00 5591 00 3070 00 Figure 4 10 Selected wells 4911 00 5011 00 5591 00 3070 00 1129 00 10 398 180i 11 MasterPlex ReaderFit www miraibio com 41 CHA PTER4 DEFINING A PLATE 4 2 Setting Standard Concentrations After you define and group the standard wells use the auto fill feature to help you automatically enter the standard concentrations Auto Fill icon is enabled when one or more standard groups are in the plate 1 Click the Auto Fill button 2 located above the well grid Alternatively Right click the standard data set and select Auto Fill from the popup menu The Auto Fill dialog box
41. that the user cannot always MasterPlex ReaderFit www miraibio com 149 APPENDIXC MODEL EQUATIONS predict the weight values because of unknown error distribution But for many measured datasets it 1s often the case that the Response Values are more accurate at their lower levels so it may be reasonable to use the 1 Y weighting method to put more weight on the lower part of the curve C 5 Results of Weighting In addition to the R Square value MasterPlex ReaderFit computes Recovery values that measure how well the calibration curve standard curve fits the observed data Recovery Observed Concentration Expected Concentration 100 The closer Recovery is to 100 the better the curve fit at that point If 9o Recovery is less than 100 the point is below the curve If it is greater than 100 the point 1s above the curve MasterPlex ReaderFit www miraibio com 150
42. with the same source plate csv txt or xls or mxqs 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 The data are added to the virtual plate and the virtual analyte filter closes MasterPlex ReaderFit www miraibio com 69 CHA PTER4 DEFINING A PLATE 4 9 Quality Control Manager Quality Control Manager helps you flag and optionally set as an outlier any wells whose value is outside of the range defined by the thresholds Thresholds can be assigned using the manual method for A single selected analyte Multiple selected analytes All analytes To open the Quality Control Manager Figure 4 40 click the Quality Control Manager button jy Quality Control Manager All analytes This tool helps you flag and optionally set as an outlier any well whose Value is outside of the range defined by the thresholds Thresholds can Analyte be assigned using the Manual method for A single selected analyte Multiple selected analytes All analytes After each change please click the Apply button to Save Flagged wells Mark as outlier _ LOD extrapolated Values Lower Upper Response Values Calculated Values Figure 4 40 Quality Control Manage
43. 00 65 00000 43 00000 38 00000 52 00000 40 00000 45 00000 E 3104 000 2990 500 41 00000 5591 000 3989 500 113 0000 38 00000 44 00000 40 00000 42 00000 D 1040 000 1094 000 45 00000 3070 000 1808 000 270 0000 49 00000 40 00000 36 00000 47 00000 E 292 0000 300 0000 39 00000 1129 000 119 0000 41 00000 42 00000 40 00000 37 00000 49 00000 F 108 5000 104 0000 45 00000 107 5000 111 0000 70 00000 41 00000 39 00000 326 0000 41 00000 G 56 00000 55 00000 51 00000 5578 000 59 00000 50 00000 38 00000 37 00000 128 5000 39 00000 H I Figure 3 5 Pasted data in the blank plate 6 To delete the copied data in the plate press DELETE key or choose Delete Selected Wells command from right click menu MasterPlex ReaderFit www miraibio com 14 GETTING STARTED 3 4 Importing Measurement Results and Analyte Assign To begin a MasterPlex ReaderFit analysis import a csv txt or xls file using toolbar menu bar or application icon Importing Scanning Results Using the File Open Menu File Open Icon or Application Icon 1 Choose File gt Open click the File Open icon gt or click the application icon The Open dialog box appears Figure 3 6 2 Enter the file path for the csv txt or xls that you want to import Look in examples A 2 ES E 10Plex csv My Recent E IL5 csv Documents Desktop g
44. 07 67122 Unina 1 5100 540 88 180 04 87132 Unkinawn 1 11200 54085 225 35275 8122 Urin 1 51100 540 85 3855 87 87122 Unina 1 501100 540 89 m 3044 48 87122 Unknown 1 5591 00 540 89 282 4418 11 671221 E4 Unknown 1 3070 00 520 88 185 254435 87122 Fa 1 112880 540 89 238 17748 E GA Unkmeawn 1 3 50 540 89 im 34185 EM 44 Unina 1 5578 00 52288 241 4407 15 sim Unina 1 82 00 540 89 24 31071 67122 a5 1 10550 540 89 200 34185 67122 1 65 00 540 89 i72 23715 mm 25 Uninen 1 3883 5 52288 m 317784 67122 E Unknown 1 1808 00 540 89 218 172264 67122 1 119 00 540 89 179 36503 87122 G5 Unin 1 111 00 540 89 211 35080 812 5 EE 53 00 540 89 am 21554 67122 Unknown 1 2426 5 540 89 218 212204 87122 B6 Unknown 1 18500 540 89 219 41188 67122 1 aim 540 89 202 12821 87122 26 Unkina 1 11300 54188 218 35458 67122 Figure A 13 Example of the text and image watermark MasterPlex ReaderFit www miraibio com 131 APPENDIXB MASTERPLEX ReaderFit 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 File Menu and Toolbar Table B 1 Main File menu and toolbar Menu Bar Main Function Command Toolbar Button File New Open blank plate for ReaderFit application File Open Displays the Open dialog bo
45. 1 1PlateFormatEndpointLMax LuminescenceRawFALS um 2 3 Temperature CC 123456789101112 2 PUP L P E L R F 1 1 lali 1 12 96 4 25 503 9633 9884 0124 0474 09882 7028 1 6628 1 5644 0823 3 T J1 2 4 5 6 7 8 10 Jaa 122 5 13 7543 7583 8583 9274 02221 59921 72521 553 9173 8753 4 2 3 Bove 4 4 4 8 8 8 4 4 3 3 6 3 7793 823 8364 044 0795 8336 0295 9024 154 1294 1254 3 3 3 Bove 4 1 2 2 2 3 3 3 3 7 3 6793 7663 9624 0844 0431 6241 691 7214 0864 064 215 3 3 3 4 4 5 6 5 4 4 4 4 8 3 5263 8513 784 1374 2540 6671 0520 6544 2734 0644 161 9 3 6844 0934 0373 8724 2320 2570 3250 2564 0834 2544 20 3 13 3144 1 naa nat Matt Ha n 10 3 713 6523 7073 9133 9180 1770 1790 2233 8233 8724 206 3 43 13 14 14 10 11 0 14 14 4 4 11 3 7063 6733 8113 9013 9650 1120 1210 1633 9313 8623 98 3 44 14 3 14 10 0 10 4 14 4 3 3 3 3 3 3 10 0 0 3 3 4 1 Bu 3 3 3 3 3 0 Devel 0 3 3 Bees O Figure 3 9 Data Delimitation Change 2 Select data format type from select data format box Figure 3 10 plate or list 2 Select Data Format Plate List Figure 3 10 Select Data Format Box 3 In case of Plate Select data area you want to import by mouse dragging Figure 3 11 2 3 4 5
46. 4 DEFINING A PLATE Use Concentration value for threshold conditions Use Error Range for threshold conditions Automatically check on off the outlier check box for the wells To check on click Set button To check off click Clear button MasterPlex ReaderFit www miraibio com 61 CHA PTER4 DEFINING A PLATE 4 7 Creating a Virtual Plate 1 Open the measured results files csv txt or xls or MasterPlex ReaderFit files mlx that are the data sources for the virtual plate 2 Click the Virtual Plate button The Virtual Plate dialog appears Figure 4 33 Virtual Plate Plate Dimensions To create a virtual plate please enter the application type and the dimensions for the virtual plate Type MasterPlex Read Ronis Columns OK b Cancel Figure 4 33 Plate Wizard Plate Dimensions tab 3 Enter the number of rows and columns for the virtual plate Click OK A module window opens and displays the empty well grid of the virtual plate Figure 4 34 MasterPlex ReaderFit www miraibio com 62 CHA PTER4 DEFINING A PLATE G H Figure 4 34 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 copies all of the analyte data a well including the computed analyte concentrations It remains loa
47. 6 00 1005 00 209 50 95 00 B 5850 50 5807 00 1297 00 4276 00 37 50 18 00 3271 00 7722 50 104 00 45 00 C Twocolrs Upper EEE 255 0 0 2656 00 3049 00 63 50 3277 00 1846 00 109 00 1957 00 6467 00 43 00 32 00 D EM 1104 50 914 00 57 50 1501 00 1842 00 138 00 7233 00 5535 50 27 50 185 00 E 300 00 404 00 1396 00 433 00 133 50 15 00 6247 50 3727 50 15 00 99 00 Click one color and select desired color for F the maximum value The color density E 143 50 116 00 7453 00 61 00 58 00 7190 00 4878 00 2840 50 779 00 70 00 decreases directl with the value 42 50 40 00 7817 00 3178 00 54 50 7301 00 3625 50 1662 50 343 00 31 00 y Ea Color 1 2 3 4 5 6 7 8 9 10 A 19 00 6903 00 48 50 116200 5573 00 2145 00 443 00 187 00 i On 9929 00 8989 50 6994 00 4487 00 64 00 179 00 4746 00 1005 00 209 50 95 00 One color 5850 50 5807 00 1297 00 4276 00 37 50 18 00 3271 00 7722 50 10400 45 00 7 it Two colors Upper L 255 0 0 2656 00 3049 00 63 50 3277 00 1846 001 109 00 1957 00 6467 00 43 00 32 00 CUM Wassussssuasauirasannuusas D 110450 91400 57 50 1501 00 1842 00 138 00 7233 00 5535 50 27 50 185 00 Lower EE 0 0 122 300 00 40400 139600 43300 133 50 1500 624750 372750 1500 99 00 Click t lors and select desired color for th IC WO COIO
48. Analytes in the I 0 1 Beta plate To turn the IL 10 display of the 1 2 ff IL 4 analyte on or off M 5 check the box next IL 6 to it TNF alpha Figure 7 8 Analyte Selector drop down list MasterPlex ReaderFit www miraibio com 105 Ed GM CSF gamma 1 1 Beta IL 10 1 2 1 4 IL 5 v IL 6 IL 8 TNF alpha Response Values 7 gamma Il 1 Beta Select the analyte and click the Down button IL 2 v IL 4 IL 5 v IL 6 IL 8 gamma Fi GM CSF v 11 1 Beta v IL 10 CHAPTER7 DATA CHARTS TNF alpha Response Values Il Figure 7 9 Change the analyte position All Analytes in the plate To select the analyte click the radio button next to the analyte name 8 GM CSF IFN gamma Il 1 Beta IL 10 TNF alpha 12000 10000 S000 6000 4000 2000 12309 0516 Il 1 Beta garam Figure 7 10 Analyte Selector drop down list MasterPlex ReaderFit www miraibio com 106 7 4 Changing the Color Palette 7 DATA CHARTS There are 30 color palettes available from Color Palette button o Figure 7 11 shows the palette names and their corresponding color patterns Apex BEE Aspect m i Black and White Chameleon Civic B Concourse Equity Flow 1
49. Chooser H 9 Best Fit q WE rics E Best Fit all columns Figure A 7 Opening the Filter Builder MasterPlex ReaderFit www miraibio com 122 APPENDIXA PREFERENCES Basic steps for constructing filter criteria with the Filter Builder 1 Right click on the grid column 2 Choose Filter Editor Filter Builder window appears Figure A 8 Click to change the current operator Cs Filter Builder add remove conditions and groups of To add filter condition click button Sample Mame Begins with enter a value conditions To remove filter condition QD And click x button Or aD Mot And QD Not Or Add Condition Add Group Clear All Figure A 8 Filter Builder Window 3 Click Sample Name and choose one of the column item 4 Click Equals and choose one of the operator Table A 3 shows the conditions you can choose from 5 Click lt Enter a value gt and enter the operand value 6 Click the Apply button to apply the filter setting to the grid If you want to apply and close the Filter Builder window click the OK button Table A 3 Available Commands for Filter Icon Operator Function Shows only Sample Name lt value gt Does not equal Shows only Sample Name value gt 16 greater than Shows only Sample Name gt lt value gt 2 15 greater than or equal Shows Sample Name lt value gt and Sample Name gt lt value gt
50. Column Remove This Column Column Chooser a Column Chooser Best Fit Best Fit Best Fit Call columns Z i Best Fit Call columns T Filter Editor 52 T Filter Editor Figure A 1 Grid Menu in the Fit Curves tab and View Results tab Table A 1 Main toolbar buttons and functions Icon Command Function A Sort Ascending Sort right clicked column by ascending Fl 7 Sort Descending Sort right clicked column by descending Clear Sorting Clear sorting of right clicked column if the MasterPlex ReaderFit www miraibio com 118 APPENDIXA PREFERENCES Group By This Column Make a group by right clicked column The group is located under the last position of current group hierarchy Hide Group By Box Display Hide Group Box Figure A 2 on the upper side of the columns It enables you to make group s by drag and drop operation Remove This Column Remove right clicked column zm Column Chooser Show Column Chooser box BEEN Adjust the width of right clicked column H r4 Clear Filter Clear filter condition of right clicked column 1f column is filtered Filter Editor Open Filter Builder window Figure A 6 Best Fit all columns Adjust the width of all columns Group Box The Group Box appears on the upper side of the column Figure A 2 by choosing the Group By Box menu from the right click menu on the grid column You can drag one of the column you want t
51. IAL DAMAGES ARISING OUT OF THE USE OR INABILITY OF SUCH 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 MasterPlex ReaderFit www miraibio com 111 LICENSE AGREEMENT The Government acknowledges HISAL s representation that the Software 15 Restricted Computer Software as that term 1s defined in Clause 52 227 19 of the Federal Acquisition 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 document
52. LOAN RESELL FOR PROFIT OR DISTRIBUTE TERM 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 15 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 15 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 t
53. LOD pop up well grid Exclude selected wells from outlier marking Select wells to be used in ULOD calculation ULOD Mean MFI of selected wells 8 Stdev Figure 4 45 ULOD tab CV Use value of the group as a threshold criterion Figure 4 46 Flag the values greater than the specified CV value cv Extrapolated Values For all analytes I Set SoC threshold as greater than 10 Figure 4 46 tab MasterPlex ReaderFit www miraibio com 73 CHA PTER4 DEFINING A PLATE Extrapolated Values Flag the values extrapolated by the standard curves Figure 4 47 Extrapolated Values For all analytes Figure 4 47 Extrapolated values tab MasterPlex ReaderFit www miraibio com T4 5 STANDARD CURVES amp CONCENTRATION 7 5 Standard Curves amp Concentrations Fit Curve tab This chapter explains how to generate standard curves and compute interpolate or extrapolate analyte concentrations from the standard curves 5 1 Go to Fit Curves Tab Click Fit Curves tab then application window displays the Fit Curves tab page Figure 5 1 Fit Curves tab Fili Oran ramum Fert Marge Gas Rampoca mam cere s mea a 1 Le Ent E agat A I 5amcirzr man Drie
54. Manager button The Template Manager appears Figure 4 23 2 Click a template the Available Templates list to view information about the template Template Manager x Available Templates Template Properties Human 10 MsSPlexSingleStd fez M5PlexSingleStd Created by Creation date 9 3 2008 Creation time 9 59 45 pM Description Mouse 5 plex Single standard set Load Export Import j Overwrite Delete Close Figure 4 23 Template Manager shows available templates MasterPlex ReaderFit www miraibio com 52 CHA PTER4 DEFINING A PLATE Click a template to view information about the template Saving a Template You 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 The Template Name and Description box appears Figure 4 24 Mame Description Cancel Figure 4 24 Template Name and Description box 2 Enter a name and descriptions for the template and click OK The new template is added to the Available Template list Loading a Template You can apply or oad 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 Load button The template is applied and the well grid shows the new well attributes well type well group and links to standard
55. Number Replicate Orientation Side by Side Sidebyside Stacked Details Dilution Direction Figure 4 13 Replicate Options Choose Replicate Number from 2 to 5 If replicate number is selected other than None Side by Side and Stacked are selectable MasterPlex ReaderFit www miraibio com 45 CHA PTER4 DEFINING A PLATE 10000 00 M 10000 00 39 06 39 06 m _ lA Replicate Number 2 977 Replicate Orientation Side by Side Dilution Direction iba 625 00 1 78 13 ak 0 31 0 31 Replicate Number 3 Replicate Orientation Stacked Dilution Direction 0 5 o o oo E sls Figure 4 15 Stacked Replicate Options MasterPlex ReaderFit www miraibio com 46 CHA PTER4 DEFINING A PLATE Input Standard Data Manually If your standard data series does not have sequential diluted values use direct edit mode on the well grid Figure 4 16 Response Val 2949 50 E Response Values Y axis Response Mean Response SD Response Waly Independent Values X exis B Calculated values 1 Calculated Mean Calculated SD 33 Calculated CV Sample Replicate Group 62 Outlier Status D Dilution Factor Analyte Name ap 7 Standard Links us Set data type as standard in the upper or Standard data is shown in the well grid lower dropdown list ITI LLL 2 00 1134000 11340 00
56. S 4 Make sure the module name you purchased and click icon you don t want to install Inziallseld Erizard Cuin fetus Custom lack Hes popan asirai pou Select the program ee eee an the lat bales ip chargg hors afarira Ciokan amines Em ut neti cg Foro Se inward Pes T zd Tia 4 Pease i T t n red ba pnilacal hard chive EJ ad al miaacurar oil be roiak an kea hard dria This eser oun SEE e your hard AT Figure 2 4 Install module selection window MasterPlex ReaderFit www miraibio com 6 CHAPTER2 INSTALLING MASTERPLEX READERFIT 5 To continue click Next The Ready to Install the Program window appears Figure 2 5 i amp MasterPlex InstallShield Wizard Ready to Install the Program The wizard is ready to begin installation Click Install to begin the installation If you want to review or change any of your installation settings click Back Click Cancel to exit the wizard Figure 2 5 Ready to Install the Program window 6 Click Install gt The Start Copying Files window appears Figure 2 6 MasterPlex ReaderFit www miraibio com 7 CHAP TER2 INSTALLING MASTERPLEX READERFIT i amp MasterPlex InstallShield Wizard Installing MasterPlex The program features you selected are
57. The analyte concentrations are interpolated from the standard curve that is 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 oF NOTE A plate can have more than one standard data set The standard data sets may have different concentrations or model equations MasterPlex ReaderFit www miraibio com 16 5 STANDARD CURVES amp CONCENTRATION Selecting a Model Equation for the Standard Data Set 1 Select an analyte from the left analyte pane 2 In the right pane select one equation from the drop down list Equation symbol is shown under the drop down list Figure 5 2 Equations A Five Parameter Logistics D F x 4 1 Select for all analytes Figure 5 2 Model Equations drop down list Model equations available for regression analysis of a standard data set 3 Select a model equation 4 To apply the selected model to all analytes choose the Select for all analytes option 5 To apply weighting during curve fitting choose the Use Weighting option and select a weighting method from the drop down list 6 To fix the lower asymptote to zero sets A 0 select the Fixed lower asymptote zero option Figure 5 3 This is an option of the Five Parameter Logistics and Four Parameter Logistics equations NOTE This feature is reasonable to use if enough background was s
58. USER MANUAL For Microsoft Windows MasterPlex ReaderFit Quantitative Analysis Module HITACHI Inspire the MaraiDBio Grou 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 33340 10201 TRADEMARKS Microsoft is a registered trademark of Microsoft Corporation COPYRIGHT 2009 2010 Hitachi Software Engineering America Ltd All Rights Reserved Ver P072010 22 MasterPlex ReaderFit 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 1s 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
59. ata point as a wrench Curve fitting can be imagined as pulling the curve line so that it 1s 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 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 MasterPlex ReaderFit www miraibio com 144 APPENDIXC MODEL EQUATIONS concentrations decreases Levenberg Marquardt algorithm LMA The Levenberg Marquardt algorithm LMA used by MasterPlex ReaderFit minimizes a function over a space of parameters of the function It 1s commonly used in least square fitting and nonlinear regression curve fitting LMA interpolates between the Gauss Newton algorithm GNA and gradient descent LMA is more robust than GNA but can be slower for very large datasets Robustness 1s a measure of how well the algorithm can
60. ation RESTRICTED RIGHTS LEGEND Use duplication or disclosure by the Government 15 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 is 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 1s 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 MasterPlex ReaderFit www miraibio com 1V LICENSE AGREEMENT 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 fur
61. ay in the well grid Make a selection from this drop down list to choose the data type displayed in the well grid Click the drop down arrow to view the list and select a data type See Table 3 1 for a description of the data types The well grid can be separated into upper and lower grids by clicking lower grid switch See Figure 3 8 for more further details Enables the lower grid data selection and display Displays the selected data type value for the active selected well Displays a list of the analytes in an assay Icons for sample marking Displays the background subtracted value Icons for operating input data tab MasterPlex ReaderFit www miraibio com 28 GETTING STARTED Table 3 1 Data Types in the well grid Description Edit Data i plate reader instruments The analyte concentration that is computed Data Type Response Values Calculated Values Independent Values Standard Links Outlier Status Sample Name Replicate Group Name Analyte Name Dilution Factor Response Mean Response SD Response 96 CV Calculated Mean Calculated SD Calculated CV Intensity interpolated or extrapolated from the user selected standard curve The dilution factor for the well Yes Shows the standard number that is linked to Yes each well or well group A check mark indicates the well is outlier and Yes the well data are not included in the calculation of concentrations User specified na
62. bio com 2 CHAP TER2 INSTALLING MASTERPLEX READERFIT 2 Installing MasterPlex ReaderFit This chapter explains the minimum hardware and software requirements needed to install and use MasterPlex ReaderFit It provides installation instructions for a computer for your analysis 2 1 Requirements For optimum performance MasterPlex ReaderFit requires hardware and software that meet or exceed the following specifications Minimum Hardware Requirements Platform CPU Intel Pentium 4 2 GHz or equivalent Intel Pentium 4 2 GHz or better recommended otorage space 120 MB available hard drive space for the installation HDD Input devices Keyboard and mouse or other pointing device Video RAM 32MB or higher Monitor resolution XGA 1024x768 pixels or higher 1280 x1024 recommended Memory RAM 512MB or higher for Windows XP Vista 7 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 XP Vista 7 Microsoft NET3 5 framework required MasterPlex ReaderFit www miraibio com 3 CHAPTER2 INSTALLING MASTERPLEX READERFIT 2 2 Installing MasterPlex ReaderFit 1 Insert the MasterPlex CD ROM in the workstation computer and double click setup exe The installation begins and the InstallShield Wizard appears Figure 2 1 iz MasterPlex InstallShield Wizard
63. but HISAL retains ownership of all 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 15 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 MasterPlex ReaderFit www miraibio com LICENSE AGREEMENT copies 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
64. d Description Well name Analyte name User specified name for the well The group number of the well Wells that belong to the same group have the same group number If this is checked this data is not included for the regression analysis The light intensity measured by the plate reader instrument Background subtracted value from Response Value Shows the Response Value average within the group Shows the Response Value standard deviation within the group Shows the Response Value CV within the group The concentration that is calculated interpolated or extrapolated from the user selected standard curve Standard data user inputted Shows the concentration average within the group Shows the concentration standard deviation within the group Shows the concentration CV within the group Residual Observed or calculated concentration Expected concentration Recovery Calculated Expected x 100 Response Value background value for the plate 4 Check the fitted curve on the standard curve chart Figure 5 12 If the standard curve uses a sigmoidal model for example the Four Parameter Logistics equation Figure 5 12 the software interpolates the analyte concentration when Highest standard Response Value lt Response Value lt Lowest standard data point The software extrapolates the analyte concentration when A lt Response Value lt Lowest standard Response Value MasterPlex ReaderFit www miraibio
65. d rectangle indicator inside the grid if the range threshold conditions meet the criteria Mark as outlier Mark flagged data as outlier Threshold Options Manual Use raw value or concentration value for threshold conditions Figure 4 43 Manual Extrapalated Values Flag the wells outside of this Lower range Response Values 150q7 Calculated Values re Combination selection is allowed Apply Figure 4 43 Manual threshold tab LLOD Lower Limit of Detection Flag the lower values than the LLOD calculation value Figure 4 44 LLOD is based on the Response Value mean value of the selected wells plus the standard deviation multiplied by the user selected number So cee oes For all analytes Select the base Calculate LLOD Response Value i wells from pop up Exclude selected wells from well grid Select wells to be used in LLOD calculation LLOD Mean MFI of selected wells E Stdey ao i Figure 4 44 LLOD tab MasterPlex ReaderFit www miraibio com 12 CHA 4 DEFINING PLATE ULOD Upper Limit of Detection Flag the upper values than the ULOD calculation value Figure 4 45 ULOD is based on the MFI mean value of the selected wells plus the standard deviation multiplied by the user selected number CV Extrapalated values Select the base For all analytes MFI wells from f Calculate LI
66. de columns selector Selector m Export to File xs Export data table by selected format Format Excel CSV PDF HTML Text Print Preview Open print preview window Table Template Open table template manager Merge Cells By BE Toggle the cell merge mode Group MasterPlex ReaderFit www miraibio com 135 APPENDIXB MASTERPLEX ReaderFit TOOLBARS B 4 Create Graphs Tab Toolbar The chart toolbar is available on the top of the chart view area in the Create Graphs tab Figure B 5 Chart toolbar Table B 5 Chart toolbar buttons and functions Menu Bar Function Command Button Replicate View Toggle replicate view mode Analyte Selector Shows drop down list for all analytes Well Selector Shows mini sized plate view Chart Gallery Color Palette Shows drop down list of available palette Shows drop down list of available chart format Chart Properties Open chart properties dialog Chart Template m Open chart template manager ally Print Preview Open print preview MasterPlex ReaderFit www miraibio com 136 APPENDIXB MASTERPLEX ReaderFit TOOLBARS B 5 Customized Report Manager Tab Toolbar The Customized Report Manager toolbar 1s available on the top of the preview area in the Customized Report Manager tab Figure B 5 Chart toolbar Table B 6 Chart toolbar buttons and functions Menu Bar Function Command Button Print Pr
67. ded until you dispense or clear the pipette oF 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 Working with the Virtual Analyte Filter on section 4 8 1 In the source plate select the 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 oF NOTE Selecting non adjacent wells is not recommended 2 Right click the selected wells and select Aspirate from the pop up menu that appears Figure 4 35 c The data for the analytes in the selected wells are added to the virtual pipette and 1s ready to dispense into a virtual plate MasterPlex ReaderFit www miraibio com 63 CHA PTER4 DEFINING A PLATE oF 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 LJ button to turn background subtraction on or off i gc 3 4 41 00000 39 00000 37 00000 112 00 A 4 A ELA C26 E4999 1 oe SALE A B C 3104 91 0 D
68. do the curve fitting with fewer data Given two points on a data grid LMA iteratively takes steps from point 1 towards point 2 adjusting its slope each time This is harder to visualize in a multidimensional space but LMA adjusts in all directions simultaneously as it fits a multivariate function LMA adjusts the parameters of the equation 1 e the Five Parameter Logistics or Four Parameter Logistics equations at each iteration of the algorithm until there 15 no improvement in the curve fit or until it reaches a maximum threshold 100 iterations in this case To accomplish the curve fitting it combines two common statistical math techniques Gradient Descent and Gauss Newton GNA Gradient Descent Take steps proportional to the negative of the gradient of the function at the current point Each step reduces the sum of squares Gauss Newton GNA Determine the amount of change in sum of squares when each parameter changes This determines the slope at that point Gradient descent usually works well in early iterations while Gauss Newton usually works well in later iterations LMA automatically switches between these two methods The result is a curve fit with a high degree of precision over a wide variety of datasets No algorithm can do curve fitting for all MasterPlex ReaderFit www miraibio com 145 APPENDIXC MODEL EQUATIONS datasets since some datasets are too sparse or have faulty data But a good algorithm can find a cu
69. e le ee Me one ete area mmm mmu mmu mto mmm mm mmn mm mm 1 10 1 10 1 10 1 11 1 10 IL 2 1 2 IL 2 1 2 2 3104 00 2990 50 4j00 559100 3989 50 113 00 38 00 44 00 40 00 42 00 D x fitt Li Ew B mem s ba 3 E 4 a y see en ew eee eee es Peewee eee ees ees 1 10 1 10 1 10 1 10 1210 IL 2 IL 2 IL 2 1 2 1 2 1040 00 1094 00 45 00 3070 00 1808 00 270 00 49 00 40 00 36 00 47 00 E Pet et at nmm unnm ee E ak Ne Ven mnt hee ee ee ee ro oe frat 7 uiam Un avo fot a 1 10 1 10 1 10 1 10 1 IL 2 IL 2 IL 2 1 2 IL 2 Analyte Pane p Well grid Figure 3 24 Input Data tab and Analyte pane MasterPlex ReaderFit www miraibio com 21 Plate View Components Well Grid Data type upper and lower drop down list Lower grid switch Display box Analyte pane Sample marking icon Subtract background Command icon GETTING STARTED A representation of a microtiter plate that displays the well contents for the analyte selected from the analyte panel and data type selected from the data drop down list Some data types can be edited see Table 3 1 Select one of the wells The wells turn gray then click the same well once again to edit mode Shows the types of data available for displ
70. e mxqs 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 txt or xls MasterPlex ReaderFit www miraibio com 11 GETTING STARTED 3 2 Starting MasterPlex ReaderFit On the desk top double click the MasterPlex icon 8 4 Alternatively you can click the Windows start menu button and select Programs gt MasterPlex gt MasterPlex The MasterPlex user interface appears and lists up all detected applications in the application pane Figure 3 1 You can import a results file csv txt or xls or paste the raw data in the blank plate from this interface 4 MasterPlex Ele Windows Tools License Help Look And Feel mai E Application Module Pane Logged onas Figure 3 1 MasterPlex user interface MasterPlex ReaderFit www miraibio com 12 PTER3 GETTING STARTED 3 3 Paste the raw data from plate reader s result file To begin a MasterPlex ReaderFit analysis open blank plate and simply paste the copied date from the plate reader result file MasterPlex ReaderFit opens 100 x 100 size blank plate
71. efine one or more groups of control wells per plate MasterPlex ReaderFit www miraibio com 36 CHA PTER4 DEFINING A PLATE oF NOTE A group can include nonadjacent wells A plate can have more than one group of standards or unknowns Grouping Wells by Pattern The purpose of pattern grouping is to provide users another way to easily and quickly make replicate groups Pattern here means two things the group type e g 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 1s especially useful when the plate has many groups replicates that 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 5 1308 00 429 00 480 50 392 00 95 66 78 79 1084 50 82 00 88 00 78 00 mmm eee Rm men 72 79 75 70 525 00 88 50 82 00 96 50 D ee es ee 79 66 76 74 201 00 of 50 38 00 37 00 81 82 85 96 66 50 32 00 32 00 34 00 80 79 74 39 00 18 00 18 00 23 00 G see ee ee 78 81 58 69 25 50 19 00 17 50 27 00 68 69 80 42 Figure 4 5 Well groups MasterPlex ReaderFit www miraibio com 3T CHA PTER4 DEFINING A PLATE 3 Move the pointer to the bottom right corner of the s
72. election When you see the pointer turn into a black cross hold down the left mouse button and drag the pointer over the selection 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 6 2 3 4 367 00 506 50 341 00 20 00 367 008 506 500 341 00 3 67 80 n 7 429 00 392 00 8 7 525 00 79 78 201 00 Figure 4 6 Making Well groups by mouse Dragging MasterPlex ReaderFit www miraibio com 38 CHA PTER4 DEFINING A PLATE 4 Once you are satisfied with the selection just release the mouse button The software will automatically finish the grouping Figure 4 7 387 00 506 50 341 00 Figure 4 7 Well groups downwards or rightwards which results in different ways to select wells To switch between the two modes just drag the pointer back into the pattern group and then drag it out in either direction So it is determined by your first move direction when NOTE When starting drag you can move the pointer you can move it either you are dragging the pointer out of the pattern group MasterPlex ReaderFit www miraibio com 39 CHA PTER4 DEFINING A PLATE 367 00 506 50 341 00 A J 1308 00 95 1084 50 367 00 67 78 1308 00 429 00 480 50 B Se ee See eee ee 95 5 9 Figure 4 8 Making Well groups by mouse Dragging Dragging downwards as the first
73. ent HT Customize Customize the printing items Open preview document files prnx MasterPlex ReaderFit www miraibio com 125 APPENDIXA PREFERENCES Save Save preview document by prnx format Open print dialog Print the preview document Print Setup Open print setup dialog Header and Footer Set up header and footer options Figure A 10 Set page scale KH HE s Es EZ B Hand Tool User hand icon AU AA User magnifier el m Zoom out the preview document 709 Set zoom size from the drop down list e Zoom in the preview document 4 First Page Show first page 4 Show previous page Next Show next page Last Show last page Multiple Pages Select multi pages to preview Color Open color picker and set the document E PE background color MasterPlex ReaderFit www miraibio com 126 APPENDIXA PREFERENCES Open water mark setting dialog Figure A 10 Export the document by selected format PDF HTML MHT RTF Excel CSV Text Image Export the document by selected format and send it via e mail PDF MHT RTF Excel CSV Text Image Close preview window Insert Header Footer To insert header footer into your printing document open Header and Footer dialog Figure A 11 Select header or footer radio button then input text or items from the header footer toolbar Table A 5 in the left center or ri
74. er click Import button The Open dialog box appears Figure 4 27 Look in 25 examples 4 gt m sample name empty 1 Recent Documents Desktop My Documents 44 Computer File name Open My Network Files of type MasterFlex Template matq Cancel Figure 4 27 Open dialog box 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 mxtq from the system 1 In the Template Manager click the template that you want to delete 2 Click Delete button MasterPlex ReaderFit www miraibio com 55 CHA PTER4 DEFINING A PLATE A confirmation box appears Figure 4 28 Warning x A Are you sure you want to delete Mb5PlexSingleStd Figure 4 28 Confirmation box 3 Click OK to delete the template The template is removed from the Template Manager WARNING This permanently removes the template from the system WARNING MasterPlex ReaderFit www miraibio com 56 CHA PTER4 DEFINING A PLATE 4 6 Preferences Preferences are user modifiable software settings They are displayed in the Preferences dialog box To open the Preferences dialog box Figure 4 29 click the Preferences button i23 Preferences Application Auto Fill Split Cell Color Automatically Popup Auto Fil
75. ght text box Header Footer toolbar Header and Footer an nn bud m 7 D 2 Oa jolly Font Microsoft Sans Serif 8 25pt Left center and right Header text box OK Cancel Figure A 11 Header and Footer dialog MasterPlex ReaderFit www miraibio com 127 APPENDIXA PREFERENCES Table A 5 Header and Footer Toolbar buttons and functions Icon Command Function Page Number Insert page number of Pages Insert current page of total pages type page number Date Printed Insert date printed Time Printed Insert time printed ya 9 i EE Insert user name who login the windows Insert the image 0 Align to Top Align all headers footers to the top level a Align to Center Align all headers footers to the center level Align to Bottom Align all headers footers to the bottom level Font Open font dialog for the header and footer font Insert Water Mark To insert water marks into your printing document open Water Mark dialog Figure A 12 There are two types of watermarks you can insert in your document one is Text and another is Picture They are separated into two tabs To insert a text watermark use Text Watermark tab To insert a picture watermark use the Picture Watermark tab MasterPlex ReaderFit www miraibio com 128 APPENDIXA PREFERENCES Watermark Text Watermark Text Direction Font Transparency 0
76. he purchase price of the media damaged by accident abuse or misapplication THE ABOVE WARRANTIES ARE EXCLUSIVE AND IN LIEU OF ALL OTHER MasterPlex ReaderFit www miraibio com 11 LICENSE AGREEMENT WARRANTIES WHETHER EXPRESS OR IMPLIED INCLUDING THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR 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 15 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 CONSEQUENT
77. igure 5 16 1333 20006 d 22988 26027 e 4 28625 GM CSF 5101001 Well B1 Data pop up window Group Std001 MFI 10475 00 Conc 10000 00 Figure 5 15 Click one of the data points on the chart Wel Sample Name Group Ignored Response Values Calculsted Indepe Y EJ Analyte IL 10 WONNENCONE HENCE TNI Std001 6152 00 4954 91 5000 00 01 541001 3104 00 2582 16 2500 00 El 541001 1040 00 1194 31 1250 00 Fi Std001 292 00 385 60 625 00 Figure 5 16 Corresponding Data is highlighted 2 Check the corresponding box in the ignored column by clicking on it Figure 5 15 Sample Name Group Ignored Response Values Calculsted Y EJ Analyte IL 10 I Bi 509001 10516 00 10023 06 10000 00 Ci 510001 6152 00 4934 91 3000 00 D1 Std001 3104 00 2582 16 2500 00 El Std001 1040 00 1154 31 1250 00 Fi 509001 292 00 585 60 625 00 Figure 5 17 Check on the box by clicking MasterPlex ReaderFit www miraibio com 85 5 STANDARD CURVES amp CONCENTRATION 5 4 Best Fit Calculation Option ReaderFit has an advanced feature called BEST FIT This feature finds the best equation model and weighting combination for your standard data automatically and does this for all analytes 1 Go to Input Data tab 2 Click the BEST FIT button c Progress window appears and MasterPlex begins searching for the best combination Figure 5 16
78. int current displayed document MasterPlex ReaderFit www miraibio com 137 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 variability heteroscedasticity C 1 Four Parameter Logistic Curve D Upper Asymptote B Slope at inflection point 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 ReaderFit www miraibio com 138 APPENDIXC MODEL EQUATIONS predicted If Response Value lt A or Response Value lt D the Response Value is out of the calculable range 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 1s the line x 0 This means the function can never reach the line where x 0 For example log 0 cannot be calculated In Figure C 1 B 1s the slope at the inflection point It is the speed of the functio
79. ion gradient configuration for the standard data set MasterPlex ReaderFit www miraibio com 43 CHA PTER4 DEFINING A PLATE 7 To specify the same starting concentration dilution factor and concentration units for all analytes in the standard data set choose the Fill for all analytes option To specify a different starting concentration dilution factor or concentration unit for a different analyte repeat step 2 through step 4 8 Click Apply button when finished entering the concentration the dilution and the dilution direction for all analytes in the standard data set If you want to close the dialog box at the same time click Fill amp Close button NOTE If you want to fill the standard value for your desired wells select wells on the well grid and choose Selected Wells at the target well selection in the Auto Fill dialog Auto Fill process is applied for only the wells you selected MasterPlex ReaderFit www miraibio com 44 Fill in for replicate standard samples CHA PTER4 DEFINING A PLATE If you have replicate standard samples in your plate and if you want to fill the same diluted standard concentration value for each replicates use replicate filling option Figure 4 13 Figure 4 14 and 4 15 shows each Side by Side and Stacked replicate example Auto Fill Dilution Parameters Analyte All Target Wells 610001 Highest Value 10000 000 Dilution Factor Unit Replicate Options Replicate
80. l Dialog es C 255 255 240 W Auto calculate after loading Plate Template Best Fit Intensity Color it Root Mean Square Error RMSE One color EN 0 0 255 i R Square Least deviation of Recovery EES Displayed Precision Lo Exampie 0 00 i Cancel Apply Figure 4 29 Preferences dialog box Application Preferences Auto Fill Automatically Popup Check this option if you want to open autofill Auto Fill Dialog dialog automatically when you mark the standard sample Auto calculate after Check this option if you want to calculate loading Plate Template automatically right after the template loading Best Fit Root Mean Square Use RMSE index to choose the best curve fit Error RMSE combination R Square Use R square to choose the best Least deviation of Use LD of Recover to choose the best Recovery MasterPlex ReaderFit www miraibio com 57 CHA PTER4 DEFINING A PLATE Split Cell Color Color lower grid by specified color 1 2 3 4 41 00 39 00 37 00 112 00 Zl 1 1H a Figure 4 30 Colored lower well grid Intensity Color One Color Use one color for representing the value shading Two Color Use two colors for representing the value shading 2 3 4 5 6 7 8 9 0 Intensity Color 2 10 00 9 00 19 00 6903 00 48 50 1162 00 5573 00 2145 00 443 00 187 00 E One color LimeGreen 9929 00 8989 50 6994 00 4487 00 64 00 179 00 474
81. lect 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 4 38 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 2 To rename an analyte so that it is consistent with the nomenclature 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 the virtual plate Figure 4 39 b Select a name from the drop down list The virtual analyte filter displays the new name for the analyte MasterPlex ReaderFit www miraibio com 68 CHA PTER4 DEFINING A PLATE Virtual Analyte Filter New Analytes Assign Aliases GM CSF Click here to assign IFM gamma Click here to assign List of analytes Il 1Beta Click here to assian in the virtual IL 10 Click here to assign IL 2 Click here to assign IL 4 Click here to assign None Place a check IL 8 Click to display mark next to an TNF alpha 27 3 ated drop down list of analyte to add 054 Mouse IL 2 it to the virtual 073 Mouse IFM gamma the virtual plate plate 077 Mouse IL 4 Select a name from the list to rename the analyte from the source plate Check all analytes Add all as new OK Cancel Figure 4 39 Virtual analyte filter 3 To save the renaming assignments for use again
82. license server License Server IP Address License Server Porti Server Mame 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 The license is installed MasterPlex ReaderFit www miraibio com 10 GETTING STARTED 8 Getting Started This chapter provides a brief overview of data analysis using MasterPlex ReaderFit It also explains how to start the software import a result file csv txt or xls from plate reader instruments and the user interface components 3 1 Overview of MasterPlex ReaderFit Analysis MasterPlex ReaderFit software analyzes results files csv txt or xls from the major plate reader instruments The analysis steps include Import a results file csv txt or xls Designate well types standard unknown background or control and well groups identify 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 ink a standard data set to an unknown group s Compute the analyte concentrations Save the results file in MasterPlex ReaderFit file format mxqs Th
83. list MasterPlex ReaderFit www miraibio com 5 STANDARD CURVES amp CONCENTRATION 5 6 Printing and Exporting the Standard Data You can print or export the chart and data table of the standard data 1 Click the Print button Print preview window appears Figure 5 21 Lo Preview File View Background Five Parameter Logistics R Square 0 9992191 RMSE 126 00284 1 03240 b D 77951 1333 20006 d 22988 26027 e 4 28625 27 Lum E i Fe i 1 in v e z LL IS T 1000 Concentrations Well Sample Mame Outlier MF Calculated Expected Recovery Analyte GM CSF Group 50001 L 105 16 000000 10429 395669 10000 UD 429 99566083 104 2939566 r 10034 5000000 6152 00000000 4908 055464 SO00 00000000 91 9445358 98 1611092836 recent erm aea ELA 1 14 dS SUL 101000000 4 4 J atata 9609 668740 10000 0000000 390 3312290 96 0966877097 Ws rn 7 pof Zoom Factor 100 Figure 5 21 Print Preview window 2 To pint this click Print amp icon from the menu bar Print setting dialog appears MasterPlex ReaderFit www miraibio com 89 5 STANDARD CURVES amp CONCENTRATION 3 To export this click Export Document gj
84. lt Shows only Sample Name lt lt value gt 15 less than or equal Shows Sample Name lt value gt and Sample Name lt lt value gt MasterPlex ReaderFit www miraibio com 123 APPENDIXA Shows only Sample Name gt lt value gt Shows only value are blank Shows only value are not blank Is any of Shows only value which has lt value gt s PREFERENCES i 15 between Shows only the value which has between first BEEN value and second value Is not between Shows only the outside value which has between first lt value gt and second lt value gt abe Shows only the value contains lt value gt acb Shows only the value does not contains value a b Shows only the value starts with value b c Endswih Shows only the value ends with value aac 0 Shows only Sample Name gt value snotlike sbank 0 Isnotblank _ snoeof Shows only value does not have lt value gt s 111 oa Fitter Builder Well Group Name RLU F HIE EJ Analyte Name IL 10 E1 Standard 1 292 00 Analyte Name Equals IL 10 Fi Standard 1 108 50 RLU Is between 100 00 and 500 00 EJ Analyte Mame IL 6 Analyte Equals IL 6 me Eq D3 Standard 3 679 00 RLU Is less than 1000 00 E3 Standard 3 194 00 F3 Standard 3 85 50 H3 Unknown 3 3 81 Shows only Analyte name is IL 10 and Response Value is between 100 and 500 or Anal
85. me for the well Yes The group name of the well Wells that belong Yes to the same group have the same group number The analyte name assigned to the well Wells Yes that belong to the same group have the same group number The dilution factor for the well Yes Shows the Response Value average within the No group Shows the Response Value standard deviation No within the group Shows the Response Value CV within the No group Shows the concentration average within the No group Shows the concentration standard deviation No within the group Shows the concentration CV within the group No Shows the Normalized data within the group No This data type is available in the lower drop down list only MasterPlex ReaderFit www miraibio com 20 3 GETTING STARTED Display Double Data Information in one Cell ReaderFit has an unique feature for data viewing on the well grid You can select two data type from various kind of data and it 1s displayed in the one cell separated into upper and lower Figure 3 8 shows how to display the double data in one cell Response Val 7 Response Val Analyte Name Analyte Name Response Val 186 00 Analyte Name IL 2 1 t1 1 2 3 4100 mc M 3i 41 00 C Response Values IL 10 I 1i ll Lm 1 20516 00 1009450 IL 10 Name IL 10 IL 1i Figure 3 25 Upper and lower
86. n as it moves away from the inflection point C is the most interesting parameter The C parameter corresponds to the x value that 1s 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 is on the x axis that produces a 50 response C 2 Five Parameter Logistic Curve Five Parameter Logistics R Square 0 9996930 RMSE 120 51352 16 70189 b 0 68911 c 1246 32313 d 28652 03187 e 4 70687 5101001 qi ro gt th t e tL Un T aa 1000 Independent Values Figure C 2 Five parameter logistic curve MasterPlex ReaderFit www miraibio com 139 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 1s not symmetrical because the lower part of the curve behaves quite differently from the upper portion The additional parameter E compensates for this asymmetric behavior and adjusts the curve Fixed Lower Asymptote Zero Usually an asymptote is estimated where a function is tending toward a constant value A fixed asymptote is a restriction that tells the function to never go beyond that value For example setting A 0 as in the Fixed lower asymptote zero option in the Fit Curves tab It 1s used with logistic functions
87. nformation window shows style sheet s information Figure 8 3 MasterPlex ReaderFit www miraibio com 113 Report Template fez 5x12 plate map HTML Custom data format2 Custom data formati Tab Format PlateFormat Report InFarmatian x Mame 812 plate map HTML Modified Time 1 11 2010 6 30 00 PM Method Html Figure 8 3 Style Sheet List and Information 8 2 Exporting a User Defined Stylesheet Export 1 Click Export button The file save dialog appears Figure 8 4 CHAPTERS EXPORT DATA MasterPlex ReaderFit www miraibio com 114 CHAPTERS EXPORT DATA Save in examples E a norm xml xsl My Recent a jall xml xsi Documents HE first_analyte_html xsl conc_unk_xmi xsl all_htrml xsl data File name My Network Save Masterlex Style Sheet x Figure 8 4 Save File Dialog 2 Set the destination and input file name then click the Save button Style sheet s name is displayed in the Style Sheet window and XSL Information window shows style sheet s information Figure 8 3 8 3 Delete Style Sheet File from Style Sheet List 1 Select the style sheet you want to delete from the style sheet list 2 Click the Delete button Delete The confirmation dialog appears Figure 8 5 3 Click OK to delete MasterPlex ReaderFit www miraibio com 115 CHAPTERS EXPORT DATA Warning X A Are vou sure vou
88. nse Values which can be MasterPlex ReaderFit www miraibio com 141 APPENDIXC MODEL EQUATIONS caused by user error MasterPlex ReaderFit provides a function to subtract the background noise from the Response Values to obtain more accurate responses If the user subtracts a reasonable amount of background it could be assumed that the lowest detectable Response Value 1s zero Note that log 0 is undefined So in some cases fixing the lower asymptote to zero 1s theoretically correct However in reality most datasets contain some slight amount of error so fixing the lower asymptote to zero can actually make it more difficult to achieve a good curve fit For this reason a slightly better R2 value is usually reported when the curve fitting is done with this feature turned off unchecked which is the default but the difference in the R2 value would be very small usually less than 1 100 00th or 0 00001 Any change the graph would be unnoticeable The difference in the interpolated concentrations would also be very small usually less than 2 if any Asymptotes can be used to gauge the similarity of functions For example if two curves calculated with the Four Parameter Logistics equation end up having very close to the same A and D parameters their interpolated concentrations would be in the same range The lower and upper asymptotes A and D of the 4PL and 5PL models usually calculated automatically during the curve fit
89. o group to this box Figure A 3 Also you can multi group by repeating same way Figure A 4 Analyte Analyte IL 1 Al 41 00 0 00 1 50001 10516 00 10516 00 Ci 510001 6152 00 6152 00 Figure A 2 Group Box MasterPlex ReaderFit www miraibio com 119 APPENDIXA PREFERENCES Sampla Name oOo Drag 55 ReNwery Well A5 51 Analyte Name sree Ay Scie sees 2 A2 si IL 2 Stand A4 51 THF alpha Stand B4 52 Conc o B2 lt 2 Conc amp CV Well Sample Name Group Name M EJ Analyte Name IL 10 1 51 Standard 1 Bis Standard 1 Figure A 3 Group by Drag amp Drop Sample Response Values gt E Analyte Analyte 1 EJ Replicate Group Name AL 41 00 2 39 00 Figure A 4 Multi Group by Drag amp Drop MasterPlex ReaderFit www miraibio com 120 APPENDIXA PREFERENCES Group Box Menu Group Box has context menu Table A 2 shows the menus Table A 2 Main toolbar buttons and functions Icon Command Function Expand all group trees s E Full Collapse Collapse all group trees Se Clear Grouping Clear group and return the group columns to the grid column Working with Column Filter There are two ways to set a filter The first way 1s by using the filter drop down menu from the column Fig
90. olumn Figure 5 11 Add column from column chooser box MasterPlex ReaderFit www miraibio com 81 Sample Name Response Values Calculated Independ Jp Mame 541001 2 1 2 1 1 Figure 5 12 Delete columns 541001 55 00 56 00 104 00 108 50 300 00 292 00 55 00 36 00 104 00 108 50 300 00 292 00 199 04 203 26 336 75 345 86 609 27 600 61 156 25 156 25 312 50 312 50 625 00 625 00 156 25 156 25 312 50 312 530 625 00 625 00 Figure 5 13 Interchange the column position Residuak 42 79 47 11 24 25 33 36 15 73 24 39 5 STANDARD CURVES amp CONCENTRATION Select the data type you want to delete in the data grid column and drag amp drop it away from the column Select the data type you want to move in the data grid and drag amp drop it to the new position oF NOTE There are some other features for the data gird to customize the gird view See appendix A section A 1 Grid Customize Menu for further details MasterPlex ReaderFit www miraibio com 82 5 STANDARD CURVES amp CONCENTRATION Table 5 1 Data Types in the well grid Data Type Well Analyte Sample Name Replicate Group Name Ignored Response Values Response Values Background Response Mean Response SD Response CV Calculated Independent Values Calculated Mean Calculated SD Calculated CV Residuals Recovery Backgroun
91. om the data type upper drop down list The well grid displays the data for the selected analyte Figure 3 24 shows the components of the Input Data tab Table 3 1 lists the MasterPlex ReaderFit www miraibio com 26 GETTING STARTED types of data available for display in the plate view 3 To view background subtracted data click the Subtract background button 1 2 The Input Data tab displays background subtracted data For more information on background calculation options see Background Type on section 4 6 Data type upper and lower drop down list Subtract Background button Lower grid switch Command icon Se ka BS Sample marking icon Response Val 200 0 fo Ka E Wis Wes IE X Analyte Name ad L 10 LE al 2 2 4 5 10 6 7 12 41 00 39 00 37 00 112 00 02 00 2426 50 43 00 36 00 48 00 80 00 k a i z rs i 5 J 1 Ey woe ech oe ey get Pd rR hae ie en Se ae a es Oh PS meer te 1 10 1 10 1 10 10 1 10 IL 2 IL 2 IL 2 IL 2 10515 00 10034 50 55 00 4911 00 105 50 186 00 41 00 38 00 45 50 55 00 B ud de 9 99 ee eee ee c ee eee ee n 10 1 10 1 10 1 10 1 1 IL 2 IL 2 n3 1 2 IL 2 6152 00 6299 00 38 00 5011 00 55 00 43 00 38 00 52 00 40 00 45 00 E g de do d 4 a n n n m n m m u m mm 5 5 4 eee eee ee 2 de d o ww e Ra ee ee e
92. on information Look and Feel Change application skins MasterPlex ReaderFit www miraibio com 133 APPENDIXB MASTERPLEX ReaderFit TOOLBARS B 2 Input Data Tab Toolbar The Input Data tab toolbar is available on the top of the well grid in the plate tab G a a Wes Figure B 2 Plate toolbar 3 SN Table B 2 Plate toolbar buttons and functions Menu Bar Toolbar Function Command Icon Background B Mark selected wells as Background Standard Mark selected wells as Standard Unknown v Mark selected wells as Unknown Control Mark selected wells as Control Unmark Unmark selected wells Subtract Toggle subtract background function Background Best Fit Start best fit mode FIT Auto Fill Open Auto Fill dialog Link Standard Make link s between standard data and others Quality Control Open Quality Control Manager Manager d MasterPlex ReaderFit www miraibio com 134 APPENDIXB MASTERPLEX ReaderFit TOOLBARS Template Open template manager Manager Plate Open plate preference dialog Preferences B 3 View Results Tab Toolbar The View Results toolbar is available on the top of the data grid the View Results tab Show Columns Selector Export to File Le Print Preview Table Template Merge Cells By Group Figure B 4 Calculation toolbar Table B 4 Calculation toolbar buttons and functions Menu Bar Function Command Button Show Hide Columns Show or hi
93. port your data table data from Export to File button 1 Click Export to File drop down button 2 Select the file format you want to export There are five file formats available Excel CSV PDF HTML and Text Figure 6 8 Figure 6 8 Export to File menu 3 File save dialog appears Set file path and input file name then click OK Figure 6 9 Figure 6 9 File save dialog MasterPlex ReaderFit www miraibio com 96 CHA 7 DATA CHARTS 4 After saving the file an Open file prompt appears Figure 6 10 If you want to open the saved file immediately using the program the files extension is associated with click Yes The saved file is opened on the program Figure 6 11 v Bo vou want to open it now PES eee demum I 7 xg Xi Esa 5 x dii Porat ai d tuia Ir We NET Bie wm e juam 14 Fike Seer bol mun H x4 swaro 1040 00 ISO anu po 31 51 TY x nmm oof 6 11 qoem in Excel MasterPlex ReaderFit www miraibio com 97 7 DATA CHARTS 6 4 Printing a Data You can preview your data with the Print Preview button 5 1 Click the Print Preview button Print preview window appears Figure 6 12 E Pru oy a Anziyte Name 1 1 Al 41 00 0 00
94. r dialog The software automatically marks wells that contain data less than the user MasterPlex ReaderFit www miraibio com 70 CHA 4 DEFINING A PLATE specified threshold with a red border Figure 4 41 To set a threshold s 1 Select analytes you want to attach the threshold criteria from the analyte pane Use All analytes check box or Ctrl key for multiple selection 2 Check Show threshold marker box and or Mark as outlier box 3 Select the threshold criterion from the threshold tab 4 Set threshold conditions and click apply button Close the dialog box red border marks wells that contain data meet the threshold criteria If you choose Mark as outlier at the same time the data are marked as outlier and outlier check boxes are checked Figure 4 42 4 5 6 4 8 9 10 1 2 3 112 00 92 00 2426 50 43 00 36 00 80 00 10516 00 4911 00 106 50 186 00 77 6152 00 6299 00 39 00 5011 00 43 00 38 00 40 00 45 00 3104 00 2990 50 5591 00 3989 50 113 00 38 00 44 00 40 00 1094 00 45 00 1808 00 270 00 49 00 40 00 36 00 47 00 300 00 39 00 1129 00 42 00 40 00 37 00 49 00 24 ine 4017 111 nn 7n nn 4 nal n nn nal Figure 4 41 Well grid JL d M ja Figure 4 42 Outlier check boxes MasterPlex ReaderFit www miraibio com T1 CHA PTERA DEFINING A PLATE Settings Flag wells outside Show re
95. rm Original Data into Your Customized Data 1 16 APPENDIX Preferences Grid Customize Menu views Se n A 1 18 Print Preview Menu TT TT Te ee Ce AE ee Tee 125 APPENDIX B MasterPlex ReaderFit Toolbars Main File Menu and Toolbar ee eT E RR 132 Input Data Tab Toolbar s 134 View Results Tab Toolbar 135 Create Graphs Tab Toolbar 8 136 Customized Report Manager Tab Toolbar 137 APPENDIX C Model Equations Four Parameter Logistic Curve eene 138 Five Parameter Logistic Curve 139 Heteroscedasticity prid va DR RU 143 Weighted Nonlinear Least Square srr 149 Results of Weighting ARCU 150 MasterPlex ReaderFit www miraibio com CHAPTER 1 WELCOME MiraiBio MasterPlex ReaderFit CHAPTER 1 Welcome to the MiraiBio MasterPlex ReaderFit User Manual MasterPlex ReaderFit software analyzes results files csv txt or xls from the plate reader instruments 1 1 About This Manual This manual explains how to use the MasterPlex ReaderFit application module to Open blank plate and then paste the raw value from result files Import results files csv txt or xls from the plate reader instruments Designate standard unknown control and background wells Generate standard curves Compute analyte concentrations Generate data chart
96. rs selec esired color tor e 143 50 116 00 7453 00 6100 58 00 7190 00 4878 00 2840 50 779 00 70 00 maximum and minimum value The color shifts 42 50 40 00 7817 00 3178 00 54 50 7301 00 3625 50 1662 50 343 00 31 00 upper to lower directly with the value Figure 4 32 Example of Intensity color H MasterPlex ReaderFit www miraibio com 58 CHA PTER4 DEFINING A PLATE Plate Preferences Preferences Plate Information Original File Mame SinglePlex csv Analyst Name N A Plate Name ReaderFit sample data csv Background Type Average P Peak Value Lowest Value Cancel Plate Information Original File Name Displays the name assigned to the result file in the plate reader software To edit the plate name enter a new name Analyst Name Displays the analyst name entered in the plate reader Plate Name Shows plate name of this file Background type Average Calculate average value in the background group Background Bkg Response Value Bkg Response Value Bkg Response Value Bkg Response Value n where n the number of background wells in the plate Peak Value Take highest value in the background group Lowest Value Take lowest value in the background group MasterPlex ReaderFit www miraibio com 59 CHA PTER4 DEFINING A PLATE Threshold You can select one of the criteria for threshold marker from Response Value Concentration and Error range Select one of them and enter a
97. rve fit for many datasets and have a degree of precision in its parameter calculations To track the data points MasterPlex ReaderFit uses a class of a nonlinear transfer function called a sigmoid function for example the Five Parameter Logistics model This typically produces a sigmoidal or S shaped curve Figure C 5 C FF C A A A GF C i N A e 6 4 2 a 4 e Figure C 5 A sigmoidal or S shaped curve MasterPlex ReaderFit uses 4 or 5 parameters models to help in the curve fitting for the nonlinear model The Five Parameter Logistics model compensates for asymmetry in the curve To a certain extent it 15 useful to have more parameters to tweak during the curve fitting process thus producing a more precise curve to fit the data This 15 why the Five Parameter Logistics model usually outperforms the Four Parameter model for most datasets MasterPlex ReaderFit includes a Best Fit feature to automatically choose a model and weighting method that produces the best curve fit However choosing a model can be a scientific decision based on chemistry or biology MasterPlex ReaderFit still provides the flexibility for the scientist to choose the model and weighting method themselves which may be desirable in some cases depending on the assay MasterPlex ReaderFit www miraibio com 146 APPENDIXC MODEL EQUATIONS Interpreting the Result One measure of goodness of the curve fit is the value
98. s and reports What s New in MasterPlex ReaderFit MasterPlex ReaderFit 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 it 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 ReaderFit software The manual uses a step format to explain the various tasks associated with MasterPlex ReaderFit A symbol may follow a step instruction It indicates the software response to the action performed by the user MasterPlex ReaderFit www miraibio com 1 CHAPTER 1 WELCOME Screen Captures Screen captures may accompany the step instructions for further illustration The screen captures 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 Tel 1 650 615 7600 Toll Free 1 888 615 9600 Fax 1 650 615 7639 E mail support miraibio com www miraibio com MasterPlex ReaderFit www mirai
99. s that are present in the virtual pipette It enables you to choose the analytes that you want to add to the virtual plate and 1f 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 the well If a plate file csv txt xls mlx or virtual contains data holes the concentrations cannot be calculated 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 ReaderFit www miraibio com 67 CHA PTER4 DEFINING A PLATE Virtual Analyte Filter New Analytes Assign Aliases EGMCSF Click here to assign IFN gamma Click here to assign Il 1 Beta Click here to assign IL 10 Click here to assign IL 2 Click here to assign IL 4 Click here to assign IL 5 Click here to assign IL 6 Click here to assign IL 8 Click here to assign TNF alpha Click here to assign Check all analytes Add all as new OK Cancel Figure 4 38 Virtual analyte filter shows the analytes in the virtual pipette Selecting and Renaming Analytes If the virtual analyte filter appears you must se
100. s x I Set X axis to log scale Set Y axis to log scale I Plot unknown wells Figure 5 5 Chart Scale option MasterPlex ReaderFit www miraibio com 78 5 STANDARD CURVES amp CONCENTRATION Generating Standard Curves 1 To generate the standard curves and compute interpolate or extrapolate the analyte concentrations click the Calculate button Calculate A message box confirms the calculations are completed Figure 5 6 Information x m 2 All calculations were completed successfully Figure 5 6 Message box Generating Standard Curves by using Best Fit 1 To generate the standard curves and compute the analyte concentrations by using Best Fit feature check Use Best Fit feature on Figure 5 7 then click the Calculate button csse A message box confirms the calculations are completed Figure 5 6 Best Fit im v Use Best Fit feature Figure 5 7 Best Fit feature check box NOTE If overall R square value is less than 0 9 a warning message box appears Figure 5 8 Information x 1 Some of the calculations did not result in good fitting RSquare 0 9 Figure 5 8 Warning Message box MasterPlex ReaderFit www miraibio com 19 5 STANDARD CURVES amp CONCENTRATION 5 3 Reviewing Calculated Standard Data 1 Standard data grid and Standard chart view are updated Figure 5 7 Well Sample Mame ignored RLU Calcula
101. served concentration is from its expected value It plots residuals against observed concentrations In Figure C 3 we can see that the deviation of the observed concentration from the expected value increases as concentration increases This means the variability 1s 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 1s illustrated 1n Figure C 4 MasterPlex ReaderFit www miraibio com 143 APPENDIXC MODEL EQUATIONS Predicted T concentration m ara a ara Expected concentration D 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 the data the curve 1s 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 represent the residuals as ropes and each d
102. t My Documents 3 My Computer i 9 pu en My Network Files of type Data Files csv xls bed Figure 3 6 Open dialog box MasterPlex ReaderFit www miraibio com 15 3 GETTING STARTED 3 Navigate to the directory of the csv txt or xls that you want to import 4 Select one or more csv txt or xls files and click Open c File import wizard appears Figure 3 7 Data Input Selection Page ud SinglePlex Wizard Data Input Selection Helps you select the correct input variables for file parsing Follow the steps on the right to correctly parse your data 1 Select Deliminator Type H Comma Space Tab Other Plate C List 3 Select Data Values Please click and drag to select the data area to be read excluding row and column headers 2 3 4 5 6 7 8 9 Row Column Figure 3 7 Data Input Selection Page 1 Select one of the deliminator type from select deliminator type box Figure 3 8 so that your result data is correctly delimited in the file display grid Data delimitation in the grid view will be changed Figure 3 9 1 Select Deliminator Type Space i amp iTab Other Figure 3 8 Select Deliminator Type Box MasterPlex ReaderFit www miraibio com 16 GETTING STARTED 1 b 1 lesBOCKS 1 10 11 12 13 14 15 16 17 18 19 2 Plate Plate 1
103. t www miraibio com 93 5 STANDARD CURVES amp CONCENTRATION 6 2 Sort or Filter the Column Data To sort by specific column click the column title Ascending and descending are changed alternatively Figure 6 5 hinan UA Response Values Response Values UU apse 2d 10034 50 36 00 6299 00 37 00 6152 00 37 00 5591 00 37 00 5578 00 38 00 5011 00 00 Ascending sort Descending sort Figure 6 5 Sort Column Data To clear the sort right click on the column you want to clear the sort select Clear Sorting from the menu Figure 6 6 SI Sort Ascending l Sort Descending Clear Sorting tm Group By This Column PU mem Ds Des Figure 6 6 Clear Sorting MasterPlex ReaderFit www miraibio com 94 5 STANDARD CURVES amp CONCENTRATION To filter by specific data in the column click upper right side of the column you want to use it as filter base Figure 6 7 Well Sample Well C Sample lH Bead Name GM CSF Bead GM CSF Al A10 B1 Ci EJ Bead Name IFN gamma A10 Bead Name 1 1 Beta A10 Figure 6 7 Filter by the data in the column NOTE There is a way to construct more complex filter conditions using Filter Builder See appendix A section A 1 Create Complex Filter Criteria paragraph MasterPlex ReaderFit www miraibio com 95 CHA 7 DATA CHARTS 6 3 Exporting a Data You can ex
104. ted Expected FW Residuals Recovery gt Analyte IL 10 Group Standard 1 Bi 10516 00 10429 40 10000 00 429 40 104 22 B2 E 10034 50 9609 67 10000 00 390 33 96 10 Ci 6152 00 4908 06 5000 00 31 54 95 16 C2 6299 00 5042 17 5000 00 42 17 100 84 D1 3104 00 2567 05 2500 00 67 05 102 68 2 2990 50 2401 46 2500 00 8 54 99 66 1040 00 1211 88 1250 00 38 12 946 95 EZ E 1094 00 1249 39 1250 00 0 62 99 95 Five Parameter Logistics R Square 0 9999550 1 632 b 0 780 c 1333 200 d 22988 260 e 4 286 IL 10 di 4 10000 Standart amp s000 2 6000 E 3 4000 gt FE 2000 cx 1000 10000 Concentrations Figure 5 9 Standard Data Table and Chart 2 To view multiple standard data select the analyte from the analyte pane during CTRL key pressing down Data table and chart are updated by the multiple standard Figure 5 8 MasterPlex ReaderFit www miraibio com 80 Analytes 1 10 4 IL 6 1 Ee TNF alpha ge 1 12 4 IMF gamma UE Standard 6 B Analyte IL 12 Well Analyte 1 10 EJ Group Name Standard 1 1 D1 x Gl Sample Name Ignored e E 3 E 5 STANDARD CURVES amp CONCENTRATION RLU 10516
105. the page by the image Set transparency for the insert image Select image position In front or Behind Set page range for the image water mark MasterPlex ReaderFit www miraibio com 130 APPENDIXA PREFERENCES 4 Best kame 2 10111 1 Background 1 41 00 189 10194 8427 81 Standard 1 30518 00 237039 10429 40 284314 Standard 1 6152 00 297033 225 4908 06 294114 01 1 310400 297033 23 2557 05 284114 E Standard 1 1040 00 237039 121188 294114 Fi Standard 1 297035 185 Gi Stendand 1 108 50 297039 190 345 86 294214 Hi Fanii 1 5500 292033 183 20336 29 314 A2 Background 1 33 00 194 847 82 Standard 1 1003450 2870 38 152 9509 57 284314 a Standard 1 250 00 297035 245 5 42 17 Standard 1 2990 50 297039 252 2481 45 EE Fanii 1 1084 00 2970 39 zn 124538 284314 Standard 1 300 00 297035 217 50827 292314 G2 Standard 1 10400 297039 258 33575 282314 H2 Standard 1 55 00 287038 18804 2843 14 Unknown 1 3700 540 89 193 46450 67122 Unknown 1 540 88 198 240 47 813 1 3900 540 89 27 64 60 87122 1 41 00 52588 183 103 94 67122 a Unknown 1 45 00 588 am 67122 Unkina 1 3900 540 89 213 64 60 87122 63 Unknown 1 45 00 520 85 207 1434
106. ther agree that it is the complete and exclusive statement of the agreement 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 ReaderFit www miraibio com V 1 1 MasterPlex ReaderFit Analysis software for cytokine data from plate reader instruments CONTENTS ve Welcome About This Manual 1 Technical Support 2 CHAPTER 2 Installing MasterPlex Requirements eme meme 3 Installing MasterPlex Readerbif sewer 4 Installing a License 10 CHAPTER 3 Getting Started Overview of MasterPlex ReaderFit Analysis 11 Starting MasterPlex ReaderFit 8 12 Paste the raw data from plate reader result 13 Importing Measurement Results And Analyte Assign T M 15 Import csv txt xls or Open mlx files by drag and drop 23 Tab categorized work flow 24 Viewing Data in the Input Data Tab 757577008 26 Saving Plate Data Sel 3 CHAPTER 4 Defining a Plate Input Data tab Designating Well Type and Group 77777068 33 MasterPlex ReaderFit www miraibio com I Setting Standard Concentration een 42 Linking a Standard Data 48 Working with Diluted Unknowns srt 49 Working With Templates 52 Pre ferences
107. ting process and are derived from the characteristics of the data The Fixed lower asymptote zero feature makes 1t possible to fix the lower asymptote to a hardcoded value of zero which 1s useful in some experiments This feature 1s reasonable to use 1f enough background was subtracted and the data has little user error But for most data sets the R2 and concentration values will not be improved with this feature on 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 15 appropriate for data that are intrinsically linear MasterPlex ReaderFit www miraibio com 142 APPENDIXC MODEL EQUATIONS C 3 Heteroscedasticity Fitting nonlinear models to observed data 1s often complicated by non constant or heterogeneous variability Heterogeneous variability or heteroscedasticity occurs 1n most types of observed data This 1s especially true for biochemical assays where concentration or dose 1s the predictor Therefore we can expect that measurement error varies with respect to the mean In the plate reader system Response Values are based on vary with the concentration In this case we expect the error in detecting Response Value to increase as concentration increases This is best seen in Figure C 3 a residual plot from a plate reader cytokines assay A residual plot is a graphical representation of how far away an ob
108. u eee ee tn ar Ae ha pennae k boron 0 et anin mnim m Ju m min m mmummhuikum d Figure 3 17 Data Selection Breyer en urn mox finde Pou at ina ibincarmact Ew wck iiim boda manam ey ee ey ante res enc oe Ehe Deo ee DO Sn nates Thee S19 poen i Ehe plate To de fina exc ete oneri dz oe rp fe 1 fam bar Ami Tou bo rug quem erp ee ey 4er chao please he humor 9 ext Miis Secrest l LI iy Been zee 5 289 aere Coke ee E umani run d Figure 3 18 Data Assignment MasterPlex ReaderFit www miraibio com 21 3 GETTING STARTED n x rabda fase Pom raceci Eua pl dia beor 1binzamach iiim do peg ey De mar ce B ndi ors Debo ces eS anc multiple 3 in the To deine esch dx eel fren fhe atm fur cct briiign mu mea ce HT
109. ubtracted and the data has little user error but for most data sets the R and concentration values will not be improved with this feature on I Use weighting Fixed lower asymptote zero Figure 5 3 Weighting and Fixed asymptote option Model equations available for regression analysis of a standard data set NOTE For more information about model equations and weighting methods see Appendix C MasterPlex ReaderFit www miraibio com TT 5 STANDARD CURVES amp CONCENTRATION 5 2 Generating Standard Curves amp Computing Analyte Concentrations MasterPlex ReaderFit 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 Points Options Figure 5 4 Individual points Displays each point in the standard data set default individually on the standard curve chart Average standards Displays averaged data points within the same standard values with an error bar amp Individual points Average standards Figure 5 4 Standard Points option Standard Points Options Figure 5 5 Set X axis to log scale Plot X axis data on the chart by log scale Set Y axis to log scale Plot X axis data on the chart by log scale Plot unknown wells Plot unknown wells on the curve Display Option
110. ure A 5 You can filter the column by selecting the specific data in the column The second way is by using the Filter Builder window Figure A 6 Well Sample Name Well Sample El Bead Name GM CSF Bead Name GM CSF Al 10 B1 C1 I Bead Name IFN gamma A10 Bead Mame Il 1 Beta A10 Figure A 5 Direct Filtering from the column MasterPlex ReaderFit www miraibio com 121 APPENDIXA PREFERENCES Create Complex Filter Criteria To construct filter criteria involving multiple columns and various comparison operators use the filter drop down list and click Custom This invokes the Custom AutoFilter dialog Figure A 6 which allows you to compare a column with one or two values To construct using more various operators and multiple conditions use the filter builder See Basic step for constructing Filter Criteria by using Filter Builder paragraph in this section Custom AutoFitter Show rows where equals IL 10 And or equals Cancel Figure A 6 Custom AutoFilter dialog Filter Builder Window By using the filter builder you can filter the data more specifically To open the filter builder window choose Filter Editor from the right click menu on the column or click Edit Filter button on the bottom of the data grid Figure A 7 Well sample Mame Sort Ascending LN Sort Descending E d c Group By This Column Group By Box B gH Column
111. us eT Five Parameter Logistes Agere gt AOH AMG z 3x SL be 00000 ce Sd H We Talore ie 1 ee IL 1b Bas m 4 i 4 E a D Eat M ig roses Pink urna Zoon 1m 3 77 foam Cub Latt Dick 4 dhe Trani 4 un ase es Pub un Tere Independent values Standard Data Standard Curve Data Grid and Chart Commands and Group pane Display Options Figure 5 1 Fit Curves page MasterPlex ReaderFit www miraibio com 15 5 STANDARD CURVES amp CONCENTRATION Each well in a standard data set represents an x y data point The Response value 16 plotted on the y axis and the concentration 1s plotted on the x axis MasterPlex ReaderFit uses nonlinear regression curve fitting analysis to fit a user specified model equation to the standard data set and generate a standard curve oF NOTE The standard curve may not pass through each point in the standard data set The software computes the R value 0 lt lt 1 for the model equation measures the goodness of fit of model equation to the standard data set where R 1 is the probability that the model predicts the data perfectly The steps to create a standard curve include 1 Mark the standard wells 2 Link the standard data set to the unknown well group s of interest
112. ve jc und ga Sad ele asas e Ma Umm Choose a model equation and set options Review regression i curve and data Make some outliers and re calculate the curve d E MasterPlex ReaderFit www miraibio com 24 PTER3 GETTING STARTED Review all data eee ren Print or export the data T Review Data by Chart Review all data on the chart Customize chart properties Print or export the chart Export Customized Data Transform the MasterPlexReaderF it xml data to original data format Import or export the style sheet data MasterPlex ReaderFit www miraibio com 25 GETTING STARTED 3 7 Viewing Data in the Input Data Tab The ReaderFit application module starts 1n the Input Data tab If any other tab page 1s displayed click the Input Data tab to display the Input Data tab as shown below Figure 3 23 Input Data tab pata lr Ara hanis see ND Bi o Ls Figure 3 23 Input Data tab page 1 If more than one application window is open select the Ej Cascade Tile Horizontal or M Tile Vertical menu from the window menu bar to arrange the application windows for easier viewing 2 To change the data displayed in the well grid a Click an analyte in the Analyte pane b Make a selection fr
113. vte name is Il and Resnonee Vale is less than Figure A 9 Example of the complex filter criteria MasterPlex ReaderFit www miraibio com 124 APPENDIXA PREFERENCES A 2 Print Preview Menu Print preview allows you to zoom navigate print out set printing options export and other useful tasks Figure A 10 Preview Menu Preview Toolbar File View Background Bo Ege e ad SpE Bal Phin e 9 ah Well Sample Name Group Name RLU RLU Average Concentration Analyte Name IL 10 1 Standard 1 10516 00 10516 00 10023 06 B1 standard 1 i 6152 00 8152 00 4334 91 ci Standard 4 3104 00 3104 00 2582 16 Di Standard 1 I 1040 00 119431 E1 Standard 1 li Preview 292 00 585 60 Fi standard 1 108 50 352 43 ci Standard 1 56 00 24625 Hil 0 00 0 00 Analyte Name IL 12 o i as Standard 5 9681 00 9681 00 0 00 10044 46 B5 Standard 5 6359 00 6359 00 0 00 4907 52 cs standard 5 3929 00 3928 00 0 00 254428 D5 Standard 5 il 2163 00 2163 00 0 00 1303 86 ES Standard 5 840 001 840 00 0 00 558 88 FS standard 5 i 336 001 336 00 0 00 298 68 _ __ CurrentPage 1 Total Pages 3 Zoom Factor 130 Preview Status Bar Figure A 10 Print Preview Window Table A 4 Preview Toolbar buttons and functions Icon Function Search specific word or value from the Jj preview docum
114. want to delete All HTML Figure 8 5 Confirmation Dialog 8 4 Including standard curve images 1 Check Include standard curve images Include standard curve images as i 64 strings Mak common The transformed data includes standard curve image data as base64 encoding 8 5 Transform Original Data into Your Customized Data 1 Select the style sheet you want to apply from the style sheet list 2 Click the Apply button The transformed data 15 shown in the preview window Figure 8 6 MasterPlex ReaderFit www miraibio com 116 CHAPTERS EXPORT DATA Flat File bv Determination FileName BunDate RunTime SampleWell AmahteName WellType SampleName 3 Ci Documents and Desktoc e 7 12 2008 6 10 PAT GM CSF Crther 4 C Dormen and ettings tshmizu Desktop examples H IP cz C Documents and _ a Desktoo examples HLI Pler 912008 8 10 PM 1 1 Beta Documents and AA _ cer 12 2008 5 10 PAY 1 10 CHher C Documents and en d les HlPlex cer 12 2008 5 10 PAY 1 2 Other C Documents and 30 TX _ Hi Settings tshimizu Desktop examples pul zc C Documents and c lex HlUPlex cer 2008 6 10 PM 1 L 5 Other 8 C Docunents aid
115. www miraibio com 48 CHA PTER4 DEFINING A PLATE 4 4 Working With Diluted Unknowns If you need to dilute a sample prior to an assay you can specify a dilution factor in the well grid MasterPlex ReaderFit can compute the diluted analyte concentration For a diluted unknown Original concentration Dilution factor Calculated concentration Editing a Dilution Factor 1 Select Dilution Factor data type from upper drop down list or lower drop down list Figure 4 18 Current dilution factor settings are shown in the plate well grid Dilution Factor 1 00 br d sampe Mame T 3 4 Ba Bn Figure 4 18 Display Dilution Factor 2 Click one of the desired well your want to set the dilution 3 Click the same well again to enter the edit mode Figure 4 19 Dilution Factor 1 Li i E pum het 4 2 2 Figure 4 19 Dilution Factor Edit Mode MasterPlex ReaderFit www miraibio com 49 CHA PTER4 DEFINING A PLATE Editing a Dilution Factor using batch input feature 1 Select multiple wells you want to set the dilution at one time 2 Right click on the well grid Context menu appears Figure 4 20 1 2 3 4 A B Aspirate c D 1 Mark Wells E Unmark F n Figure 4 20 Dilution Factor Menu 3 Select Plate Dilution menu Plate Dilution dialog appears Figure 4 21 A Please enter dilution factor
116. x so that a results file csv txt or xls MasterPlex ReaderFit file mxqs may be opened File Close Close currently opened plate data file zh x File gt Save Save currently opened plate data File gt Save as Save currently opened plate data as different file name File gt Recent Files List up the files recently opened File gt Exit Close MasterPlex application Analyte Filter Valid analyte filter feature Virtual Plate Generate virtual plate It opens plate dimension input dialog Windows gt Show Set tab style window display Tab style Windows gt Arrange the window by cascade style Cascade Dover e Windows gt Tile Arrange the window by horizontal style MasterPlex ReaderFit www miraibio com 132 APPENDIXB MASTERPLEX ReaderFit TOOLBARS Horizontal Windows Tile Arrange the window by vertical style Vertical Tools User Open user management dialog Management For 21 CFR Part 11 module only Tools Log Viewer Open log viewer For 21 CFR Part 11 module only Open verification checker dialog For 21 CFR Part 11 module only Open application license information dialog Tools gt Verify files License Applications License gt Plugins Open plugin license information dialog Help Manuals Open manuals folder Help Tutorial Open video tutorial Help Online Open support URL by the default browser Support Help About Display splash screen with application versi
117. x y chart 3D bar style x y chart 3D bar style x y z chart Point Point style x y chart Line interpolated style x y chart Step line interpolated style x y chart Spline interpolated style x y chart 3D line interpolated style x y chart Step Line 3D 3D step line interpolated style x y chart Spline 3D 3D spline interpolated style x y chart Are painted style x y chart Spline area painted style x y chart 3D area painted style x y chart Spline Area 3D 3D spline area style x y chart Pie 520 Pie style circular chart 3D pie style circular chart Doughnut style circular chart 3D doughnut style circular chart Line interpolated style radar chart Point plotted style radar chart POOOOCCRE MasterPlex ReaderFit www miraibio com 104 CHA 7 DATA CHARTS 7 3 Analyte Selector In the Well Group chart Analyte Selector allows you to change the analytes position in the chart and allows you to display on off setting for each analyte To move the analyte position 1 Click Analyte Selector drop down list Figure 7 8 2 Select the analyte you want to move 3 To move the analyte to the left click Up button To move the analyte to right click Down button The analyte moves one position to the left or right from its current position Figure 7 9 In the Single Analyte chart the Analyte Selector allows you to select the analyte you want to display in the chart Figure 7 10 gamma All

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