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Revelation Program User Manual MRX

1. ccceseeeeeeseseeeeeseeeeeeeeseeneeeeseeeeeeeeseeenens B 1 Bil OVeGrvidWesscsses i sissetsisass a Ret neaees tad Ae ea ats ets E og Mea et eee ETE B 1 B 2 Lam ar Regressio niera sire settee vaedes EE ra er E E S EEN E eE Er Eeh opsae B 2 B 3 Polygon Fitting neser eanes ner erea EEA A EEES EEE ne E EE EEEO TETEA EEES B 4 B 4 Quadratic Cubic and Quartic Regression ssseessseeseiesssreeesrerrsserresrsreeresreersserrrererrreresrent B 5 B 5 C bic Spline Curve Fitting srasni o rear cl een wa ERE dine anus ENSS B 7 B6 Akima Fit ee hinged e E a aia aa elses tee E adn ee eee B 8 B 7 Extrapolation of Non Linear Curves 0 e cece cee cseecseeeeeeeeeeeeeeseceaeceaecsaecneeeaeseaeeeeeeereneeens B 9 B 8 Sigmoid Equations iren aoin arare e ieee anes ads leet A SERR IRERE IS ESEAS B 10 B 9 Michaelis Menton Curve Fit seseeessesssreesesreersstsressesresrrsrerreseerinestentestenresrerrerentsseeeresreees B 11 B 10 Using Logarithmic Curve Fitting seeeseseeeesseeseresesreeresresrssrerrserrentrsrenresrerresenrrnresrenesee B 13 B20 Semi 0 8 Fit n a heh dea a EE e ia as ae ee siete B 13 Bi10 2 Log Lot Fitria tity hal dean Ade ae ed vain ele Aiea B 13 NNO E E A E A NTE E EE N E E ETT l 1 vi MRX Revelation Manual Chapter 1 Introduction 1 1 Overview Chapter 1 Introduction 1 1 Overview The Revelation application program is used with the DYNEX Technologies MRX Microplate Absorbance Reader Figure 1 1 This instrument
2. MRX Revelation Manual r R9232 Port Parity C COM1 T Rone O N Ee C com C Odd Baud Rate Data Bits 110 C 150 On 300 Stop Bite 600 E 1200 2 C 2400 4800 9600 OK Cancel Apply Help Figure A 5 The Setup External MRX Dialog Box RS232 Tab A 5 Setting the System Configuration Appendix A Using an External Computer Then establish Communications between a MRX Reader and an External Computer 1 Use the RS232 cable provided with the system This cable should be connected between the 9 pin serial port item 6 Figure A 1 and the serial port on the computer the computer may have more than one serial port and the mouse may be connected to a serial port 2 Power up the reader and allow it to perform the self test The MRX will indicate Endpoint on the left and Setup on the right on the display Press the arrow under the word Endpoint The display should indicate Endpoint Ready Start Recall Utility Program CE 3 Power up the computer close applications that may have been loaded during bootup e g anti virus programs network interfaces etc and launch Revelation 4 Close any self test that may be indicated on the monitor 5 Select Reader Configuration on the Utility menu No password is needed 6 Adialog box listing various configurations is presented Figure A 6 Reader Type x Select a reader OK Internal MAX Figure A 6 The Reader Type
3. 05 5 s ssscssccssseseescosesseeshsgstes vnposscesbeusees cbecpsdeasvencpesscevsnesbenseeeses 3 2 3 3 The Main Window sei sses sosessesbsisees visesses shsucees capsssesebauiees VE io SAE SEEE DEE r ETE EErEE ESE 3 5 e A EA E S E E EE 3 6 Chapter 4 Commands aa r ae aa iaa a a aa a aar a aa rer aaae aa ae arrana E aon aai dindan 4 1 A A OVERVIEW i n Seed dese eis ea A E E EE E E A E 4 1 42 The MENU Balaren e a a a e a e E A S T 4 2 AZ The Fle Men sor na r e E EA A E i E T 4 2 AD Dade INGW ASSAY r on o a a EE OS AEE E EE 4 2 4 2 12 Opein e oe Ae MANN Aa aks aed 4 3 ADA 3 Closes tikes sie d ee tite AAR SE AGES SARA E aaes 4 3 ADA Save Assay civics chek vibie dink ai uiteesh elena ME ae Ration oe 4 3 ADS Save Assay A Surio eect detent DRUG bleep esha te bbl EEE Greene needs 4 3 4 2 1 6 AMpOrt oo a ack Ss bie A AANA en EER ees 4 4 AD lad Run Plate eos ocak es seis suse suites cboebunadaenetes T A E ET 4 4 AD BRS AN PHU ARE AONE PAEA EEEN EE E E E E E ORE tees 4 4 4219 Pint Previ EW n e E AEE EEN R ER A 4 4 A ALO PUNE SOUP LENEE E EEE E ETE E O 4 4 AD A D S S A EE E E E E OE EO E Gana 4 5 422 Th Settings MON soreer eeoa ree eere e E ee a areas EEEN S EE SE SREE EENE 4 5 423 The View Men en eee area A A E a a e i 4 5 42A The Utility Menu ee eeraa nE E r A E S AEE eE ERESSE 4 5 ASDA Plate Plate Stati Sts ev renens a a a er i 4 6 4 2 4 2 Plate Reproducibility vepren perena i RE IS Oin E RA 4 7 E A SA WAN QUA VEA e AAE
4. Figure 3 2 The Reader Control Dialog Box Reader Option Tab The Reader Control dialog box will be described as follows Read Mode a Single Dual Wavelength used to collect a Multi Wavelength used to collect a Agglutination T G C used to collect Multi Wavelength Mode a Linear Regression describes how data is processed 5 There are several configurations of the system Some features are optional and are not included in all system configurations e g Agglutination This manual describes a complete program and indicates when a feature is available only in some configurations 6 Some features are not active in a given situation e g the radio buttons in the Multi Wavelength Mode region are not active unless a previous command has been selected Multi Wavelength in Read Mode 7 Some dialog boxes include tabs e g Figure 3 1 and edits can be made on various tabs Any change to the parameters will not be entered until the OK button is pressed to close the dialog box simply moving to another tab will not enter the data 3 4 MRX Revelation Manual Chapier 3 Introduction to the Operating Program 3 3 The Main Window 3 3 The Main Window The Main window of the program is presented in Figure 3 3 TITLE BAR MENU BAR TOOL BAR WORKSPACE Endpoint Untitled Assay Laboratory information Header information Lot specific data Removed outliers Edited wells Calculation mode Blan
5. Replicates The Replicates feature is used to indicate that a given sample is to be placed in two or more wells The number of wells that the sample is to be deposited in is indicated in the number field As an example if you indicate 4 replicates the Column orientation see below the Standard radio button and click in A1 wells A1 B1 C1 and D1 will be indicated S1 The number of replicates can range from 1 96 Replicates can be averaged into a single sample value or treated separately by selecting the appropriate option in the Output Format dialog box Chapter 10 The Define button which is only active for Agglutination assays is discussed in Chapter 9 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 4 Template Window To assign a sample with random replicates 1 2 3 4 Select the well type Select the number of replicates Select Random orientation Click on the well locations for each replicate Ee Note Samples are assigned according to available space If you select a format that does not fit into the available space Revelation will not assign the samples Orientation Orientation used to indicate the desired orientation of replicates if a sample is arranged in a group of more than one replicate Fill Direction Fill Direction used with the Fill command to define the direction in which the program will fill the template Fill places a sample in each position starti
6. 1 0 mm Add All Add Remove Position 1 2 8 mm Position 2 2 6 mm Position 3 2 4 mm Position 4 2 3 mm Position 5 2 1 mm Position 6 1 9 mm Position 7 1 7 mm Position 8 1 5 mm Position 9 1 4 mm Position 10 1 2 mm Position 11 1 0 mmi Position 12 0 8 mm Position 12 0 8 mm LJP eteentats Ose E S on 13 0 6 mm Clear Position 13 0 6 mm 100 well Bi Prsitinn 14 0 5 mm Z mi Prisitinn 14 N F mm Reader Options Start Mode Agglutination Parameters Agglutination Options Transmission Graphs Data Log r Readings Used C No Select Check list o o Cancel aw Figure 9 8 The Reader Control Dialog Box Agglutination Option Tab 9 11 9 3 Generating an Agglutination Assay Chapter 9 Agglutination Analyses and TCG Mode 9 12 Data Log The Data Log field will be active if the formulas or rules options have been selected on the Agglutination Parameters tab No data will not be presented in tabular format Columns displays the information in a column by column format Rows displays the information in a row by row format ae Note The data log may be useful when deciding the rules or formulae to set as it lets you see the importance of each parameter Blanking Options Point Point subtracts the blank from every agglutination reading across the well Well Well subtracts the blank from the calcul
7. Curve Fit x Fits Standards Fit Type Graph Sample Selection Dilutions Fit Q C Calculated Q C Data Conversion Results Flagging Output Format Equation Functions Units Figure 10 7 The Curve Fit Dialog Box Data Conversion Tab Curve Fit Data Conversion equations are prepared in the same manner as Ratio Equations see Section 10 5 1 10 4 4 The Curve Fit Dialog Box Results Flagging Tab The Curve Fit dialog box Results Flagging tab Figure 10 8 is used to specify a phrase that either replaces a numerical result or is displayed next to the result and is accessed by selecting the Results Flagging tab The phrase will be initiated whenever the numerical result meets the criteria that are specified 10 18 MRX Revelation Manual Chapier 10 Incorporating Decision Making Protocols into the Assay 10 4 Curve Fit Curve Fit Ed Fits Standards Fit Type Graph Sample Selection Dilutions Fit Q C Calculated Q C Data Conversion Results Flagging Output Format Condition Result Flag Functions E o WWA To Sample checks Move Up mE _Move Down Down Remove Geer Cancel Apply Help Figure 10 8 The Curve Fit Dialog Box Results Flagging Tab a Condition is used to indicate the Results Flagging expression Result is used to indicate if a phrase to be reported rather than a numerical result a Flag is used to enter the phrase if the numerical result is to be acc
8. 3 6 11 11 Exit File Menu 4 5 Exporting Data 11 4 11 9 Export to File Tab Report Options Dialog Box 11 6 External Computer A 1 Configuration A 2 Extrapolation of Non Linear Curves B 9 F Features 1 3 File Menu 4 2 File Types 3 6 Fill Direction Template Window 6 11 Filter Test 2 10 4 10 Fit QC Tab Curve Fit Dialog Box 6 32 Fits Tab Curve Fit Dialog Box 6 21 10 17 Fit Type Tab Curve Fit Dialog Box 6 24 Fit Types 6 25 B 1 Font Dialog Box 4 16 Formatting Reports 11 1 Formula Agglutination Parameters Tab 9 9 Formula Calculation Mode Agglutination Mode 9 4 G Generating an Assay Endpoint Analyses 7 2 General Information 6 1 Kinetic Analyses 8 2 Getting Started 2 1 Graph Curve Fit Dialog Box 6 28 H Header Footer Tab Report Options Dialog Box 11 10 Help Menu 4 23 I Import File Menu 4 4 Inhibitor Fit Type tab Curve Fit Dialog Box 6 27 Initial Testing 2 10 Initiating a Run 5 8 Agglutination Analysis 9 14 Endpoint Analysis 7 4 Kinetic Analysis 8 11 Installing Software External Computer A 5 Interfacing External Computer A 3 Introduction 1 1 to Operating Program 3 1 K Kinetic Analyses 1 3 8 1 During the Assay 8 15 Kinetics Graphs Reader Control Dialog Box 8 8 Kinetic Time Interval Reader Control Dialog Box 8 3 L Laboratory Information 11 2 Language Utility Menu 4 8 Linearity Testing 2 10 4 9 Linear Regression B 2 Logarithmic Curves B 13 Lot Spe
9. A sample Event Log is shown in Figure 11 8 All Worklists All Plates 8 29 59 AM 2 16 00 Revelation started Heaters turned off Loading assay C Program Files Revelation Assays od muin asy Heaters turned off Readine 405 Readine 405 Readine 405 Readine 405 Readine 405 8 32 30 AM od Readinge 405 8 32 34 AM od Reading 405 Loading assay C PROGRAM FILES REVELATION ASSAYSUOSETEST ASY Heaters set to 37 0 C 00 Readine 405 10 00 12 AM Heaters turned off 10 00 29 AM Loading assay C PROGRAM FILES REVELATION ASSAYSVOSETEST ASY Figure 11 8 An Event Log An event log can be viewed by 1 Selecting Open on the File menu 2 Select Event Log in the Files of type field The display will list the sample logs as shown in Figure 11 9 MRX Revelation Manual 11 11 11 4 The Event Log File Chapter 11 Formatting Reports and Exporting Data Look in a Event Logs aa 6 02110101 03140101 03140102 03190101 03190102 03190103 03190104 03190105 x 03190106 03200101 x 03200102 x 03200103 8 03260101 03260102 03260103 03280101 x 03280102 ws 03280103 Figure 11 9 Event Log Listing 3 Select the desired log Each log name is derived from the date and the number of times the reader was powered up that day e g 03200103 refers to the third time the reader was powered up on March 20
10. Move Up Move Down Remove V Full QC report I Suppress results if OC fails Clear J Lot specific checking of control values ww SAN HO Cancel Apply Figure 10 4 The Threshold Dialog Box Threshold Q C Tab Editing of this dialog box is identical to that for the Quality Control dialog box Section 10 2 10 12 MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 3 Threshold 10 3 5 Output Format The Output Format tab Figure 10 5 is used to determine what the format of the printout should be Threshold Ea Threshold Equations Pos Neg Q C Threshold Q C Output Format Output Options Mean r Scientific Notation M Matix M Average Replicates Arithmetic P Enabled G Free format T Table Decimal Places 3 C Geometric tulipes of 3 Fived at power ot E Result Order r Normal Ranges M Sample ID noma Renges M WellLocation s S Beo M Replicate Value oe eee M ean value 7 gt High Value 2999 933 Vi SD V Cy P SEH F Dilution kacir Table Options Printout Mode last ye First ve last ve First pve Last ve C Last ve OK Cancel Apply IE Figure 10 5 The Threshold Dialog Box Output Format Tab Output a Matrix outputs the data as a matrix which displays the values obtained from each well location in terms of its position on the plate in an 8 x 12 ar
11. asy Clear Bradford Proteins as R z Orientation CAT asy CMVIGG ASY Columns C Rows I Search for sample IDs First well location Number of wells 95 Cancel Help Figure 9 10 The Run Plate Dialog Box COLBLNK ASY CRYPTO ASY f Plate ID Plate ID A plate ID is used as a temporary filename for the plate data file which is created when the assay ends If you have selected Default Plate Ids in the assay a default ID will appear in the Plate ID box otherwise you will have to assign the ID manually Plate IDs can be assigned either by typing a name into the Plate ID text box or by scanning a barcode on the side of the microplate 9 14 MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode 9 4 Starting an Assay If you are using the Barcode option you should make sure the scanner is set up to decode the correct type of barcode Refer to Barcode Setup or refer to the Barcode Program User Manual Scan Bar Code before run To automatically read the plate barcode before the plate is run place a check mark in the Scan Barcode before run box As an alternative you can read the barcode at anytime by pressing the Read Barcode Now button Options Auto save plate data as The Auto save plate data as command is linked to the plate ID option When you enter a plate ID or scan a barcode Revelation displays the resulting filename in the Options box along with the default desti
12. 1 3 1 3 Principle of Operation sisin ninsis pes oiron aee E chante E EO EE E EESE NE rE E 1 4 1 4 MRX Revelation Application Software esssssseeesesesssesrseereseeststsreessesesrerreteeeseseererrees 1 5 1 5 Contents ofthis Mantal iensen ianen a E N E E N 1 6 Chapter 2 Getting Started ccssecesseeeeseeseseeeenseeeesseeeseeeeenseeeeseeseseesenseeeseeseseeesnseeaeeeas 2 1 Deli OVERVIEW iiei a rE E E oes aueadaa E E E eed Senso ecbetep tot E TRET 2 1 2 2 Powering Up the System isiessebsssisses cacesscoeeeas es vheaseavese sien chpeseadebuaioes sapestssesebeasoeesheeseedebasoe say 2 2 2 3 Setting the System Configuration eee cee eeseeseceseceecesecseecaeecseeeseseeeseeeeeeesseeseeeseeeeeeseens 2 5 2 3 1 Setting the Reader Type cee ceeeseeeceeeessceescesecesecssecaecaecsaecseecaeseseseeeeeeeeeeees 2 5 2 3 2 Configuring an Internal MRX Reader oo ee eeeceeeeceseceeeecseeeeeeaeeecsaeceeeseeneeeeenee 2 5 2 3 2 1 The Reader Tabien ienr a a a 2 6 23 2 2 The Maintenance Ta Desist ea a o aE e SEE 2 7 2 32 30 The RS 232 Ta Deseci oee o reee ae aiea ee na bes O ERE Ansa es 2 9 2 4 Initial Testing of the System oo cece cece creeeeeeseecseeeaeeeeeeseceseesseesecssecsaeesecesecaeenaes 2 10 Chapter 3 Introduction to the Operating Programm cesss esse seessnessesseesseneenseennens 3 1 Bill OVELVICW 5 decarns tiedigeaverecbenti ed Sovsvaceansees Uh eenwenevhes np aaa aaa ye seersenn ee a n 3 1 3 2 Conventions 1n this Manual
13. 8 Agglutination Analysis 9 14 Endpoint Analysis 7 4 Kinetic Analysis 8 11 Status Bar 4 26 Suppress Results if QC Fails 10 3 T Template Window 6 11 Test Report 2 12 Threshold Dialog Box 10 8 Threshold Equations Tab Threshold Dialog Box 10 9 Equations 10 9 Threshold Q C Tab Threshold Dialog Box 10 12 Tissue Culture Growth Measurements 9 6 Tool Bar 4 24 Transmission Graphs Tab Reader Control Dialog Box 9 13 MRX Revelation Manual ince U Use Original Assay Plate Plate Statistics Dialog Box 4 6 Use Readings Reader Control Dialog Box Calculation Mode Tab 8 5 Utility Menu 4 5 V Verification Plate 2 11 View Menu 4 5 W Wavelength Read Mode Tab Reader Options Tab 6 6 Well Type Template Dialog Box 6 12 Window Menu 4 22 MRX Revelation Manual l 5
14. D axis may be typed in In the Maximum box a higher O D or standard concentration number can be entered Tick marks there may be between 2 and 10 tick marks divide the axis evenly between the minimum and maximum entered values 6 8 6 The Curve Fit Dialog Box Sample Selection Tab The Curve Fit dialog box Sample Selection tab Figure 6 12 is used to indicate the samples that should be included in the curve fit calculation All the samples are shown in the Samples box The samples which are included in this curve fit are shown in the Check list box By default all test samples will be calculated against every curve fit MRX Revelation Manual 6 29 6 8 Curve Fit Chapter 6 Generating and Editing Assays Curve Fit Calculated Q C Data Conversion Results Flagging Output Format Fits Standards FitType Graph Sample Selection Dilutions Fita c m Select Samples Samples Check list Add All Add Remove Clear bh Tl Display Points on Graph Cancel Apply Help Figure 6 12 The Curve Fit Dialog Box Sample Selection Tab The Display Points on Graph check box is used if you want each point to be displayed on the plot in addition to the selected curve format 6 8 7 The Curve Fit Dialog Box Dilutions Tab The Curve Fit dialog box Dilutions tab Figure 6 13 is used to indicate if any of the samples have been diluted 6 30 MRX Revelation Manual Chapter 6 Generating and E
15. Dialog Box 7 Select the External MRX option and press Setup The Setup External MRX dialog box will be presented 8 Select the RS232 tab to present Communication Options Figure A 7 A 10 MRX Revelation Manual Appendix A Using an External Computer A 5 Seiting the System Configuration Setup External MRX x Reader R5232 mi 5232 Port Parity ne or r None C COM3 Even Default coma Odd Baud Rate m Data Bits 110 s 7150 o C 300 Stop Bits C 600 E 1200 Ca C 2400 C 4800 9600 Cancel Apply Help Figure A 7 Setup External MRX RS232 Tab Set the appropriate transmission parameters The Default button selects the configuration that is most frequently employed for serial communication it should not be necessary to change the Baud Rate Parity Data Bit and Stop Bits selections e COMI e Baud Rate 9600 e 1 Stop Bit e Parity Even e 8 Data Bits 9 Press OK The computer should link to the reader If the message Command Time Out or Reader not Detected is presented 10 Change the setting to COM 2 and press Verify If this does not solve the problem try COM 3 and COM 4 IEE Note Make certain that the COM port assignment does not conflict with any other serial port assignment on the computer MRX Revelation Manual A 11 A 5 Setting the System Configuration Appendix A Using an External Computer 11 Determine that the port is operating proper
16. Factor disables the function 6 22 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 8 Curve Fit a Additive used to increment each successive value by a specified amount To apply the additive function 1 Click on Additive and type in the incremental value If for example your standard concentrations are 1 5 9 13 17 pg ml standards then the additive number to enter is 4 2 Click on the first standard and type in its value in the box labelled Concentrations In this example that number would be 1 3 Click on the first item and type in its value 4 Press Enter on the keyboard to apply the addition to each successive item a Multiplier used to multiply each successive value by a specified amount To apply the multiplier function 1 Click on Multiplier and type in the multiplication value If your concentrations are 0 5 1 2 4 8 then the multiplier number is 2 If your concentrations are 100 50 25 12 5 6 25 and so on then the multiplier value is 0 5 2 Click on Standard one to highlight it and type in its concentration 3 Press Enter on the keyboard to apply the multiplier to each successive item a Percentage Response The Percent Response option allows the operator to use specified Standards as references for 0 or 100 or both By default the program uses the lowest Standard sample O D min or as a reference point for 0 and the highest Standard sample O D max as a reference point f
17. Generating and Editing Assays 6 3 8 Agglutination Options The Reader Control dialog box Agglutination Options tab is used to set a variety of data presentation parameters for agglutination measurements This tab is discussed in Section 9 3 4 6 3 9 The Reader Control Dialog Box Transmission Graphs Tab The Reader Control dialog box Transmission Graphs tab is used to specify that an Agglutination graph is desired for a given well s This tab is discussed in Section 9 3 5 6 10 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 4 Template Window 6 4 Template Window The Template window Figure 6 4 is provided to allow the user to indicate the sample positions and types of samples that will be contained by the plate Template for 96 wells x z ma N a a E N EA S Well Type Blank B1 C NCI1 C 5 SC1 Standard 1 C 2 PC1 C 6 AC1 Control C1 C 3 C01 C 7 N1 C Test T97 C 4 PRI C 8 HS1 C Delete Change Label Clear Undo Figure 6 4 The Template Dialog Box The template is used to indicate the type of sample that will be placed in the various wells To define the type of sample that will be placed in a specific well click on the radio button for the desired well type then double click on the well position If a given position is not to be used select the Delete radio button and click on the well Before a well type can be assigned to any well the existing templ
18. Print results automatically I Search for sample IDs m Assays Select Apoptosis asy Bradford Proteins as CAT asy CMVIGG ASY COLBLNK ASY CRYPTO ASY cet ew Open Selected Assayfs Save a Add Remove Clear Orientation Columns C Rows 406a asy First well location Number of wells 95 Figure 8 6 The Run Plate Dialog Box MRX Revelation Manual 8 11 8 3 Starting an Assay Chapter 8 Kinetic Analyses Plate ID Plate ID A plate ID is used as a temporary filename for the plate data file which is created when the assay ends If you have selected Default Plate Ids in the assay a default ID will appear in the Plate ID box otherwise you will have to assign the ID manually Plate IDs can be assigned either by typing a name into the Plate ID text box or by scanning a barcode on the side of the microplate If you are using the Barcode option you should make sure the scanner is set up to decode the correct type of barcode For more information refer to the Barcode Program User Manual Scan Bar Code before run To automatically read the plate barcode before the plate is run place a check mark in the Scan Barcode before run box As an alternative you can read the barcode at any by pressing the Read Barcode Now button Options Auto save plate data as The Auto save plate data as command is linked to the plate ID option When you enter a plate ID or sca
19. Result Sample construct to designate the calculated concentration of a sample instead of the absorbance values that were obtained For example the expression Result Sample lt 123 can be created to set a lower limit of 123 mg dL for the calculated value for any test sample Then whenever the value of a sample is less than 123 mg dL the designated phrase will be displayed and or operators Use the and or or operator in a Results Flagging expression to test multiple conditions MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 5 Ratio 10 5 Ratio 10 5 1 Ratio Equations The Ratio processing dialog box Ratio Equations tab Figure 10 9 is used to convert raw data into data with a different scale Ratio data is output as a matrix or table of converted results To enter a ratio equation click on the Enable box Ratio and data conversion equations may consist of up to 50 characters and can only include items shown in the table below Equations may not contain any spaces Ratio Equations Results Flagging Output Format Ratio Equation pl Units Data Equation Units Functions Cancel Apply Help Figure 10 9 The Ratio Processing Dialog Box a Ratio Equation used to convert raw assay data into a different scale Every value on the plate is fed into the equation The resulting value replaces the original valu
20. Stored Curve No Inhibitor Data Source Fit Fi 7 C Stored GUWE Browse OK Cancel Apply Figure 6 10 The Curve Fit Dialog Box Fit Type Tab Select the fit type by clicking on the desired radio button The various options are described in Table 6 4 along with the minimum number of points that must be provided for the fit type A discussion of each fit type is presented in Appendix B MRX Revelation Manual 6 25 6 26 Chapter 6 Generating and Editing Assays Table 6 4 Fit Types Fit Type Description of Standards Linear Linear polynomial regression 2 Quadratic Quadratic polynomial regression 3 Cubic Cubic polynomial regression 4 Quartic Quartic polynomial regression 5 Polygon Point to point straight line fit 3 Cubic Spline Smooth curve cubic spline fit 3 Lineweaver Burk Hyperbolic fit transformed axes kinetic 3 Michaelis Menten Hyperbolic fit kinetic only 3 Sigmoid S shaped curve fit four definable 4 parameters Log Logit S shaped curve fit with fixed parameters Akima Smooth curve passes through all data 3 points Parallel Line Bioassay Parallel line bioassay 4 Stored Curve Curve saved from a previous test 0 a c Sigmoid Parameters activates the Sigmoid Parameters Maximum Iterations The value entered here is the maximum number of times for Revelation to attempt to construct a satisfactory plot before abortin
21. T represents the mean of all the Test well values T1 represents the mean of the replicates of T1 if T1 contains multiple replicates or the individual value of T1 T1 1 represents the value of the first replicate of T1 Tli represents any individual replicate of test one 4 The following logarithmic functions can be used Log Base 10 logarithm Alog Inverse base 10 logarithm Ln Natural logarithm Exp Inverse natural logarithm 5 For the Minimum Maximum of a group of values MIN or MAX represents the highest or lowest value on the plate or within a group of values The group of values may be a well type or a sample if it contains multiple replicates As examples MIN PC is the lowest value of all the Positive Control samples MIN is the lowest value on the plate 10 4 MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 2 Quality Conirol CV is used to represent the coefficient of variation within a group of values The group of values may be a well type or a sample if it contains multiple replicates As an example CV T1 is the value inserted into the equation for the coefficient of variation between every replicate of Test 1 SD is used to represent the standard deviation within a group of values The group of values may be a well type or a sample if it contains multiple replicates As an example SD T is the value inserted into the equation for the standard deviation between every tes
22. across the well with a T less than 100 9 2 MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode 9 1 Agglutination CELLS DISPERSED 100 T O READINGS 31 Figure 9 3 Cells Disperse 3 The cells coagulate and settle to the bottom of the well in a central stain and the rest of the sample is clear close to 100 T The profile shows 100 T at the sides of the well with a small T in the region of the dark stain CELLS COAGULATE 1002T O READINGS 31 Figure 9 4 Coagulated Cells 4 Ifthe cells do not agglutinate but settle to the bottom of the well in a ring and the rest of the sample is clear close to 100 T The profile shows high transmission in the clear areas of the well but shows two troughs that correspond to a cross section through the ring MRX Revelation Manual 9 3 9 1 Agglutination Chapter 9 Agglutination Analyses and TCG Mode RING 1002 T O READINGS 31 Figure 9 5 Ring Formation 9 1 3 Agglutination Calculations The user can generate four parameters which can be used to calculate well profiles from the data obtained by scanning the well When you are creating agglutination assays you must specify how the reader is to determine if the results are positive or negative There are two general approaches that can be used a Enter a formula based on the four parameters that are used to define positive and negative threshold ranges a Establish a list of rules to which results must
23. bottom region indicates all open windows and the check mark indicates the active window 42200 NRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar 4 2 6 Help The Help menu Figure 4 22 accesses the on line help program and a dialog box with version number information Help Help Topics About Revelation Figure 4 22 The Help Menu In addition you can get help by clicking F1 or by dragging the Help icon Section 4 3 MRX Revelation Manual 4 23 4 3 The Tool Bar Chapter 4 Commands 4 3 The Tool Bar The Tool Bar Figure 4 23 contains a series of icons that are used to rapidly access various features of the program Bist 34 SIL Figure 4 23 The Tool Bar Accesses the Assay Type dialog box Section 4 2 1 1 Accesses the Open dialog box Section 4 2 1 2 Accesses the Save Assay dialog box Section 4 2 1 4 Initiates the Run Plate activity Section 4 2 1 7 Prints the run Section 4 2 1 8 Shows the Print Format on the display Accesses the Assay Wizard Section 6 1 Accesses the Assay Title dialog box Section 6 2 Accesses the Reader Options dialog box Section 6 3 1 Accesses the Template tab Section 6 4 JESORe BEB ee Accesses the Blanking dialog box Section 6 5 A240 MRX Revelation Manual 4 3 The Tool Bar Chapter 4 Commands e cge y z Sees MRX Revelation Manual Accesses the Quality Control dialog box Section 10 2 Access
24. convert OVER or UNDER readings into values that can be input into quality control equations check this box OVER Value Enter the value that all OVER values will be converted to this field is active only if you checked the OVER UNDER Conversion box The range is from 0 000 to 9 999 UNDER Value Enter the value that all UNDER values will be converted to this field is active only if you checked the OVER UNDER Conversion box The range is from 9 999 to 0 000 Miscellaneous Options Repeatability Mode The value for Repeatability Mode indicates the number of times Revelation will read each individual well If the value is greater than 1 Revelation will average the repeated readings into a single result for each well This means that the result is slightly more accurate but it does lengthen the reading time significantly The range is from no repeats to 4 Sleep Mode The sleep mode interval determines how long the plate carrier can be left out before it is automatically retracted Automatic retraction prevents the plate carrier from collecting dust and being damaged It will not work if the reader failed any part of its self test The range is 0 minutes no automatic retraction to 32767 minutes 2 3 2 2 The Maintenance Tab The Setup Internal MRX dialog box Maintenance tab is presented in Figure 2 6 MRX Revelation Manual 2 7 2 3 Setting the System Configuration Chapter 2 Getting Started Setup Internal MRX x
25. have been artificially coated with antigen can also be used Various types of particles are commonly used including turkey red cells which work well because they are large and sediment quickly bentonite polystyrene and latex Agglutination tests are commonly carried out in the liquid phase When the test is performed two responses are possible a No cross linking has occurred cells fall to the bottom of the well a Cross links formed cells form a lattice structure in which they are evenly divided 9 1 2 The Agglutination Measurement The reader measures the extent of the agglutination reaction by taking 32 readings across the well as shown in Figure 9 1 The reader measures T the fraction of light that is transmitted through each point in the well MRX Revelation Manual 9 1 9 1 Agglutination Chapter 9 Agglutination Analyses and TCG Mode il NUMBER OF 1 READINGS 52 Figure 9 1 Measuring the Agglutination Reaction The reader plots the T against the well position to form a well profile If a well contains a clear area and a dark area the clear area will have a high T and the dark area will have a low T Figures 9 2 through 9 5 show typical results of four agglutination conditions 1 An empty well transmits all the light giving a uniform profile of 100 T EMPTY WELL 1002T O READINGS 31 Figure 9 2 An Empty Well 2 The cells agglutinate to form a murky liquid and light is transmitted at an even level
26. in Section 11 3 3 To automatically export data as a part of an assay 1 Select the Run Plate command from the File menu to present the Run Plate dialog box Figure 11 6 Run Plate Ea Plate ID Options Plate ID IV Auto save plate data as I Scan Barcode before wn CAPROGRAM FILES REVELATION Plate T Print results automatically Head Barcode Wow Operater I Search for sample IDs Operator Assays Select Open Selected Assay s asy 96 Al 406a asy Save Add Remove Apoptosis asy Clear Bradford Proteins as CAT asy Orientation 7 i CMVIGG ASY Qn First well location COLBLNK ASY Number of wells 35 Cancel Help Figure 11 6 The Run Plate Dialog Box CRYPTO ASY m Rows 2 Enable the Auto save plate data as check box 3 Enter the Plate ID information in the Plate ID field The data will be saved using that file name When the plate is run the data will be saved in the appropriate directories as a dat file which is read by Revelation and a txt file which can be read by external programs The data will be the same but will be in different formats To see the txt file it is necessary to leaveRevelation either by minimizing or closing the program After you have left Revelation open the program with which you want to edit the text and then open the file MRX Revelation Manual 11 9 11 3 Report Options Chapter 11 F
27. is an initial installation or you want to change various configuration parameters select Configure Reader on the Utility menu to present the Reader Type dialog box Figure 2 4 You MUST close out of Self Test all assays and all data screens so that only the top task bar is showing with the rest of the window blank before Revelation will allow you to chose the correct reader type and then change the activate the setup button Reader Type x Select a reader OK External MFX External MAX Figure 2 4 The Reader Type Dialog Box Select the reader type Internal MRX and press Setup to access the Setup Internal MRX dialog box see Section 2 3 2 2 3 2 Configuring an Internal MRX Reader Once the parameters have been set it will not be necessary to configure the system unless you need to change a parameter To access the Setup Internal MRX dialog box Figure 2 5 highlight Internal MRX and press Setup ae Note If this is an original installation a message will be presented to alert the user to enter an integer between 2 and 6 instead of the above dialog box This message is used to indicate that the present value for the Number of Filters field is not valid and must be edited Click OK to access the dialog box which will include the value of 0 for the Number of Filters field MRX Revelation Manual 2 5 2 3 Setting the System Configuration Chapter 2 Getting Started 2 3 2 1 The Reader Tab The Setup Internal MRX dialo
28. is user defined 9 6 MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode 9 3 Generating an Agglutination Assa 9 3 Generating an Agglutination Assay 9 3 1 Basic Assay Information To generate an Agglutination assay 1 Select New Assay on the File menu 2 Select Endpoint on the Assay Type dialog box 3 Select Agglutination T C G in the Read Mode field of the Reader Options tab of the Reader Control dialog box 4 Develop the assay as described in Chapter 6 Several dialog boxes on the Reader Control dialog box are used when Agglutination assays are employed 9 3 2 The Reader Control Dialog Box Start Mode Tab The Start Mode tab on the Reader Control dialog box Figure 9 6 is provided to indicate when the O D of the sample should be read Reader Control xi Agglutination Options Transmission Graphs Reader Options Start Mode Agglutination Parameters Start Mode Immediate C When ri reaches oD When ri reaches OD min After fi minutes Pees Cancel Apply Figure 9 6 The Reader Control Dialog Box Start Mode Tab MRX Revelation Manual 9 7 9 3 Generating an Agglutination Assay Chapter 9 Agglutination Analyses and TCG Mode Four options are provided Immediate the data is acquired as soon as the assay is initiated When T1 reaches xxx OD do not use When T1 reaches OD min do not use After xxxx minutes the data is collected after the indicated tim
29. it from the other well values on the plate The program always subtracts the Blank value from individual well values before any other processing calculation is made If any of the non Blank samples are arranged in multiple replicates the blank value is subtracted before the average of the replicates is calculated The program allows you to choose from nine different blanking modes but some of them require a certain amount of Blank samples on the template In these cases Revelation will allow you to select a Blank mode which requires Blank samples before these samples have been defined However the blanking operation will not work unless you create these samples before running the assay MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 5 Blank Mode a None indicates that the transmission of air should be used as the reference level for 100 transmission This is the default blanking mode and Blank samples do not need to be defined in the template a Previous Each time the system finishes reading a plate it stores the last Blank value in memory Previous blanking mode uses the last value stored as a Blank for every well on the current plate If the last plate did not use any blanking then the transmission of air is the blank value This value will be used as the previous blank value No Blank samples are required for this mode a Plate the data matrix from a previously read plate is used as a blank matrix for th
30. levels Test Bulb Test ilter motor Test nsmission levels Test ilters Self Diagnostic Summary Reader Status Ready Figure 2 2 Sample Self Test Report MRX Revelation Manual 2 3 2 2 Powering Up the System Chapter 2 Getting Started We recommend that you print a copy of the report on a periodic basis e g once a month by selecting the Print command on the File menu and save the reports to monitor the performance of the reader on a long term basis 7 Check that the time and date is properly indicated on the report If the time date are not correct click on the time on the bottom right corner to present the Date Time Properties dialog box Figure 2 3 and edit these parameters as appropriate Date Time Properties 24 x Date amp Time Time Zone 7 B 9 13 14 15 16 17 20 21 22 23 24 27 28 fir 25 40am H Current time zone Eastern Standard Time Figure 2 3 Date Time Properties Dialog Box 8 The system is now ready to collect data a If this is the initial use of the system or it is necessary to change the system configuration e g you have installed a new filter refer to Section 2 3 If you want to use the verification plate to check the system see Section 2 4 otherwise continue to Chapter 3 2 4 MRX Revelation Manual Chapter 2 Getting Started 2 3 Setting the System Configuration 2 3 Setting the System Configuration 2 3 1 Setting the Reader Type If this
31. mode one reading is taken in Single mode where a test filter is used to read the plate In Dual mode a test filter and a reference filter are used for extra accuracy and reduction of minor background contribution by the microplate In Multi Wavelength Mode the plate is read at two different wavelengths If any O D in the primary reading is above the OVER limit Section 2 3 2 1 the reading from the secondary test filter reading will be used and the secondary value will replace the OVER value in the results This value is converted into the primary reading range using one of three calculation modes a In Agglutination mode a filter is used in the same manner as in Single Dual Wavelength mode see Chapter 9 for details MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 3 The Reader Control Dialog Box 6 3 2 2 Multi Wavelength Mode The Multi Wavelength Mode field is activated when the Multi wavelength radio button is selected in the Read Mode field This mode is used to read the absorbance peak if wells are very dark in color or if it is likely that the O D will be over the set O D limit It provides three options a Linear Regression indicates that Revelation uses a line of best fit calculated from the two sets of data a Well Type uses the ratio between the primary and secondary readings for a specified sample The user indicates the sample a Value multiples the readings by a fixed value The user can enter
32. of Results Flagging expressions can be defined for an assay Table 6 5 Results Flagging Elements Type Use the Type keyword to designate the well type Standards Controls Tests etc that a Results Flagging equation is applied to For example the expression Type C 0 means that this equation applies only to the control wells Sample for Use the Sample keyword to designate the absorbance of a particular O D values sample replicate or well type in the same manner as used in Q C Raw Data equations Result Sample for calculated values Use the Result Sample construct to designate the calculated concentration of a sample instead of the absorbance values that were obtained For example the expression Result Sample lt 123 can be created to set a lower limit of 123 mg dL for the calculated value for any test sample Then whenever the value of a sample is less than 123 mg dL the designated phrase will be displayed and or operators Use the and or or operator in a Results Flagging expression to test multiple conditions MRX Revelation Manual 6 37 6 8 Curve Fit Chapter 6 Generating and Editing Assays 6 8 12 Curve Fit Output Format Tab The Curve Fit dialog box Output Format tab Figure 6 18 is used to indicate the format of the report for the Curve fit Curve Fit a Vv M Ta JM a m J ir m Figure 6 19 The Curv
33. or formulae The four agglutination parameters are derived for each well using the well s transmission profile The reader calculates the parameter values using a complex algorithm MRX Revelation Manual 9 5 9 2 Tissue Culture Growth T C G Measurements Chapter 9 Agglutination Analyses and TCG Mode 9 2 Tissue Culture Growth T C G Measurements 9 2 1 Introduction to Tissue Culture Growth Measurements An example of Tissue Culture Growth Measurements is the cultivation of viruses using a thin layer of animal tissue The spread of the virus is artificially limited leading to local destruction of cells The culture is then stained with a dye which distinguishes living tissue from dead tissue and the plaques of dead tissue can be clearly seen The reader measures the contents of the well by taking a series of readings across the base of the well in a manner similar to that for agglutination measurements When Tissue Culture Growth measurements are made a flat bottom well is normally used If a well contains a clear area e g dead cells and a dark area e g live cells the clear area will transmit a lot of light and the dark area will transmit little light The reader will express this area on the well profile as regions of high transmission and low transmission The reader then takes the T from each well and averages them to give an O D for each well and this O D is used to calculate a threshold matrix the range for this matrix
34. place the assay in wells Al to H1 and A2 to D2 if you select Rows Revelation will place the assay in wells Al to A12 ae Note The selected orientation applies to the whole plate and not just the highlighted assay If you change the orientation when some assays have already been positioned on the plate you will have to change the first well assignments as these depend on the template orientation MRX Revelation Manual 8 13 8 3 Starting an Assay Chapter 8 Kinetic Analyses IEE Note Do not combine different assay types on the same plate For example do not combine kinetic assays and endpoint assays or agglutination assays and non agglutination assays on the same plate To initiate the run press Start In a few instances the assay will not be automatically initiated a If you inserted a Shake operation the program will shake the plate before reading it a If the assay requires that the assay temperature is not the ambient temperature the reader will check that the plate is as at the appropriate temperature If the temperature is not correct a dialog box will be presented indicating that the present temperature and programmed temperature The operator will be given the option to Read Now or Abort the Assay The temperature will be driven to the set point and you can wait until the present temperature is at near the desired temperature before starting the assay a In some cases the analyst will be prompted to e
35. sample before running the assay a When the rate of change of the O D for a specific sample exceeds a user specified rate the reading of the plate will begin a After a fixed period of time range 1 3599 minutes the reading of the plate will begin Stop mode is similar to Start mode and is used to indicate when the measurement should stop The After readings entry is used to indicate that the measurement should stop after the specified number of readings have been taken 8 2 3 The Reader Control Dialog Box Kinetic Time Interval The Reader Control dialog box Kinetic Time Interval tab is presented in Figure 8 2 MRX Revelation Manual 8 3 8 2 Generating an Assay for a Kinetic Analysis Chapter 8 Kinetic Analyses 8 4 Reader Control E x Reader Options Start Stop Mode Kinetic Time Interval Calculation Mode Kinetic Graphs m Time Interval Variable Time Interval C ariable Reading 1 00 00 00 Fixed 00 01 00 Fixed Time Interval e0 Seconds Auto Increment feo SECS Eill OK Cancel Figure 8 2 Reader Control Dialog Box Kinetic Time Interval Apply The Kinetic Time Interval tab is used to define the time interval s between the kinetic readings Time Interval The Time Interval selection is used to indicate if the time interval between readings should be the same Fixed number of minutes or different between different readings Variabl
36. the beginning or at the end will not be used in the calculations of changes in O D over time The readings outside the range will not be processed but they will used to plot the Kinetic graphs All no readings will be deleted Range indicates points or data from which numbered readings are to be included Result Format Result Format used to indicate if the data should be reported as O D or mO D milli values If the results are reported as mO D the reader will multiply each O D by 1000 and remove the decimal point and decimal places This option is similar to the Results Scaling option see below except that the results are reported in different units Cut off used to eliminate kinetic readings toward the end of the assay The operator can enter the desired cut off value in O D Any reading for an individual well above this cut off value will not be processed and will not be displayed in the Kinetics graph for that well The reader will continue to read the plate until all wells have reached this value or the run is aborted Ie Note If the data is decreasing with time the cut off value will represent a lower limit The Cut off can be absolute or relative to the first data point Click on Absolute or Relative and enter the cut off value Miscellaneous Options Data Increase with Time If a calculation mode with an O D limit is used this field is used to indicate if the O D value should be increasing or decr
37. the MRX is performing according to manufacturer specifications a Alignment Consists of three alignment checks X alignment Y alignment and Skew which are directions of movement of the plate carrier An instrument passes this test if the x alignment is 0 6mm y alignment is 0 8mm skew is 0 8mm a Linearity An area of a test plate is set aside for a series of wells that should in theory give a straight line plot of O D against concentration The Linearity test measures the deviation of these O D readings from this straight line To determine Linearity Revelation calculates the slope y intercept and t value of the line where t value is a statistical measure of how well the plate readings match the theoretical straight line High t values indicate good linearity at high O D s low t values indicate that the instrument will only have good linearity at low O D s An instrument passes this test if the t value for each filter is greater than or equal to 8 61 for filters that transmit wavelengths above 400 nm a Filter Test This test verifies proper filter installation in the filter holder This test only determines an incorrect filter set up if the filters installed in an instrument differ by more than 60 nm from the filter test wells on the plate The data will indicate if a filter is failing a Optic Channel Matching Readings are taken for each optical channel and are compared with each other to check for consistent performa
38. the equations are not valid an error message telling you which equation is invalid is presented Most errors are due to small errors such as spelling the order of arithmetic operators etc 10 2 3 Typical Equations This section includes a few examples for a template that includes 4 NC s negative controls and 3 PC s positive controls 1 If you want a criteria that if any NC is greater than 25 higher or lower than the mean of all the NC s it must be rejected the following equation would be used 0 75 NC lt NCi lt 1 25 NC 2 If you want a criteria that if one NC fails the first quality control equation the test must be rejected the following equation would be used valid NC gt 3 3 If you want a criteria that if the average NC must not be greater than 0 2 but if the average PC is greater than 0 7 average NC s of up to 0 3 will be accepted the following equation would be used If PC gt 0 7 then NC lt 0 3 else NC lt 0 2 4 If you want a criteria that if at least 2 of the PC samples must be within 10 of the mean of the other PC values the following equation would be used Verify PC 10 1 gt 2 5 If you want the average of the Negative Controls to be more than 0 5 O D then enter NC gt 0 5 6 Ifthe average of the Positive Control O D s must be less than 20 of the average of the Negative Control O D s then type PC lt 0 2 NC MRX Revelation Manual 10 7 10 3 Threshold Chapier 10 Incorporating Decision M
39. the plate barcode before the plate is run place a check mark in the Scan Barcode before run box As an alternative you can read the barcode at any by pressing the Read Barcode Now button 2 Select the Desired Option s Auto save The Auto save command is linked to the plate ID option When you enter a plate ID or scan a barcode Revelation displays the resulting filename in the Options dialog box along with the default destination The default destination is determined by the Options command on the Utility menu If you intend to recall plate data at a later date you should always assign the ID as a permanent filename Print Results If you do not want the data to be printed automatically un check the Print Results check box stored data can be printed using the Print command on the File menu after collection Search If you created a sample ID file using the Sample ID SID entry on the Utility menu Revelation can automatically search for this file Sample ID files are saved by default in the SID directory If you want to search for the file automatically you should make sure it has the same name as the plate ID To link sample IDs to the template click on Use Template MRX Revelation Manual 5 5 5 3 Selecting the Assay Conditions Chapter 5 Running an Assay 5 6 3 Select the Desired Assay An assay list is used to indicate the assay s to be run Assay lists may contain a single assay or a number of assays If you pla
40. time period and is calculated by subtracting the O D of the reading at the first time point from the O D of the reading at the last time point which is then the average value over the whole of all the readings OD min Vmax measures the rate when the reaction rate is greatest Vmax by measuring the change in O D between any two time points OD min by Linear Regression measures the rate by drawing the best least squares linear fit between the O D s at all the time points Time options measure the time taken for the reaction to reach a particular point Time to Max OD measures the absolute time that the reaction takes in each individual well to reach to the maximum constant O D Time to Max OD min measures the time required for the maximum change in O D as a function of time OD min It is the point where the change in O D divided by the time is at its greatest or steepest part of the curve Time to defined OD measures the time to the user selected O D which is entered in the field adjacent to the entry Time to defined OD min measures the time to the user selected O D min which is entered in the field adjacent to the entry Time to relative OD min measures the time to the user selected O D min which is entered in the field adjacent to the entry The measurement is made relative to the first reading taken to relative O D The relative value is entered in the field adjacent to the entry Moni
41. will operate on the last option s processed data IEE Note You cannot pass a Parallel Line Bioassay to another processing option 6 13 4 Assay Options Dialog Box Sample ID Setup Tab The Assay Options dialog box Sample ID Setup tab Figure 6 23 is used to assign sample ID s to the various wells on the plate Assay Options Ea Area Statistics Processing Order Sample ID Setup Figure 6 23 The Assay Options Dialog Box Sample ID Setup Tab 6 44 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 12 Assay Options By default every sample and replicate is selected and indicated by a diamond adjacent to the sample Clear All is used if you do not want SIDs to be assigned to any of the wells or if you want to start again from scratch Undo is used to undo any changes you have made either since you selected the Sample ID s tab or since you last pressed the Undo button Select All is used to assign sample IDs to every well on the plate click on Select All Split Replicates is used if you want sample IDs to be applied to whole samples regardless of the number of replicates they contain un check the Split Replicates box If you want the sample IDs to be assigned to every replicate check the Split Replicates box MRX Revelation Manual 6 45 Chapter 7 Endpoint Analyses 7 1 Overview Chapter 7 Endpoint Analyses 7 1 Overview In an endpoint analysis the reaction forming the chromophore color
42. will place the assay in wells Al to A12 ae Note The selected orientation applies to the whole plate and not just the highlighted assay If you change the orientation when some assays have already been positioned on the plate you will have to change the First well assignments as these depend on the template orientation Ie Note Do not combine different assay types on the same plate For example do not combine kinetic assays and endpoint assays or agglutination assays and non agglutination assays on the same plate 9 16 MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode 9 4 Starting an Assay Press Start to initiate the run In a few instances the assay will not be automatically initiated a If you inserted a Shake operation the program will shake the plate before reading it a If the assay requires that the assay temperature is not the ambient temperature the reader will check that the plate is as at the appropriate temperature If the temperature is not correct a dialog box will be presented indicating that the present temperature and programmed temperature The operator will be given the option to Read Now or Abort the Assay The temperature will be driven to the set point and you can wait until the present temperature is at or near the desired temperature before starting the assay a In some cases the analyst will be prompted to enter additional information before proceeding e g sample names
43. 2001 11 12 MRX Revelation Manual Appendix A Using an External Computer A 1 Overview Appendix A Using an External Computer A 1 Overview In most instances the Revelation software is used with the personal computer that is installed inside the MRX reader The system is also available for use with an external computer This appendix describes how an external unit is interfaced to the reader and the differences in operation between the internal and external computer configuration MRX Revelation Manual A 1 A 2 The Personal Computer Configuration A 2 The Personal Computer Configuration Appendix A Using an External Computer As a minimum the personal computer should have the configuration indicated in Table A 1 Table A 1 Minimum Computer Configuration Component Configuration Computer Pentium processor 233 Hz Operating System Microsoft Windows 98 NT Hard disk space At least 1OMB of free space Monitor An SVGA Color graphics card and compatible color monitor Monochrome CGA EGA or VGA calibrated monitors are not supported Mouse A Windows compatible mouse or other pointing device Memory At least 64 MB of RAM MRX Revelation Manual A 3 Interfacing the Computer to the MRX Reader A 3 1 Electrical and Communications Interface IEE Note The computer and MRX reader should not be powered up while the communications cable is being installed To interface the comp
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45. ANNOT draw a curve unless the wells used to create the x and y axis have been labelled with an S on the template see Section 6 4 When Curve Fit processing is used the program measures the O D for a series of standard samples of known concentrations and uses the readings to generate a graph which is used to calculate the concentrations of test samples experimental unknowns The numbers entered for the known standards are used to draw the x axis of the graph and the O D s from the known standards are used to draw the y axis The Curve Fit dialog box Figure 6 8 includes ten tabs which are used to present options for various aspects of the curve fitting protocol Curve Fit Ed Calculated Q C Data Conversion Results Flagging Output Format Fits Standards FitType Graph Sample Selection Dilutions Fito c Enter Fit Name Fte Fits Remove Fit Copy Fit OK Cancel Apply Help Figure 6 8 Curve Fit Dialog Box Fits Tab MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 8 Curve Fit 6 8 2 The Curve Fit Dialog Box Fits Tab The Fits tab is used to insert or select a curve in the assay All existing fits are indicated in the listing area in the Fits field To select the desired fit click on it a Fit Name by default curves are named Fit1 Fit2 and so on When you open the Curve Fit dialog box Revelation offers Fit 1 You can either enter a new na
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48. Assay Wizard command on the Settings menu The Assay Wizard presents a series of dialog boxes that the analyst completes to indicate the desired parameters for the assay As an alternative an assay can be generated by selecting each setting parameter separately from the drop down Settings menu and saved If desired you can edit an existing assay by selecting Open from the File menu and then access the desired dialog box es via the Settings menu After an assay has been created or edited it can be saved via the Save command on the File menu Assays can be retrieved via the Open command on the File menu Section 3 4 1 and edited via the various commands on the Settings menu In order to execute an assay select the Read Plate command on the File menu MRX Revelation Manual 6 1 6 2 Generating a New Assay Assay Title Dialog Box Chapter 6 Generating and Editing Assays 6 2 Generating a New Assay Assay Title Dialog Box When the Assay Wizard command is selected the Assay Title dialog box Figure 6 1 is presented Assay Title x Enter Assay Title Untitled Assay Written by m Password Enter Password Retype Password Assay Code Prefix Suffix Finish Next gt Cancel IE Figure 6 1 The Assay Title Dialog Box The Assay Title dialog box includes the following fields a Assay Title The name of the assay which can be any alphanumeric name that you like This title can bu
49. Blank highlighted in the Blanks list box Assigning a Blank to a Sample 1 Highlight the required Blank 2 Highlight the samples you want to assign the Blank to 3 Click on Add The samples will appear in the Check list box and will continue to be displayed until you click on a different Blank MRX Revelation Manual 6 17 6 6 Quality Control Raw Data Chapter 6 Generating and Editing Assays 6 6 Quality Control Raw Data Quality Control Q C equations are used to place criteria on the raw assay data threshold data or curve fit data If the criteria are not met the data will be processed as normal but the results report will contain a message showing that the data failed Q C Quality Control Calculations are described in Chapter 10 6 18 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 7 Threshold 6 7 Threshold The Threshold dialog box is used to indicate ranges for samples that are going to be qualitatively determined to be positive or negative according to the assay parameters If there is a large neutral area between positive and negative you may define segments or break points within this neutral area This feature is described in Section 10 3 MRX Revelation Manual 6 19 6 8 Curve Fit Chapter 6 Generating and Editing Assays 6 8 Curve Fit 6 20 6 8 1 Introduction to Curve Fitting Standard wells MUST be designated on your template before a standard curve can be set up Revelation C
50. Box 6 2 Assay Wizard 6 1 Assigning Assay Buttons 4 17 Automatic Entry Standards tab Curve Fit Dialog Box 6 22 Automatic Outlier Removal QC Tab Curve Fit Dialog Box 6 32 B Blanking Option Agglutination Options Tab Reader Control Dialog Box 9 12 Blank Mode 6 14 Blank Subtraction Kinetics Mode 8 7 Buttons Tab Options Dialog Box 4 17 MRX Revelation Manual C Calculated Q C Tab Curve Fit Dialog Box 6 34 Calculation Mode Reader Control Dialog Box 6 9 8 5 Calculation Options Fit Type Tab Curve Fit Dialog Box 6 27 Calibration Plate Dialog Box 2 11 Calibration Protocol 4 10 Change Label Dialog Box 6 12 Close File Menu 4 3 Colors Tab Options Dialog Box Commands 4 1 Configure Reader Utility Menu 4 8 Contents of Manual 1 6 Conventions 3 2 Creating Quality Control Equations 10 4 Cubic Regression B 5 Cubic Spline Fitting B 7 Curve Fit Dialog Box 6 20 10 17 Custom Tab Blank Mode Dialog Box 6 17 Cut off Reader Control Dialog Box Calculation Mode Tab 8 5 D Data Conversion Tab Curve Fit Dialog Box 6 35 10 18 Data Log Agglutination Options Tab Reader Control Dialog Box 9 12 Decision Making Protocols 10 1 Default Plate ID Preferences Tab Options Dialog Box 4 18 Diagnostics 2 3 Dilutions Tab Curve Fit Dialog Box 6 28 Directories Utilities Dialog Box 4 21 E Editing Assays 6 1 Endpoint Analyses 1 3 7 1 Generating 7 2 Report 7 3 Event Log Files log
51. CE OF THE SOFTWARE PROGRAM IS WITH YOU NO LIABILITY FOR CONSEQUENTIAL DAMAGES DYNEX TECHNOLOGIES SUPPLIERS SHALL NOT BE HELD TO ANY LIABILITY FOR ANY DAMAGES SUFFERED OR INCURRED BY THE END USER INCLUDING BUT NOT LIMITED TO GENERAL SPECIAL CONSEQUENTIAL OR INCIDENTAL DAMAGES INCLUDING DAMAGES FOR LOSS OF BUSINESS PROFITS BUSINESS INTERRUPTION LOSS OF BUSINESS INFORMATION AND THE LIKE ARISING FROM OR IN CONNECTION WITH THE DELIVERY USE OR PERFORMANCE OF THE SOFTWARE PROGRAM Special Provisions This Software Licence Agreement and Warranty are governed by the laws of the States of Washington U S A and Virginia U S A IMPORTANT WARRANTY INFORMATION FOR REVELATION MRXS Do not install any applications other than Revelation on a Revelation Reader with a built in PC as this may affect the reliability of assays and will invalidate the warranty Do not attach non DYNEX Technologies approved monitors keyboards or mice to the Reader as this will invalidate the warranty ii MRX Revelation Manual Table of Contents Table of Contents Revelation Software License Agreement eseerersreeersererererereesrerersreersrerersreeeseereroreseeneusrererssrerses i Chapter T IMtrOGuctionn a ra a cada aaraa a a aaa pa aara cate didcceeteadesadaesaccecnccces aedecddctazecee 1 1 DV OVE VIC W AA A san levine cpubeysceavse ceduaobaus 1 1 1 2 Modes of Operation and System Features 00 cece eeeeeeeeeeeeeceeeeesecnsecesecsaecsecaeeaeeeneees
52. DY NE X Absorbance amp Plate Washing wee Revelation Program User Manual Automated for MRX Software DYNEX Technologies Inc 14340 Sullyfield Circle Chantilly VA 20151 1683 Phone 703 631 7800 FAX 703 803 1441 www dynextechnologies com This page is intentionally left blank Revelation Software Users Guide IMPORTANT Please read this manual carefully before using the program Notice The Revelation application program is covered by a software license a copy of which is enclosed in this manual Part Number 99001830 Rev 10 19 04 DYNEX Technologies Inc reserves the right to make technical improvements to this program and documentation without notice as part of a continuous program of product development This manual supersedes all previous editions If this manual is provided to an end user it is with the understanding that the material included herein is proprietary to DYNEX Technologies Inc The user may not provide this material to a third party without the written permission of DYNEX Technologies Questions or comments regarding the content of this manual can be directed to DYNEX Technologies 14340 Sullyfield Circle Chantilly VA 20151 1683 Phone 800 288 2354 Toll Free 703 631 7800 Fax 703 803 1441 Internet www dynextechnologies com Part Number 99001830 2001 DYNEX Technologies Inc This document is the copyright of DYNEX Techno
53. E EEE ANETE E EE EEEE EEE EEE E 4 8 4 2 AA Comliputte Reader cor e henee ne e Ene TREE E ae a 4 8 EPE oA Plate a PERAE EE E EA E E E 4 9 424 6 d eO 1i E E E E seicesies tadstestistavasse dies Wayeesde 4 9 4 24 7 Read Calibration Plate n aiene aie eea enairt Ees N EEE E a E Aoi 4 9 MRX Revelation Manual iii Table of Contents A DAS SOMES E rane n E E E R el EA E AS E E ES 4 11 4 249 SPECTO e a e aS sees esha setae E S tas aseteueess 4 12 4 24 10 Sample ID ENY arti a s E E A S 4 13 AD AVA Results POD sass ss Sachi tases rey a E e a S 4 16 4 2 AN2 OPTIONS serro eese r EE E EEEa EE a ase seete ae 4 16 A D AAS Password tssctestissscuvespesseesssaseeg veessetiasisons EE EEE E EREE EEEE E E 4 22 ADS WdoWa coin che erase Eaa sashes E TEE EE EE TREE ESTERE 4 22 ADO Help orena e rar E o E E TTE E T EREE EE RS 4 23 4 3 The Tool Bat sssssczseicess ca ssstssctestiasen tayt ss teeitascg esse pe toast inte seedy cbs eeieaies pede codec tates E i E aE ESS 4 24 AA Stats Balsi aeneo o tates ceeass Ea bap EE EE TO as o E EE ETE TO ES 4 26 Chapter 5 Running an ASSAY cccseeeeeseceeneeeeeeeeeeeeeeesneeseseeeeseaeseseaesnsneeeneeeseseaeenseeeeeees 5 1 DN Over E Wie es cos elite ges i Ried EE EE Mile montis etnies E S E EE 5 1 5 2 Powering Up the Systeme vic eesotsas oee ereo irs E eare En EESE E EOE E SEEE S 5 2 5 3 Selecting the Assay Conditions seesseseerersererseeserseesterenstssreenensteresseversrererorrsrsnensrsreressersee
54. E is owned by DYNEX Technologies or its suppliers and is protected by United States copyright laws and international treaty provisions and all other applicable national laws Therefore you must treat the SOFTWARE like any other copyrighted material e g a book or musical recording 4 U S GOVERNMENT RESTRICTED RIGHTS The SOFTWARE and documentation are provided with RESTRICTED RIGHTS Use duplication or disclosure by the United States Government is subject to restrictions as set forth in subparagraph c 1 ii of The Rights in Technical Data and Computer Software clause at DFARS 252 227 7013 or subparagraphs c 1 and 2 of the Commercial Computer Software Restricted Rights at 48 CFR 52 227 19 as applicable Manufacturers of the respective software are Microsoft Corporation One Microsoft Way Redmond WA 98052 6399 and DYNEX Technologies 14340 Sullyfield Circle Chantilly VA 20151 Please use the Warranty and Special Provisions for information concerning governing law Product support for the SOFTWARE is not provided by Microsoft Corporation or its subsidiaries DY NEX Technologies only provides support for DY NEX Technologies software products Should you have any questions concerning this Agreement or if you desire to contact DYNEX Technologies for any reason please refer to the address listed in this documentation FOR THE LIMITED WARRANTY AND SPECIAL PROVISIONS PERTAINING TO YOUR COUNTRY PLEASE REFER TO THE WARRANTY AND SP
55. ECIAL PROVISIONS SHEET INCLUDED IN THIS PACKAGE MRX Revelation Manual i Revelation Software License Agreement Warranty and Special Provisions Limited Warranty LIMITED WARRANTY DYNEX Technologies Inc warrants that 1 the SOFTWARE will perform substantially in accordance with the accompanying written materials for a period of ninety 90 days from the date of receipt Any implied warranties on the SOFTWARE are limited to ninety 90 days Some states jurisdiction do not allow limitations on duration of an implied warranty so the above limitation may not apply to you CUSTOMER REMEDIES DYNEX Technologies and its suppliers entire liability and your exclusive remedy shall be at DYNEX Technologies option either 1 return of the price paid or 2 repair or replacement of the SOFTWARE that does not meet the above Limited Warranty and which is returned to DYNEX Technologies with a copy of your receipt This Limited Warranty is void if failure of the SOFTWARE has resulted from accident abuse or misapplication Any replacement SOFTWARE will be warranted for the remainder of the original warranty period or thirty 30 days whichever is longer NO OTHER WARRANTIES THE MICROSOFT SOFTWARE PROGRAMS ARE PROVIDED TO THE END USER AS IS WITHOUT WARRANTY OF ANY KIND EITHER EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE THE ENTIRE RISK OF THE QUALITY AND PERFORMAN
56. F Data rinse i etek E E S A E 11 10 11 3 4 The Report Options Dialog Box Header Footer Tab eee eee eee ereeeee 11 10 11 4 The Event Los Flener nnee a aE Man avedeeneontidaveceunninn E vanced Leasee cwuntbens 11 11 Appendix A Using an External Computer cccsccssscesseeeeeeeeesseeseseeeeneeeeeseeeseseaeeneeeaes A 1 ABRONA AATA E ee a he E A EE eh aes A 1 A 2 The Personal Computer Configuration sesesssessseeesssreereserreerrsresresrerreserrrnestenesrentesrerrssee A 2 A 3 Interfacing the Computer to the MRX Reader nw eee ecesesessseceeeeecnereecaeeecaecaseeeaeeaeeaes A 3 A 3 1 Electrical and Communications Interface 0 00 eeeceseceeecneeceeeeeeeeseeeeeeeeeeeeees A 3 A A Installing the Software 220 0 ssesssessesscesseosneesecsseeseceseesuaesocvenevesevseesensessenssensnensecsseesaeensens A 5 A 5 Setting the System Configuration 0 0 0 0 eee eee esecesecssecnseceeceeecaeeeseseeeseeeeeeseaeseeeseeeserenseens A 6 A 5 1 Selecting the External Configuration 0 cee cesecssecseecneecseceecaeeeseeeseseeeeeeeeeeenes A 6 A 5 2 Setting System Parameters eee eeceseeeeceeecesecesecssecsecsaecsaecsaecseseseseaeeeeeeeeegs A 8 A521 The Reader Tabizs c cscecseon fic cscay cout res oere eoe oee S eiea scenes eG A 8 Asd 2 2 The RS232 Tab erosin sree erete nn dt sets leobecha that tebe EE EOE A 9 A 6 Differences Between Internal and External Operation ee ee eeeecesecesecneeceeeeeeneeees A 13 Appendix B Mathematical Relationships
57. Filter z animea P i o Jt Seconda hel atens 3 z Frequency He fis Cancel Apply Help Figure 6 2 The Reader Control Dialog Box Internal MRX System Endpoint Assay 6 4 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 3 The Reader Control Dialog Box Table 6 1 Configurations of the Reader Control Dialog Box Tab Reader Options Section 6 3 2 Endpoint Assay Kinetic Assay Start Mode Section 7 2 Start Stop Mode Section 8 2 Kinetic Time Interval Section 8 2 3 Calculation Mode Section 8 2 4 Kinetic Graphs Section 8 2 5 Agglutination ae gi Parameters Section 9 3 3 Agglutination F T Options Section 9 3 4 Transmission x Graphs Section 9 3 5 Some commands in the Reader Control dialog box are general and are used in most assays while others are used for specific activities e g endpoint analyses or generating reports and exporting data This chapter describes the general commands commands that are used in specific activities are considered in separate chapters as described below MRX Revelation Manual Endpoint Analyses Chapter 7 discusses the details of the generation of an assay for an endpoint analysis Kinetic Analyses Chapter 8 discusses the details of the generation of an assay for a kinetic analysis Agglutination Analyses Chapter 9 discusses the
58. Information Chapter 11 Formatting Reports and Exporting Data 11 2 Laboratory Information Laboratory information e g the name address phone and fax number of the laboratory can be printed at the top of each report This information is entered in the Laboratory Information tab of the Options dialog box Figure 11 1 which is accessed via the Utility menu Options x Buttons Preferences Colours Directories Laboratory Information Name Address Cancel Apply Help Figure 11 1 Options Dialog Box Laboratory Information Tab 11 2 MRX Revelation Manual Chapter 11 Formatting Reports and Exporting Data 11 3 Report Options 11 3 Report Options The Report Options dialog box is used to indicate the desired format for a given assay and indicates the data selections to be included in the report The report options feature is designed to allow you to choose the contents and order of the report to define data matrix settings and to specify special export formats When you select a tab the dialog box changes to display the options and default settings for that tab Any new settings you select will take effect when you close the dialog box 11 3 1 The Report Options Dialog Box Report Layout Tab The Report Options dialog box Report Layout tab Figure 11 2 is used to define the content of the report and the order of items on the report The selection list on the left column lists all the possible items in the repor
59. Reader Maintenance Extemal RS232 Self Test J Retest Before Every Read Maintenance Frequency None Daily Weekly Monthly C Quarterly Cancel Apply Help Figure 2 6 The Setup Internal MRX Dialog Box Maintenance Tab Self Test The Print Self Test check box is used to indicate that the self test report is automatically printed The Retest Before Every Read check box is used to indicate that the self test should be performed prior to reading the plate This provides greater assurance that the data is valid but is time consuming Maintenance Frequency The Maintenance Frequency region is used to allow the user to indicate how often the maintenance reminder is displayed on power up Click the desired radio button Maintenance procedures are described in the MRX manual MRX Revelation Manual Chapter 2 Getting Started 2 3 Setting the System Configuration 2 3 2 3 The RS232 Tab The Setup Internal MRX dialog box RS232 tab Figure 2 7 is generally not used for internal computer systems If you install an external computer click the Enabled check box to activate the various selections then configure the computer communications mode that you will use See Appendix A The Default button selects the configuration that is most frequently employed for serial communication ae Note Make certain that the COM port assignment does not conflict with any other serial port assignment on the com
60. Up System 2 2 MRX Revelation Manual Preferences Tab Options Dialog Box 4 18 Principle of Operation 1 4 Print File Menu 4 4 Print Preview File Menu 4 4 Print Self Test 2 8 Print Setup File Menu 4 4 Processing Order Tab Assay Options Dialog Box 6 43 Q Quadratic Regression B 5 Quality Control 10 2 Dialog Box 10 2 Equations 10 3 10 5 Functions 10 3 Reports 10 3 Quality Control QC Tab Curve Fit Dialog Box 6 32 Quartic Regression B 5 R Ratio 10 21 Ratio Equations Tab ratio Dialog Box 10 21 Raw Data Tab Report Options Dialog Box 11 4 Read Calibration Plate Utility Menu 4 9 Read Mode 6 6 Reader Control Dialog Box Agglutination Mode 9 7 General Information 6 4 Kinetics Mode 8 2 Reader Serial Number Dialog Box 2 2 5 2 Reader Tab Setup Internal MRX Dialog Box 2 6 Reader Type Dialog Box 2 5 Readings Used Agglutination Options Tab Reader Control Dialog Box 9 12 Repeatability Mode Setup Internal MRX Dialog Box Reader Tab 2 7 Report Layout Tab Report Options Dialog Box 11 3 Report Options 6 4 10 3 Reader Options Tab Reader Control Dialog Box 6 4 Replicates Read Mode Tab Reader Options Tab 6 6 Results Font Utility Menu 4 16 Result Format Reader Control Dialog Box Calculation Mode Tab 8 5 Results Flagging Tab Curve Fit Dialog Box 6 36 10 18 Results Flagging Tab Ratio Dialog Box 10 23 Retest before Every Read 2 8 Revelation Application Software 1 5 RS232 Tab Int
61. aces the highlighted assay in the Select field into the Selected Assays field MRX Revelation Manual 9 15 9 4 Starting an Assay Chapter 9 Agglutination Analyses and TCG Mode Remove Removes the highlighted assay from the Selected Assays field Clear deletes all the assays from the Selected Assays field First well Select an assay in the Check list box and type in the well location when you want this assay to start Number of wells Enter 96 for this value for each assay ae Note The program allows you to assign fewer wells to an assay than were originally programmed into the assay template For example if your assay contains 96 Test samples but this run only requires 10 test samples you can assign 10 wells to the assay Revelation will reduce the template to fit into 10 wells The program reduces templates by removing Test samples from the end of the template until the required number of wells is left All non Test samples are left in the same positions relative to the end of the plate If it is not possible to reduce the template the program prints an error message and will not reduce the template Orientation If you have assigned less than 96 wells to any assay on the plate you must select Columns or Rows to specify the plate orientation As an example if Assay contains 12 wells and starts at Al and you select Columns the program will place the assay in wells Al to H1 and A2 to D2 if you select Rows Revelation
62. age Checking Alignment will be presented The plate carrier will move at 4 times less than its normal speed into the optical reading chamber After the alignment tests are completed the message Reading wavelength x which will show all filter wavelengths being checked will be presented When Revelation is finished reading the verification plate the plate carrier will be ejected and a report with data with pass fail notations will be presented e g Figure 2 9 MRX Revelation Manual 2 11 2 4 Initial Testing of the System 2 12 Calibration Plate 212 055 Reader Serial 1CXD3086 Time 10 43 15 AM Date 249101 X Alianment 0 23mm Y Alianment 0 01mm Skew 0 14mm Test Linearity Slope Intercept tvalue Filter Optic Channel 1 Optic Channel 2 Optic Channel 3 Optic Channel 4 Optic Channel 5 Ontic Channel 6 Optic Channel 7 Optic Channel 8 Ontic Channel 9 Optic Channel 10 Optic Channel 11 Figure 2 9 Test Report 405nm 0 606 0 727 55 594 Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed 450nm 0 556 0 675 54 023 Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed 490nm 0 561 0 674 54 772 Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed Passed 630nm 0 556 0 664 54 184 Passed Passed Passed Passed Passed Passed Pas
63. ains multiple replicates As an example valid PC gt 3 means that the number of valid PC samples must be greater than or equal to 3 or the data fails Q C 12 An equal comparison is represented by It indicates that one value must be equal to another As an example NC1 1 2 953 means that the first replicate of NC1 must equal 2 953 or the data fails Q C MRX Revelation Manual 10 5 10 2 Quality Control Chapter 10 Incorporating Decision Making Protocols into the Assay 10 6 13 An assignment expression is represented by It indicates that depending on certain criteria a well type sample or replicate should be assigned a certain value As an example the expression If NCi lt 0 0 then NCi 0 0 is used to indicate that if every NC value is less than 0 0 then let every NC value be assigned a value of 0 0 14 The conditions If then and else conditions are used to set criteria for Q C As an example the expression If PC lt 0 2 then NC gt 0 5 else NC gt 0 1 has the following interpretation If the average PC value is less than 0 2 then the average NC value must be more than 0 5 else the average NC value must be more than 0 1 If these conditions are not met the data will fail the Q C test 15 Verify Number of Values verify represents a verify values expression This indicates that a specified number of values within a group must be within specified limits otherwise the data fails Q C The group of values may be a
64. aking Protocols into the Assay 10 3 Threshold 10 3 1 The Threshold Dialog Box The Threshold dialog box Figure 10 2 is used to indicate ranges for samples which are going to be qualitatively determined to be positive or negative according to the assay parameters If there is a large neutral area between positive and negative you may define segments or break points within this neutral area Threshold x Threshold Equations Pos Neg O C Threshold Q C Output Format m Options l Enabled Aistogam Label Functions Equation e Variable Equation S Ce Equation D p Equation p Hooo Log Equation 0 Ps Equation i Number of Segments fi fo Figure 10 2 The Threshold Dialog Box Threshold Equations Tab 10 8 MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 3 Threshold 10 3 2 Threshold Equations Tab The Threshold dialog box Threshold Equations tab Figure 10 2 is used to generate various equations to define threshold conditions which will determine if a sample is positive or negative Equations based on numerical O D or on controls may be written to define the threshold limits A negative limit is set using the equation and a positive limit is set using a equation A positive and negative limit will always be required but three extra positive levels and are also available Threshold limits can be entered direc
65. alculations that were included in the assay In addition data will be saved and or exported as indicated in the assay MRX Revelation Manual 7 7 Chapier 8 Kinetic Analyses 8 1 Overview Chapter 8 Kinetic Analyses 8 1 Overview Continuous sampling of reactants as they are consumed or products as they are formed measured without interfering with the progress of the reaction is defined as a kinetic analysis In a kinetic analysis the rate of change in the O D is measured and related to the concentration of the compound of interest Measuring the optical density is an ideal way to measure changes in reaction rates because the Beer Lambert law states that this change is directly proportional to concentration The user can select the time interval for data collection and view the data as it is being collected It should be noted that the final form of the data is not in absolute optical density but reflects the increase or decrease in optical density as it changes over time according to the user selected calculation mode This chapter includes a Generating an Assay for a Kinetic Analysis Section 8 2 a Starting an Assay Section 8 3 a During the Assay Section 8 4 MRX Revelation Manual 8 1 8 2 Generating an Assay for a Kinetic Analysis Chapter 8 Kinetic Analyses 8 2 Generating an Assay for a Kinetic Analysis 8 2 1 Basic Assay Information To generate a Kinetic Analysis assay 1 Select New Assay on the File menu 2 Sele
66. applied to every well to interpret the data MRX Revelation Manual 9 9 9 3 Generating an Agglutination Assay Chapter 9 Agglutination Analyses and TCG Mode Agglutination Formula equations may contain any number of characters but they may not contain any spaces and standard agglutination formula equation components as well as arithmetic operators exponent which are entered via the keyboard Other equation components can also be entered using the Functions list box To enter a function double click on it A detailed discussion of equation writing is presented in Section 10 2 2 When you have finished entering the equation click OK to exit the dialog box Revelation checks each equation Typical equations are Example 1 All values must be multiplied by the average of parameters A and B The formula should read A B 2 Example 2 All values must be multiplied by the sum of parameters A and B minus the sum of parameters C and D The formula should read A B C D Rules The Rules field is used to establish positive and negative rules which will be used to calculate results and is activated if Rules is selected in the Calculation Mode field Four positive and four negative rules can be set one for each of the four Agglutination parameters A D At runtime Revelation will calculate values for the Agglutination parameters for every well The rules will then be applied to each well to determine whether i
67. ate mode and then click on any replicate in the sample To view a graph showing each well in a column click on the column label a To view a graph showing each well in a row click on the row label a To view a matrix showing the current O D for each well select Display Values MRX Revelation Manual 8 15 8 4 During the Assay Chapter 8 Kinetic Analyses Ie Note Do not alternate between the viewing modes while a reading is being taken as this may interfere with the assay timings At the conclusion of the assay the desired report will be presented in the desired format along with the calculations that were included in the assay In addition data will be saved and or exported as indicated in the assay 8 16 MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode 9 1 Agglutination Chapter 9 Agglutination Analyses and TCG Mode Ie Note Agglutination Analysis and TCG Mode are only available with MRX Revelation systems which are configured with an internal computer 9 1 Agglutination 9 1 1 Introduction to Agglutination Testing An agglutination test is used for microbial identification monitoring cell growth or determining red blood cell types Antibodies cause the agglutination reaction by fusing with antigens on the surfaces of cells Since most cells repel each other because of their electrical charge large numbers of antibody links are needed for agglutination As an alternative particles which
68. ate is in the reader a delay time could be programmed to ensure that the reaction is allowed to go to completion 7 2 MRX Revelation Manual Chapter 7 Endpoint Analyses 7 2 Generating an Assay for an Endpoint Analysis A typical assay is presented in Figure 7 2 Assay type Assay title Password Written by Prefix Suffix Report layout Header information Footer DYNEX REVELATION 4 22 Endpoint RP 606 R Patrick 123 789 Laboratory information Header information Removed outliers Edited wells Calculation mode Blank mode Q C equations Filename Date Plate ID Assay title Page Q C summary Test wavelength 410 nm Ref wavelength oe Initial shake 10 Seconds Mode 1 Start mode Immediate Calculation mode Endpoint Results format 0D 1 2 3 4 5 6 7 8 9 10 11 12 A S1s TQs Cis T25 S T41s C2s T57s S T7 C3s T8Qs B T2s T10s T18 T26s T34 T4 T5 T5 T66 174 T82s T90s c Tas T115 T1 T27s T35 T4 TSts T5 T67s T7 amp T83s T91s D T4 T15 T20s T2 T36s T4 T5 T6 T6 T76s T84 T95 E S amp T1 C6s T2 S amp T45 C5s T61s S6s T77s C4s T93s F T s 1145 T2 T30s T388 T46s T54 T6 T70s T7 T86s T94 G T7s T15 T2 T31s T39s T47s T55 T63 T71s T7 T875 T95 H S7s T16s T24 T3 S amp T48s T56s T64 S T80 T88 T96s s indicates that a sample ID is required for this weli location Blank mode Individual Q C equations Full Q C Report Yes Suppress results No Lot specific c
69. ate must be CLEARED by clicking on the Clear button at the bottom right of the dialog box MRX Revelation Manual 6 11 6 4 Template Window Chapter 6 Generating and Editing Assays 6 12 Well Type When a well type is defined the well will be labelled to denote the type and the lowest unused number for that type As an example in Figure 6 4 all positions are defined as Tests T1 T96 If position Al should be a blank click on the Blank radio button and then click on position Al The position will be indicated B1 and the Blank entry on the Well Type field will indicate B2 indicating that the next Blank to be assigned will be B2 In most cases a fairly structured order is used e g every 24 sample is a blank every 8 sample is standard every 16 sample is a control etc but the template allows the operator to define the wells on a completely random basis If desired you can change the labelling for a well type by selecting the well type to be edited and then selecting the Change Label button to present the Change Label dialog box Figure 6 5 Select the desired format 1 2 3 or A B C and then indicate the label type As an example if you wanted to use the label XX select A B C and enter XX in the Label field Change Label x m Labeling Eam Cancel 1 2 3 Help See e Figure 6 5 The Change Label Dialog Box Standard Control and Test types are standard well types whose names cannot be changed
70. ated final result for each well Miscellaneous Noise Filtering smoothes out the well profile by averaging each data point with the two points on either side Percentage Mode calculates all the results as a percentage of a specific well The reader will take the transmission of the specified well as 100 and 0 0 as 0 and measure all other results against this To use this option check the box and type in the sample ae Note The reference well must exist on the template You cannot specify individual replicates of samples Readings Used The program uses all the readings specified in the Readings per Well field or any selection of readings you choose Any readings that are deleted will be discarded and will not be used when calculating the results for that well The Select column includes all of the readings that could be used in the calculation and the Check list columns list the readings that have been selected Add All places all entries in the Select column field into the Check list field Add places the highlighted reading in the Check list field MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode CCM CT Val veclilare EUR e TECE Remove removes selected readings from the Check list field and from the calculations Clear removes all readings from the Check list field and from the calculations 9 3 5 The Reader Control Dialog Box Transmission Graphs Tab The Reader Control dialog
71. ation Manual 10 23 10 6 Spreadsheet Chapier 10 Incorporating Decision Making Protocols into the Assay 10 6 Spreadsheet 10 24 10 6 1 Spreadsheet Dialog Box Results Flagging Tab The Spreadsheet dialog box Results Flagging tab Figure 10 11 is used to specify a phrase that either replaces a numerical result or is displayed next to the result select the Results Flagging tab The phrase will be initiated whenever the numerical result meets the criteria that are specified A detailed discussion of the Results Flagging tab is presented in Section 10 4 4 Spreadsheet Ea Spreadsheet Results Flagging Output Format Condition Result Flag Functions If then Sample checks Cancel Apply Help Figure 10 11 The Spreadsheet Dialog Box Results Flagging Tab MRX Revelation Manual Chapter 11 Formatting Reports and Exporting Data 11 1 Overview Chapter 11 Formatting Reports and Exporting Data 11 1 Overview The Revelation program generates a hard copy report which presents the analytical results in a user definable format The analytical results can also be exported in a format which can be imported into other programs e g a spreadsheet such as Microsoft Excel or a text editor This chapter describes how to define the format of the printed report and the exported data In addition it describes a number of other reports that are generated by the reader MRX Revelation Manual 11 1 11 2 Laboratory
72. based on the selection made in the assay a For systems in which the computer is external the computer will initiate communication with the reader but this assay option is not available MRX Revelation Manual 9 17 9 5 9 18 During the Assay Chapter 9 Agglutination Analyses and TCG Mode 9 5 During the Assay While the reader is performing the agglutination measurements you can a Plot a graph of the data from a single well Check the well location box on the matrix a Plot a graph of each well in a column or row Check the Row or Column check box Revelation plots a series of transmission graphs showing the data for each individual well in the row or column a Plot graphs of each individual well on the plate Check the 96 Well Plot box Revelation creates a matrix of 96 miniature individual graphs showing every well location MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 1 Overview Chapter 10 Incorporating Decision Making Protocols into the Assay 10 1 Overview Revelation allows the analyst to develop protocols that can be used to determine and report acceptable unacceptable data in the report These protocols are available for a large number of assays and in general follow a common format MRX Revelation Manual 10 1 10 2 Quality Control 10 2 Quality Control Chapter 10 Incorporating Decision Making Protocols into the Assay 10 2 1 Role of Quality Contr
73. box This topic is discussed in detail in Formatting Reports and Exporting Data Chapter 11 MRX Revelation Manual 6 41 6 12 Assay Options Chapter 6 Generating and Editing Assays 6 12 Assay Options 6 12 1 Assay Options The Assay Options Tab is used to establish parameters for a number of miscellaneous parameters and includes three tabs Area Statistics Processing Order and Area Statistics 6 12 2 The Assay Options Dialog Box Area Statistics Tab The Assay Options dialog box Area Statistics tab Figure 6 21 is used to indicate that various statistical processes should be carried out for different areas of the plate An area is the collection of wells for which the statistical analysis is desired The program will calculate the mean standard deviation standard error mean and coefficient of variation for each area neat seta Ed Area Statistics ics ae ina o der Sample D s i ag Titi is fen E Cancel Ces f Help Figure 6 21 The Assay Options Dialog Box Area Statistics Tab 6 42 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 12 Assay Options Up to 96 areas labelled 0 to 95 can be created Each area may contain as many wells as you wish from anywhere on the plate A well can be in one and only one area The following operations can be done to define the area a To place a single well location in an area Type a number into the relevant box a To place a row in an area Typ
74. box Transmission Graphs tab Figure 9 9 is used to specify that an Agglutination graph desired for a given well s is available Reader Options Start Mode Agglutination Parameters Aagglutination Options Transmission Graphs 96 Well _ Clear o gt Sec gt Figure 9 9 The Reader Control Dialog Box Transmission Graphs Tab To plot a graph of the data from a single well check the well location box on the matrix Multiple plots can be obtained by selecting the appropriate well location boxes MRX Revelation Manual 9 13 9 4 Starting an Assay Chapter 9 Agglutination Analyses and TCG Mode 9 4 Starting an Assay Open the desired assay If a password has been assigned to the assay a password dialog box will be presented The password must be entered if you want to edit the assay If you simply want to run the assay it is not necessary to enter the password and the Cancel button should be pressed To run an assay press Run Plate on the File menu or click on the Run Plate button icon on the Tool Bar to present the Run Plate dialog box Figure 9 10 Run Plate x m Plate ID Dptions Plate ID JARE IV Auto save plate data as C PROGRAM FILES REVELATION Plate T Print results automatically Scan Barcode beroetan Head Barcode Wav Operator Operator Select Open Selected Assay s 96 Al 406a asy Save Add ACCKinetic asy ANTICAR ASY Remove APOB ASY Apoptosis
75. by pressing F1 or pressing the Help button on a dialog box 1 6 MRX Revelation Manual Chapter 2 Getting Started 2 1 Overview Chapter 2 Getting Started 2 1 Overview This section describes how to power up an MRX Reader with Revelation application software The application software is loaded on the hard drive of the internal computer and the system will automatically start Revelation when the reader is powered up If the system includes an external computer refer to Appendix A MRX Revelation Manual 2 1 2 2 Powering Up the System Chapter 2 Getting Started 2 2 Powering Up the System To Power the System Up 1 Make sure that the instrument is switched off The power switch is ON when it is flush with the front panel and OFF when it stands out from the panel Turn on the power switch for the printer and verify that the printer completes its self test successfully Turn on the power switch on the front panel of the MRX reader Click on the Revelation icon on the Windows desktop The monitor will present a welcome screen and the Reader Serial Number dialog box Figure 2 1 Enter the serial number and press OK Reader Serial Number Ed Serial No Cancel Figure 2 1 The Reader Serial Number Dialog Box 6 Aseries of self tests will be automatically performed and the results will be indicated in the workspace of the Revelation screen The self test checks the items indicated in Table 2 1 and a sample report i
76. ce more than one assay on a plate you can either over lap some assays so that more than one assay will run on the overlapped wells or you can run each assay on different wells The Select list box contains a list of all your assays Open Presents the Open dialog box with a listing of all previously saved assay checklists Save Saves an assay checklist Checklists are saved by default in the main Revelation directory using the LST extension If the checklist has not been saved before Revelation displays the standard Save As dialog box Add Places the highlighted assay in the Select field into the Selected Assays field Remove Removes the highlighted assay from the Selected Assays field Clear delete all the assays from the Selected Assays field First well location Select an assay in the Selected Assay s field and type in the well location when you want this assay to start Number of wells Enter 96 for this value for each assay Ee Note The program allows you to assign fewer wells to an assay than were originally programmed into the assay template For example if your assay contains 96 Test samples but this run only requires 10 test samples you can assign 10 wells to the assay Revelation will reduce the template to fit into 10 wells The program reduces templates by removing Test samples from the end of the template until the required number of wells is left All non Test samples are left in the same po
77. ceecesecesecssecnseceeecaeesaeseseseeeeeeeeseenes 9 1 9 1 2 The Agglutination Measurement 00 0 0 ee ee ceeecesecseecseeceeeeeseeeeeeeeeeeseeesecaecsaessaeaee 9 1 9 1 3 Agglutination Calculations occ eeceecesecesecssecsseceeeeseeeeeceeeesecsecsaecsaecsaecnaeeneeegs 9 4 9 1 4 Formula Calculation ooieoe secors sos ee Ee ea eera EEE Eea Eroa 9 4 9 1 5 Rules Calc latioN sissies eo eniro eeno reae es E aE eA eee EE 9 5 9 1 6 Setting Rules and Formulae se seseseseesseeeesssterrsstsrreresreetsserrrsereenresrenreseerrsserrrsresrent 9 5 9 2 Tissue Culture Growth T C G Measurements cccccccessesccceessececeeececessececeeseeeeseseeeenenee 9 6 9 2 1 Introduction to Tissue Culture Growth Measurement 00 eee eeeeeceseceseceseeeeeenee 9 6 9 3 Generating an Agglutination ASSay 0000 0 eeeeceeecesecesecnsecaeecaeecneeeseseaeeseeeeeeeseeeeeeseneensees 9 7 9 3 1 Basic Assay Information cece ceeeeeeeeceescesecesecesecsaecsaeceaeeseeeeeeseesecsaeesaecsaesaes 9 7 9 3 2 The Reader Control Dialog Box Start Mode Tab oo eee ee ceeeceeeceecnseeneeeneeene 9 7 9 3 3 The Reader Control Dialog Box Agglutination Parameters Tab eee 9 8 9 3 4 The Reader Control Dialog Box Agglutination Options Tab eee eee 9 11 9 3 5 The Reader Control Dialog Box Transmission Graphs Tab 9 13 9 4 Startins an Assay ann n R E Sov AA E Sethian diees E 9 14 9S D ne the Assay anen a a A a aa Sune bia E E 9 18 Chapter 10 Incorporating Decision Mak
78. cific Checking 10 3 M Maintenance Frequency 2 8 Maintenance Tab Setup Internal MRX Dialog Box 2 7 Mathematical Relationships B 1 Main Window 3 5 Menu Bar 4 2 Michaelis Menton Curve Fit B 11 Miscellaneous Options Reader Control Dialog Box Calculation Mode Tab 8 5 Modes of Operation 1 3 MRX Revelation Application Software 1 5 MRX Revelation Manual Multi Wavelength Mode Reader Control Dialog Box 6 7 N New Assay File Menu 4 2 O Open File Menu 4 3 Optic Channel Matching 2 10 4 10 Options Dialog Box 4 16 Orientation of Run 5 5 Orientation Template Window 6 13 Outlier Removal QC Tab Curve Fit Dialog Box 6 32 Output Format Tab Curve Fit Dialog Box 10 13 Output Format Tab Threshold Dialog Box 10 13 OVER Limit Setup Internal MRX Dialog Box Reader Tab 2 6 OVER UNDER Conversion Setup Internal MRX Dialog Box Reader Tab 2 7 OVER Value Setup Internal MRX Dialog Box Reader Tab 2 6 P Password 4 22 6 2 Percentage Response Standards Tab Curve Fit Dialog Box 6 23 Plate Data files dat 3 6 Plate ID Sample ID Entry Dialog Box 4 14 Plate In Utility Menu 4 9 Plate Orientation 5 7 Plate Out Utility Menu 4 9 Plate Plate Statistics Utility Menu 4 6 Plate Reproducibility Plate Plate Statistics Dialog Box 4 6 Plates Plate Plate Statistics Dialog Box 4 6 Polygon Fitting B 4 Powering Up the System 5 3 Pos Neg Q C Tab Threshold Dialog Box 10 11 Powering
79. cond assay Read your second plate Your data will be calculated using the stored curve fit Graphs and concentrations will appear again as when the first plate was read MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 8 Curve Fit 6 8 4 1 Calculation Options Average Standard Replicates average the replicate values for individual standards before they are used for curve fitting Extrapolate Data extends the graph beyond the O D for either the highest or lowest known data points Revelation does this by calculating extra data points based on the known data Extrapolation is usually valid scientifically off a linear regression curve fit but not off a sigmoidal curve fit Sigmoid Parameters are used to define the conditions when a Sigmoid fit is used Fix Parameters All four parameters of the Sigmoid curve are user definable Check the box of any parameter you want to define and type a number in the box Maximum iterations the maximum number of times for the program to attempt to construct a satisfactory plot before aborting the operation The default value of 20 is suitable for most applications the range is from to 29999 Results Format Values for the Standard samples can be entered as concentrations or as titer values Select the required units Inhibitor used to examine the effect of an inhibitor when using Lineweaver Burk or Michaelis Menten fit types If an inhibitor is present 1 Place a c
80. conform to determine if a result is to be considered as positive or negative 9 1 4 Formula Calculation The Formula Calculation mode allows you to enter equations using any or all of the agglutination parameters After the plate has been read your formula is applied to the four agglutination parameters to produce a formula value In this approach a threshold matrix must be defined in formula mode see Section 9 1 6 The formula that you construct depends on the type of test that you are running so it is best to calibrate your formula for every new test that you perform 9 4 MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode 9 1 Agglutination 9 1 5 Rules Calculation Rules calculation lets you enter sets of rules that the reader uses to interpret results It is necessary for you to enter one set of rules for positive results and one set for negative results Once the plate has been read each well result is compared to the positive and negative rules If the result meets the criteria set by the positive negative rule it is considered as a positive negative If the result does not meet either criterion it is described as questionable 9 1 6 Setting Rules and Formulae The rules and formulae that you enter will change to suit the test that you are performing and the relative importance of the four agglutination parameters You should be aware of the way that the parameters are affected by the test when you set rules
81. ct Kinetic on the Assay Type dialog box 3 Develop the assay as described in Chapter 6 Several dialog boxes on the Reader Control dialog box are used when kinetic assays are employed as described below 8 2 2 Start Stop Mode The Reader Control dialog box Start Stop Mode tab is presented in Figure 8 1 Reader Control E x geoegeccessescesocscsocecseesssesssseesesoea Reader Options Start Stop Mode Kinetic Time Interval Calculation Mode Kinetic Graphs r Start Mode Immediate f When fri reaches 0 0D C When T reaches OD min C After fi minutes Stop Mode After 2 readina s When E reaches 0 oD When fr reaches OD min C After fi minutes Cancel Figure 8 1 Reader Control Dialog Box Start Stop Mode 8 2 MRX Revelation Manual Chapter 8 Kinetic Analyses 8 2 Generating an Assay for a Kinetic Analysis The Start Stop Mode tab is used to indicate when the program should take the first reading relative to the start of the assay the assay starts when you click on Start in the Run Plate dialog box and the last reading There are four options for the Start time a Immediate the first reading is taken when the assay is initiated a When a specified sample Tx reaches a user specified O D the entire plate will be read ae Note Revelation will allow you to enter a sample which does not exist but the measurement will not be performed unless you create the
82. cted an endpoint assay the Reader Control dialog box Figure 6 2 will include a Start Mode tab which includes parameters that are used to indicate when data collection should begin This tab is discussed in Section 7 2 If you have selected a kinetic assay the Reader Control dialog box Figure 6 2 will include the Start Stop Mode tab which includes parameters that are used to indicate when data collection should begin and end This tab is discussed in Section 8 2 6 3 4 Kinetic Time Interval The Reader Control dialog box Kinetic Time Interval tab is used to indicate when data should be collected when a Kinetic Assay is being performed This dialog box is described in detail in Section 8 2 3 6 3 5 Calculation Mode The Reader Control dialog box Calculation Mode tab is used to specify the calculation mode and other miscellaneous calculation options for kinetic assays This tab is discussed in Section 8 2 4 6 3 6 Kinetic Graphs The Reader Control dialog box Kinetic Graphs tab is used to define the kinetics plots that will be output in the results and presents a matrix denoting the plate wells This tab is discussed in Section 8 2 5 6 3 7 Agglutination Parameters The Reader Control dialog box Parameters tab is used to set a variety of operating parameters for measuring the agglutination of red blood cells This tab is discussed in Section 9 3 3 MRX Revelation Manual 6 9 6 3 The Reader Control Dialog Box Chapier 6
83. d store them in a secure location To install the Revelation software 1 Access the Windows desktop If any programs are open close them at this time 2 Insert Installation Disk 1 into the floppy disk drive 3 Press Start to access the task bar and then select Run from the task bar to present the Run dialog box is presented 4 Type x setup in the Run dialog box and press OK x is the drive designator for the floppy drive and is almost always a 5 Follow the directions provided by the installation it is recommended that you use the default directory 6 After the first diskette is copied you will be prompted to insert the second diskette Insert the diskette labelled Disk 2 into the diskette drive and select OK 7 When the installation is complete the message Setup is complete is displayed Remove the second installation diskette If desired you can create a shortcut on your windows desktop 1 Open Windows Explorer a Locate the Revelation shortcut in the directory in which the program was installed if you have used the default directory during installation the shortcut will be in c Windows Start Menu Programs Revelation b Drag the Revelation shortcut to the desktop IEE Note Be sure to drag the Revelation shortcut not the Revelation Help shortcut 2 Close Microsoft Windows Explorer MRX Revelation Manual A 5 A 5 Setting the System Configuration Appendix A Using an External Computer A 5 Setting the System C
84. details of the generation of an assay for an agglutination analysis Incorporating Decision Making Protocols into the Assay Chapter 10 describes how you can develop protocols to determine and report acceptable unacceptable data in the report Generating Reports and Exporting Data Chapter 11 explains how you can customize the report format and export data to an external program 6 3 The Reader Control Dialog Box Chapter 6 Generating and Editing Assays 6 6 6 3 2 Reader Options Tab The Reader Control dialog box Reader Options tab is presented in Figure 6 3 Reader Control x Kinetic Graphs Agglutination Parameters Agglutination Options Reader Options Start Stop Mode Kinetic Time Interval Calculation Mode Multiwavelength Mode r Shake Single Dual Wavelength Linear Regression T Enable Shaking C Multiwavelength Wel Type Re inital Stake jo Seconds C Agglutination T C G C Yalue fi Repeat Shake Duration Fivediat fi Seconds C Gontnuoue Primary Test Filter b Modes Mode 1 14 Hz 1 7 mr z Primary Ref Filter bi ere eee ingens Seconda lest rite z Amplitude mm E m Wavelengths seconda Her iten y r Frequency Hel fis Cancel Apply Help Figure 6 3 Reader Control Dialog Box Reader Options Tab 6 3 2 1 Read Mode The Read Mode field is used to indicate the type of measurement to be made a In Single Dual Wavelength
85. dicated on the Start Stop tab Figure 8 1 8 2 4 The Reader Control Dialog Box Calculation Mode Tab The Reader Control dialog box Calculation Mode tab Figure 8 3 is used to specify the calculation mode and other miscellaneous calculation options Reader Options Start Stop Mode Kinetic Time Interval Calculation Mode Kinetic Graphs Calculation Mode Blank Subtraction Result Format av lal Intec op C OD min max Post Kinetic Calculations C mOD OD min by Linear Regression Use Readings Cut off Time to max OD All 2 5 0D Time to max OD min Range Absolute Time to defined OD fo aa to eo Relative to First Time to defined OD min pooo Miscellaneous Options Time to relative OD fo IV Data increase with time C Monitor Im to fo IV Display Well mE l DataLog Moving Average 1 Points Result Scaling fo OK Cancel Apply Help _ Cancel e Figure 8 3 The Reader Control Dialog Box Calculation Mode Tab Calculation Mode Calculation Mode describes how the data is to be processed in any of the programmable options The operator should choose one option from the following OD min options measures the rate of the reaction via the rate of change of O D as a function of time MRX Revelation Manual 8 5 8 2 Generating an Assay for a Kinetic Analysis Chapter 8 Kinetic Analyses OD min Total measures the rate of the reaction over the entire
86. dited An assay that has been edited should be saved before it is used MRX Revelation Manual 4 3 4 2 The Menu Bar Chapter 4 Commands 4 2 1 6 Import Import presents the Import Data Files dialog box which is used to import a file from an external software package If the reader is connected to a sample preparation device the program can periodically interrogate the device software for sample IDs or data files This feature is initiated via the Polling button that presents the Define Polling Options dialog box Figure 3 6 Check the Enable Polling box type in the anticipated location of the file s and enter a polling interval 1 30000 minutes Define Polling Options Ea I Enable polling for Hamilton File Directory c Every fi minute s Figure 4 3 The Define Polling Options Dialog Box 4 2 1 7 Run Plate Run Plate initiates the run using the active assay A detailed description of how an assay is run is presented in Chapter 5 4 2 1 8 Print Print presents the Print dialog box that is used to select the print conditions the nature of this dialog box is dependent on the printer connected to the system 4 2 1 9 Print Preview Print Preview displays the report on the monitor 4 2 1 10 Print Setup Print Setup presents the Print Setup dialog box the nature of this dialog box is dependent on the printer connected to the system B40 MRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar 4 2 1 11 Exit Exit c
87. diting Assays 6 8 Curve Fit Calculated Q C Data Conversion Results Flagging Output Format Fits Standards FitType Graph Sample Selection Dilutions FtO C Samples Automatic Entry Enter Dilution il No Factor Additive fi Multiplier 2 I Split replicates b ee ad Cancel Apply Help Figure 6 13 The Curve Fit Dialog Box Dilutions tab Samples The Dilutions list box lists all of the Test and Control samples on the template If you have diluted any of these type the dilution factor in the Enter Dilution field The factor will not automatically appear To apply the value for a given sample press Enter on the keyboard or click on the sample again a Split Replicates used to indicate that the Test and Control samples contain multiple replicates and you want to assign separate dilution values to these replicates Automatic Entry Automatic Entry is used to enter dilution values automatically No Factor Switches automatic entry off Additive used to increment each successive value by a specified amount MRX Revelation Manual 6 31 6 8 Curve Fit Chapter 6 Generating and Editing Assays To apply the additive function 1 Click on Additive and type in the incremental value 2 Click on the first item and type in its value 3 Press Enter on the keyboard to apply the addition to each successive sample Multiplier used to multiply each successi
88. e a Fixed activates the Fixed Time Interval field The analyst should indicate the desired Fixed Time interval in the field immediately below the Time Interval area a Variable activates the Variable Time Interval field The analyst can enter the time for the first reading and for each successive reading The time between readings may vary if the kinetic reaction starts off very fast in which case a very short time interval is needed and then tapers off to a slow rate of O D change in which case the time interval can be increased By default the program lists the kinetic reading intervals in the Variable Time Interval list box at 60 second intervals If you want to edit an individual interval click on the interval type the new interval into the Reading field and then click on any other reading to assign the interval To edit a group of intervals click on the interval you want to start at enter the new time interval in the Auto increment box and click Fill The maximum interval is 1 hour and the minimum interval for the Internal MRX is 4 seconds for the External MRX the minimum interval is 12 seconds MRX Revelation Manual Chapter 8 Kinetic Analyses 8 2 Generating an Assay for a Kinetic Analysis a The Auto Increment field is used if you want to have a reading at a fixed time increment after you have set the times they can be edited as described above IEE Note The number of points that should be collected can be in
89. e current plate The plate ID for the plate you wish to use is indicated in the field adjacent to this option The program subtracts the 96 values in the data file from the 96 wells on the current plate on a well for well basis No Blank samples are required for this mode a Individual involves defining a number of blanks on the plate When the software encounters a blank it updates the stored blank value This value is subtracted from all subsequent readings until the program finds another Blank or until the whole plate has been read It is possible to arrange the template so that the program reads one Blank value straight after another for example if a Blank sample contains multiple replicates arranged in columns If this happens the program stores only the last blank value it reads It will read the first replicate and update the blank value move to the next replicate and update the blank value again This will continue until it reaches a well containing a non Blank sample It will then subtract the Blank from the reading for this well and move on to the next well a Average requires one or more Blank sample on the template All the Blank ODs are averaged and the averaged value is subtracted from all other readings on the plate a Row treats each row separately If there are any Blank samples in a particular row the program averages all the Blank values into a single value and subtracts this value from every reading in the row I
90. e Fit Dialog Box Output Format Tab 6 38 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 9 Ratio The Ratio processing dialog box Ratio Equations tab Figure 6 20 is used to convert raw data into data with a different scale Ratio data is output as a matrix or table of converted results This topic is discussed in detail in Section 10 5 2 Ratio Equations Results Flagging Output Format Enabled Ratio Equation 0 ee Units mean Data Equation ee SE eee Units eee Functions Figure 6 20 The Ratio Dialog Box Ratio Equations Tab MRX Revelation Manual 6 39 6 10 Spreadsheet Chapter 6 Generating and Editing Assays 6 10 Spreadsheet 6 10 1 Spreadsheet Mode Spreadsheet Tab The Spreadsheet Mode is used to establish a spreadsheet so that equations and formulas can be entered into individual wells for non standard data manipulations This topic is discussed in detail in Formatting Reports and Exporting Data Chapter 11 6 40 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 11 Report Options 6 11 Report Options The report options feature is designed to allow you to choose the contents and order of the results report to define data matrix settings and to specify special export formats When you select a tab the dialog box changes to display the options and default settings for that tab Any new settings you select will take effect when you close the dialog
91. e ID file was created in the Utility menu Averaging Options Average Replicates used to indicate that the average of the raw data replicates should be reported Arithmetic Mean Reports the arithmetic mean or average of replicate determinations Geometric Mean Reports the geometric mean the antilog of the mean of the logarithms of replicate determinations Reporting the geometric mean is useful when the imprecision is directly proportional to the level Decimal Places Free format Results are displayed in the most appropriate format Fixed at Allows the analyst to enter the desired number of decimal places Table Options The Table Options field is used to indicate the information that should be included with the results value for each sample when the table format is selected The items are summarized in Table 11 1 Table 11 1 Table Options Option NONE Sample ID Sample ID a created sample ID file Well Location Well location on the template Replicate Value Raw data or OD value of each sample Mean Value Mean value of the whole well type S D Standard deviation value of the whole well type C V Coefficient of variation of the whole well type S E M Standard error mean of the whole well type MRX Revelation Manual 11 5 11 3 Report Options Chapter 11 Formatting Reports and Exporting Data Ee Note Some table options are not available for certain data processing options whi
92. e an area number into the first well in the row and click on the letter next to the row a To place a column in an area Type an area number into the top well in the column and click on the number above the column To clear a single well location from an area Delete the area number from the relevant box using the Delete key To clear a row Delete the area number from the first well in the row and click on the letter next to the row a To clear a column Delete the area number from the top well in the column and click on the number above the column To clear all the area assignments Click Clear 6 12 3 Assay Options Dialog Box Processing Order Tab The Assay Options dialog box Processing Order tab Figure 6 22 is used to indicate in what order the operations programmed in other screens will be performed Assay Options Ea Area Statistics Processing Order Sample ID Setup Spreadsheet Curve Fit Clear List Cancel Apply Figure 6 22 Assay Options Dialog Box Processing Order Tab MRX Revelation Manual 6 43 6 12 Assay Options Chapter 6 Generating and Editing Assays The buttons on the left will be active if the option is enabled e g if you have enabled Curve fit the Curve Fit button will be active To activate an option click on the button and it will appear in the list ae Note If you select more than one data processing option their order in the assay is very important as each option
93. e at each point is determined by these polynomials The Akima fit is based on the assumption that each data point is determined by five points the point of interest and two points on either side of it For additional information refer to A New Method of Interpolation and Smooth Curve Fitting Based on Local Procedures J Assoc Comp Machinery 17 4 589 602 Oct 1970 B 8 MRX Revelation Manual Appendix B Mathematical Relationships B 7 Extrapolation of Non Linear Curves B 7 Extrapolation of Non Linear Curves In many instances a data point is observed outside the range of the curve that is used to determine the concentration The analyst should note that some curve fit functions may not behave as expected if they are extrapolated beyond the range defined by the data points For example the readings for a set of Standards increase in a non linear fashion with increasing concentration for a given test right side of curve in Figure B 7 and the user determines that a quadratic curve fit might give the best fit for such data However at concentrations lower than the concentration of the lowest Standard the curve may turn sharply upwards as shown in the left side of the curve Absorbance 4 3 10 30 100 200 Concentration Figure B 7 Non Linear Curve Fit MRX Revelation Manual B 9 B 8 Sigmoid Equation Appendix B Mathematical Relationships B 8 Sigmoid Equation Many immunoassays are characterized by an S shaped or si
94. e goodness of the fit is determined by the value of the linear correlation coefficient R Since the linear correlation coefficient can be either positive or negative the value of R is normally reported When comparing two sets of data fits the fit with the larger value for R is a better fit for the data set if R 1 all data points lie along the line of best fit and if R 0 the data points are highly scattered and there is a poor correlation Once the linear relationship best fit has been established for a set of known standards the concentration of an unknown sample can be determined via equation B 1 An example of a linear relationship is presented in Figure B 1 B 2 MRX Revelation Manual B 2 Linear Regression Appendix B Mathematical Relationships 0 350 0 300 0 250 0 200 0 150 0 100 Absorbance 0 050 0 000 0 050 0 100 0 500 1 500 2 500 Figure B 1 Linear Relationship MRX Revelation Manual Concentration 3 500 B 3 Appendix B Mathematical Relationships B 3 Polygon Fitting B 3 Polygon Fitting Polygon Fitting of the data involves joining successive data points using straight lines as shown in Figure B 2 Absorbance Concentration Figure B 2 Polygon Fitting of Data MRX Revelation Manual Appendix B Mathematical Relationships B 4 Quadratic Cubic and Quartic Regression B 4 Quadratic Cubic and Quartic Regression Polynomial regressions are similar to a linear regressi
95. e has elapsed This option can be used if a delay in the reading is desired 9 3 3 The Reader Control Dialog Box Agglutination Parameters Tab The Reader Control dialog box Agglutination Parameters tab Figure 9 7 is used to set a variety of operating parameters for agglutination measurements Reader Control Ea Reader Options Kinetic Graphs m Parameters Plate Speed pg Readings well 32 m Calculation Mode Average C Sum C Area under curve C Formula C Rules Agglutination Parameters Start Stop Mode Kinetic Time Interval Calculation Mode Agglutination Options Formula Formula Rules a ec weifB E weit K o ve if D nl vei ve if B vei C ve if D Cancel Apply Help Figure 9 7 The Reader Control Dialog Box Agglutination Parameters Tab 9 8 MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode 9 3 Generating an Agglutination Assa Parameters Plate Speed the agglutination plate speed setting determines the distance between the readings across a well If a low setting is selected the distance between the readings will be larger and the read will be faster Plate speed settings are calculated via equation 9 1 Plate Speed Setting 9 017 distance between readings mm 9 1 The minimum setting is 8 and the maximum setting is 50 0 18 mm between readings Readings well the number of agglutination readings per w
96. e in the ratio matrix or table a Data Equation used to convert raw assay data into different units Every value on the plate is fed into the equation The resulting value replaces the original value in the data conversion section of the ratio results MRX Revelation Manual 10 21 10 5 Ratio 10 22 Chapter 10 Incorporating Decision Making Protocols into the Assay a Units used to indicate the units you wish to use for each set of calculated data Units may consist of up to eight characters a Functions equation components can be selected by double clicking on the required function These are standard functions described in Section 10 2 2 Examples of equations 1 To convert all values into a percentage of the maximum value on the plate enter 100 sample max in the Ratio field 2 If there are three replicates of each test to be ratioed against a positive control from which a negative or non reactive background has been subtracted then use this equation sample1 sample2 sample3 3 NC PC NC 3 Convert all values to a ratio of the average Positive Controls enter sample PC in the Ratio field 4 To convert all values into units which are 15 times smaller than the current units enter sample 15 in the Data Conversion field Item Example INumbers Type 0 9 Standard Arithmetic Operators Type Brackets up to 6 levels of parentheses From 1 to 5 variable values can be in the ratio Type Var
97. easing towards this limit A check mark indicates that results are increasing with time MRX Revelation Manual 8 7 8 2 Generating an Assay for a Kinetic Analysis Chapter 8 Kinetic Analyses Results Scaling If the difference between the O D for each point is expected to be small this command allows for scaling to view the data more easily Minimum scaling x 1 enter 1 Maximum scaling x 29999 enter 29999 Data Log Not used for kinetic assays Display Well used to indicate that a specific well should be indicated on the display throughout the assay this option is useful if you are running a particularly long assay and you want to make sure the reaction is progressing as expected 8 2 5 Kinetic Graphs Tab The Reader Control dialog box Kinetic Graphs tab Figure 8 4 is used to define the kinetics plots that will be output in the results and presents a matrix denoting the plate wells Reader Control a xi Reader Options Start Stop Mode Kinetic Time Interval Calculation Mode Kinetic Graphs 96 Well Plot S O mai mio o s gt J Combine individual graphs Cancel Apply Help Figure 8 4 The Reader Control Dialog Box Kinetic Graphs Tab Mode Mode The Mode selection is used to indicate if the plate location should be indicated in each box on the plot 8 8 MRX Revelation Manual Chapter 8 Kinetic Analyses 8 2 Generating an Assay for a Kinetic Analysis Location displays the plate ma
98. eeeeees 6 30 6 8 8 The Curve Fit Dialog Box Fit Q C Tab wee ceeecesecesecneeceecaeeeeeseeeeeeeennees 6 32 6 8 9 The Curve Fit Dialog Box Calculated Q C Tabu eee ceecesecsecseeereeeeeeeeeeeeeees 6 34 6 8 10 The Curve Fit Dialog Box Data Conversion Tab 00 eee ee eesee cee ereeereeeeeeeeees 6 35 6 8 11 The Curve Fit Dialog Box Results Flagging Tab ooo eee cee ereeeeeeeeeeeeees 6 36 6 8 12 Curve Fit Output Format Tab oo ee eecesecesecesecssecaecseecseseaeseeeeeeeeeeeeeeens 6 38 69 RATIOS nieas te atoen tin op igs tae a E E E Sulout E O S 6 39 6 10 Spreadsheet nenien a N E a E N E E 6 40 6 10 1 Spreadsheet Mode Spreadsheet Tab ee eceesceesecesecnseceeceeeseeeeeeeeeeereeeeens 6 40 6L Report OptrOns ciisescesad tari cteveccesseith iectestceran ul bawee roersen Subsea N E ute E s 6 41 6 12 Assay Options Zosi nia E A E A E A dd aerate neers 6 42 iv MRX Revelation Manual Table of Contents 6121 ASsay Options onien E a a a A E A A A 6 42 6 12 2 The Assay Options Dialog Box Assay Statistics Tab eee eee eeeeeeeeeeee 6 42 6 12 3 Assay Options Dialog Box Processing Order Tab eeseeeeeeeeseereereereerrereesrese 6 43 6 13 4 Assay Options Dialog Box Sample ID Setup Tab eee eeeeeeeeeeeeeeees 6 44 Chapter 7 Endpoint Analyses cccssccsseeeeseeeeeeeeeseaeeneeeeeeeeeeseaeseseeeseseeesseaeseseneneeeeeeas 7 1 TA OV CEVICW isi costes alee Se sig ti vig R cea gu eee ee 7 1 7 2 Generating an Assay for an Endpoint
99. eereeeeeeees 10 17 MRX Revelation Manual v Table of Contents 10 4 4 The Curve Fit Dialog Box Results Flagging Tab eseeseseeeseesrsreerssrsrresrsreesesee 10 18 105 RANO ie a A a A A E R E a A R 10 21 10 5 1 Ratio Equations en ies an S S A SE E E 10 21 10 5 2 Ratio Dialog Box Results Flagging Tab sseesseeessereesesrsrreresreeresrrrrssrrrrrerssene 10 23 10 6 Spreadsheet ives erir ckcascansebaye os tapace ses usens sasuhsenshibss d esvasetebsasves sheesseeaneadeasoe 10 24 10 6 1 Spreadsheet Dialog Box Results Flagging Tab eee ee eecesecesecneecneeeeeene 10 24 Chapter 11 Formating Reports and Exporting Data nsnnnnnnusnnnnrnnnnnnnnnnnnnnnnnnnnn ennen 11 1 TII OVERVIEW v2 sess RE aed ee Stes ate as eee Ps tee 11 1 11 2 Laboratory Information eee ri E EE E aE i ERa 11 2 11 3 Report Options seeen R A aia ee E TR aSa 11 3 11 3 1 The Report Options Dialog Box Report Layout Tab sseesseesesseesereersreeresresrreees 11 3 11 3 2 The Report Options Dialog Box Raw Data Tab eseseessseesseeeessrsrrerrsreeresererssess 11 4 11 3 3 The Report Options Dialog Box Export to File Data Tab eee 11 6 11 331 AS CU ROrmat 32 38 coe cites ssa dese yaa tedous sos tees Sliven chbvadyyevbiauwhoucnates E i 11 7 11 332 SS AN BUI eneee aa eyecbands Gal E E E E ct sonal E EEE EEES 11 8 11 3 3 Exporting Data Using Revelation ssessseeeseereesesrsreerrsreerrstrrrsserrenresreeresresreeess 11 9 133 1 Retrieval o
100. ell is directly related to the plate speed setting For example if you set the plate speed setting at 8 1 13mm between reads you can not set the number of readings any higher than 8 because the well is only 7mm wide Enter the number of readings you wish to take across the well The minimum is 4 readings and the maximum is 32 or the plate speed setting whichever is lower Agglutination Calculation Modes ae Note Before selecting Calculation mode we suggest that you run a calibration test with the Data Log feature switched on to determine which mode will be most useful and how the Agglutination parameters will affect your results Average takes the mean of all the readings taken across the well Sum adds together all the readings taken across the well Area measures the area under the O D plot for the well Formula calculates the result for each well using a formula which you enter using the agglutination parameters Generation of the formula is presented below Rules calculates the result for each well and whether it is positive or negative using user defined rules based on the agglutination parameters as described below Formula The Formula field is used to enter the desired formula for agglutination measurements and is activated if Formula is selected in the Calculation Mode field At run time Revelation will calculate values for the Agglutination parameters for every well The formula equation will then be
101. emoval Limit The maximum number of values that can be removed If you require that at least one replicate be retained for each standard level click on the Leave at least one replicate check box MRX Revelation Manual 6 33 6 8 Curve Fit Chapter 6 Generating and Editing Assays 6 8 9 The Curve Fit Dialog Box Calculated Q C Tab The Curve Fit dialog box Calculated Q C tab Figure 6 15 is used to develop quality control equations that are used to place criteria on samples calculated from a curve fit Quality control criteria are placed on wells after the curve fit has been drawn and concentrations or titers have been calculated If the criteria are not met the data will be processed as normal but the results report will contain a message showing that the data failed Q C Fits Standards Fit Type Graph Sample Selection Dilutions Fit O C Calculated Q C Data Conversion Results Flagging Output Format Equation S Functions Failure description P O C equations Move Up me _Move Down Down Remove IV Full QC report I Suppress results if OC fails __ fea T Lot specific checking of control values Figure 6 15 The Curve Fit Dialog Box Calculated Q C Tab Quality control equations are established in the same manner as equations for Q C Raw Data a detailed description is presented in Section 10 2 2 for Q C Raw data and is applicable to curve fitting of data 6 34 MRX Revelation Manual C
102. eport Options Ea Report Layout Raw Data Export To File Header Footer Matris Options Averaging Options gt Decimal Places Free Format Fixed at Be I Calculated Data J Average Replicates Sample IDs Arithmetic Mean Geometric Mean Table Options Result Order lV Sample ee T T V Well Location z V Replicate Value C MV Mean Value NC M SD PC Vv CV co VV SEM PR lear List OK Cancel Apply Help Figure 11 3 The Report Options Dialog Box Raw Data Tab Output Matrix displays the values obtained from each well location in terms of its position on the plate in an 8x12 array that shows the grid and is labelled Table presents the values obtained from each sample replicate in terms of its well type in a column format 11 4 MRX Revelation Manual Chapter 11 Formatting Reports and Exporting Data 11 3 Report Options Ie Note If you have included a number of different processing options and used a number of tables in the output the program may overwrite sections of data in the output If this happens try using matrices instead of tables or try removing one of the processing options Matrix Options Calculated Data box should be checked if the values calculated from a curve fit or ratio equation should be displayed Sample ID displays the identification code for each sample beside the calculated data if a sampl
103. er 5 Running an Assay 5 2 Powering up the System To Power up the System 1 Turn on the power switch for the printer and verify that the printer completes its self test successfully 2 Turn on the power switch on the front panel 3 Click on the Revelation icon on the Windows desktop 4 The monitor will present a welcome screen and the Reader Serial Number dialog box Figure 5 1 Enter the serial number and press OK Reader Serial Number Ed Serial No Cancel Figure 5 1 The Reader Serial Number Dialog Box 5 A series of self tests will be automatically performed and the results will be indicated in the workspace of the Revelation screen The self test checks the items indicated in Table 5 1 and a sample report is presented in Figure 5 2 the report is presented in the workspace of the main Revelation window which is described in Chapter 3 Ee Note When the reader is operating under the control of an internal computer the keyboard on the front panel reader will not operate The display will indicate the serial number of the instrument and the version of the firmware 5 2 MRX Revelation Manual Chapter 5 Running an Assay 5 2 Powering up the System Table 2 1 Self Test Diagnostics Test Action A D Status Begins conversion and checks that the status line goes high and low Verifies that hardware can be detected and chosen Plate Motor Moves the plate to Out position and then to the In position Verifies that the
104. ernal MRX 2 9 Rules Agglutination Parameters Tab Reader Control 9 10 Rules Calculation Agglutination Mode 9 5 Running an Assay 5 1 Run Options 5 5 Run Plate File Menu 4 4 Run Plate Defaults Preferences Tab Options Dialog Box 4 18 Run Plate Dialog Box 5 5 S Sample ID Entry Dialog Box 4 13 Sample ID Files sid 3 6 Sample ID Sample ID Entry Dialog Box 4 14 Sample ID Setup Tab Assay Options Dialog Box 6 42 Sample Selection Curve Fit Dialog Box 6 29 Sanguin Export Format 11 8 Save Assay File Menu 4 3 Save Assay As File Menu 4 3 Selecting Assay Conditions 5 5 Selecting Default Directories 4 21 Select New Assay Plate Plate Statistics Dialog Box 4 6 Self Test Diagnostics 2 3 5 3 Self Test Print 2 8 Self Test Utility Menu 4 11 Self Test File tst 3 6 Self Test Report 2 3 Setting System Configuration External Computer A 6 A 8 Internal MRX Reader 2 5 Settings Menu 4 5 Setup Internal MRX Dialog Box Reader Tab 2 6 Shake Read Mode Tab Reader Options Tab 6 6 Sigmoid Equation B 10 Sleep Mode Setup Internal MRX Dialog Box Reader Tab 2 7 Spectral Response Curve Dialog Box 4 12 Spectrum Utility Menu 4 12 Spectrum files spc 3 6 Spreadsheet 10 24 Standards Tab Curve Fit Dialog Box 6 21 10 17 Start Mode Reader Control Dialog Box General Information 6 9 Agglutination Mode 9 7 Start Stop Mode Reader Control Dialog Box Kinetic Mode 8 2 Starting a Assay 5
105. es the Threshold dialog box Section 10 3 Accesses the Curve Fit dialog box Section 10 4 Accesses the Ratio dialog box Section 10 5 Accesses the Spreadsheet dialog box Section 10 6 Accesses the Report Options dialog box Section 11 3 Accesses the Assay Options dialog box Section 6 12 Accesses the About Revelation dialog box Section 4 xx Provides Help drag to item where you want help 4 25 4 4 Status Bar Chapter 4 Commands 4 4 Status Bar The Status Bar Figure 4 24 is located at the bottom of the window CAPS SCROLL lack Por Help press FI AP r AL MESSAGE TEMPERATURE NUMBER BAR CONTROL LOCK STATUS Figure 4 24 The Status Bar The Message Bar region presents information about the current pull down menu option or the status of the reader The Temperature Control region indicates the temperature of the thermal chamber if you are running a Temperature Control Option Caps Lock Number Lock and Scroll Lock are standard commands selected by the keyboard 420 NMRX Revelation Manual Chapter 5 Running an Assay 5 1 Overview Chapter 5 Running an Assay 5 1 Overview In this chapter we describe the steps that the analyst should perform so that data can be collected on a routine basis This discussion assumes that the appropriate assay and related information has been established if this is not the case refer to Chapters 6 10 MRX Revelation Manual 5 1 5 2 Powering up the System Chapt
106. est a Kinetic measurements the reaction forming the chromophore takes place in the well The change in O D as a function of time is used to determine the concentration of the compound of interest When an assay type is selected the temporary name Assayx where x is the lowest unused number e g Assay1 is assigned The default conditions for the selected assay type are set and the assay is displayed in the workspace Figure 3 3 The conditions for the assay can be changed as described in Chapter 6 11 4 2 1 2 Open Open presents a standard Windows dialog box which is used open various file types including Assay Files asy Plate Files dat Spectrums spc Self Tests tst Sample ID Files sid and Event Logs log The desired file is selected in the same manner as other Windows files e g Microsoft Word 4 2 1 3 Close Close shuts the open file e g the Assay file If any changes have been made to the file the user will be prompted to save the changes 4 2 1 4 Save Assay Save Assay is used to save the active assay using the present name It is active only when you have made a change to the active assay and should be employed before you run an assay that you have edited 4 2 1 5 Save Assay As Save Assay as presents a standard Windows Save As dialog box to allow the user to assign a name to the file rather than the default name In addition it can be used to assign a new name to an assay that has been e
107. f no blanks were assigned to a row Revelation does not use any blanking Column treats each column separately If there are any Blank samples in a particular column the program averages all the Blank values into a single value and subtracts this value from every reading in the column If no blanks were assigned to a column Revelation does not use any blanking a Paired used when each Test Standard or Control sample uses an individual blank i e the number of Blank samples on the template must equal the total number of all the other samples MRX Revelation Manual 6 15 6 5 Blank Mode Chapter 6 Generating and Editing Assays Blank samples are assigned to different well types in the following order 1 2 Tests Standards Controls Positive Controls Negative Controls Cut off Controls User Well Types 1 to 8 As an example If a plate contains 3 Standard samples and 8 Test samples 11 Blank samples are required The blanking process will be performed in the following manner e Revelation will assign the Blanks to the other samples Blanks 1 8 will be assigned to Test samples 1 8 Blanks 9 11 will be assigned to Standard samples 1 3 e If the Blank samples are arranged in multiple replicates Revelation will average the replicates to produce a total of 11 Blank values e The Blank values will be subtracted from the assigned samples Blank value 1 will be subtracted from Test 1 Blank value 2 will be sub
108. formation is separated by a comma All data will be put into the same cell on a spreadsheet as a long string of numbers separated by commas Space information is separated by a space All data will be put into the same cell on a spreadsheet Semicolon information is separated by a semicolon All will be put into the same cell on a spreadsheet Format Format is used to specify the way in which the results will be displayed Table puts data into columns Matrix puts data into rows with header information for each well and row Listing exports data in the 8 x 12 array with no column or row header information Sort Table On Sort Table On active only if Table format is selected is used to indicate how you want the variables on which you can sort the data for presentation order Place an x in the Jn Descending Order check box to sort in descending order Select Data to Export is used to indicate the data which should be exported Ee Note Raw Data MUST be checked if you want to export raw O D data from individual reads in a kinetic assay 11 3 3 2 Sanguin If the Sanguin format is selected a dialog box to enter the Over Under Unused and Invalid values will be presented 11 8 MRX Revelation Manual Chapter 11 Formatting Reports and Exporting Data 11 3 Report Options 11 3 4 Exporting Data Using Revelation If you want to export data activate the Export feature and select the appropriate parameters as described
109. g box Reader tab is presented in Figure 2 5 Setup Internal MRX g x Reader Maintenance External RS232 Filters Data Conversion Number of Filters E OVER Limit 25 J OVER UNDER Conversion Fiter1 810 nm oyen Value Filter 2 450 nm UNDER Value 5595 Filter 3 490 nm m Miscellaneous Options Filter 4 550 z i Repeatability Mode ft Filter 5 s 570 nm Sleep Interval 5 Mins Filter 6 630 Cancel Apply Help Figure 2 5 The Setup Internal MRX Dialog Box Filters Indicate the number of filters that you have installed in the reader The corresponding number of filter fields will be activated and you can enter the wavelength for each filter ae Note Filters must be installed in increasing order If desired the other options on this dialog box can be set at this point they can be set at any time Data Conversion OVER Limit The OVER limit entry is used to create an upper and lower range for expected readings For example if you enter an OVER limit of 3 500 Revelation will classify any readings above 3 500 as OVER and any readings below 0 100 as UNDER 2 6 MRX Revelation Manual Chapter 2 Getting Started 2 3 Setting the System Configuration OVER UNDER Conversion If O D values fall outside the OVER or UNDER limits they cannot be passed to QC equations see Section 10 2 unless they are converted into numerical values If you wish to
110. g the operation The default value of 20 is suitable for most applications Minimum iterations 1 Maximum 29999 A Log Logit curve fit produces an S shaped curve similar to a Sigmoid curve defined by four parameters A to D The program sets the A parameter to the lowest Standard O D and sets the D parameter to the highest Standard O D It then attempts to fit all the other points into an S shape The curve may be quite irregular and will not be as accurate as if you had used a Sigmoid curve If you want to produce an S shaped curve it is normally better to use a Sigmoid curve unless a Log Logit curve is specifically requested Parallel Line Bioassays require special numbers of Standard and Test samples Standard samples At least two Standard samples in multiple replicates Test Samples The number of Test samples must be a multiple of the number of Standard samples and they must be arranged in the same number of replicates Stored Curve accesses a dialog box that lists a previously stored curve and Fit data The program uses the curve file to generate the axis fit the curve fit type and standard concentrations or the slope and y intercept You do not have to enter any of these values for a stored curve calculation Curves are stored after the test has been run using the Fit Information box To use this fit type 1 Create your first assay by using either the Assay Wizard or individual selections in the drop down Settings
111. gmoid curve Figure B 8 Absorbance C Concentration Figure B 8 Sigmoid Curve The sigmoid curve is described by equation B 11 ails B 11 L where a is the minimum response b is the shape factor determines the gradient of the curve c is the response midway between the maximum d and the minimum a response d is the maximum response The above definition assumes a positive value for b If b is negative the definition of parameter a and d are reversed Revelation contains an efficient algorithm for estimating the four parameters and determining the best fit for the sigmoid curve The curve requires at least four standards data points one for each of the four parameters It is recommended that at least eight Standards are defined on a plate to ensure satisfactory statistical significance Ie Note The sigmoid curve algorithm is iterative requiring many complex calculations and Revelation may take some time to process the results If the curve cannot be drawn or data cannot be processed a Windows Illegal Operation shut down error message will occur If this happens examine your data carefully to determine if the number of Standards should be altered or a different type of curve fit should be used MRX Revelation Manual Appendix B Mathematical Relationships B 9 Michaelis Menton Curve Fit B 9 Michaelis Menton Curve Fit The Michaelis Menton curve fit equation is normally used to describe enzyme kine
112. hange an ID click on the ID type a new ID into the Enter Sample ID box and then press the Enter button on the keyboard Sample IDs may be any number of characters in length but in printouts or results in matrix format long sample IDs will be shortened to fit into the available space Use Assay Template is used to enter the ID s on a sample by sample basis as opposed to a well by well basis The program will change the list on the right of the sample IDs list box to show samples instead of well locations By default the program displays a list of 96 test samples To change to a different assay template click on Change Assay Revelation displays the Open dialog box allowing you to select the required assay Auto Increment is used to automatically increment the sample IDs To use this option 1 Highlight the sample ID where you want to begin incrementing 2 Check the Auto Increment box 3 Type the first sample ID into the Enter Sample ID box and then press the Enter key on the keyboard Each time you press Enter the cursor will move down one sample ID and increment the end number 4 When the sample IDs are correct click on OK If you have made changes to any of the sample ID files without saving them the program will prompt you if you wish to save or lose these changes MRX Revelation Manual 4 15 4 2 The Menu Bar Chapter 4 Commands 4 2 4 11 Results Font The Font dialog box Figure 4 14 is used to select the desired Font Fo
113. hapter 6 Generating and Editing Assays 6 8 Curve Fit 6 8 10 The Curve Fit Dialog Box Data Conversion Tab The Curve Fit dialog box Data Conversion tab Figure 6 16 is used to assign data conversion equations to the curve fit data tab Data conversion equations are used to convert the data into different units by multiplying or dividing by some number or calculated sample value Fits Standards Fit Type Graph Sample Selection Dilutions Fit Q C Calculated Q C Data Conversion Results Flagging Output Format Equation Functions Units Cancel Help Figure 6 16 The Curve Fit Dialog Box Data Conversion Tab You can enter the desired units e g mg dL by typing them in the units field The functions used to generate equations are entered as indicated in Section 10 4 2 MRX Revelation Manual 6 35 6 8 Curve Fit Chapter 6 Generating and Editing Assays 6 8 11 The Curve Fit Dialog Box Results Flagging Tab The Curve Fit dialog box Results Flagging tab Figure 6 17 is used to specify a phrase that either replaces a numerical result or is displayed next to the result and is accessed by selecting the Results Flagging tab The phrase will be initiated whenever the numerical result meets the criteria that are specified Curve Fit x Fits Standards Fit Type Graph Sample Selection Dilutions Fit Q C Calculated Q C Data Conversion Results Flagging Output Format Conditio
114. he assay Initial Duration indicates the initial shake time which is the duration of the shake before an endpoint read The Range is from 0 seconds no shake to 59 seconds If this is a kinetic reading it indicates the shake time before the first reading Repeat Shake Duration defines the shake time between kinetic readings Click on Fixed to set a specific time or click on Continuous to shake the plate continuously between kinetic reads Shake Modes allow the user to select the plate frequency and amplitude The standard modes are described in Table 6 2 and are accessed by clicking on the down arrow adjacent to the field Table 6 2 Standard Shake Modes 1 14 Hz 4 2Hz 1 7 mm 9 Hz 2Hz 3 0 mm 8 Hz 4 2Hz 3 0 mm a Custom allows the user to select the shake amplitude range 1 10 mm and frequency 5 20 Hz manually Ie Note Custom shake mode is intended for experimental purposes and may not 6 8 produce the exact amplitude and frequency specified It will however produce reproducible shaking for a given unit If the plate carrier is heavily loaded and the amplitude or frequency is very high the plate carrier may stall The analyst should always test run Custom Shake Mode settings before using them in actual assays MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 3 The Reader Control Dialog Box 6 3 3 Start Mode Start Stop Mode Tab If you have sele
115. he reader or perform basic operations e g moving the plate in or out of the reader a Commands that are specific to a given analytical procedure these commands are incorporated into an assay To employ an assay the user accesses it and then runs it In this manual we will assume that the user is familiar with the general mode of Windows operation For the sake of brevity this manual will not describe a Standard Windows operations e g it will be assumed that the OK button on a dialog box will perform the desired operation a Windows dialog boxes that perform standard Windows operations e g the Open dialog box is similar to that employed with other Windows programs MRX Revelation Manual 1 5 1 5 Contents of this Manual Chapter 1 Introduction 1 5 Contents of this Manual This manual is divided into two sections Basic Operation of the Reader Using Revelation Software describes how the system is used on a routine basis to collect data using an existing assay a Installation Chapter 2 describes how the software is configured to meet the needs of the laboratory and includes a quick test to ensure that it is working in an acceptable manner a The Main Window Chapter 3 introduces the user to the operating program describes the contents of the Main Window and explains how you navigate between various elements of the program a Commands Chapter 4 describes the command structure of the program a Running an Assay Cha
116. heck No Output format Matrix Average replicates No Mean Arithmetic Area statistics No Export to file No Dynex Technologies Figure 7 2 A Typical Endpoint Assay 7 3 MRX Revelation Manual 7 3 Starting an Assay Chapter 7 Endpoint Analyses 7 3 Starting an Assay Open the desired assay If a password has been assigned to the assay a password dialog box will be presented and the password must be entered if you want to edit the assay If you simply want to run the assay it is not necessary to enter the password and the Cancel button should be pressed To run an assay press Run Plate on the File menu or use the Run Plate icon on the Tool Bar to present the Run Plate dialog box Figure 7 3 Run Plate x r Plate ID r Options Plate ID sft K IV Auto save plate data as I Scan Barcode before un CAPROGRAM FILES REVELATIONS Plate PeBe J Print results automatically Operator I Search for sample IDs Operator m Assays Select Open Selected Assayfs 96 Al 406a asy Save Add Remove Apoptosis asy Clear Bradford Proteins as A Orientation CAT asy CMVIGG ASY Columns C Rows First well location COLBLNK ASY i CRYPTO ASY Number of wells 95 cet ew Figure 7 3 The Run Plate Dialog Box 7 4 MRX Revelation Manual Chapter 7 Endpoint Analyses 7 3 Starting an Assay Plate ID a Plate ID A pla
117. heck mark in the present check box 2 Enter the concentration of the inhibitor 3 Specify the data source which does not contain inhibitor by selecting the appropriate radio button e Fit Select another fit within the same assay e Stored Curve Select a stored Michelis Menten or Lineweaver Burk curve use the Browse button to search for a file MRX Revelation Manual 6 27 Chapter 6 Generating and Editing Assays 6 8 Curve Fit a Axes Select the desired axes not active for Lineweaver Burk Michaelis Menton Sigmoid and Log Logit options by clicking on the radio button When a curve fit is chosen Revelation will gray out any axes fits which are not available for that type For example if a sigmoidal fit is chosen all selections are grayed out and the fit is automatically drawn on a semi log scale 1 Lin Lin both the x and y axes are on a linear scale 2 Log log Both axes use logarithmic scales 3 Log Lin the x axis is graphed on a log scale while the y axis O D s are graphed on a linear scale 4 Auto Revelation selects the best axes fit automatically 6 8 5 The Curve Fit Dialog Box Graph Tab 6 28 The Curve Fit dialog box Graph Tab Figure 6 11 is used to include a concentration graph in the results and define how it will be output Calculated Q C Data Conversion Results Flagging Output Format Fits Standards FitType Graph Sample Selection Dilutions FitQ C M Produce a Gra
118. hether or not to continue the assay a Finish indicates that all desired editing of the assay has been completed The assay will be presented in the workspace but is not saved at this point If you want to save the assay select Save Assay on the File menu and enter the desired name which can but need not be the same as the assay title MRX Revelation Manual 6 3 6 3 The Reader Control Dialog Box Chapter 6 Generating and Editing Assays 6 3 The Reader Control Dialog Box 6 3 1 Configuration of the Tabs on the Reader Control Dialog Box The Reader Control dialog box Figure 6 2 is used to set a broad range of parameters for the assay This screen tells the reader how and when to read your plate The tabs that are presented in this dialog box depend on the assay type Figure 6 2 describes the dialog box for an endpoint assay The various configurations for this dialog box are summarized in Table 6 1 A discussion of systems with external control is presented in Appendix A Agglutination Options Transmission Graphs Reader Options Start Mode Agglutination Parameters Multiwavelength Mode r Shake linear Hearession T Enable Shaking C Well Type tr Initial Sheke 0 SECONAS C Aggluination T C G C vaue f Repeat Shake Duration Fived at fi Seconds m Wavelengths Gontnucus Primary Test Filter ind j ModeE Mode 1 14 Hz 1 7 mr 7 Primary Ref Filter x A Shake Mis Secondary Mast
119. iablel All variables must be numbered equation to be entered at run time Every individual well on the plate Use the word Sample Replicate X of each sample on the plate Use the words Sample 1 Sample Sample by well type and number of replicates IT1 S2 CO2 indicates all wells with the same label and number Notation for an individual replicate T1 1 T1 2 NC2 1 NC2 3 Average well type value Type the well label T NC PC All wells with this label will be averaged in the calculations IMaximum of a group of values Imax Minimum of a group of values min Coefficient of Variation for a well type ICV well type Standard deviation for a well type SD well type MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 5 Ratio 10 5 2 Ratio Dialog Box Results Flagging Tab The Ratio dialog box Results Flagging Tab Figure 10 10 is used to specify a phrase that either replaces a numerical result or is displayed next to the result select the Results Flagging tab The phrase will be initiated whenever the numerical result meets the criteria that are specified A detailed discussion of Results Flagging is presented in Section 10 4 4 Ratio Equations Results Flagging Output Format Condition Result Flag Functions If fi then Sample checks Cancel Apply Help Figure 10 10 The Ratio Dialog Box Results Flagging Tab MRX Revel
120. ick on the time on the bottom right corner to present the Date Time Properties dialog box Figure 5 3 and edit these parameters as appropriate Date amp Time Time Zone Date Time F Se Je ont a a a a a 6 7 B 9 10 a 13 14 15 16 17 oe a 20 21 22 23 24 atone as 27 28 S fir zs qoam Current time zone Eastern Standard Time Figure 5 3 Date Time Properties Dialog Box 7 Select the Run Plate command on the File menu The Run Plate dialog box is described in Section 5 4 MRX Revelation Manual Chapter 5 Running an Assay 5 3 Selecting the Assay Conditions 5 3 Selecting the Assay Conditions To select assay conditions press Run Plate on the File menu to present the Run Plate dialog box Figure 5 4 Figure 5 4 The Run Plate Dialog Box To run an assay 1 Enter the Plate ID if applicable The plate ID is used as a temporary filename for the plate data file which is created when the assay ends If you have selected Default Plate IDs in the assay a default ID will appear in the Plate ID field box otherwise you will have to assign the ID manually Plate IDs can be assigned either by typing a name into the Plate ID text box or by scanning a barcode on the side of the microplate If you are using the Barcode option you should make sure the scanner is set up to decode the correct type of barcode For additional information refer to the Barcode Program User Manual Scan Bar Code before run To automatically read
121. ing After 2 readings 771 No ix No Post kinetic calculations 4 5 6 7 8 T25s T33s Ths T4 T57s T26 T34 T42s T50 T58 T27s T35s T43s TS1s TS T2 amp T36s T4 T5 T60s T2 T37s T45 T5 T61s T30s T38 T46s T54 T62 T31s T3 T47s T55 T63s T3 T40s T48s TS6s T64 T65 T66s T675 T68 T6 T7 T71s T7 s indicates that a sample ID is required for this well location None Yes gt No No Matrix No Arithmetic No No Dynex Technologies 10 T7 T74 T75 T76s T77s T78s T7 T80 T8is T82s T83s T84 T85 T86s T87s T88 12 T8 T90 T915 192s T9 T94 T95 T96 Figure 8 5 A Typical Kinetic Assay MRX Revelation Manual Chapter 8 Kinetic Analyses 8 3 Starting an Assay 8 3 Starting an Assay Open the desired assay If a password has been assigned to the assay a password dialog box will be presented The password must be entered if you want to edit the assay If you simply want to run the assay it is not necessary to enter the password and the Cancel button should be pressed To run an assay press Run Plate on the File menu or click on the Run Plate button icon on the Tool Bar to present the Run Plate dialog box Figure 8 6 Run Plate x r Plate ID Plate ID sft K r Operator Operator fo Scan Barcode betore min Head Barcode Woy r Options IV Auto save plate data as C PROGRAM FILES REVELATION Plate J
122. ing Protocols into the Assay 0 ss 0 10 1 10 1 OVERVIEW iso sessing Beet he Bees TE ity Ay eesti Sor ETE EEr EE deena dures 10 1 10 2 Quality Control ois scseg coh elisa a tia ease ceased ee tea chad ed acted dee eaters 10 2 10 2 1 Role of Quality Control oo eee nni es eie ire nis ari iS 10 2 10 2 2 Creating Quality Control Equations cece csc cseeeseeeeeeeeeeeeeeerecaecaecaeeneeenes 10 4 10 2 3 Typical Equations sen enee e aie atin aaa 10 7 10 3 Threshold et asinen ink deans e ese Ee eden adie teas Sek Waa edd 10 8 10 3 1 The Threshold Dialog BoX ecceeesceseeeseeseeseeeeeeeseeeeceseceecesaecseecaeseaeseeeeeeeeereeaeens 10 8 10 3 2 Threshold Equations Tab cece ceeceseceseeeseeeeeeeeeeeeeeeceecesecsaecaeceeeseeeeereeereeaeens 10 9 10 3 2 1 4 4 4 4 Equations cece eecesecesecsecssecseeceeeseseeeeeseeeeeeseesaees 10 9 10 3 3 The Pos Nes Q C2 Tabira eiere eR E pE ASEE S EEE IERE EE R SSK 10 10 10 3 4 Threshold QC mei As in eh edt eae eee 10 12 10 3 5 Output Format s c 35 cscs ciecescdecccbucnseecs fesseccatte sie EREE E E AVERSE I ES eE VIRS TE ARE EE 10 13 104 Curve Fitts a R din eed S A E E A EREE E tie 10 16 10 4 1 Introduction to Curve Fitting eeesseesseeeessseeeessesreereserersseeressesrenresrerrssrnrrnseeseeeeset 10 16 10 4 2 Curve Fit Dialog Box Calculated Q C Tab eecesecssecsseensecseeeeeeneeees 10 16 10 4 3 The Curve Fit Dialog Box Data Conversion Tab 000 0 eee eeceeeceseeese
123. inish lt Last Apply Help t e Figure 3 1 The Reader Options Dialog Box Report Layout Tab e Commands will be indicated in bold face while dialog boxes and fields within a dialog box will be indicated in italics 4 In some dialog boxes the commands are organized in a region that relates to a specific task As an example Figure 3 2 includes boxes around parameters that relate to the Read Mode Multi Wavelength Mode etc In this manual these regions will be called out by an underline e g Read Mode and each of the commands selections etc in the region will be indicated in bold face and described immediately below the descriptor of the region MRX Revelation Manual 3 3 3 2 Conventions in this Manual Chapier 3 Introduction to the Operating Program Reader Control x Agglutination Options Transmission Graphs Reader Options Start Mode Agglutination Parameters Multiwavelength Mode gt r Shake I Enable Shaking lritiel Shake fi Seconds Repeat Shake Duration Fived at fi Seconds Continuous Mode Mode 1 14 Hz 1 7 mr r Custom Shake Mode Aripltude rim E Frequency Hela lo heer Regression Welljppe fr C Yanes aa C Multiwavelength C Agglutination T C G Wavelengths Primary Test Filter H Primary Ref Filter e Seconda lest Filter Ed Seconda HER Filter es Cancel Apply Help
124. ion of bacteria and determining red cell types Agglutination is caused by the addition of antibodies to the sample to fuse with the antigens on the cell surface The O D is measured at different areas of the well to collect information about the cell Features of the System include m Quick read times less than 4 seconds for a 96 well plate a Dynamic Range 0 1 to 4 0 O D a Excellent Linearity and Precision a Built in temperature control optional from ambient to 63 C A verification plate optional is used to test for a Plate alignment X axis Y axis and Skew a Linearity of O D measurements a Filter accuracy a Optical channel matching Photometric accuracy MRX Revelation Manual 1 3 1 3 Principle of Operation Chapter 1 Introduction 1 3 Principle of Operation The reader is a filter photometer that measures the optical density of each well in a plate Figure 1 2 SOURCE FILTER PLATE DETECTOR Figure 1 2 Block Diagram of Reader Components The source is a 75 W Tungsten Halogen lamp that provides a wavelength range from 340 to 850 nm The filter selects the wavelength used for the measurement Up to six different filters can be installed in the system so that you can measure a broad range of different types of assays The detector measures the amount of light that is transmitted through each well Since wells can contain samples blanks or standards concentration of the compound of interest can be de
125. is allowed to go to completion before the plate is read The O D Optical Density is measured and then used to determine the concentration of the compound of interest This chapter includes e Generating an Assay for an Endpoint Analysis Section 7 2 e Starting an Assay Section 7 3 MRX Revelation Manual 7 1 7 2 Generating an Assay for an Endpoint Analysis Chapter 7 Endpoint Analyses 7 2 Generating an Assay for an Endpoint Analysis To generate an Endpoint Analysis 1 Select New Assay on the File menu 2 Select Endpoint on the Assay Type dialog box 3 Develop the assay as described in Chapter 6 The Start Mode tab on the Reader Control dialog box Figure 7 1 is provided to indicate when the O D of the sample should be read Reader Control Ed Agglutination Options Transmission Graphs Reader Options Start Mode Agglutination Parameters Start Mode Immediate C When ri reaches 0 0D When jTi reaches 0 OD min After fi minutes OK Cancel Apply i Figure 7 1 The Reader Control Dialog Box Start Mode Tab Four options are provided Immediate the data is acquired as soon as the assay is initiated a When T1 reaches xxx O D do not use a When T1 reaches O D min do not use a After xxx minutes the data is collected after the indicated time has elapsed This option can be used if a delay in the reading is desired As an example if the chromophore is developed while the micropl
126. is an automated microplate reader that is designed to automatically read the absorbance of 96 well plates in research and clinical applications Figure 1 1 The DYNEX Technologies MRX Microplate Absorbance Reader MRX Revelation Manual 1 1 1 1 Overview Chapter 1 Introduction The reader is computer driven and includes Revelation application software to establish assays as well as to collect process and report data in a format that meets the needs of the laboratory An MS DOS computer is integrated into the MRX optical compartment The user plugs the mouse keypad and printer into the instrument Ie Note The MRX Reader can be interfaced to an external computer via an RS 232 port A discussion of use of the reader with external control is presented in Appendix A 1 2 MRX Revelation Manual Chapter 1 Introduction 1 2 Modes of Operation and System Features 1 2 Modes of Operation and System Features Revelation provides the following modes of operation a Endpoint measurements the reaction forming the chromophore is allowed to go to completion before the plate is read The observed O D is used to determine the concentration of the compound of interest Kinetic measurements the reaction forming the chromophore takes place in the well The change in O D as a function of time is used to determine the concentration of the compound of interest Agglutination measurements are used for monitoring cell growth identificat
127. k mode Q C equations Data matrix leader information Filename Date Plate ID Assay title Page Q C summary 410 nm ef wavelength a STATUS e Figure 3 3 The Main Window The Main Window includes a Title Bar indicates the name of the assay and its path a Menu Bar contains a series of drop down menus that contain commands Section 4 2 a Tool Bar icons that serve as short cuts for commonly used commands and assays Section 4 3 a Workspace region for display of dialog boxes and data when the program is opened the workspace will be empty a Status Bar indicates the present status of the system Section 4 4 MRX Revelation Manual 3 5 3 4 File Types Chapter 3 Introduction to the Operating Program 3 4 File Types A number of file types are associated with the Revelation program Some files e g the assay file is user generated while others are automatically created edited by the operating system Each file type can be opened via the Open dialog box accessed from the File menu 1 3 6 Assay files asy An assay file include the complete description of the protocol that should be used when a series of samples is analyzed The assay file includes data collection data processing and reporting parameters A detailed discussion of the assay is presented in Chapters 6 11 Plate Data files dat A plate data file contains the analytical results from a given plate These files canno
128. l ACwels CO wels Nwels O PR wells HS wells OK Cancel Apply i Figure 10 3 The Threshold Dialog Box Pos Neg Q C Tab This option allows you to assign a result or to each well type All samples within the well type must fall into the required result range or the well type will fail Q C and a warning will be displayed in the results Alternatively you can view the O D with the results so that you can manually check its accuracy To do this type O D in the box ae Note If you changed any of the threshold labels you must use the new label in the Pos Neg Q C boxes not the default labels MRX Revelation Manual 10 11 10 3 Threshold Chapier 10 Incorporating Decision Making Protocols into the Assay 10 3 4 Threshold Q C The Threshold dialog box Threshold Q C tab Figure 10 4 is used to generate Quality Control Q C equations which place criteria on the threshold data If the criteria are not met the data will be processed as normal but the Results report will contain a message showing that the data failed Q C In addition note that when the Q C Raw Data command is selected from the Settings menu the Threshold Q C dialog box which is identical to this tab is presented Threshold Ea sogesseeeeeeeeeseeceeseeeeeseeesosesssg Threshold Equations Pos Neg Q C Threshold C Output Format Equation OT Functions Failure description CY O C equations New
129. late type is saved via the Save button it will be indicated in the large box as shown above Plates can be saved deleted etc As an alternative click Barcode to scan the barcode on the microplate if a barcode reader is present a Delete removes the currently open SID file that is shown in the Enter Plate ID box Click on Delete to delete this SID file Delete All removes all SID files Import presents a dialog box to import a file from an external system Figure 3 4 After the file type has been selected a dialog box is presented to allow for selection of the specific file see Section 4 2 16 IEE Note Revelation may not be able to import files from external software if they do not follow certain file format conventions MRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar Sample ID Sample IDs can be assigned in two different ways a By well Revelation displays all the wells and you can assign sample IDs to as many of the wells as desired a By sample The program displays the assay template sample by sample and you can assign IDs to as many of these as you want To use this option check the Use assay template box see Use Assay Template below The Sample IDs list box contains two columns The left column shows the well locations or sample ID s depending on how you are assigning ID s and the list on the right shows the assigned sample ID s By default Revelation assigns the IDs as T1 96 To c
130. le some are obligatory Options which are not available for a given situation will be greyed out Options which are obligatory for a given situation will be in bold type and the corresponding check box will be checked If Table Output is selected you can place the data in order according to well type To delete the current order and create a new one select Clear List and click on the well types in the order you want them to appear To move a well type to the top of the list double click on it You must add every well type to the list before leaving the dialog box even if you are not using every well type 11 3 3 The Report Options Dialog Box Export to File Data Tab The Report Options dialog box Raw Data tab Figure 11 4 is used to indicate that the results of the test should be exported as an ASCII text file and assign the desired format Report Options Ed Report Layout Raw Data Export To File Header Footer IV File Export Enabled File Format Sanguin Mikrotek Concentrations RES OK Cancel Apply Help Figure 11 4 The Report Options Dialog Box Export to File Tab 11 6 MRX Revelation Manual Chapter 11 Formatting Reports and Exporting Data 11 3 Report Options In the File Format dialog box select the format of the file to be exported ae Note If you plan to ever export data as an ASCII file a plate ID must be entered in the Plate ID field in the Run Plate dialog box before the plate read o
131. letes all the assays from the Selected Assay s field a First well location Select an assay in the Selected Assay s field and type in the well location where you want this assay to start a Number of wells Enter 96 for this value for each assay IEE Note The program allows you to assign fewer wells to an assay than were originally programmed into the assay template For example if your assay contains 96 Test samples but this run only requires 10 test samples you can assign 10 wells to the assay Revelation will reduce the template to fit into 10 wells The program reduces templates by removing Test samples from the end of the template until the required number of wells is left All non Test samples are left in the same positions relative to the end of the plate If it is not possible to reduce the template the program prints an error message and will not reduce the template a Orientation If you have assigned less than 96 wells to any assay on the plate you must select Columns or Rows to specify the plate orientation As an example if Assay contains 12 wells and starts at Al and you select Columns the program will place the assay in wells Al to H1 and A2 to D2 if you select Rows Revelation will place the assay in wells Al to A12 ae Note The selected orientation applies to the whole plate and not just the highlighted assay If you change the orientation when some assays have already been positioned on the plate y
132. logies and must not be reproduced in any form without prior consent MRX is a registered trademarks of DYNEX Technologies Revelation is a trademark of DYNEX Technologies Microsoft Windows and Excel are registered trademarks of Microsoft Corporation Revelation Software License Agreement Revelation Software License Agreement This software license agreement including the Warranty and Special Provisions set forth on the separate sheet included in this package is a legal agreement between you either an individual or an entity hereinafter End User and the manufacturer DYNEX Technologies Inc of the system containing software product By using the system in which software program s have been preinstalled SOFTWARE you are agreeing to be bound by the terms of this agreement 1 GRANT OF LICENCE This License Agreement permits you to use the SOFTWARE as preinstalled on the system 2 INTELLECTUAL PROPERTY MRX Revelation contains intellectual property i e software programs that are licensed for the end user customer s use hereinafter End User This is not a sale of such intellectual property The end user shall not copy disassemble reverse engineer or decompile the software program 3 COPYRIGHT Windows SOFTWARE is owned by Microsoft Corporation or its suppliers and is protected by United States copyright laws and international treaty provisions and all other applicable national laws Revelation SOFTWAR
133. loses the program If you have made any changes to the assay a dialog box will be presented to ask if you want to save the changes IEE Note The last four assays that have been accessed are presented on the File menu for convenience in retrieving recently used assays 4 2 2 The Settings Menu The Settings menu is activated when an assay has been opened either an existing assay or a new assay and is used to generate edit the assay protocol A detailed description of the Settings menu is presented in Chapters 6 11 4 2 3 The View Menu The View menu Figure 4 4 is used to configure the Main window View Toolbar Status Bar Figure 4 4 The View Menu The Toolbar and the Status bar can be presented on the Main window by clicking on the appropriate command When the item is selected a checkmark is placed to the left of the command A typical status bar is described in Section 3 6 4 2 4 The Utility Menu The Utility Menu Figure 4 5 is used to access a variety of commands that relate to system configuration system testing etc MRX Revelation Manual 4 5 4 2 The Menu Bar Chapter 4 Commands Utility Plate Plate Statistics Plate Reproducibility Select Language Configure Reader Plate In Plate Out Read Calibration Plate Selftest Spectrum Sample ID Entry Results Font Options System Password Figure 4 5 The Utility Menu Note If an external reader is used the Plate I
134. ly This can be done by checking the properties for each port via the Device Manager To access the Device Manager a Click on the My Computer icon on the main window minimize Revelation b Select the Control Panel icon c Select Device manager on the Control Panel d Select Communication Port COM1 on the Ports entry e Click on the Properties tab Check the settings and verify that it is working correctly f Repeat this for all ports to verify that they are working properly g Return to Revelation and try to link the reader to the computer h Change the cable and the adapter independently if possible 1 Check the RS232 port using another RS232 device j Change the computer and reload the software k Call your local DYNEX Technologies service representative for assistance Please have the serial number of the system version number of the software the version number of the firmware in the reader and a description of the computer that you are using available MRX Revelation Manual Appendix A Using an External Computer A 6 Differences Between Internal and External Operation A 6 Differences Between Internal and External Operation The general mode of operation of a system with an internal computer and that with an external computer is very similar The user should note the following differences 1 Agglutination does not function when an external computer is used 2 With an internal computer the communicati
135. mal results to fall The program will flag any results which fall outside this range with a character used with Curve fit only Printout Mode used to identify the cross over between positive and negative in a dilution block You can flag the change from positive to negative or from negative to positive the last positive result or the last negative result The Threshold output is active only when a dilution block has been defined using the Replicates Define option MRX Revelation Manual 10 15 10 4 Curve Fit Chapter 10 Incorporating Decision Making Protocols into the Assay 10 4 Curve Fit 10 4 1 Introduction to Curve Fitting When Curve Fit processing is used the program measures the O D for a series of standard samples of known concentrations and uses the readings to generate a graph which is used to calculate the concentrations of Test experimental unknowns samples The numbers entered for the known standards are used to draw the x axis of the graph and the ODs from the known standards are used to draw the y axis The Curve Fit protocol is described in detail in Section 6 8 In this section we describe the selections which can be incorporated into the data is to be fit i e the equations to be used 10 4 2 Curve Fit Dialog Box Calculated Q C Tab The Curve Fit dialog box Calculated Q C tab Figure 10 6 is used to develop quality control equations that are used to place criteria on samples calculated from a curve fi
136. mation that should be included with the results value for each sample when the Table format is selected The items are summarized in Table 10 1 Table 10 1 Table Options OPTION DISPLAYS Sample ID Sample ID if one was entered Well Location Well location Replicate Value Raw data value Mean Value Mean value of the whole well type S D Standard deviation value of the whole well type C V Coefficient of variation of the whole well type S E M Standard error mean of the whole well type Dilution Dilution of every sample if one was entered IEE Note Some table options are not available for certain data processing options while some are obligatory Options which are unavailable will be greyed out for a given situation Options which are obligatory will be greyed out and the corresponding check box will be checked MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 3 Threshold a Results Order If Table Output is selected you can place the data in order according to well type To delete the current order and create a new one select Clear List and click on the well types in the order you want them to appear To move a well type to the top of the list double click on it You must add every well type to the list before leaving the dialog box even if you are not using every well type Normal Ranges used to set a range within which you expect all nor
137. me for Fitl or retain it and enter another name for the next fit in the Enter Fit Name field and then press the Add Fit button when a new Fit is created a You can enter the desired name in the Enter fit name field and then press the Add fit button when a new Fit is created The name entered for a given fit is typically related to the kind of fit it is or how many standards are in that fit i e Linear or S1 5 To include a fit into the assay the settings must be defined in the other tabbed folders and then the Add button should be clicked a Add Fit The name of the current curve fit is shown in the Enter Fit Name box To add this fit to the list of curves or to save changes to a fit click on Add Fit a Remove Fit used to delete the highlighted fit from the list of fits a Copy Fit used to copy a fit from the list of fits i e if you want to make a small change to a present fit To copy a fit enter a name for the copied fit in the Enter Fit Name box then and click on Copy Fit The copied fit will be added to the list under the new name 6 8 3 The Curve Fit Dialog Box Standards Tab The Curve Fit Dialog Box Standards tab Figure 6 9 is used to define the various standards that are used to generate the fit MRX Revelation Manual 6 21 6 8 Curve Fit Chapter 6 Generating and Editing Assays Calculated Q C Data Conversion Results Flagging Output Format Fits Standards Fit Type Graph Sample Selectio
138. menu Your first assay will have your standards in the template and the curve fit programmed in Run your plate Data will appear on your monitor The top task bar will have the words File Data View Utility Window Help Click on the word File then click on Save Data As Save your data in a file with a dat extension Click on the word Data in the top task bar Click on the selection Fit Information A dialog box will appear Make sure the fit you want to save is highlighted Enter any information about the x or y value you wish Click on the Save button A different dialog box labelled Save As will appear Type in the file name you wish this curve saved under Make sure the extension is cur and that it is being saved in the Curves folder in the top Save in rectangle box Click on the Save button The Fit Information box will come back up Click on the Close button Create a second assay In the second assay there will be no wells labelled as standards in your template In the Curve Fit screen under the assay Settings go to the tabbed folder marked Fit Type At the very bottom of the list on the left labelled Fit type there is a selection for Stored Curve Click on it A dialog box will appear labelled Use Stored Curve Highlight with the one in the list of fits that you want to use and click OK Click OK on the Curve Fit screen and finish programming your se
139. n Dilutions Fit Q C Select Check list Automatic Entry 51 Add All S No Factor S2 S2 ED 3 Add S3 C Additive fi 2 Remove C Multiplier 5 Oea m Percentage Response 0 Ret J Percent Response P 1010 Lou Hef hep S mn M LESO Clear Refs 100 Reh Sime E esi Enter Concentrations jo I Variable Std Multiplier Enter Units J Enter concentrations at run time Apply Help Figure 6 9 The Curve Fit Dialog Box Standards Tab To use the Curve Fitting function you must create a Standards checklist that contains at least one Standard standards are entered into the program via the Template dialog box Section 6 4 The Standards indicated in the Check list are used to create the standard curve which is highlighted in blue in the Fits tabbed folder The actual number of Standards required depends on the Curve Fit Type The Select field contains all the Standard samples and the Check list shows all the Standards that have been selected Add All used to indicate that all standards in the Select list should be moved to the Check list a Add adds the Standard highlighted in the List box to the bottom of the Check list a Remove removes the highlighted Standard from the Check list a Clear clears all the Standard samples from the Check list a Automatic Entry allows you to enter values automatically such as dilution factors or evenly spaced concentration values a No
140. n Plate Out Read Calibration Plate and Selftest entries are not presented 4 2 4 1 Plate Plate Statistics Plate Plate Statistics presents the Plate Plate Statistics dialog box Figure 4 6 which is used to compare data from one plate to data from another plate and use the plate data from one test with the data processing options of another test The Plate Plate statistics command allows you to compare plate data to find out the mean standard deviation coefficient of variation and standard error mean for designated types or areas of wells Plate Plate Statistics xi a Plates Select Check list Bradford_Proteins DA Gk Examplel DAT s Cancel Add Add All Remove Clear Figure 4 6 The Plate Plate Statistics Dialog Box C Use Original Assay Select New Assay Pekel 460 S MIRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar Assay The information in data files is stored as raw Endpoint data or Kinetic data When you open a file the program provides the option of processing the data using the assay that was originally run to create the file or via another assay Use Original Assay opens all the data files on the checklist using the original assays that were used to create them Select New Assay lets you open a file and process the data using the blanking options and calculation mode options from a different assay Ie Note Revelation cannot reprocess an Endpoint data file
141. n Result Flag Functions If then Sample checks Move Up m _Move Down Down _ Bemove Gee Cancel Apply Help Figure 6 17 The Curve Fit Dialog Box Results Flagging Tab a Condition is used to indicate the Results Flagging expression a Result is used to indicate if a phrase is to be reported rather than a numerical result a Flag is used to enter a phrase if the numerical result is to be accompanied by a phrase in the Flag box An example of the Results Flagging feature would be to report the phrase lt 20 mg dL instead of the actual numerical result whenever results are less than 20 mg dL As an alternative the phrase Abnormal High can be displayed to flag any results that are higher than a specified level The phrase will be initiated whenever the numerical results meet the criteria that are specified 6 36 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 8 Curve Fit Results Flagging Equations are used to specify criteria for generating the phrase or flag Although several unique elements are used for constructing a Results Flagging expression the expression is created in the same way as Raw Data Q C s The unique elements for Results Flagging are described in Table 6 5 The Sample Checks field presents a listing of the various conditions that have been generated and the Function field lists the operators that can be used see Chapter 10 for information An unlimited number
142. n a barcode Revelation displays the resulting filename in the Options box along with the default destination The default destination is determined by the Options command on the Utility menu If you intend to recall plate data at a later date you should always assign the ID as a permanent filename Print Results If you do not want the data to be printed automatically un check the Print Results check box stored data can be printed using the Print command on the File menu after collection Search If you created a sample ID file using the Sample ID Entry on the Utility menu Revelation can automatically search for this file Sample ID files are saved by default in the SID directory If you want to search for the file automatically you should make sure it has the same name as the plate ID To link sample IDs to the template click on Use Template Assays An assay list is used to indicate the assay s to be run Assay lists may contain a single assay or a number of assays If you place more than one assay on a plate you can either overlap some assays so that more than one assay will run on the overlapped wells or you can run each assay on different wells 8 12 MRX Revelation Manual Chapter 8 Kinetic Analyses 8 3 Starting an Assay The Select list box contains a list of all your assays Open Presents the Open dialog box with a listing of all previously saved assay checklists Save Saves an assay checklist Checklists are sa
143. n displays the spectral response curve with suggested test and reference filters A typical report is shown in Figure 4 12 Note At least two filters must be installed for this test 4120 NRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar Revelation Spec1 del x Lii File View Utility Window Help lej x 0 150 a 0 100 0 050 400 500 600 700 wavelength inm Suggested test wavelength 405 nm Suggested ref wavelength 630 nm Fa 4 gt A Start Revelation Spec1 3 49 PM Figure 4 12 Spectrum Report 4 2 4 10 Sample ID Entry Sample ID Entry Sample ID files are stored in the SID directory by default and are identified by the SID file extension When you run a plate the program can be set up to automatically search for a sample ID file to attach to the data For this to work the sample ID file must have the same name as the plate ID The Sample ID Entry dialog box is presented in Figure 4 13 MRX Revelation Manual 4 2 The Menu Bar Chapter 4 Commands Sample ID Entry Ea CAIs I Ce Enter Plate ID fi 2 Barcode SIE aS Delete Help Delete All Import T Auto Increment I Use assay template Cancel HEE Sample ID Enter Sample ID Columns C Rows Well Orientation Figure 4 13 The Sample ID Entry Dialog Box Plate ID The Plate ID fields are used to identify the plates that can be used A number of plate ID s can be identified When a p
144. n the operation click OK or press Enter on the keyboard The program will read the plate and calculate a mean standard deviation and coefficient of variation value for each well and for each the readings The individual well results are displayed as a matrix showing either the mean the standard deviation SD or the coefficient of variation CV You can switch between these viewing modes using the View menu The mean SD and CV values for all the plates are shown below the matrix 4 2 4 3 Language Language presents a dialog box to be used to select the choice of language At the present time English is the only option This option is presented only if no assays are open 4 2 4 4 Configure Reader a Ifno assays are open e g the system has just been powered up and the initial self test is closed the Reader Type dialog box Figure 4 8 is presented the user should check that the appropriate system type is selected and then press Setup Reader Type Ea Select a reader OK External MFX Internal MAX Figure 4 8 The Reader Type Dialog Box 48 0 NIRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar The Setup button accesses the Configuration Information dialog box This dialog box is described in Section 2 7 these parameters are normally set during installation and rarely changed once they are established a Ifan assay has been opened the Configuration Information dialog box is presented This dialog b
145. nation The default destination is determined by the Options command on the Utility menu If you intend to recall plate data at a later date you should always assign the ID as a permanent filename Print Results If you do not want the data to be printed automatically un check the Print Results check box stored data can be printed using the Print command on the File menu after collection Search If you created a sample ID file using the Sample ID Entry on the Utility menu Revelation can automatically search for this file Sample ID files are saved by default in the SID directory If you want to search for the file automatically you should make sure it has the same name as the plate ID To link sample IDs to the template click Use Template Assays An assay list is used to indicate the assay s to be run Assay lists may contain a single assay or a number of assays If you place more than one assay on a plate you can either over lap some assays so that more than one assay will run on the over lapped wells or you can run each assay on different wells The Select list box contains a list of all your assays Open Presents the Open dialog box with a listing of all previously saved assay checklists Save Saves an assay checklist Checklists are saved by default in the main Revelation directory using an LST extension If the checklist has not been saved before Revelation displays the standard Save As dialog box Add Pl
146. nce The printout will indicate if this test produces a failure An overall test summary will be given a Accuracy Determines if an instrument is within specification by comparing the readings with pre determined factory values printed on the test plate calibration certificate included with every plate at time of manufacture or yearly re certification The values in the plate data should be compared to those on the certificate of calibration 2 10 MRX Revelation Manual Chapter 2 Getting Started 2 4 Initial Testing of the System To use the Verification Plate Select Plate Out on the Utility menu The plate carrier is ejected from the instrument Insert the 12 way verification plate into the plate carrier with the numbers on the white tag on the left arrow pointing up Select Read Calibration Plate from the Utility menu Revelation presents the system password dialog box Enter the password the default password is DYNEX and press OK The Calibration Plate dialog box Figure 2 8 will be presented This dialog box is used to verify the serial number of your MRX reader and the serial number of the 12 way verification plate you will be using Make any changes in either serial number Calibration Plate Ed Reader Serial No e ais Read Plate Plate Serial No fi 2 055 Cancel Help Figure 2 8 The Calibration Plate Dialog Box 5 Click the Read Plate button A dialog box indicating Status with the mess
147. ng in the upper left corner A1 and finishing in the lower right corner H12 The type of sample is dependent on the radio button selected in the Well Type field The orientation row or column is dependent on the selection in the Fill Direction field Randomize places a sample in each position similar to fill but the locations are set up on a random basis The type of sample is dependent on the radio button selected in the Well Type field Renumber numbers all samples on a sequential basis if there are gaps due to deletion of samples Clear clears all the existing well assignments so that you can redefine the template from scratch MRX Revelation Manual 6 13 6 5 Blank Mode Chapter 6 Generating and Editing Assays 6 5 Blank Mode The MRX reader automatically uses the transmission of air as a reference for 100 transmission For certain applications however other reference levels may be more appropriate The Blank Mode dialog box Figure 6 6 is used to select the definition for blanking Blank mode x Blank Mode Custom C Previous Plate Individual Select C Average C Row Column Paired C Custom Cancel Apply Help Figure 6 6 The Blank mode Dialog Box Blank Mode Tab Blanking is useful if you want to eliminate the absorbance of a reagent solution from the result The program can store the O D value of this reagent solution in memory as a Blank value and subtract
148. nt Style Font Size etc for the report This is a standard Windows dialog box and Windows fonts will be presented additional fonts may be presented if other programs are loaded on the computer Font Font Font style facial Regular Arial Black O Arial Narrow O Arial Rounded MT Bok T Arial Unicode MS O Baskerville Old Face F Batang ne Bold Italic Effects Sample I Strikeout F Underline aBbYyZz Color MM Black Western Figure 4 14 The Font Dialog Box 4 2 4 12 Options The Options dialog box Figure 4 15 is used to allow the operator to customize a number of features 4160 MIRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar Buttons Preferences Colours Directories Laboratory Information IV Enabled Buttons Add Move Up Move Down Remove Clear aoa e asss Button Test Assay Worklist Name Select E Cancel C E Figure 4 15 The Options Dialog Box Buttons Tab 1 Buttons Tab The Buttons tab is provided to assign a short cut button for an assay The button s will appear directly below the tool bar To create a short cut assay button 1 Click Enabled 2 Enter the name of the desired button e g 111 in the Button Text field The name will be presented in the highlighted line of the Buttons field as you enter the name 3 Click Select to access the Select Assay Worklist dialog box that is
149. nter additional information before proceeding e g sample names based on the selection made in the assay a For systems in which the computer is external the computer will initiate communication with the reader MRX Revelation Manual Chapter 8 Kinetic Analyses 8 4 During the Assay 8 4 During the Assay Revelation will display and update the data each time a reading is taken A typical display is presented in Figure 8 7 1 z 4 6 7 8 9 10 iw A 0 002 0 000 0 000 0 000 0 000 0 000 0 000 0 000 0 019 sale 0 0 HILI KKK ele se 1 1 a 0 0 000 sii 001 0 000 HEHE E REN acs AHA HEE HBB HHH REETA TETT fo 5 o p C 5 o 1 o o io o C Z oS i gt 5 o o o v bei vp x Reading 22 of 25 in 11 seconds T1 0 234 0 D Figure 8 7 Typical Display During a Kinetic Analysis IE Note The lag of a few seconds for display of kinetic data is due to the requirement that data be transmitted and processed A number of modes are available for viewing the data m Revelation shows a matrix of 96 miniature graphs one for every well location default mode a To view an individual well click on the well location required a To view a graph for a entire sample when replicates are used select Replic
150. ogs log The desired file is selected in the same manner as other Windows files e g Microsoft Word 2 We will not describe standard Windows commands such as OK Cancel Apply and Open unless they provide a unique feature in the program 3 Standard Windows operations will not be described These include e Selection of the order of items in a list via the Move Up and Move Down buttons e Insome dialog boxes e g the selection of Report Options Figure 3 1 a list of all options is present in a field the Check list field The operator can select the desired items via the Add button or Add All button to place them in a list the Check list field In the same vein items can be deselected by the Remove or Clear button 3 2 MRX Revelation Manual Chapter 3 Introduction to the Operating Program Report Options x 3 2 Conventions in this Manual Report Layout Raw Data Export To File Header Foater Select from Check list Laboratory information Add Laboratory information Header information Header information Lot specific data Lot specific data Removed outliers Removed outliers Edited wells Edited wells Calculation mode Insert Calculation mode Blank mode Blank mode O C equations Remove O C equations Data matrix Data matrix Area statistics Area statistics Spreadsheet Spreadsheet Ratio Ratio Curve Fit Move Down Threshold Page break Clear Add All Move Up EEE F
151. ol Quality Control Q C equations are used to place criteria on the raw assay data threshold data or curve fit data that validate the assay that was performed In this mode Well O D s are accepted or rejected using criteria such as comparison against a control well or against a numerical value If the user defined criteria are not met the data will be processed as normal but the results report will contain a message showing that the data failed Q C When the Q C Raw Data command is selected from the Settings menu the Quality Control Raw Data dialog box Figure 10 1 is presented Quality Control Raw Data x O C Equations Equation Functions Failure description PO O C equations M Full QC report MV Suppress results if OC fails Clear IV Lot specific checking of control values New Move Up Move Down Remove Cancel Apply Help Figure 10 1 The Quality Control Raw Data Dialog Box 10 2 MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 2 Quality Control a Equation the Equation field is used to enter the desired equation using the operators indicated in the Function field A detailed discussion is presented in Section 10 2 2 a Failure Description indicates the error message that is to be displayed if the Q C test is not passed a Q C Equations lists the Q C equations that have been generated The equations are processed in the o
152. old Chapier 10 Incorporating Decision Making Protocols into the Assay Eqn everything greater than or equal to this entry yet less than the values will be labeled or denoted as positive Typical equations include 1 If values above the result of Cut off Control 1 plus 0 4 are positive For the positive equation enter CO1 0 4 2 Values above the result of Cut off Control 1 plus 0 6 are double positive For the positive equation enter CO1 0 6 3 If values below the result of Cut off Control 1 minus 0 4 are negative For the negative equation enter CO1 0 4 4 Aresult is positive if it is equal to the Positive Control minus the Negative Control then enter PC NC 5 O D s above 1 00 must be recorded as positive enter the number 1 00 The Threshold Histogram check box to is used to indicate that a histogram should be included in the results showing all the different threshold ranges and how many wells are in each The Apply button is used to apply the current settings to the other tabs This option is only available for certain configurations 10 3 3 The Pos Neg Q C Tab The Threshold dialog box Pos Neq Q C tab Figure 10 3 is used to assign simple quality control to the threshold results 10 10 MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 3 Threshold Threshold Ed Threshold Equations Pos Neg Q C Threshold Q C Output Format NC wels scwis Pewe
153. ompanied by a phrase in the Flag box An example of the Results flagging feature is to report the phrase lt 20 mg dL instead of the actual numerical result whenever results are less than 20 mg dL As an alternative the phrase Abnormal High can be displayed to flag any results that are higher than a specified level The phrase will be initiated whenever the numerical result meets the criteria that are specified Results Flagging Equations are used to specify criteria for generating the phrase or flag Although several unique elements are used for constructing a Results Flagging expression the expression is created in the same way as Raw Data O K The unique elements for Results Flagging are described in Table 10 2 An unlimited number of Results Flagging expressions can be defined for an assay MRX Revelation Manual 10 19 10 4 Curve Fit 10 20 Table 10 2 Results Type Chapter 10 Incorporating Decision Making Protocols into the Assay Flagging Elements Use the Type keyword to designate the well type Standards Controls Tests etc that a Results Flagging equation is applied to For example the expression Type C 0 means that this equation applies only to the control wells Sample for OD values Use the Sample keyword to designate the absorbance of a particular sample replicate or well type in the same manner as used in Q C Raw Data equations Result Sample Use the
154. on equation B 1 but use quadratic cubic and quartic equations to relate the observed O D to the concentration A quadratic equation is presented in equation B 5 a cubic equation is presented in Equation B 6 and a quartic equation is presented in equation B 7 Quadratic Equation Y a bX cX i B 5 Cubic Equation Y a bX cX dX B 6 Quartic Equation Y a bX cX 7 dX eX B 7 The procedures used to fit these functions to experimental data are similar to that for linear regression and have a similar mathematical derivation A polynomial regression tends to minimize the deviations of the data points from the polynomial equation The squaring cubing etc of the deviations tends to minimize them since positive and negative deviations tend to cancel out The regression coefficient is handled in a similar manner as with a linear relationship When R 1 all the data points have been fitted to the curve when R 0 the data points are scattered Examples of Quadratic and Quartic fits are shown in Figure B 3 and B 4 respectively 2 000 1 500 1 000 Absorbance 0 500 0 000 00 6 000 2 600 4 006 6 0900 Concentration Figure B 3 Quadratic Fit MRX Revelation Manual B 5 B 4 Quadratic Cubic and Quartic Regression Appendix B Mathematical Relationships 2 500 2 000 1 500 Absorbance 1 000 x 0 500 0 000 0 500 1 000 3 000 5 000 7 000 Concentration Figure B 4 Quartic Fit B 6 MRX Re
155. on link test and the self test are performed during power up In contrast the communication link and self test are tested when a command is given by the external computer rather than during power up MRX Revelation Manual A 13 Appendix B Mathematical Relationships B 1 Overview Appendix B Mathematical Relationships B 1 Overview Revelation allows the analyst to use a broad range of mathematical relationships to determine the concentration of the compound of interest In this appendix we describe the basis of each of the methods to assist the analyst in more fully understanding the nature of the data reduction processes MRX Revelation Manual B 1 B 2 Linear Regression Appendix B Mathematical Relationships B 2 Linear Regression A Linear Regression is used to put a best fit straight line through a set of data points Equation B 1 describes a linear relationship between the measured variable and the concentration of the compound of interest Y mX b B 1 where m the slope b the y intercept defining the line Y the value for measured variable O D X the value for the concentration The linear regression formulae for determining the best straight line through a series of data points are provided in equations B 2 and B 3 m N2dxiyi Xi i B 2 NEx _ Ex b Y mean m X mean B 3 The linear regression coefficient R is defined by equation B 4 R N x iy x Xy B 4 NEx Ex NEy Ey Th
156. onfiguration A 5 1 Selecting the External Configuration When Revelation is installed the software is set up for an internal computer This section describes how to change the configuration for an external computer and change communication parameters if necessary To set the system configuration 1 Open Revelation A welcome window and a dialog box that requests the serial number for the system are presented Enter the serial number which is displayed on the display of the reader module 2 The message box shown below Figure A 2 will be presented since the computer is not yet configured to communicate with the reader Revelation AN Hardware link transmit error Check cables and port setting Figure A 2 Hardware Link Error Message Box 3 Click OK to accept the error message The monitor will present the self diagnostic sequence report which will indicate that the system was not tested 4 Close this window it is not necessary to save the test data 5 Select Configure Reader on the Utility drop down box to present the Configure Reader dialog box Figure A 3 MRX Revelation Manual Appendix A Using an External Computer A 5 Setting the System Configuration Reader Type External MF External MAX Internal MAX Figure A 3 The Configure Reader Dialog Box 6 Select External MRX and click Setup to present the Setup External MRX dialog box Figure A 4 Setup External MRX E P Use reader
157. or 100 When you use this option the concentration graph is reported in terms of percentages rather than well absorbance values If you want to use different Standards for 0 or 100 1 Place a check mark in the Percent Response box 2 Select the Standard you want to use for 0 and press the 0 Ref button The standard will be displayed next to 0 Ref 3 Select the Standard you want to use for 100 and press the 100 Ref button The standard will be displayed next to 100 Ref If you want to reset the references to the default values press the Clear Refs button MRX Revelation Manual 6 23 6 8 Curve Fit Chapter 6 Generating and Editing Assays If you want to report 10 50 and or 90 lethal concentration values 1 Place a check mark in the LC10 LC50 and or LC90 box a 0 Ref used in Percent Response Calculations see above a 100 Ref used in Percent Response Calculations see above a Clear Refs used if you want to reset the references to the default values a Enter Concentration used to enter a concentration or titer value for the Standard highlighted on the Check list The value will not automatically appear To apply the value press Enter on the keyboard or click on the Standard again a Enter units used to enter the units for the x axis of the graph You may use up to eight letters or symbols a Variable Standard Multiplier A variable standard multiplier is a multiplication factor which is applied
158. ormatting Reports and Exporting Data To change a binary data file to a text format 1 Open the data file 2 Select View Assay from the View menu The assay settings will be presented 3 Select Report Options on the Settings menu 4 Enable the Export to File option select the desired format and data listings and save the assay 5 Select the data file that you have opened via the Windows menu and select Refresh on the Data menu The data will be reprocessed according to the change in the assay settings and saved as a txt extension and can be opened in the desired application 11 3 4 The Report Options Dialog Box Header Footer Tab The Report Options dialog box Header Footer tab Figure 11 7 is used to indicate the information that should be included in the report header and footer Report Options Ea Report Layout Raw Data Export To File Header Footer m Header V Filename V Time amp Date V Page Number IV Plate ID IV Assay Title I Q C Summary Footer I Filename T Time amp Date J Page Number T Plate ID T Assay Title P Q C Summary J Figure 11 7 Report Options Dialog Box Header Footer Tab To indicate the information that should be presented check the appropriate box es 11 10 MRX Revelation Manual Chapter 11 Formatting Reports and Exporting Data 11 4 The Event Log File 11 4 The Event Log File An Event Log lists the various activities of the reader including error messages etc
159. ou will have to change the First well assignments as these depend on the template orientation Ie Note Do not combine different assay types on the same plate For example do not combine kinetic assays and endpoint assays or agglutination assays and non agglutination assays on the same plate 7 6 MRX Revelation Manual Chapter 7 Endpoint Analyses 7 3 Starting an Assay To initiate the run press Start In a few instances the assay will not be automatically initiated a If you inserted a Shake operation the program will shake the plate before reading it a If the assay requires that the assay temperature is not the ambient temperature the reader will check that the plate is as at the appropriate temperature If the temperature is not correct a dialog box will be presented indicating that the present temperature and programmed temperature The operator will be given the option to Read Now or Abort the Assay The temperature will be driven to the set point and you can wait until the present temperature is at near the desired temperature before starting the assay a In some cases the analyst will be prompted to enter additional information before proceeding e g sample names based on the selection made in the assay a For systems in which the computer is external the computer will initiate communication with the reader At the conclusion of the assay the desired report will be presented in the desired format along with the c
160. ow 3 500 as UNDER OVER UNDER Conversion If O D values fall outside the OVER or UNDER limits they cannot be passed to Q C Equations see Section 10 2 unless they are converted into numerical values If you wish to convert OVER or UNDER readings into values that can be input into quality control equations check this box MRX Revelation Manual Appendix A Using an External Computer OVER Value A 5 Seiting the System Configuration Enter the value that all OVER values will be converted to this field is active only if you checked the OVER UNDER Conversion box The range is from 0 000 to 9 999 UNDER Value Enter the value that all UNDER values will be converted to this field is active only if you checked the OVER UNDER Conversion box The range is from 9 999 to 0 000 The Verify button is used to check if the computer and the reader can communicate If the computer presents an error message or cannot communicate with the reader it is possible that a The communication cable is not securely fastened to either the computer or the reader Check the cable connection a COM Port 1 which is the default communications port is assigned to another device or the communication parameters are incorrectly set for your system A 5 2 2 The RS232 Tab If the computer cannot communicate with the reader and the cable appears to be connected click on the RS232 tab Figure A 5 Setup External MRX Ea Reader R5232
161. ox is described in Section 2 7 these parameters are normally set during installation and rarely changed once they are established 4 2 4 5 Plate In Plate In draws the plate into the reader 4 2 4 6 Plate Out Plate Out ejects the plate from the reader 4 2 4 7 Read Calibration Plate Read Calibration Plate performs the system calibration using the 12 way verification plate optional Role of the Calibration Plate The Calibration plate consists of neutral density and colored glass that extend over one or more wells which are used to carry out five different tests Alignment Linearity Filter Test Optical Channel Matching and Accuracy A verification plate is used as an external source of verification that the MRX is performing according to manufacturer specifications Alignment Consists of three alignment checks X alignment Y alignment and Skew which are directions of movement of the plate carrier An instrument passes this test if the x alignment is 0 6 mm y alignment is 0 8 mm skew is 0 8 mm Linearity An area of a test plate is set aside for a series of wells that should in theory give a straight line plot of O D against concentration The Linearity test measures the deviation of these O D readings from this straight line To determine Linearity Revelation calculates the slope y intercept and t value of the line where t value is a statistical measure of how well the plate readings match the theoretical st
162. ph I Eror Bars I Combine with other graphs Title Horizontal Axis Label Auto Scaling Manual Scaling Minimum fi Maximum fi Vertical Axis Lael Auto Scaling Manual Scaling Minimum jo Maximum fi Tick Marks fio Tick Marks fi 0 Cancel Apply Help Figure 6 11 The Curve Fit Dialog Box Graph Tab MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 8 Curve Fit a Produce a Graph indicates if a graph is to be included in the Results report A graph is always included by default a Error Bars indicates if bars which show the variation in O D for each point are to be included on the graph There must be at least two replicates of each point for error bars to appear a Combine with other graphs if more than one fit has been programmed into the assay this option allows more than one fit to be shown on the same graph To combine graphs 1 Select the Fits tab 2 Select the fit for each fit you want to include 3 Click on the Graphs tab and check the Combine with other graphs check box a Title used to name the graph 6 8 5 1 Horizontal Axis Vertical Axis If Autoscaling is selected the program automatically sets the scaling based on the range of data Manual Scaling allows definition of the axes scaling according to the numbers entered by the user In the Minimum box the lowest desired value for either the concentration or O
163. plate carrier position sensors are operating correctly Checks motor and sensors Background Light Checks for light leakage into the reading compartment Bulb Verifies that current is flowing through the bulb when it is switched on Checks that the bulb has not blown Filter Motor Moves the filter motor to the first position Checks the filter motor and optical alignment Transmission Verifies that all filters are capable of transmitting at least Levels 33 of the maximum transmission level Checks for blocked channels or broken diodes Filters Checks that gains can be selected for all filters Checks for damaged or missing filters SELF DIAGNOSTIC SEQUENCE Reader Serial 2CXC2549 Technologist Date 2 T Dek Time 11 15 02 AM Test A D Test Plate motor Tesi PAE kground light levels Test Bulb Test Filter motor Test Transmission levels Test Filters Self Diagnostic Summary Reader Status Ready Figure 5 2 Sample Self Test Report MRX Revelation Manual 5 3 5 2 Powering up the System Chapter 5 Running an Assay We recommend that you print a copy of the report on a periodic basis e g once a month by selecting the Print command on the File menu and save the reports to monitor the performance of the reader on a long term basis 6 Check that the time and date is properly indicated on the report If the time date are not correct cl
164. printer P J Figure A 4 The Setup External MRX Dialog Box Reader Tab MRX Revelation Manual A 7 A 5 Setting the System Configuration Appendix A Using an External Computer A 5 2 Setting System Parameters The Setup External MRX dialog box is used to set a variety of system parameters It consists of two tabs the Reader tab Section A5 2 1 and the RS 232 tab Section A5 2 2 A 5 2 1 The Reader Tab The Reader tab is used to set various instrument parameters Filters Indicate the number of filters that you have installed in the Reader The corresponding number of filter fields will be activated and you can enter the wavelength for each filter The wavelengths entered should correspond to the wavelengths indicated on the black casing around each filter that is physically present in the filter wheel Each filter should be located in the position of the filter wheel indicated in the table ae Note Filters must be installed in increasing order Options Indicate the options that are installed on the reader by checking the boxes Data Transmission Mode Fast Transmission Mode If you want to transmit data at an enhanced speed check the Fast Transmission Mode check box OVER Limit The OVER limit is used to create a range for expected readings The value entered is also used as an UNDER limit For example if you enter an OVER limit of 3 500 Revelation will classify any readings above 3 500 as OVER and any readings bel
165. pter 5 discusses the steps used to initiate an assay and the nature of the report that is generated Generating an Assay Reporting and Exporting Data describes how the analyst develops an assay to collect process and report analytical data In addition this section describes how data reports can be generated and how data can be ported to external programs General Assay Information Chapter 6 describes the structure of an assay and the role of the Assay Wizard which leads the analyst through the generation of an assay a Endpoint Analyses Chapter 7 discusses the details of the generation of an assay for an endpoint analysis a Kinetic Analyses Chapter 8 discusses the details of the generation of an assay for a kinetic analysis Agglutination Analyses Chapter 9 discusses the details of the generation of an assay for an agglutination analysis Incorporating Decision Making Protocols into the Assay Chapter 10 describes how you can develop protocols to determine and report acceptable unacceptable data in the report a Generating Reports and Exporting Data Chapter 11 explains how you can customize the report format and export data to an external program Appendices which describe use of the reader with an external computer and a mathematical discussion of data reduction are included In addition to the printed manual an extensive on line help system is included with the program Context sensitive help can be obtained
166. puter Setup Internal MRX x Reader Maintenance External RS232 Data Format m Baud Rate RS232 Port p Parity oie f COMA None MAO 9 1z C COM2 e Even Cl Eytended ou Gore oda C MRX ae C coms TZ C 2400 Data Bits r Stop Bits Enabled 4600 97 9l G Sean g Oc Default OK Cancel Apply i Figure 2 7 The Setup Internal MRX Dialog Box External RS232 Tab Set the appropriate transmission parameters The Default button selects the configuration that is most frequently employed for serial communication Ie Note Make certain that the COM port assignment does not conflict with any other serial port assignment on the computer MRX Revelation Manual 2 9 2 4 Initial Testing of the System Chapter 2 Getting Started 2 4 Initial Testing of the System An MRX is ready to operate after the self test has been completed and you have set up the system To verify that the system is functioning properly DYNEX Technologies offers a verification plate accessory at an additional charge part number 20098 visible range which can be used as described below The optional verification plate which is used to check that various aspects of the system conform to specifications consists of neutral density filters and colored glass filters that extend over one or more wells A test plate is used to carry out the following tests and can be used as an external source of verification that
167. raight line High t values indicate good linearity at high O D low t values indicate that the instrument will only have good linearity at low O D An instrument passes this test if the t value for each filter is greater than or equal to 8 61 for filters that transmit wavelengths above 400 nm MRX Revelation Manual 4 9 4 2 The Menu Bar Chapter 4 Commands Filter Test This test verifies proper filter installation in the filter holder This test only determines an incorrect filter set up if the filters installed in an instrument differ by more than 60 nm from the filter test wells on the plate The data will indicate if a filter is failing Optic Channel Matching Readings are taken for each optical channel and are compared with each other to check for consistent performance The print out will indicate if this test produces a failure An overall test summary will be given Accuracy Determines if an instrument is within specification by comparing the observed readings with pre determined factory values printed on the test plate calibration certificate included with every plate at time of manufacture or yearly re certification The values in the plate data should be compared to those on the certificate of calibration b The Calibration Protocol To use the calibration plate 1 Select Plate Out on the Utility menu to drive the plate carrier from the instrument 2 Insert the 12 way verification plate into the plate carrier with the numbe
168. ray a Table outputs the data in columns which shows the values obtained from each sample replicate in order by well type ae Note If you have included a number of different processing options and used a number of tables in the output the program may overwrite sections of data in the output If this happens try using matrices instead of tables or try removing one of the processing options MRX Revelation Manual 10 13 10 3 Threshold Chapier 10 Incorporating Decision Making Protocols into the Assay Options a Average Replicates replicates will be averaged before any other calculations take place a Decimal Places indicates the required number of decimal places for data calculations The default number is three this is suitable for most applications the range is 0 6 Mean a Arithmetic Reports the arithmetic mean of replicate determinations a Geometric Reports the geometric mean the antilog of the mean of the logarithms of replicate determinations Reporting the geometric mean is useful when the imprecision is directly proportional to the level Scientific Notation If scientific notation is desired check the enabled box a Free format Results are displayed in the most appropriate format a Multiples of 3 Limits the number of significant figures to multiples of three a Fixed at Allows you to enter an exponent manually Table Options The Table Options field is used to indicate the infor
169. rder they appear on this list Up to 12 quality controls can be used for a given assay ae Note The order of executing the equations can be very important For example equations containing validity expressions are used to check that a previous equation has not eliminated too many values An equation of this type must be placed below the equation it is checking or the Q C will not work a Functions items that are used to generate an equation To add one of these functions into the equation double click on the required item a New used to create a new equation The newest equation is placed at the end of the list When this button is pressed a cursor will appear in the Equations field allowing the equation to be typed As the equation is being generated it will appear in the Equations field as well as in the Q C equations field When the equation is complete press New again to enter another equation or press OK to close the dialog box a Move Up Down used to change the order of the equations in the Q C equations list To move an equation select it in the Q C equations list box and then select Move Up or Move Down a Full QC Report indicates that all of the Q C data equations values etc should be included in the results report If this option is desired place a check mark in the Full Q C report check box a Suppress Results if QC Fails indicates that results should not be printed if Q C fails If this option is de
170. re appropriate to the curve type will be activated Expressions which are not available for a given curve fit are grayed out a Min R Squared Enter range 0 0 to 1 0 The R squared value is an indication of how closely the curve fit is drawn between all points used If the R value 1 the curve has been drawn perfectly through all points on the graph A value of 1 is only possible to obtain when a spline point to point fit has been used a Min Slope Minimum slope value for a linear fit The slope of a line is the O D or value on the y axis concentration or the value on the x axis a Max Slope Maximum slope value for a linear fit a Min Y Intercept minimum intercept of the plot through the y axis or point where the line crosses the y axes a Max Y Intercept maximum intercept of the plot through the y axis Automatic Outlier Removal Automatic Outlier Removal is used to automatically remove outlier values when the standard curve is calculated The criteria for removal of outliers are established by entering the R Squared Limit the Residual Limit and the Removal Limit a R Squared Limit The proportion of the variance of values that can be attributed to the linear regression of the standard values R squared can range from 0 to 1 0 If R Squared is 1 0 there is no scatter in values Residual Limit An estimate of the error of each observation The residuals are expressed as the percentage of the value a R
171. rs on the white tag on the left arrow pointing up 3 Select Read Calibration Plate on the Utility menu 4 The Password pop up box will be presented to allow the user to enter the system password the default password is DYNEX and can be changed as described in Section 4 2 4 14 5 The Calibration Plate pop up box will be presented which is used to indicate the serial number of your MRX reader and the serial number of the 12 way verification plate you will be using Make any changes in either serial number 6 Click Read Plate The Status pop up box will be presented with the message Checking Alignment The plate carrier will move at 4 times less than its normal speed into the optical reading chamber The message Reading wavelength x will then appear which will show all filter wavelengths that are being checked When Revelation is finished reading the verification plate the plate carrier will come out and data with pass fail notations will be presented Figure 4 9 410 MRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar Revelation Cal2 iaf x wz File View Utility Window Help 18 x Slt 34 Sia 21x DYNEX REVELATION 4 22 Calibration Plate Reader Serial 1CXD3086 2 41 59 PM 2 2 01 ime Date Alignment 4 0 23mm Alianment 0 01mm 0 11mm 405nm 450nm 490nm 630nm Slope 0 609 0 561 0 560 0 553 Intercept 0 724 0 668 0 675 0 665 t
172. s presented in Figure 2 2 the report is presented in the workspace of the main Revelation window which is described in Chapter 3 IEE Note When the reader is operating under the control of an internal computer 2 2 the keypad on the front panel reader will not operate MRX Revelation Manual Chapter 2 Getting Started 2 2 Powering Up the System Table 2 1 Self Test Diagnostics Test Action A D Status Begins conversion and checks that the status line goes high and low Verifies that hardware can be detected and chosen Plate Motor Moves the plate to Out position and then to the In position Verifies that the plate carrier position sensors are operating correctly Checks motor and sensors Background Light Checks for light leakage into the reading compartment Bulb Verifies that current is flowing through the bulb when it is switched on Checks that the bulb has not blown Filter Motor Moves the filter motor to the first position Checks the filter motor and optical alignment Transmission Verifies that all fibers are capable of transmitting at least Levels 33 of the maximum transmission level Checks for blocked channels or broken diodes Filters Checks that gains can be selected for all filters Checks for damaged or missing filters SELF DIAGNOSTIC SEQUENCE Reader Serial 2CXC2549 Technologist Date 2 17 01 Time 11 15 02 AM Test Test Plate motor Test zol kground light
173. sed Passed Passed Passed Passed Passed Passed Chapter 2 Getting Started MRX Revelation Manual Chapier 3 Introduction to the Operating Program 3 1 Overview Chapter 3 Introduction to the Operating Program 3 1 Overview The Revelation operating program controls all aspects of the DYNEX Technologies MRX Microplate Reader This chapter describes the overall structure of the program and explains how the program is used Other chapters in this manual describe the use of the program from an operational perspective and will show how you can run an assay process stored data and perform a variety of other operations MRX Revelation Manual 3 1 3 2 Conventions in this Manual Chapier 3 Introduction to the Operating Program 3 2 Conventions in this Manual This program is a Microsoft Windows application and closely follows the general approach of commonly used Windows applications In this manual we assume that the reader has some general understanding of the use of Windows For the sake of clarity and brevity we have employed the following conventions 1 We will mention but will not describe dialog boxes that have a format that is similar to that of commonly used Windows applications such as Microsoft Word As an example the Open dialog box allows the operator to select a broad range of files including Assay Files asy Plate Files dat Spectrum Files spc Self Tests tst Sample ID Files sid and Event L
174. sed to assign colors to the various well types indicated in the table Options xi Buttons Preferences Colours Directories Laboratory Information Red Blue Green Blank Text Test Text Standard Text Control Text C User Text User 2 Text C User 3 Text User 4 Text C User 5 Text C User 6 Text C User Text C User 8 Text e Aane 2255 OK Cancel Apply Figure 4 18 The Options Dialog Box Colours Tab To select a color 1 Click the radio button corresponding to the item for which you want to change the color 2 Move the slide bars for Red Blue and Green The color of the word Text will change to show the currently selected color of the well type The program will display the colors you have chosen on the template and in printouts if you have a color printer If you do not have a color printer all colors will be printed as black ae Note Windows normally has use of a limited number of colors depending on the graphics board that is installed in the computer so it will pick the closest color to the one you have created 420 NMRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar 4 Directories The Directories tab Figure 4 19 is used to indicate the default directory for each type of file that is used in the program Buttons Preferences Colours Directories Laboratory Information Assay Directory Aimee A EEEE Browse Plate Data Directory C Program Files Re
175. sired place a check mark in the Suppress results if Q C fails check box a Lot Specific Checking of Control Values used to indicate if the program should check calculated values on controls against lot specific values found on the reagent container or package insert If this option is desired place a check mark in the Lot specific checking of control values check box MRX Revelation Manual 10 3 10 2 Quality Control Chapter 10 Incorporating Decision Making Protocols into the Assay 10 2 2 Creating Quality Control Equations The following rules must be used when creating Quality Control equations 1 Equations may contain only standard Quality Control equation components and they may not contain any spaces except in conditional expressions 2 When conditions are used you must insert a space after IF before ELSE and before and after THEN 3 Numbers arithmetic operators exponent brackets up to six levels of brackets and well values can be entered using the computer keyboard Other components of the equation can also be entered using the Functions list box or via the computer keyboard To enter a function double click on it Well Values can be entered as the average value of a well type or sample or as the individual value of a sample replicate For an endpoint assay the value in the well will be in O D units For a kinetic assay the value will be in O D min units or a time value Typical well values are
176. sitions relative to the end of the plate If it is not possible to reduce the template the program prints an error message and will not reduce the template Orientation If you have assigned less than 96 wells to any assay on the plate you must select Columns or Rows to specify the plate orientation As an example if Assay contains 12 wells and starts at Al and you select Columns the program will place the assay in wells Al to H1 and A2 to D2 if you select Rows Revelation will place the assay in wells Al to A12 ae Note The selected orientation applies to the whole plate and not just the highlighted assay If you change the orientation when some assays have already been positioned on the plate you will have to change the First well assignments as these depend on the template orientation MRX Revelation Manual Chapter 5 Running an Assay 5 3 Selecting the Assay Conditions 5 4 To Initiate the Run 1 Place the microplate in the reader 2 Press Start to initiate the assay In a few instances the assay will not be automatically initiated a Ifa Shake operation is in included in the assay the program will shake the plate before reading it a If the assay requires that the assay temperature is not the ambient temperature the reader will check that the plate is at the appropriate temperature If the temperature is not correct a dialog box will be presented indicating that the present temperature and programmed temperat
177. specific to a given analytical procedure these commands are incorporated into an assay To employ an assay the user accesses it and then runs it This chapter describes the various commands that are included in the program Chapter 5 describes how the commands are used to perform a series of analyses using an assay while Chapter 6 10 describes how an assay is generated MRX Revelation Manual 4 1 4 2 The Menu Bar Chapter 4 Commands 4 2 The Menu Bar 4 2 1 The File Menu The File menu Figure 4 1 includes a variety of commands related to opening generating and executing assays File New Assay Ctrl N Open Ctrl O Close Save Assay Ctrl S Save Assay As Import Ctrl I Run Plate Ctrl R Print Ctrl P Print Preview Print Setup 1 C Program Filesi Assay2 2 C Program Files PU 22 3 C Program Files 41234 4 C Program Files Untitled Exit Figure 4 1 The File Menu 4 2 1 1 New Assay New Assay accesses the Assay Type dialog box Figure 4 2 which is used to select the desired type of assay Assay Type x Select Assay Type Kinetic Figure 4 2 Assay Type Dialog Box 4200 WRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar a Endpoint measurements the reaction forming the chromophore is allowed to go to completion or may be stopped before the plate is read The observed O D is used to determine the concentration of the compound of inter
178. t Quality control criteria are placed on wells after the curve fit has been drawn and concentrations or titers have been calculated If the criteria are not met the data will be processed as normal but the results report will contain a message showing that the data failed Q C 10 16 MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 4 Curve Fit Fits Standards Fit Type Graph Sample Selection Dilutions Fit O C Calculated Q C Data Conversion Results Flagging Output Format Equation a Functions Failure description Q C equations Move Up a Move Down Bown Remove NM Full QC report I Suppress results if OC fails lt lt gt gt Geer T Lot specific checking of control values 0K ca toy EE Figure 10 6 The Curve Fit Dialog Box Calculated Q C Tab Quality control equations are established in the same manner as equations for Q C Raw Data A detailed description is presented in Section 10 2 2 10 4 3 The Curve Fit Dialog Box Data Conversion Tab The Curve Fit dialog box Data Conversion tab Figure 10 7 is used to assign data conversion equations to the curve fit data tab Data conversion equations are used to convert the data into different units by multiplying or dividing by some number or calculated sample value MRX Revelation Manual 10 17 10 4 Curve Fit Chapter 10 Incorporating Decision Making Protocols into the Assay
179. t The Check list shows the current order of the items By default all items are included in the checklist even if they are not programmed into the assay Report Options Ea Report Layout Raw Data Export To File Header Footer Select from Check list Laboratory information Add Laboratory information Header information Header information Lot specific data Lot specific data Removed outliers Removed outliers Edited wells Edited wells Calculation mode Insert Calculation mode Blank mode Blank mode O C equations Remove O C equations Data matrix Data matrix Area statistics Area statistics Spreadsheet Spreadsheet Ratio Ratio Curve Fit Move Down Threshold Page break Clear Add All Move Up kekkel OK Cancel Apply Figure 11 2 The Report Options Dialog Box Report Layout Tab MRX Revelation Manual 11 3 11 3 Report Options Chapter 11 Formatting Reports and Exporting Data If you do not want to include an item in the report highlight it and press the Remove key The order of items in the report can be changed by highlighting an item and using the Move Up Move down button Items that should not be presented in the report can be taken off the Check list column by highlighting them and pressing Remove 11 3 2 The Report Options Dialog Box Raw Data Tab The Report Options dialog box Raw Data tab Figure 11 3 is used to indicate how the raw data is presented R
180. t be edited Spectrum files spc A spectrum file contains data obtained from absorbtion measurements at different wavelengths see Section 4 2 4 9 and is used to find the optimum wavelength for a given analysis Self Test files tst A self test file is the report that is generated when the instrument is powered up The file can be saved by the user via the Save Save As command It is worthwhile to save these files as an ongoing demonstration that the system is working on an acceptable basis Sample ID files sid A Sample ID file presents the information that you have used to define a sample ID see Section 6 1 3 4 This file is not editable from this screen Event Log files log An event log file is automatically generated every time the system is powered up and includes a list of all activities The file name is created from the date and the number of times that the system has been powered up on that day e g 03010101 refers to the first time the system was powered up on March 1 2001 MRX Revelation Manual Chapter 4 Commands 4 1 Overview Chapter 4 Commands 4 1 Overview The Revelation menu bar is used to access all commands that are included in the program Two types of commands are provided a General system commands which are used to set parameters that are global in nature e g configure the reader or perform basic operations e g moving the plate in or out of the reader a Commands that are
181. t is positive or negative To enter a rule for a parameter select an inequality symbol and then a number The following symbols can be used equal to gt greater than or equal to lt less than lt less than or equal to gt greater than lt gt not equal to X the value of the parameter is not important and will not be used when calculating the results MRX Revelation Manual Chapter 9 Agglutination Analyses and TCG Mode 9 3 Generating an Agglutination Assa Example All results with an A parameter of more than 1 0 and a D parameter of greater than or equal to 1 0 should be recorded as positive All results with an A parameter of less than 0 5 should be recorded as negative For the Positive Rules enter A gt 1 0 BX CX D gt 1 0 For the Negative Rules enter A lt 0 5 BX CX DX 9 3 4 The Reader Control Dialog Box Agglutination Options Tab The Reader Control dialog box Agglutination Options tab Figure 9 8 is used to set a variety of operating parameters for agglutination measurements Reader Control Ea In Columns In Rows Blanking Options C Well Well Point Point Miscellaneous IV Noise Filtering MRX Revelation Manual Position 1 2 8 mm Position 2 2 6 mm Position 3 2 4 mm Position 4 2 3 mm Position 5 2 1 mm Position 6 1 9 mm Position 7 1 7 mm Position 8 1 5 mm Position 9 1 4 mm Position 10 1 2 mm Position 11
182. t need not be the filename that is used to save and retrieve the assay a Written By The name of the individual who created the assay a Password The password is an alphanumeric series of characters e g ABC123 which is required to edit the assay When an assay is opened a dialog box is presented that requests the operator to enter the password If the user wants to execute the program but does not want to edit it it is not necessary to enter the password a discussion of the Password dialog box is presented in Section 4 2 4 13 When the assay is set up the password is entered twice to ensure that the desired series of characters is entered While a password can contain an unlimited number of characters it is best to keep them quite short so they will be easier to remember In order to maintain security the password is indicated as on the assay report regardless of the actual password length MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 2 Generating a New Assay Assay Title Dialog Box a Assay Code used to indicate the plate code for plates to be used with this assay If you entered a prefix the program will check the start of the barcode or plate ID against the prefix at run time If you entered a suffix the program will check the end of the barcode or plate ID against the prefix at run time If the prefix or suffix is not correct the program will display a warning message asking w
183. t sample Variables represent values which must be entered at runtime Five variables are allowed per assay As an example variablel could be used For each sample of a well type the expression 1 is used As an example the relationship 0 75 C lt Ci lt 1 25 C is used to indicate that each control sample must be within 25 of the mean of the Control samples or that well is removed from the calculations ae Note Expressions containing i often lead to values being excluded from the calculations If every value in a certain well type is excluded and this well type is used in other Q C equations the Q C operation will not work properly To solve this each equation containing i should be accompanied by a Validity expression This will ensure that if too many values are rejected the test will fail 10 Median represents the value in the middle of the list of values If all values in the group are listed the median is the value in the middle of the list If the list contains an even number of values the median is the mean of the middle two values The group of values may be a well type or a sample if it contains multiple replicates As an example Median C is used to calculate the median of all the Control values 11 Valid Number of Values valid indicates that a specified number of values within a group must pass the other Q C equations otherwise the data fails Q C The group of values may be a well type or a sample if it cont
184. te ID is used as a temporary filename for the plate data file which is created when the assay ends If you have selected Default Plate IDs in the assay a default ID will appear in the Plate ID field box otherwise you will have to assign the ID manually Plate IDs can be assigned either by typing a name into the Plate ID text box or by scanning a barcode on the side of the microplate If you are using the Barcode option you should make sure the scanner is set up to decode the correct type of barcode For additional information refer to the Barcode Program User Manual a Scan Barcode before run To automatically read the plate barcode before the plate is run place a check mark in the Scan Barcode before run box As an alternative you can read the barcode at any time by pressing the Read Barcode Now button Options a Auto save The Auto save command is linked to the plate ID option When you enter a plate ID or scan a barcode Revelation saves your data in a file using the plate ID as the filename with a file extension of dat The default destination is in the Plate Data folder To change the default destination go to the Directories tabbed folder in the Options command on the Utility menu If you intend to recall plate data at a later date you should always assign the ID as a permanent filename a Print Results If you do not want the data to be printed automatically un check the Print Results check box stored data can be prin
185. ted using the Print command on the File menu after collection a Search If you created a sample ID file using the Sample ID SID entry on the Utility menu Revelation can automatically search for this file Sample ID files are saved by default in the SID directory If you want to search for the file automatically you should make sure it has the same name as the plate ID To link sample IDs to the template click on Use Template Operator Indicate the name of the individual performing the assay Assays An assay list is used to indicate the assay s to be run Assay lists may contain a single assay or a number of assays If you place more than one assay on a plate you can either over lap some assays so that more than one assay will run on the overlapped wells or you can run each assay on different wells MRX Revelation Manual 7 5 7 3 Starting an Assay Chapter 7 Endpoint Analyses The Select list box contains a list of all your assays a Open Presents the Open dialog box with a listing of all previously saved assay checklists m Save Saves an assay checklist Checklists are saved by default in the main Revelation directory using an LST extension If the checklist has not been saved before Revelation displays the standard Save As dialog box a Add Places the highlighted assay in the Select field into the Selected Assay s field a Remove Removes the highlighted assay from the Selected Assay s field a Clear de
186. termined by Beer s Law equation 1 1 O D k log I D k log abc 1 1 Where O D is the optical density k is the absorptivity coefficient I is the intensity of light when a blank is read I is the intensity of light when the sample is read a is a constant that is dependent on the nature of the compound b is the path length which is constant for all samples c is the concentration of the compound of interest The system determines the constant a by measuring the O D for standards and then uses that value to determine the concentration of the compound of interest in the samples from the O D reported for the samples 1 4 MRX Revelation Manual Chapter 1 Introduction 1 4 MRX Revelation Application Software 1 4 MRX Revelation Application Software The MRX Revelation application software is a Microsoft Windows 95 98 NT application that is used for general system control data acquisition data processing data storage and reporting The program follows general Windows protocols ae Note There are small differences in the mode of operation between the use of an external computer and an internal computer The body of this manual describes the use of an internal computer A discussion of the use of an external computer is presented in Appendix A Operation of Revelation includes two types of commands General system commands which are used to set parameters that are global in nature e g configure t
187. the multiplier 6 3 2 3 Wavelengths The MRX can read in single wavelength mode using one filter or in dual wavelength mode using two filters The first filter is the test filter the second is the reference filter A reference filter is commonly used to reduce errors caused by dirt and scratches on the bottom of the wells but a single filter is suitable for most applications a If Single Dual Wavelength mode is selected a primary test filter and if necessary a primary reference filter must be selected a If Multi Wavelength mode is selected a primary and a secondary test filter and if necessary primary and secondary reference filters must be selected a If Agglutination T C G mode is selected a primary test filter must be selected Agglutination T C G assays cannot be run with a reference filter To select the appropriate filter s click the drop down menu and select the desired filter from the list If no reference filter is required select the Single Dual Wavelength option and run the test in single wavelength mode IEE Note The list of available filters is generated from values that you entered using the Configure Reader Filters dialog box Section 2 3 2 MRX Revelation Manual 6 7 6 3 The Reader Control Dialog Box Chapter 6 Generating and Editing Assays 6 3 2 4 Shake The Shake field is used to define the parameters that define the shaking action Enable Check this box to insert a shake operation into t
188. therwise the default export file name text txt will be used The plate ID that is entered becomes the file name for the text file If you have selected ASCII or Sanguin format the option button leads to a dialog box to set various parameters 17 3 3 1 ASCII Format If the ASCII format is selected the ASCH Options dialog box Figure 11 5 will be presented when you press Options Format Sort Table On eo ene Sample IDs 7 Matrix r a Descending Arder Listing ae C Semicolon r Select data to export IV Read Date Rew Deta IV Read Time I OD OC equations OK IV Assay Title IV OD Results IV Kit Plate Lot Data IV Threshold Q C V ReagentLotData M Threshold Cutoffs Cancel M User Name IV Threshold Results J Well Positions MV Curve Fit Q C IV Well Labels IY Curve Fit Results Help IV Sample IDs IV Ratio Results IV Area Statistics IV Spreadsheet Results Figure 11 5 ASCII Options MRX Revelation Manual 11 7 11 3 Report Options Chapter 11 Formatting Reports and Exporting Data Separator The Separator selection is used to describe how data in the export file should be separated The option depends on the requirement of the program that you intend to export to In many instances e g Microsoft Excel when the data file is opened a wizard will be presented to assist you in formatting the data in the program Tab put each well s information into its own cell on a spreadsheet Comma in
189. tics but it can be used in other applications where a rectangular hyperbolic relationship is observed A typical plot is shown in Figure B 8 Vmax 1 Vmax Absorbance Concentration Figure B 8 Michaelis Menton Plot The relationship between the O D and the concentration x can be written as described in equation B 12 ah B 12 x b where a and b are constants It can be shown that a equals the limiting O D as x goes to infinity and b is the concentration at 2 OD max The equivalent formula applied to enzyme reaction rates is shown in B 13 2 V max S B 13 S Km where S is the enzyme concentration Vmax is the maximum reaction velocity Km is the concentration where the reaction velocity is 2 Vmax The constant Km is called the Michaelis constant MRX Revelation Manual B 11 B 9 Michaelis Menton Curve Fit Appendix B Mathematical Relationships In typical usage the Michaelis Menton equation is converted into a linear form which is commonly known as the Lineweaver Burke equation equation B 14 1 1 Kn 1 B 14 OD OD max OD max x To use the Lineweaver Burke equation 1 OD is plotted against 1 x A straight line is obtained with a slope of Km ODmax This line has an intercept at 1 OD and an intercept on the 1 x axis of 1 Km The reader performs this calculation automatically and determines the best straight line fit by using a linear regression OD max printed as Vmax and K are e
190. tly as absolute numbers In other words a result will be recorded as positive or negative if it falls above or below a certain number In other cases it may be necessary to introduce conditions into the equation For example Test samples could be qualified as positive or negative by comparing them to a Control sample or the average of a number of samples The value resulting from the equation is called the value The value resulting from the equation is called the value If the value is lower than or equal to the value then O D s below the value are negative O D s above the value are positive If the value is higher than the value then O D s below the value are positive O D s above the value are negative 10 3 2 1 Equations Type the required equations into the Equation text boxes The and equations are optional but the and equations are necessary To enter equations click on the Enabled check box to activate the equation boxes labels and functions The general approach to editing the threshold equations is identical to the editing of the Q C raw data equations Section 10 2 2 although there are fewer functions Eqn everything less than this entry will be labeled as a negative sample Eqn everything greater than or equal to this entry yet less than a higher level entry will be labeled or denoted as positive MRX Revelation Manual 10 9 10 3 Thresh
191. to all the Standard samples when the curve fit data is calculated at runtime When the program processes the data it asks you to enter this value The value can be changed after the plate has been run using the Data menu Variable Parameters command a Enter Concentrations at Run Time used to indicate that concentrations of the standards should be entered when the plate is run If this option is chosen a dialog box for entry of standard concentration values is displayed when the plate is run 6 8 4 The Curve Fit Dialog Box Fit Type Tab The Curve Fit dialog box Fit Type Tab Figure 6 10 is used to select how the data is to be fit i e the equations to be used 6 24 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 8 Curve Fit Calculated O C Data Conversion Results Flagging Output Format Fits Standards Fit Type Graph Sample Selection Dilutions Fit Q C Fit Type Calculation options Results Format Linear Regression IV Average Standard Replicates Concentration C Quadratic Regression Extrapolate Data Titer 2 eal ait Sigmoid Parameters Axes rs ee Regression I Fiv Parameter A T G Lin Lin olygon ele i c i F Cubic Spine J Fir Parameter B fo Log Lin Lineweaver Burk a Fry Parameter IE fi C Log Log Michaelis Menten Fie eremeter 0 fi C Auto rR Sigmoid Maximum iterations 20 Log Logit ou m Inhibitor f Parallel Present Concentration fo C
192. to print enabled IV Search for Sample IDs enabled T Auto barcode read enabled I Ask for lot specific data M Multiple assays T Orientation Lock Columns only C Rows only Ok Cancel Apply IE Figure 4 17 The Options Dialog Box Preferences Tab 4180 8 MRX Revelation Manual Chapter 4 Commands 4 2 The Menu Bar Run Plate Defaults a Auto save enabled plate data is automatically saved as a file with the file name being the plate ID entered in the Run Plate dialog box which pops up right before the plate is read To switch this option off un check the Auto save enabled box a Auto print enabled plate data is automatically printed as soon as the plate is read when the data file is created To switch this option off un check the Auto print enabled box a Search for Sample ID s enabled the program automatically searches for a sample ID file when it runs the plate The Sample ID file MUST have been previously created and stored as a sid file The plate ID entered into the Run Plate dialog box MUST match the name of the sample ID file a Auto barcode read enabled the plate barcode is automatically read when the plate is run if an internal barcode readser is installed on the instrument To switch this option off un check the Auto barcode read enabled box a Ask for lot specific data data that has been entered for specific lots of reagents are automatically retrie
193. tor monitors a specified sample e g T1 When the observed O D reaches the O D limit all the other readings are taken The specified sample should be entered in the first field and the O D limit in the second field Note Revelation will allow you to enter a sample as the reference which does y p not exist but the measurement will not be performed unless you create the sample before running the assay Moving Average allows for averaging of each data point obtained with a specified number of points on either side before plotting the graph This can smooth out the graph by reducing the effect of excessively high or low data points that would otherwise have caused bumps in the curve Minimum 1 no smoothing Maximum One less than the number of kinetic readings MRX Revelation Manual Chapter 8 Kinetic Analyses 8 2 Generating an Assay for a Kinetic Analysis Blank Subtraction Blank Subtraction describes the desired mode of blank subtraction which is used to eliminate the O D of reagents which should not be included in the final O D Blank wells B MUST be defined on the template before blank subtraction can occur Blanks may be single wells or an average of wells Intrarecord subtracts the blank from every kinetic reading Post Kinetic subtracts the blank from the calculated final result for each well Use Readings Use Readings allows the analyst to select a range of readings so that readings at
194. tracted from Test 2 etc If any of the Test or Standard samples contain more than one replicate the Blank value will be subtracted from each replicate rather than from the whole sample average a Custom similar to Paired blanking except that you can assign Blank samples to whichever non Blank sample you want To define the Custom blank mode settings select the Custom radio button and access the Custom tab Figure6 7 6 16 MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 5 Blank Mode Blank mode Ea Blank Mode Custom Blanks Subtract From Check list Add All a Cancel Apply Figure 6 7 The Blank Mode Dialog Box Custom Tab Help Each Blank sample is listed in the Blanks list box and every non Blank sample is listed in the Subtract list box a Add All assigns the Blank highlighted in the Blanks list box to all samples on the plate all samples will be displayed in the Check list box when this Blank is highlighted to indicate that a blank has been assigned a Add assigns the Blank highlighted in the Blanks list box to the sample highlighted in the Subtract list box This sample will be displayed in the Check list field when the specified blank is highlighted a Remove clears the sample highlighted in the Check list box and returns the sample to the Subtract From list box for the Blank highlighted in the Blanks list box a Clear clears all samples in the Check list box for the
195. trix in terms of well locations Replicate display the plate matrix in terms of the samples you defined using the Template command Section 6 4 Clear Clear deletes all the currently selected graphs A typical assay is presented in Figure 8 5 MRX Revelation Manual 8 9 8 2 Generating an Assay for a Kinetic Analysis Chapter 8 Kinetic Analyses 8 10 Assay type Assay title Password Written by Prefix Suffix Report layout Header information Footer Test wavelength Ref wavelength Start mode Duration of kinetic Time interval Reading range Caiculation mode Cut off Data Stop mode Display well Data log Results scaling Kinetic graphs Blank subtraction Results format 1 A Tis B T Cc T3s D T4 E TS F T s G T7s T8s Blank mode Q C equations Full Q C Report Suppress results Lot specific check Output format Average replicates Mean Area statistics Export to file T T10 T11s T12s T1 T14 T15 T16s T17s T18 Ti T2 T21s T22s T23 T24 DYNEX REVELATION 4 22 Kinetic Kinetic 44 gt R Jones 1123 789 Laboratory information Header information Lot specific data Removed outliers Edited wells Calculation mode Blank mode Q C equations Data matrix Filename Date Plate ID Assay tite Page Q C summary 410 nm Immediate 00 01 00 60 Seconds Alt Total OD min 2 500 Increas
196. ure The operator will be given the option to Read Now or Abort the Assay The temperature will be driven to the set point and you can wait until the present temperature is at near the desired temperature before starting the assay a In some cases the analyst will be prompted to enter additional information before proceeding e g sample names based on the selection made in the assay At the conclusion of the assay the desired report will be presented in the desired format along with the calculations that were included in the assay In addition data will be saved and or exported as indicated in the assay MRX Revelation Manual 5 7 This page is intentionally left blank Chapter 6 Generating and Editing Assays 6 1 Overview Chapter 6 Generating and Editing Assays 6 1 Overview An assay is a complete set of instructions for the operation of the microplate reader It includes information about the various data collection calculation and reporting processes to be performed on a microplate If you want to develop a new assay select New Assay on the File menu A dialog box will be presented to select if you want to perform an Endpoint assay single read or a Kinetic assay multiple readings over a period of time After you have selected the desired mode of operation the menu bar will include two additional items View and Window A new assay can be readily generated using the Assay Wizard which is accessed by the
197. used to select the assay for which the button should refer 4 Select the desired assay e g Assay 2 and click Open on the Select Assay Worklist dialog box The Options dialog box button tab will be presented again The selected button name and assay will be indicated e g 111 Assay2 in the Button field A button will be presented on the main window Figure 4 16 when you close the dialog box MRX Revelation Manual 4 17 4 2 The Menu Bar Chapter 4 Commands File Settings View Utility Window Help pisla s all Lan Figure 4 16 The Assay Short Cut Button When the button labelled 111 is pressed Assay 2 will be automatically loaded You can create up to eight different short cut buttons which will be displayed just below the Tool Bar When you click on a short cut button the program displays the Run Plate dialog box To run a plate you normally have to create an assay checklist but if you program the assays into a short cut button they will automatically appear on the checklist saving you the time and trouble of opening up the required assays 2 Preferences Tab The Preferences tab Figure 4 17 is used indicate a broad range of miscellaneous items as described below Options xi Buttons Preferences Colours Directories Laboratory Information r Default Plate ID I Enabled Plate ID format IV Display overwrite warning Reset Counter To Zero Run Plate Defaults I Auto save enabled I Au
198. using a Kinetic assay or a non Agglutination data file using an Agglutination assay Plates Select lists all of the data files in the active directory Check list includes the data files that have been selected for the statistical analysis To compare a set of plates 1 Select the plate data file 2 Click Select New Assay and select the new assay you want to use 3 Click OK The program will open each data file and calculate the results using the blanking options and calculation mode options of the new assay It will then calculate the mean standard deviation standard error mean and coefficient of variation for these results and display them on the screen 4 2 4 2 Plate Reproducibility Plate Reproducibility presents the Plate Reproducibility dialog box Figure 4 7 which allows you to run a plate several times and check the reproducibility of results for each well MRX Revelation Manual 4 7 4 2 The Menu Bar Chapter 4 Commands Number of readings m Test Wavelength jno Ref Wavelength lt F Cancel Help Figure 4 7 The Plate Reproducibility Dialog Box Number of readings refers to the number of times the test should be performed The default value 20 is a reasonable value Test Ref Wavelength refers to the test filter wavelength and the reference filter wavelength if employed The installed filters can be viewed via the drop down list an asterisk indicates that no filter is employed To begi
199. uter and reader 1 The personal computer should be connected to the Serial Port item 6 Figure A 1 on the rear panel of the reader using the RS 232 cable provided The reader is normally connected to a COM port on the personal computer If COM port 1 is used by another device use a different com port it will be necessary to change the system configuration as described in Section A 4 2 The mouse printer display and keyboard should be connected to the personal computer since all control of the reader is via the external computer 1 Mains Inlet 8 Monitor Port 2 Voltage Selector 9 Optics Door 3 Monitor Mains Socket 10 Optics Cooling Vents 4 Keyboard Socket 11 Serial Number Plate 5 PS 2 Mouse Socket 12 PSU Cooling Vents 6 Serial Port 13 Main Enclosure 7 Printer Port Figure A 1 The Rear Panel MRX Revelation Manual A 3 A 3 Interfacing the Computer to the MRX Reader Appendix A Using an External Computer 3 Plug the mains power cable for the reader into the mains outlet Item 1 Figure A 1 and into the line 4 Plug the mains power cable for the personal computer into the line 5 Turn on the power switch on the front panel of the MRX 6 Turn on the computer The computer should boot directly to Windows A 4 MRX Revelation Manual A 4 Installing the Software Ie Note Before installing the software make a copy of the diskettes for archival purposes an
200. valuated and printed out MRX Revelation Manual Appendix B Mathematical Relationships B 10 Using Logarithmic Curve Fitting B 10 Using Logarithmic Curve Fitting In most instances linear scales are used to plot the concentration X axis and the measured variable Y axis If desired a semi log fit or a log log fit can be used B 10 1 Semi Log Fit When a semi log fit is used the X axis presents log concentration To obtain the concentration of a sample equation B 14 is used Concentration antilog y c m B 15 B 10 2 Log Log Fit When a log log fit is used the X axis presents the log concentration and the Y axis presents log measured variable To obtain the concentration of a sample equation B 15 is used antilog Concentration antilog y c m B 14 IEE Note These examples use linear regression but Log Log and Semi Log Axes fit may be used with other curve fitting routines as well MRX Revelation Manual B 13 Index A Accuracy Testing 2 10 Agglutination Calculations 9 4 Creating an assay 9 7 During the Assay 9 18 Initiating the assay 9 14 Rules Calculation 9 5 Testing 9 1 Agglutination Options Tab Reader Control Dialog Box 9 11 Agglutination Parameters Tab Reader Control Dialog Box 9 8 Akima Fit B 8 Alignment Testing 2 10 4 9 Area Statistics Tab Assay Options Dialog Box 6 42 ASCII Text File Export 11 7 Assay Code 6 3 Assay File asy 3 6 Assay Options Dialog Box 6 42 Assay Title Dialog
201. ve value by a specified amount To apply the multiplier function 1 Click on Multiplier and type in the multiplication value 2 Click on the first item and type in its concentration 3 Press Enter on the keyboard to apply the multiplier to each successive sample 6 8 8 The Curve Fit Dialog Box Fit Q C Tab 6 32 The Curve Fit dialog box Fit Q C tab Figure 6 14 is used to indicate quality control information about the plot and to indicate how outliers should be treated Curve Fit Ea Calculated Q C Data Conversion Results Flagging Output Format Fits Standards Fit Type Graph Sample Selection Dilutions Fit Q C m Quality Control m Automatic Outlier Removal y I Enabled Minimum R Squared fi R Squared Limit fp Minimum Slope fi Residual Limit p ve Maximum Slope fi Removal Limit l reps Minimum Y Intercept fi J Leave at least i replicate Maximum Y Intercept fi soy iN Cancel Apply Help Figure 6 14 The Curve Fit Dialog Box Fit Q C Tab MRX Revelation Manual Chapter 6 Generating and Editing Assays 6 8 Curve Fit Quality Control This field is used to indicate the quality control parameters to be used for the curve if the curve fit does not meet the requirements of the expressions a message will be output with the results showing which parameters failed and why If the Curve Fit Quality Control check box is selected those expressions that a
202. ved by default in the main Revelation directory using an LST extension If the checklist has not been saved before Revelation displays the standard Save As dialog box Add Places the highlighted assay in the Select field into the Selected Assays field Remove Removes the highlighted assay from the Selected Assays field Clear deletes all the assays from the Selected Assays field First well Select an assay in the Check list box and type in the well location when you want this assay to start Number of wells Enter 96 for this value for each assay ae Note The program allows you to assign fewer wells to an assay than were originally programmed into the assay template For example if your assay contains 96 Test samples but this run only requires 10 test samples you can assign 10 wells to the assay Revelation will reduce the template to fit into 10 wells The program reduces templates by removing Test samples from the end of the template until the required number of wells is left All non Test samples are left in the same positions relative to the end of the plate If it is not possible to reduce the template the program prints an error message and will not reduce the template Orientation If you have assigned less than 96 wells to any assay on the plate you must select Columns or Rows to specify the plate orientation As an example if Assay contains 12 wells and starts at Al and you select Columns the program will
203. ved when the assay is run To switch this option off un check the Ask for lot specific data box a Multiple assays More than one assay can be run per plate To switch this option off un check the Multiple assays box a Orientation Lock fills replicates for all well types in the same direction Select the direction in which they are to be filled Columns only or Rows only Default Plate IDs Each time you run a plate you should assign a plate ID to the data under which the file can be saved If you do not want to type in a plate ID or scan a barcode every time you run a plate you can create default IDs a Enabled To switch default ID on and enter the plate ID format in the text box The program uses the same format for every plate ID and increments the end number with each new data file For example if you set the default ID format as plt_001 the first plate ID Revelation offers will be plt_000 the second will be plt_001 and so on a Display overwrite warning the program can store a maximum of 1000 default plate IDs before it has to start overwriting IDs and the data they hold If you want the program to warn you before it begins overwriting data check the Display overwrite warning box Reset Counter to zero used to reset the default plate ID counter to 0 by pressing the Reset Counter to zero buttons MRX Revelation Manual 4 19 4 2 The Menu Bar Chapter 4 Commands 3 Colours The Colours tab Figure 4 18 is u
204. velation Manual Appendix B Mathematical Relationships B 5 Cubic Spline Curve Fitting B 5 Cubic Spline Curve Fitting When Cubic Spline Curve fitting is used a curve is passed through all the data points and equations B 8 to B 10 are employed to determine the concentration y a a x o B 8 Fid where xax Xa X bx 2 o PoI 00 aw B 9 0 x x x 4 3 i _ x la x2 x B 10 ae Note Cubic Spline curve fitting is not a best fit algorithm as it assumes that each data point is correct If the data is erratic if it is known that the data has significant errors or if the concentration absorbance has many inflection points this method can lead to significant errors in the reported concentration At high concentrations the Cubic Spline Curve Fitting method may break up e g Figure B 5 In some cases a semi log or log log axis instead of a linear fit will improve the quality of the data Absorbance curve break up Concentration Figure B 5 Cubic Spline Curve Fitting MRX Revelation Manual B 7 B 6 Akima Fit Appendix B Mathematical Relationships B 6 Akima Fit The Akima fit constructs a smooth curve through the data points The fitted curve appears smooth and approximates a manually drawn curve Figure B 6 Absorbance Concentration Figure B 6 Akima Fit The Akima curve fit involves applying a set of polynomials that are applied to the data points and the slope of the curv
205. velation Plate Browse Curve Directory C Program Files Revelation Curve Browse Text Directory C Program Files Revelation Text Browse Sample ID Directory C Program Files Revelation Samp Browse Selftest Directory C Program Files Revelation Sellte Browse Spectrum Directory C Program Files Revelation Spect Browse Event Log Directory C Program Files Revelation E vent Browse Cancel Apply Help Figure 4 19 The Options Dialog Box Directories Tab To select the desired directory type in the name or use the Browse button to view the directories a Laboratory Information The Laboratory Information tab Figure 4 20 is used to include identification to be placed on the report MRX Revelation Manual 4 21 4 2 The Menu Bar Chapter 4 Commands Buttons Preferences Colours Directories Laboratory Information Name l Address Cancel Apply Help Figure 4 20 The Options Dialog Box Laboratory Information Tab 4 2 4 13 Password Password accesses a dialog box which is used to enter a password to lock the assay If the password is not entered when the assay is opened the operator can use the assay but cannot edit it 4 2 5 Window The Window menu Figure 4 21 is a standard Windows feature which is used to format the workspace Window New Window Cascade Tile Arrange Icons 1 Testi 2 Assay1 1 v 3 Assay1 2 Figure 4 21 The Window Menu The
206. well type or a sample if it contains multiple replicates Verify expressions take the form Verify value limit mode Value The value may represent A well type in which case Revelation checks the number of verified samples in the specified well type a A sample in which case Revelation checks the number of verified replicates in the specified sample Limit The limit determines the range into which values must fall to be verified A limit may be a Anumber For example if the value is PC the mode is 0 and the limit is 0 2 Revelation checks that every PC sample is within 0 2 of the average of all the samples a A percentage For example if the value is PC the mode is O and the limit is 20 Revelation checks that every PC sample is within 20 of the average of all the samples a A standard deviation For example if the value is PC the mode is 0 and the limit is 1 5 SD Revelation checks that the standard deviation of the PC samples is within 1 5 SD MRX Revelation Manual Chapter 10 Incorporating Decision Making Protocols into the Assay 10 2 Quality Control As an example the relationship verify T 10 2 gt 3 means that the number of verified Test samples must be greater than or equal to 3 A verified sample is one which falls within 10 of the mean of all the other Test samples When you have finished entering equations and click OK to exit the dialog box the program checks each equation If any of
207. yalue 55 367 55 857 57 418 55 298 Passed Passed Passed Passed Filter Passed Passed Passed Passed Optic Channel 1 Passed Passed Passed Passed Optic Channel 2 Passed Passed Passed Passed Optic Channel 3 Passed Passed Passed Passed Optic Channel 4 Passed Passed Passed Passed M gt For Help press F1 e A Start Revelation Cal2 zam Figure 4 9 The Verification Report 4 2 4 8 Selftest Selftest performs the self diagnostic sequence and presents the results in the workspace Figure 4 10 MRX Revelation Manual 4 11 4 2 The Menu Bar Chapter 4 Commands SELF DIAGNOSTIC SEQUENCE Reader Serial 2CXC2549 Technologist Date 2 17 01 Time T1 15 02 AM Test A D Test Plate motor Test Background light levels Test Bulb Test Filter motor Test Transmission levels Test Filters Self Diagnostic Summary Reader Status Ready Figure 4 10 Sample Self Test Report 4 2 4 9 Spectrum Spectrum is used to produce a spectral response curve for a specified well and suggests appropriate test and reference filter wavelengths When this option is selected the Spectral Response Curve dialog box Figure 4 11 is presented Spectral Response Curve Ed Well Location A1 Cancel Help Figure 4 11 The Spectral Response Curve Dialog Box The operator should enter the well location that should be used When OK is pressed the program reads the well using all the installed filters and the

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