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1. eSPL1 SPL2 SPL3 SPL4 SST DIr es NN n N User s Guide to KC4 PowerReports e 303 KC4 generates temporary curve s using fictitious data After the plate is read KC4 rebuilds the curve s with real plate data Certain characteristics of this curve can be modified including colors and labels Double click anywhere on the graph to enter the editing mode a shaded box should appear around the graph When this box appears click the right mouse button once to open a menu of items that can be edited Well Zoom Curve s Well zoom curves are available for kinetic spectrum and scanning protocols Up to five curves can be overlaid in one object To insert a Well Zoom curve object click on the Curves button in the RSO toolbar then click on Well Zoom The Well Zoom Properties dialog appears Well Zoom Properties Selected Curves a si J H1 Meas OD SPLE DataType Meas OD C HE Meas OD SPLA4S ell H12 Well Settings WelllD SPL96 Conc Qu Remove Cancel Help The Selected Curves box lists all wells that will be overlaid in the curve object e To add an item to the Selected Curves list Choose a Data Type from the drop down list The contents of this list depend on the current protocol parameters Data types can represent raw blank plate and corrected data Enter the Well coordinates The corresponding Well ID and Conc Dil value will appear if applicable Click
2. User s Guide for KC4 Signature and KC4 v3 4 User s Guide to KC4 v3 4 and KC4 Signature March 2005 Copyright 2005 PN 5291000 Revision E Bio Tek Instruments Inc Notices All Rights Reserved Trademarks BIO TEK INSTRUMENTS INC P O Box 998 Highland Park Winooski Vermont 05404 0998 USA Customer Service amp Sales Phone 888 451 5171 toll free in the U S 802 655 4740 outside the U S Fax 802 655 7941 E Mail customercare biotek com Internet http www biotek com Service Technical Assistance Center TAC Phone 800 242 4685 toll free in the U S 802 655 4740 outside the U S Fax 802 655 3399 E Mail tac biotek com European Coordination Center Bio Tek Instruments GmbH Kocherwaldstrasse 34 D 74177 Bad Friedrichshall Germany Phone 49 0 7136 9680 Fax 49 0 7136 968 111 E Mail info biotek de Copyright 2005 Bio Tek Instruments Incorporated This publication is protected by copyright and all rights are reserved No part of this manual may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying and recording for any purpose other than the purchaser s personal use without written permission of Bio Tek Instruments Inc Printed in the United States of America Bio Tek Part Number 5291000 Bio Tek is a registered trademark and KC4 KC4 v3 4 and KC4 Signature are trademarks
3. Cancel Help Data to Process This option allows the selection of the type of data to be analyzed 1 e the measured response value for kinetic analysis The drop down list displays the available data types Typical uses of this feature e To blank data prior to the kinetic slope calculation First define one or more blank wells in the Plate Layout and make sure Blank Wells Subtraction is enabled in the Raw Data Correction dialog Then select lt wavelength gt Corr from the Kinetic Analysis Data to Process pull down list e With some readers it is possible to perform kinetic reading with multiple filters or wavelengths In this case two levels of Data to Process options can be selected e g M 1 405 and M 2 550 to perform and compare kinetic calculations Note When two data sets are selected the kinetic curves are automatically overlaid in the kinetic graphs and in the kinetic zooms User s Guide to KC4 Protocol Menu e 131 Mean V Mean V is the calculated value of the mean slope KC4 determines the mean slope by calculating a linear regression on points that are in the calculation zone To set the calculation zone select Mean V then click on Calculation Options Kinetic Analysis Mean Y Calculation Zone a From t 00 00 00 Tot 02 00 00 i From 1 Tot E Unit i mob C oOo Per Min caca t Calculation Zone Typically the Calculation Zone is adjusted to
4. Creating and Modifying User Accounts Administrator To create or modify a user account 1 Login as the System Administrator 2 Select System Security and click New or highlight the desired account name and click Edit The KC4 User Settings dialog will appear KCA User Settings ID Ma Full Hame Mary Ann Jones Group Membership Password Standard User l Curent password Can not have access to Protected Functions Power User New password Can have access to Protected Functions but can not administrate Users Administrator Has complete access to all functions Status meuding Ween administration Midas Protocols Folder DB Protocols Mary Jones Browse Cancel Help 3 Enter a unique ID using 1 to 16 alphanumeric characters The user will enter or select this ID when logging into KC4 and when signing files 4 Enter the user s Full Name This name will be associated with events logged by this user s actions and with signatures applied by this user 5 Choose a Group Membership Standard Users are denied access to protected functions The System Administrator determines which functions shall be protected from use by Standard Users Power Users are permitted access to protected functions Power Users cannot administer user accounts or modify Security settings 6 Enter a temporary password for the user in the New and Confirm fields Share the password with the user with instructions
5. Data Menu e 41 Working with Data Files This section describes accessing generating modifying and saving files that contain plate measurement data Where and How is Plate Data Stored In KC4 there are two types of files used for storing plate data e Plate data files contain raw measurement values plate description information prompts comments date and time of reading and sample names Plate data files are identified by the extension PLA DNA Quant pla e Global data files contain plate data and protocol information The GLB file format is offered as an alternative to maintaining two separate protocol and plate files and is the required data file format for electronic signature support Global data files are identified by the extension GLB DNA Quant glb File storage options vary depending on site requirements and the current KC4 configuration e Files may be stored individually on the computer s hard drive on a CD or diskette or on a network to which the computer is attached This is the storage method that users of KC4 version 3 0x and earlier are familiar with Windows Explorer or a similar application can be used to view the file names and their locations and to move copy rename and delete files e KC4 Signature Files may also be stored inside a secure shared access database This database named KCShared mdb can be stored on a user s computer or on a shared access computer KC4 provides a spec
6. Options The FLx800 and Synergy HT can take measurements with up to six uniquely defined Filter Sets for endpoint reads and up to two for kinetic reads There are several applications for multiple filter sets including e Assays that have more than one fluorophore in the well e Comparison of results between the top and bottom optics positions e Comparison of results with varying sensitivity settings To define Filter Sets e Select either the Fluorescence or Luminescence detection method e Select Endpoint or Kinetic reading type e Click the radio button corresponding with the number of Filter Sets required Endpoint reads without area scanning to 6 Endpoint reads with area scanning 1 or 2 Kinetic reads 1 or 2 Dispense feature enabled 1 or 2 e Define the wavelengths Fluorescence detection method For each filter set use the drop down lists to select the Excitation and Emission wavelengths Fluorescence detection method with Time Resolved enabled Synergy HTTR For each Excitation filter set enter a wavelength value between 200 and 999 nm The bandpass 1s not variable it is pre defined to be 10 nm For each Emission filter set use the drop down lists to select the wavelengths Luminescence detection method For maximum sensitivity select Lum E to indicate an empty position in the filter wheel and a plug in position E in the excitation wheel For each filter set use the drop down lists to
7. User s Guide to KC4 PowerReports e 299 Protocol Data Audit Trail Protocol and Data Audit Trail information can be included on reports and in export files Note The Protocol Audit Trail is only available if the Preferred Data File Format setting is PLA To create a Protocol or Data Audit Trail table click the Tables button in the RSO toolbar then click on Protocol Audit Trail or Data Audit Trail The table is automatically inserted into the document the example below shows two separate tables Ed Microsoft Excel Worksheet in lt KC4 gt B File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves E Close Statistics Well IDs Fitting Results Interpolation Results Data Audit Trail Calculation Log Report Ei Microsoft Excel Worksheet in lt KC4 gt 1647 File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves x Clase na Protocol Audit Trail A A pe a p TW pa 44 bl Sheeti Jaf wife Ready E HA A A oa pp A ee 300 e PowerReports User s Guide to KC4 Calculation Log Report A Calculation Log Report can be included on reports and in export files To create a Calculation Log Report table click the Tables button in the RSO toolbar and then click on Calculation Log Report The table is automatically inserted into the document Ed Microsoft Excel Worksheet in lt KC4 gt 157 File Edit View Insert Format Tools Data Window
8. CUTOFFS Concentrations Value Formulae 0 6116 ST02 0 0 17 076 oT 4 61 007 oT06 1 2 e Inthe example above cutoffs were based on the Concentrations Sample Name List Report The Sample Name List contains all sample names defined for the plate sample Name List E SMPODOS 8 smpPoms 3 SMPOOOS 180 e Protocol Menu User s Guide to KC4 Plate vs Table Format Depending on the nature of the item chosen under Available Data information can be reported either in Plate format or in Table format log Plate ot Table Statistics Controle Standards Pe Wveraw wells Samples e With the Plate format selected data will print in a matrix format corresponding with the plate layout e Data for individual kinetic reads can be printed in the Plate format Select the type of data e g Meas OD Delta OD then set the range of reads to include in the report using the From and To fields e The Table format is available for kinetic and scanned data All kinetic or scanned readings for one or multiple wells Set the range of wells to include in the report using the From and To fields Statistics Report Statistical expansion of the data can be reported This information is specifically related to the Identifiers e g controls standards or samples e To specify a Statistics report select an item from the Available Data box click the Controls Standards or Samples radio button in the St
9. KC4 Data Protocol GLB All Files e If an existing file name is entered KC4 will provide a warning to prevent an accidental overwrite Click Yes to continue with the overwrite or No to rename the file before saving to open it and view its contents Closing Data Files Select Data Close to close the current data file If the file has not already been named the Save As dialog will appear requesting entry of a file name Note Clicking X in the upper right corner of the Results window does not close the data file User s Guide to KC4 Data Menu e 47 Reading Plates Once the new data file has been created see Creating a New Data File on page 43 select Data Read Plate to initiate a plate read or to enter data manually or read data from a file If no protocol is open a Reading parameters dialog appears Parameters available within this dialog are reader specific Reading J Reading Type End Point r Reader ELs806 1 on COM2 Kinetic kinetic En r empa Lorm ves Pre Heating i No Temperature 37 E ts Refr Filter ezo y sE t Meas Filter 405 Shaking A Intensity z aar 30 3 M Pre Read Blank Plate Before every reading D Read Barcode I C Before first eaten T Monitor wells tiare m Plate AAA First well t Last well m 2 Cancel Help Lag Time 00 00 00 e Define the required Readi
10. Setting Protocol Specific Options If you have opened a protocol and want to change the reporting or exporting engine but do not want to change the global default settings select Protocol Options or click the Options toolbar button Click the Report or Export tab as appropriate Raw Data Report Esport Calculation Engine KC4 E Microsoft Word Microsoft xcel Formatting Standard haster without font customization W Automatic Print after reading Keep Open after printing Protect Options Ed Raw Data Report Export Calculation Engine KCA Microsoft y ord Microsoft E scel Export Director C KCa v3 15E sport Files Browse Formatting Enhanced slower with font customization File Format Microsoft Excel Format id Automatic Export after reading W Keep Open after exporting Protect Cancel Help Select the desired Report and or Export Engines and modify other parameters as necessary User s Guide to KC4 PowerReports e 277 Important If you change the engine after a document has been created for the protocol the current document will be discarded and a new one must be created BIO Windows Application Ea A The report engine has been changed The report settings will be reset Cancel Click OK to approve the selection of a new report or export engine The current document if one exists will be discarded Click Cancel to keep the previ
11. The Purchaser must notify Bio Tek in writing within ninety 90 days after the date of delivery of the Software to the Purchaser of any claim under this Limited Warranty This 90 day period shall not be extended by the delivery or receipt of any subsequent modifications to the Software Modifications to Software within a version Updates are expressly excluded from the terms of this Warranty If the Software is found to be defective by Bio Tek Bio Tek s sole obligation under this Warranty is to use efforts consistent with Bio Tek s regular business practices to attempt to remedy such defect In no event shall Bio Tek s liability under this warranty exceed the original purchase price of the Software This is a limited warranty This warranty contains the entire obligation of Bio Tek No other warranties express implied or statutory are given The implied warranties of merchantability and of fitness for any particular purpose are expressly excluded and do not apply to the software Under no circumstances shall Bio Tek be liable to the purchaser or any other person for any direct incidental or consequential damages whether arising out of breach of warranty or otherwise This warranty shall not be changed or modified in any way without the express written permission of an officer of Bio Tek Instruments Inc This Warranty shall be NULL AND VOID if any modifications are made to the Software by the Purchaser Correction of dif
12. Excitation Optics Position Top nd Sensitivity 62 Fu Fu 7 185 20 e 485 20 528 20 e 528 20 5 50 Auto Options Options Plate Type 96 WELL PLATE v Size 8x 12 Cies E PreHeating First well fan Last well H12 No Temperature C Temperature Control Lag Time 00 00 00 m Shaking Intensity 0 Duration fo s Pre reading Before every readina O Before firstreading l Pre Read Blank Plate General Options Eject between Filter Set Head Top Probe Vertical Offset fh mm Monitor Wells Erie Pathlenath Correction Wavyelenaths Bio Cell 141 pe pios A A ee a En 10 11 12 13 14 15 16 Cancel Help Select a Detection Method of Absorbance Fluorescence Luminescence or Multi Mode Select a Reading Type of Endpoint Kinetic or Spectrum Optional For a fluorescence or luminescence protocol check Dispense Optional For a fluorescence protocol enable the Time Resolved option For a fluorescence luminescence or multi mode protocol define the Filter Sets For a Dispense protocol click Read amp Dispense to define additional parameters For an absorbance protocol define the Wavelengths and Read Mode to be used Calibrate the wavelengths if necessary Select a Plate Type and define the First and Last Wells For a kinetic protocol click the Kinetic button to define t
13. MAX lt XxX gt lt y gt lt Z gt 170 e Protocol Menu Description Addition Subtraction or negation Division Multiplication Represents inclusion The value of a specific well The mean of the specified well identifier The standard deviation of the specified well identifier The coefficient of variation of the specified well identifier Represents the LOG10 function The value of lt x gt raised to the power of lt y gt The square root of lt x gt The minimum of the defined variables The maximum of the defined variables Example CTL1 0 100 BLK 0 010 ETLI STLA STDL OO CPEL CTE2 100 A1 B1 MEAN POS1 SD BLK CV Sfp LOG SPL10 POW STD1 3 SORT A1 B1 MEN EVTETEL NS CV ICETLZ MAX A1 B1 C1 User s Guide to KC4 Validation Validation Validation is a set of determinant criteria used to assess the suitability of calibrators for the inclusion in subsequent calculations Validation criteria or Conditions are expressions based on threshold limits previously determined from known standardized conditions Select Protocol Validation with the Protocol Menu in Advanced mode to define Validation Sets and Conditions Validation El Validation SetHt T Data Type Delta DD ReadingH fal MH 405 MH 630 Delta OD Concentrations 0 000 STOT 0 400 1 000 S702 1 500 2 000 6703 2 500 1 2 3 4 5 6 T i Y Cancel
14. Single filter set protocols If light blocking is enabled a blocking filter Plug must be placed in the excitation filter wheel in one of the two positions next to the excitation filter that 1s specified in the protocol Dual filter set protocols If light blocking is enabled two blocking filters Plugs must be placed next to each other in the excitation filter wheel e Click OK to save the settings and add the Read step to the list User s Guide to KC4 Protocol Menu e 127 Raw Data Correction Raw data or the optical density or fluorescence unit values retrieved from a microplate reader can be corrected using Blank Plate Subtraction Blank Wells Subtraction and or Pathlength Correction The advanced protocol option Raw Data Correction controls the use of these correction options Raw Data Correction Ea W Blank Plate Subtraction Cancel W Blank wels Subtraction MESM Help e Pathlength Correction e Blank Plate Subtraction is accessible if Pre Read Blank Plate is selected in the Reading parameters dialog e Blank Wells Subtraction is accessible if one or more blank wells are defined under Plate Layout e Pathlength Correction is accessible if it is selected in the Reading parameters dialog e Raw Data Correction calculations are performed in the order Blank Plate Subtraction Blank Wells Subtraction then Pathlength Correction The following example describes the plate processing and calculation steps
15. 136 e Protocol Menu User s Guide to KC4 Spectrum Analysis KC4 provides the option for performing additional calculations on spectral data This is available for Spectrum reading types only Open the Spectrum Analysis dialog by selecting Protocol Spectrum Analysis Spectrum Analysis El Data to process 1 Meas OD Calculation Type Meas OD Corr h e Winiar C Formula Result Mame Calculation Options caca 00 Data to Process Select up to two levels of data to be used in calculations e When two data sets are selected the spectrum curves are automatically overlaid in the spectrum graphs and in the spectrum zooms Min Max Select Min Max to determine the minimum and maximum values for the points defined within the calculation zone This is the default setting Upon completion of a read the following is reported for each well e Minimum OD and Wavelength at Minimum OD e Maximum OD and Wavelength at Maximum OD If the Calculation Type is set for Min Max certain calculation parameters may be customized Access the Spectrum Analysis Min Max dialog by clicking the Calculation Options button User s Guide to KC4 Protocol Menu e 137 Spectrum Analysis Min Max el Calculation Zane io From ww 400 nm Tow 500 nm o From f Tot ji Cancel Help From w and To w allow the specification of a range of wavelengths upon which to perform calculations The default values correspond with
16. Brev pla Editing report Data Edit Protocol Display System Fleport Options 12 1 410 E Ob 0 069 0 861 0 695 0 413 0 80 0 475 0 366 0814 0 636 0 646 1 073 i OBS 0 071 0 847 0 797 0 664 0 913 0 608 0 64 0 934 0 841 0 837 n um x 3 0425 0503 1 069 1 113 1105 1014 1 100 1 068 1 030 1 096 1597 0835 1 077 1122 1 081 1 092 1107 1 073 1 019 Previewing Options When preparing to print a newly created or recently edited report it s a good idea to preview it first to check column widths page breaks fonts colors and so on If a change is required before printing two options are available e Modify the report document and go through the print preview process again This is the recommended option because changes will be saved with the protocol e Make a temporary change in the preview document The Protocol Options parameter Keep Open After Printing must be enabled 306 e PowerReports User s Guide to KC4 Special Notations When displaying results or when reporting results using the standard KC4 reporting engine KC4 uses various font characteristics to denote masked wells and biased values When exporting results or when reporting results using the Excel and Word reporting engines KC4 uses some special characters to denote masked wells and biased values This is to avoid conflict with any special font characteristics that may be assigned by the report export file designer e To denote a m
17. Point to Point Linear interpolation between standard points The minimum number of standards is 2 Linear Regression Linear regression y ax b y represents unknown response x represents concentration a represents the slope of the linear regression b represents the intercept to the y axis e The linear regression fit uses the least squares technique The better the quality of the fit the more the absolute value of R tends to 1 This type of curve fitting technique can be used when you think the data will fall in a predictable linear pattern It may be necessary to transform the x and or y components using Log axes e Click the Options button to enable or disable the averaging of standard replicates before curve calculation By default average replicates is enabled Curve Fit Options xl M Average replicates Cancel Polynomial Regression This type of regression fitting 1s an extension of the linear regression equations y a X a X a X tap n represents the degree of the polynomial regression Note The degree must be less than or equal to the number of standards minus 2 The unknown concentrations are calculated by using an approximate calculation method linear interpolation between 200 points evenly spaced on the x axis e Certain assays require forcing the curve through the origin KC4 provides a feature that allows you to specify the point at which the curve should meet the Y axis Click the Options butt
18. 1 0000 1 0000 H Meg Mec 1 0000 1 0000 Dilutions Only Unda Print i Cancel Help 142 e Protocol Menu User s Guide to KC4 For readers that support multiple plate formats make sure that the correct plate type is selected in the Reading parameters dialog before creating the plate layout Use the Well Settings options to select a well type customize the text and color of 1ts IDentifier s and define dilutions or concentrations 1f required Enter the number of Replicates then select the replicate mapping direction Horizontal or Vertical Check an Auto Selection option to automatically increment well ID group numbers and or concentration or dilution values Select the group mapping direction Horizontal or Vertical Highlight one or more or wells in the grid to assign the identifier s Click the Undo button to undo the last action Click the Print button to print the current plate layout If the plate contains 384 wells the printout will cover four pages one per plate quadrant The Plate Layout dialog can be maximized to fill your entire screen Click the maximize icon in the upper right corner of the Plate Layout dialog Within the grid rows and columns can be resized to see all wells of a 384 well plate for example Place the cursor over a row or column separator until 1t changes shape Click and hold the mouse then drag the cursor up or to the left to make rows or columns smaller Drag the cursor dow
19. 162 Single Plate 159 Transmission speed 195 Trial mode 18 U Unauthorized users Protected functions 218 Protocol security options 184 Uneven kinetic interval 121 123 Uninstall notes 10 Universal plate test 205 207 Unmasking wells 252 Upgrade notes 10 Upgrading databases 15 236 uQuant 89 196 Use barcode as filename 185 Use first filter set sensitivity 92 Use out of range values in statistics 188 230 User accounts 211 Creating 24 212 Deleting 215 Locking or unlocking 215 Modifying 214 Status 215 User ID 213 User notification options 220 Vv Validation 171 Validation report 180 Validation results 192 295 Index e 323 W Wavelength Selection 118 Filter Sets 95 Filters 99 Wavelengths using monochromators 118 Wavelengths Defining in protocol 118 Spectrum reads 118 Weighting of standard deviation 188 230 Well analysis results 293 Well IDs 143 144 Well mode 324 e Index FL600 104 SynergyHTTR with Injectors 120 Well settings 143 Well zoom curves 304 Well zoom report 179 Wells to monitor 108 WL 95 Word Reporting or exporting engine 276 Requirement for PowerReports 276 Using to design reports and export files 273 User s Guide to KC4
20. 2688 2469 2240 0522 2577 2 J Symbols s 229 Chane P Mask Statistics EL 808 on COM poo A e Click the Data drop down arrow to view a different data set e Click Statistics to view statistical information for the identifiers assigned to this plate e Check Change to modify one or more raw data values then click OK to recalculate and revalidate results Note The System Administrator can protect the use of the Change function User s Guide to KC4 Results e 251 252 e Results Check Mask to mask one or more wells to temporarily exclude them from calculations Click on the desired well s then click OK to recalculate and revalidate results To unmask a well click Mask click on the well then click OK Results will automatically recalculate Important Masking wells may significantly change or even invalidate the results The System Administrator can protect the use of this function To copy data to another application highlight the desired wells then select Copy from the Edit menu Switch to the target application and select a Paste option conditions occurs The plate layout specifies the contents of a well that is not read The absorbance value for the well cannot be calculated absorbance reads only The RFU RLU value exceeds 99998 fluorescence and luminescence reads only Tip If well that is not read or if the absorbance value for the well cannot be calculated absorbance reads only s
21. 30000 0000 hu 4 a F i z 2 a Well B7 10 Time 2 Selected Curve M Ane 520 p7 Recalculate Close Mean Y 3 420E 8 mF U mn H Coeff Reg 0 9530 M 465 526 Te User s Guide to KC4 Results e 259 Spectral Scan Data The default view for spectral scan data is Spectrum Curves eae RESULTS Data Spectrum Curves Template Spectrum Curves h p E 7 a g 10 11 12 Min OD Max OD M at blas OD Min w 400 rim Min y D Scales Calculations Mas B00 nm Mas y 24660 e Each well displays a graph of the measurement values taken across a wavelength range e Hold down the Ctrl key and click on up to 8 wells to overlay the spectrum curves in one display e Click the Scales button to adjust the data points displayed for each well The current scaling parameters are shown in the lower left corner of the Spectrum Curves display e Click the Calculations button to alter the spectrum calculation zone for all wells 260 e Results User s Guide to KC4 e Click on a well to enlarge the view of its spectrum curve E Well F5 well FS 450 500 Wavelength nm Selected Curve Meas OD F5 d Recalculate Min OD 0 08500 wi at Min OD 600 00 H S Meas OD 2 909 3 W at Max OD 425 00 Mas OD 3 5490 at Max wt 450 nm The spectrum curve initially contains all values measured at all wavelengths as defined in the protocol Note Ifa data point is missing for a p
22. Cancel 4 APIP Sheet1 A e Change the current folder and file name if desired then click Save to complete the export e Ifthe Protocol Options parameter Keep Open After Exporting is enabled the document stays open after the export is complete This allows you to for example print the contents of the file e See Special Notations on the previous page for information on how KC4 denotes masked wells and biased values in export files Direct Export to Excel KC4 provides a way to directly export data to Excel without first having to create an export template To export plate data directly to Excel 1 Under Protocol Options or under System Setup if no protocol is open set the export engine to Microsoft Excel Read the plate Select DataJExport KC4 will launch Excel and insert the RSO toolbar Insert objects Field Plate Table and or Curve as explained throughout this chapter Click Save As to export the data A a 308 e PowerReports User s Guide to KC4 Appendix A Records Submission Records Submission When preparing electronic records for submission to the FDA contact Bio Tek Instruments Marketing amp Sales Department for guidance and to obtain a demonstration copy of KC4 to include with your package The FDA will need KC4 to view and print your records Contact information Phone 888 451 5171 toll free in the U S 802 655 4740 outside the U S Fax 802 655 7941 E mail customerc
23. Help e Before defining validation criteria ensure that all identifiers have been selected and defined under Protocol Plate Layout e Define up to 5 different Validation Sets each containing up to 10 Conditions e The Data Type setting represents the set of data to which the Conditions equations will be applied e The Conditions fields allow fixed thresholds and or algebraic formulas to be used in validating data to satisfy known conditions of the defined assay A Condition typically combines well identifiers see Plate Layout with the following symbols lt less than lt less than or equal to gt greater than gt greater than or equal to User s Guide to KC4 Protocol Menu e 171 Example 1 Conditions to be met 1 During the first of several kinetic readings the mean value of STD1 must fall between 0 200 and 0 300 OD and the mean value of STD2 must fall between 0 900 and 1 300 OD Validation Set i Data Type E 405 Reading i 0 200 6701 0 300 0 900 STO 1 300 2 For each kinetic reading point the mean value of POS1 must be less than the mean value of POS2 Validation Set 2 Data Type M 405 Reading ja POS POS Example 2 Condition to be met The mean of every sample replicate group must be greater than 0 Validation Set H i Data Type M 405 O SPL The pound sign is a generic symbol that can be used to represent all indices o
24. It restarts with the first color when the whole list of colors has been used To remove the last color of the current sequence set it to End of Sequence the last item in the list User s Guide to KC4 Protocol Menu e 147 Standard Wells Standard ID Prefix STO E Conc 10 000 Uml Standard wells are used to generate a standard curve on which data will be interpolated Expected concentrations must be defined for each standard group Unique IDs must be used to distinguish between the different standard groups The minimum number of standard groups that can be defined is 2 and the maximum number is 20 Note Each curve fit type has its own required minimum number of standards The default ID prefix is STD and the default IDs are STD1 STD2 STD3 etc The default color for all standard wells is dark blue To change the ID prefix and or color click the box to the right of the ID field Standard STD Standard Well En e Enter an ID Prefix using 1 to 7 characters The prefix can contain letters numbers and underscores It cannot contain any spaces and it cannot end in a number If the prefix is currently used in the grid its text can be changed here The change will automatically be reflected in the grid e Optional Enter a descriptive Name using up to 20 alphanumeric characters This name can be included on reports e Click on the Color box to open a drop down list showing all of th
25. Programs gt KC4 3 4 Rev gt KC4 KC4 s opening dialog appears KC4 Signature users will see a different picture ATTENTION The Evaluation Period for KC4 will Expire in 11 days Register your copy now Register C4 2 Click Run KC4 in Trial Mode 18 e Installation and Configuration User s Guide to KC4 KC4 s main dialog appears a KC4 NewPlate Biel About E El Control Data Edit Protocol Display System E A LI Note KC4 may attempt to communicate with an attached microplate reader If you have not yet attached a reader do not be concerned at this time 1f messages resembling NO READER RESPONSE and Error when getting wavelength table from reader appear These messages will disappear when a reader is properly connected and configured KC4 Signature users will be presented with a Login dialog Please enter pour login information User Administrator kaj Full Mame E stem Administrator Password quexca Hep Enter admin in the Password field and click OK The dialog will disappear and System Administrator will appear in KC4 s status bar at the bottom of the dialog User s Guide to KC4 Installation and Configuration e 19 TplRep_1 Error Message Under certain circumstances an error message resembling TplRep_1 xla could not be found may appear in KC4 It is most likely to appear if an earlier version of KC4 was uninstalled and th
26. Protocol Description W Assay Description Selected Data Template Plate Kinetic curves 14 405 Form Feed Kinetic curves Plate Template Mean Y Mean Y Coeff Reg Coett Reg Protocol Audit Trail Forn Feed Data Audit Trail Well zoom From 41 To 44 Oyverlaid Wells Calculation Log Report Well zoom From B To B4 Oyverlaid Wells Sample names Well zoom Protocol Audit Trail Sample name list e Form Feed Format Wells for kinetic zoom 5 Plate Eo Bi lt lt Remove k I Table Ingert gt gt To Ba Statistics E fontrole a Standards i mrena el Samples Help e Check Header and enter the text to appear at the top of every report e Check Protocol Description and Assay Description to include this information on reports e The Available Data window displays the data and information available for reporting Available Data selectable items are dependent upon the type of protocol selected as well as the defined Reading Parameters Click the left mouse button to select an individual item To select groups of like items hold the Ctrl key down while highlighting them Click the Add gt gt button to append the item s to the Selected Data list User s Guide to KC4 Protocol Menu e 177 Click the Insert gt gt button to insert item s into the Selected Data list Highlight the item in the Selected Data list above which the inserted item should appear You can drag and dr
27. Refer to the Clarity Operator s Manual for complete priming instructions To prime the injectors 1 Fill the reagent bottle s with the fluid to be dispensed 2 Access the Priming Parameters dialog All installed injectors are displayed Injectors marked by an asterisk have yet to be initialized Initialization takes place automatically when the injector is used 3 Click on the injector s check box The drop down list boxes Direction Volume pl and Strokes are enabled User s Guide to KC4 System Menu e 249 4 Inthe Direction list box select To Mpl In the Volume pl box specify the injection volume and in the Strokes box specify the number of cycles The total liquid volume is displayed in the Total Volume column 5 Click lt Prime gt to prime the injector s Heating The Clarity s optional incubator is controlled via software Click the Heating button to access the Temperature Control dialog Refer to the Clarity Operator s Manual for complete heating instructions 250 e System Menu User s Guide to KC4 Results Overview When plate reading is complete or after data has been entered manually or read from a file KC4 displays the Results as demonstrated below Es KC4 Quantitativel 4 prt Quantitative pla Data Edit Protocol Display System About Wizard i revi ings Layout Options Biograph a RESULTS Data Delta 0D gt Delta O mas Delta AD Corr a 1755 1440 1051
28. Shared database Explained 233 File maintenance 226 Location 233 Renaming 233 System audit trail 224 Used for file storage 222 Signature reasons Applying 54 73 Defining 221 Signing files Global data files 54 Protocol files 73 Simulation data 50 Single Plate Transformations 159 Formula syntax 160 Overview 159 Skip reference wells 100 Software Trial mode 18 Software installation 11 Software version and revision numbers 4 SP data 129 Spectral scanning 118 Spectrum analysis 137 261 Data to process 137 Formula 137 138 Min Max 137 Spectrum read Selecting in a protocol 113 Spectrum reading parameters Scales 114 Wavelengths 118 Spline curve fit 164 168 SQRT function 161 Standard curve printing 302 Standard deviation 160 Standard deviation weighting of 188 230 Standard users 322 e Index Group membership 213 Protocol level protected functions 184 System level protected functions 218 Standard wells 143 148 Standards defining 155 Static Mode 104 Statistics 181 230 251 264 Statistics report 181 286 Status bar 31 Stop bits 195 Stop monitoring when 108 Stored curves 266 Sweep Mode 112 Symbols 160 163 171 Defining for cutoffs 175 How they are assigned 176 Method of assignment 256 Out of range 176 OUT indication 256 Viewing results 256 SynergyHT 86 199 200 SynergyHT HTTR Detection method 112 Measurement Options 104 Reader Configuration 201
29. The use of certain functions is restricted when running multiple instances of KC4 These restrictions are in place to maintain integrity with respect to communication data retrieval data reduction and reporting When running multiple instances of KC4 Individual KC4 instances must control unique reader models For example KC4 cannot control two ELx808 instruments at the same time KC4 can control an ELx808 instrument and an ELx808 instrument at the same time Under System Readers a new Current Reader can be set but its communication and filter wavelength parameters cannot be changed Software filter wavelengths tables cannot be updated at any time For example clicking the Calibrate button from within the Protocol Reading dialog generates an error message System Setup parameters cannot be modified System Users parameters cannot be modified System Plate Formats parameters cannot be modified Only one PowerReports document can be edited printed at a time 204 e System Menu User s Guide to KC4 Diagnostics The Optics Test and Universal Plate Test should be performed routinely to validate and document the consistency of a reader s optical and electronic performance alignment accuracy reproducibility and linearity Optics Test The Optics Test also referred to as the System Test validates and documents the consistency of reader optical and electronic performance For most readers this test can be performed fro
30. These features function similarly to functions of the same name in KC4 See Curves under Protocol Menu for more information User s Guide to KC4 Biograph e 269 Overlaying Graphs Biograph allows you to view multiple graphs in the same session Overlaying Fa Overlaid curves e The graphs must be open before they can be overlaid e Upto 8 graphs can be overlaid regardless of curve fit type but all must have the same axes either all linear or all logarithmic The curve overlay will display in a new window labeled OVERLAYED CURVES fig BIOGRAPH Windows Application OVERLAID CURVES File Edit View Graph window About O tae e e el 181 The curves in this window cannot be edited New interpolations can however be imposed Interpolations cannot be adjusted within this window make adjustments in the individual curve windows However any changes which you make to an individual curve or curves are reflected immediately in the shadow curve in the OVERLAYED CURVES window once you have calculated the change using Graph Recalculate 270 e Biograph User s Guide to KC4 Curve Window The replicate of each data point is displayed with a green square and the mean replicate value is displayed in a red circle Ra BIOGRAPH Windows Application STANDARD CURYE d Eile Edit View Graph Window About Hae E a 12 4 Parameters y a d 411 x c b d a 2 209 b 4 585 c 35 68 d 0 05681 R
31. To locate the installed KCShared mdb file and copy it to the network 1 In KC4 select System Setup and click the Database tab Protocol Defaults Formats Startup Database Shared database This database mainly includes Security and Plate Format information It may algo include Protocol and Data files See Security Settings It can be shared by several networked PCs Path CAD ocuments and Settings All Users4pplication Data Bio T ek Restore Lonnecton Repair Compact 2 The Path field in the Shared Database section contains the current location of KCShared To see the full pathname click once in the Path field and press the right arrow key 3 Launch Microsoft Explorer and go to this location Tip With some operating systems KCShared mdb 1s installed in the Application Data folder under Documents and Settings All Users If you are looking for the Application Data folder on your computer and can t find it it is probably hidden To reveal this folder open Microsoft Explorer and select Tools Folder Options View Make sure that Show hidden files and folders is enabled 4 Copy KCShared mdb to the clipboard and paste it in a secure shared access location on the network 5 Return to KC4 22 e Installation and Configuration User s Guide to KC4 To configure KC4 to use the KCShared mdb file on the network 1 Within the System Setup Database dialog click the Browse button A standard Windows styl
32. and saving assay protocols je KC4 NewProtocol Data Edit Protocol Display System Her b Wizard Wizard Oper Close Save Save Ag Sign Plate Description Reading New Create a new protocol Wizard Launch the Wizard for help with creating protocols Open Open an existing protocol PRT file Close Close the current protocol file If the file has not yet been saved KC4 provides the option to save before closing Save Save the current protocol PRT file Save As Save the open file under a new name Sign Electronically sign the protocol file Plate Description Define the names of up to six free text fields to appear at read time to allow the operator to enter relevant information such as the site name and lot number User s Guide to KC4 Getting Started e 33 Reading Raw Data Correction Kinetic Analysis Spectrum Analysis Scanning Analysis Plate Layout Single Plate Transformations Multi Plate Transformation Curves Validation Cutoffs Report Export Audit Trail Security Options Advanced 34 e Getting Started Define the reading parameters such as reading type filters wavelengths shaking and incubation instructions and plate type Enable Disable corrections to raw data Options can include Blank Plate Subtraction Blank Wells Subtraction and Pathlength Correction Select the primary and optional secondary set of data for kinetic analysis as well as
33. and then re import the file back into the shared database This will both preserve your current plate format information and add the custom plate formats 2 Import the plate database KC4 32 pld from the earlier KC4 version Warning This will overwrite the current plate format information in the shared database 242 e System Menu User s Guide to KC4 To manually update KC4 with the custom plate formats from the earlier KC4 version option 1 1 Locate the KC4_32 pld file containing the desired custom plate formats this file can be found in the root KC4 3 0x 2 x folder Open this file using a text editor such as Notepad Keep this file open in the background Launch KC4 v3 4 KC4 Signature Select System Plate Formats and click Export The Export plate database as dialog will appear Tip If the Plate Formats option is not available in the System Menu this means that the currently selected reader does not support customizable plate formats Select System Readers and choose a more appropriate reader Export plate database as Save ihr a RCA 43 1 da E Backup Office OLE Client Samples File name Flate atabase Oct31 2002 tut Save as type Text Files Ext Cancel wi 4 Using the file tree choose a target location Enter an identifying file name and click Save Open this file using a text editor such as Notepad Switch to the open KC4 32 pld file Locate the custom plate format it will resemble the
34. from secured file storage When Secured Protocol and Data File Storage is enabled the user cannot delete or overwrite existing folders or files within the System Maintain Files dialog Move Folder File in secured file storage When Secured Protocol and Data File Storage is enabled the user cannot move folders or files within the System Maintain Files dialog Import File to secure file storage When Secured Protocol and Data File Storage is enabled the user cannot import paste from clipboard or drag and drop from another location folders or files into the System Maintain Files dialog Export File from secured storage When Secured Protocol and Data File Storage is enabled the user cannot export folders or files out of the System Maintain Files dialog User s Guide to KC4 System Menu e 219 Audit Trail Notification Options This feature only applies to KC4 Signature Select System Security Audit Trail to enable or disable the appearance of an Audit Trail Notification dialog each time an event is logged to the Data Protocol or System Audit Trail Only the System Administrator is authorized to modify these options Users Lagin Protected functions Audit Trail Signature Reasons Files User Notification M Data Audit Trail Protocol Audit Trail e System Audit Trail Enabling the User Notification options allows users to enter comments for an event For example if a user modifies a pr
35. highlights all text within the current field or cell For example place the cursor in a formula cell within the Single Plate Transformation dialog click the right mouse button and click Select All to select the entire formula Undo Ctrl Backspace Erase the last unsaved change made and restore any items that had been modified by that last change For example if within the Plate Layout dialog you mistakenly applied Sample well identifiers atop existing Standard well identifiers choosing Undo will restore the Standard wells User s Guide to KC4 Edit Menu e 65 66 e Edit Menu User s Guide to KC4 Protocol Menu Overview A Protocol contains instructions for controlling a microplate reader and for analyzing and reducing plate measurement data Protocol parameters can be stored in a protocol file which has a PRT extension or in a global data file which has a GLB extension The topics in this chapter describe how to create and customize protocols Customizable protocol parameters include the following Detection method Absorbance Fluorescence or Luminescence Reading type Endpoint Kinetic or Spectrum Read mode Normal Rapid or Sweep Read Dispense parameters for instruments with fluid injection Wavelength selection Linear and area scanning Plate type and reading zone selections Blank plate well subtraction Pathlength correction Well monitoring Kinetic spectrum and scanning analysis Plate layou
36. options for Word 277 Dose response curves 303 Duration plate shaking 116 E Eject between filter set read FLx800 95 Eject between Filter Set Read FL600 100 SynergyHT HTTR 101 Eject between well FL600 104 Electronic signatures Data audit trail 63 Global data files 54 Protocol audit trail 183 Protocol files 73 ELx800 78 196 ELx808 79 196 Emission filter wheel 197 200 Emission wavelength 95 97 Endpoint read Selecting in a protocol 113 Engine Export 187 Report 186 Error when getting wavelength table from the reader 69 Index e 315 Excel Reporting or exporting engine 276 Requirement for PowerReports 276 Using to design reports and export files 273 Excitation filter wheel 197 200 Excitation wavelength 95 97 Experimental wells 91 Export engine 187 Exporting a plate database 242 Exporting events 225 Exporting files from the shared database 227 Exporting plate data Automatic export after reading 187 Direct export to Excel 308 Excel 187 308 Exporting sample names 61 62 KC4 182 187 Keep open after exporting 187 Protecting the document 187 Selecting a text separator 231 Selecting data sets for export 57 Selecting the exporting engine 187 230 Word 187 308 Extrapolation factor 165 254 F Failed login attempts 216 Failed to open document 242 Fax numbers Bio Tek 4 Field objects in PowerReports 279 282 File format options 222 316 e Inde
37. plugs and empty locations depending on assay requirements Use this dialog to specify the exact contents of each filter wheel The settings here will be made available as default selections within the Protocol Reading Parameters dialog Wavelengths Reader FLe800 on COM Cancel rr Excitation Emission Center Bandwidth Center Bandwidth Help E feo jjo 460 O Get Wavelenaths fas 0 528 0 Send Wavelenaths PLUG PLUG a a E Important Itis essential that you conform to the specific Bio Tek instrument procedures for altering the filter wheel configuration The FLx800 does not automatically detect which filters 1f any are installed Therefore it is your responsibility to ensure that KC4 s wavelength table exactly matches the contents of each filter wheel For instructions for physically changing filters see the FLx800 operator s manual Note To exchange wavelength information between KC4 and the reader the two must be communicating The currently selected reader and COM port are displayed at the top of this dialog If you need to change the COM port click Cancel to exit this dialog now and then click the Port button To change the current settings and download them to the instrument e Enter wavelength values in the Center fields or use the drop down boxes to select PLUG or HOLE PLUG indicates a solid plug in the filter wheel A plug in the Excitation filter wheel is typical for
38. the probe the instrument waits 100 milliseconds before taking the measurement This delay is to allow for more complete fluid settling e Rapid mode is faster than Normal mode because the instrument does not wait before taking the measurement e Sweep mode is the fastest of the three available modes the instrument does not stop before taking measurements Reader Read Method FLs800 on COM Fluorescence Luminescence The Reading parameters dialog displays the name of the currently selected Reader and the computer port to which it is attached This information is configured under System Readers The available Read or Detection Methods are reader dependent If more than one option 1s presented select the desired method by clicking its radio button This selection instructs KC4 to represent measured values as optical densities OD relative fluorescence units RFU or relative luminescence units RLU It also customizes the Reading parameters dialog to present further options specific to the read method 112 e Protocol Menu User s Guide to KC4 Reading Direction Reading Direction Horizontal Vertical Some readers also support a Reading Direction setting Choose to read in a horizontal direction A1 A12 B1 B12 H1 H12 or in a vertical direction A1 H1 A2 H2 A12 H12 e The faster method for reading a full plate is Horizontal e The faster method for reading a single column is Vertical Re
39. you cannot change the font style or size for example Word If your computer has Microsoft Word 2000 installed on it you can design the report using Word and take advantage of all its formatting features Excel If your computer has Microsoft Excel 2000 installed on it you can design the report using Excel and take advantage of all of its formatting features In addition you can create custom Excel formulas that reference KC4 data If you chose Word as the engine choose a Document Formatting option to tell KC4 how to prepare documents for printing and print previewing If Standard is selected document formatting is faster because KC4 does not apply any custom font characteristics from the Plate Layout or Cutoffs dialogs Well IDs and cutoff symbols are printed using just the default font characteristics If Enhanced is selected document formatting is slower because KC4 does apply custom font characteristics to Well IDs and cutoff symbols Select Automatic Print after reading to automatically print reports when the plate read is complete This is available for all reporting engines Select Keep Open After Printing to keep Word or Excel open after the print preview or after printing to allow for temporary formatting adjustments Note To make permanent changes modify the report via Protocol Report IfKeep Open After Printing is enabled the Protect option can be enabled to prevent unauthorized changes to the docume
40. 1 000 R 1 000 er 0 00 2461 e Click on an individual data point to temporarily exclude it from further calculations then select Graph Recalculate button to recalculate the curve without the point Important Modified data may adversely affect the final results Changes made here are not automatically transferred over to KC4 To make permanent curve fitting changes return to KC4 and select Protocol Curves e To restore an excluded data point click on the point then select Graph Recalculate e Select File Print to print the graph User s Guide to KC4 Biograph e 271 272 e Biograph User s Guide to KC4 PowerReports High Powered Reporting Since its inception KC4 has helped users with designing professional high quality reports tailored to meet their specific needs KC4 now takes information presentation to an even higher level with PowerReports PowerReports links KC4 with Microsoft Excel Word 2000 providing you with a new format for creating custom report and export documents With PowerReports you can e Take full advantage of Excel Word s document customization features to generate publication quality reports e Include pictures drawings and company logos in reports e Create custom formulas in Excel to perform additional calculations on KC4 data e Take advantage of Excel s charting capabilities to create bar graphs pie charts and more e Perform a direct export of plate data to Excel without fi
41. 1 0000 1 0000 1 0000 a SFL1 SPL2 SPL2 SPL3 SPLA SPL4 SFL4 SPLO 5 SFL PLE 2 5000 UI 2 5000 2 UU 2 5000 2 5000 2 5000 2 5000 2 5000 E 2 5000 25000 el i D al pE RT ee a A an O Dan ee L l aa at ha a ta A A A A A PA A A A PA A A Pome Pat Pa d a et A in a en Pt At C A SPL2 SPL2 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 10 000 ELO n SPL3 40 000 40 000 40 000 40 000 40 000 40 000 40 000 40 000 40 000 40 000 40 000 40 000 SPL2 160 00 160 00 160 00 160 00 160 00 160 00 160 00 160 00 160 00 160 010 160 00 E wi Laa lt E aa a o a E nes Lal Concentrations Only Undo Print a cee Cancel Help pa E F Da a i ttt e Concentrations are defined as 1 2 5 5 10 20 40 80 and 160 e Replicates is set to 2 and both Auto Select options are selected e Wells Al through H12 are highlighted to assign the concentrations and IDs 158 e Protocol Menu User s Guide to KC4 Single Plate Transformations KC4 provides for up to three levels of Single Plate Transformations The three different levels of transformations may be interdependent or completely independent of each other Typical uses include e Using specific wells as local blanks for other wells e Calculating ratios between wells The Single Plate Transformation dialog functions similarly to a spreadsheet The data entry template resembles a microplate in row x column
42. 4 mm DD 4712 x 34 850 mm Y 26 740 mm Picture Mode the default view is a graphical representation of all measurements taken across the well Place the cursor over any point in the well to see its value and its X Y location on the plate Click Data Mode to view the measurement values for all points To exclude restore a point from the scanning calculations click on the desired point and then click Recalculate 262 e Results User s Guide to KC4 Linear Scan Data The default view for linear scan results contains the graphical representation of the measurements taken across each well s RESULTS Scanning Curves Mean OD A Std Dev OD ICY OD Min x 15 Min y 0 02526 Scales Calculations Mak x 15 hax y 5 0000 e To view a different set of data click the Data drop down arrow and select Mean OD Std Dev OD or CV OD e Click on a well to zoom in on it m Well ES Well ES Reading Position Selected Curve 405 E5 Options Recalculate Close Mean OD 2 8515 Cv OD 71 139 Std Dev OD 2 0285 M 405 3 984 x 7 Place the cursor over any point in the well to see its measurement value and its X location in the well Ifthe graph contains more than one curve use the Selected Curve menu to choose a different curve and view its Mean OD Std Dev OD and CV OD values Click Options to adjust the Calculation Zone then click Recalculate to update the ca
43. About Bio Tek Home Page 4 e Introduction User s Guide to KC4 Installation and Configuration Important There 1s a certain sequence of events that must be followed to ensure that KC4 is properly installed and configured This chapter describes the essential installation and configuration tasks Review the installation options provided on the following pages and choose the one required by your site to determine which tasks must be performed Turn to the appropriate page number for detailed instructions on each task User s Guide to KC4 Installation and Configuration e 5 Special note for Clarity Microplate Luminometer users The Clarity PC software must also be installed on the PC s Install Clarity before performing the step Connect reader s to the computer and establish communication KC4 v3 4 Single PC with one or more users Step Task Page l Ensure that the computer meets the minimum system requirements 9 2 Install KC4 and restart the computer 11 3 Start KC4 and log in as the System Administrator 18 4 Optional Change the System Administrator s password 21 5 Optional Copy KCShared mdb to a secure network location 22 6 Required if step 5 was performed Configure KC4 to use the 23 KCShared mdb file on the network 7 Connect reader s to the computer and establish communication 25 KC4 v3 4 Multiple PCs in a network environment Step Task Page l Ensure that all computers meet the minimum
44. Added information for handling an unexpected termination of a kinetic read Updated the system requirements for using PowerReports to include Microsoft Office XP and Office 2003 Updated information on COM port selection now supports up to nine ports Additional cosmetic changes throughout Revision D1 one page insert removed revision letter changed as a result Contents INTRODUCTION 00022 as ed nie eee ede 1 IO A SO Hutt rd is l Technical SUPPO sra a E E N ee 4 Phone SUPPO oasian ae a eee 4 Facsimile SUPPO eree E EEN 4 Written Communica oikene ean ee e E EAE E idas 4 Electronic Oma Mica Om A A A os 4 INSTALLATION AND CONFIGURATION occcccccccccccccccnononnnnnnanannnnnnnnnnnnnnnnnnnnnrrrnnnnnnnnnnanannss 5 POr eea N E eae hana annie 5 Appointing a System Administrator ccccccessssssssssssssssssssssessseessessssssesseseeseesseeeeeegs 8 Administrator REGUIFEMICIIUS ten cfarsethe arne E A TO eas 8 Verifying Minimum System Requirement a aE 9 Upgrading from an earlier version ERA A Ati 10 DiS Ca ONGC sarria EE N E EEE T E E E E 11 Staru nae K C Acopiara a A 18 Changing the System Administrator s Passwold ooooooooooonnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnos 21 Copying KCShared mdb to a Secure Network Location ooccccccccncccccnnncnononnnnnnnnncnnnoss 22 creatino New User ACCOUN S tt 24 Connectino Reader syto the Compute did 25 Where togo Ton Nereari n E eee ET ETT TOO EO OA 26 GETTING S
45. Blank Plate Subtraction Blank Wells Subtraction and Pathlength Correction enabled fs RESULTS Data M 405 Corr MHI 405 Blank MHS 630 Blank M 977 SP and M 900 SP contain the values of the Sample Plate read at 977 nm and 900 nm respectively necessary for pathlength correction calculation Measurement numbers M 1 M 2 M 3 correspond with the wavelength selection order in the Reading parameters dialog M 1 405 M 2 450 and M 3 630 contain the raw Sample Plate values at 405 nm 450 nm and 630 nm M 1 405 Blank M 2 450 Blank and M 3 630 Blank contain the values calculated by subtracting the Blank Plate readings at the specified wavelength from the raw Sample Plate readings at the specified wavelength These values do not represent Blank Well Subtraction M 1 405 Corr M 2 450 Corr and M 3 630 Corr contain the final values after all Raw Data Correction options have been applied Blank Plate Subtraction With most readers the Pre Read Blank Plate option is available within the Reading parameters dialog If this option is selected a blank plate is read before the sample plate at the wavelengths defined in the protocol KC4 then calculates the difference in measurements between the two sets of data sample blank Note The blank plate can represent an empty dry plate or a plate filled with solution for blanking If Blank Plate Subtraction is enabled Endpoint read For each wavelength defined in the pr
46. Help Fields Plates Tables Curves gt Close Statistics Well IDs Fitting Results Interpolation Results Protocol Audit Trail Data Audit Trail Calculation Log Report EA Microsoft Excel Worksheet in lt KC4 gt EJ File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves v Y Close Po _ _ emmemm nn ee mes E Calculation Log Report i K Calculation warnings J6 X 14 4 55 Sheeti Jat o a Ready al INUM ve User s Guide to KC4 PowerReports e 301 Inserting Curve Objects A Curve Object inserts a graph showing one or multiple overlaid curves To insert a Curve Object click the Curves button in the RSO toolbar A drop down list showing the available curve type will appear ia Document in lt KC4 gt Microsoft Word File Edit wiew Insert Format Tools Table Window Help Normal Times Mew Roman 10 Fields Plates Tables CURVES pr x Close Standard Curve e This list contains all available curve types based on the current protocol settings Available curve types can include Standard Dose Response and Well Zoom Standard Curve A standard curve is generated by a protocol that has Standard wells defined in the Plate Layout To insert a Standard Curve click on the Curves button in the RSO toolbar then click on Standard Curve to insert the object i Document in lt KC4 gt Microsoft Word Times New Roman 10 B Z U
47. KCShared mdb can be stored on a user s computer or on a shared access computer KC4 provides a special file maintenance utility for viewing the file names and their locations and for moving copying renaming deleting importing and exporting files 28 e Getting Started User s Guide to KC4 Record Retention and Archival With KC4 users create and save protocol plate data and global data files containing valuable assay information and test results KC4 does not automatically delete or archive any of these records It is therefore the site s responsibility to establish a procedure and schedule for record retention and archival Important Regardless of license type KC4 stores information using three different databases KCShared mdb KCLocal mdb and CALIB mdb See Database in System Menu to learn more about their functions and locations Because file or network problems can potentially create unrecoverable database errors these databases should be backed up on a regular basis e KC4 v3 4 Users Protocol plate data and global data files are stored individually on a user s computer or on a network computer to which the user has access Windows Explorer or a similar application can be used to backup archive files by copying them to the desired medium e KC4 Signature Users Protocol PRT plate data and global data files are typically stored inside KCShared mdb they can also be stored outside this database see
48. Nalda Oo all lemicad s 171 COUOT S re AN ett Pern ats A A Eat ee R E tte dad 173 Selecta the Data CO iii 173 WSO Cot stilo titi 174 Detiino Symbol iia 175 Cutolis Evaluace id 176 REPOT A A he enw etn ctnatda E 177 Audit Tra RCD OU Si ii ia 178 Calculation Los Report lla 178 kinene CUE RED ias 179 W cll ZOOM o o 179 Valdai on RCP A EIE A 180 Sic REPO es ass ales A aceee ceeds ec mee 180 Sample Nam e160 RCM OU ionis rore aen r R a teaa sedans 180 Plate ys Lable FONI a E 181 SASE S ea 181 EXPO aeea e o nn A E 182 User s Guide to KC4 Contents e v POOL AA A crue a nt En oN er e 183 BN MOY ii oliendo 184 0 oer 0 BO 6 18 611 Laermer ot id 185 LE A nee eee eee eC 185 FRCP PP e 5 E cessualaaace 186 EXPO dd E E EEE E A EA AEE dd cidade 187 CO a ea T Ba Miter ier ne ne are 188 DISPELA MEN U ne de nl ad e 189 SS ete RR O na et A TE RR Ee A S et Pe A tee 189 CU io 189 Calculation Warn GS rosso paa 190 Y MAA ON RESUMS eR ee E ee ate ee 192 SYSTEM MENU sscccectcoznecsccouniecutracstecaosntscatconssacoseusacseaewleenesaud aai 193 OPT A a sass teaces nha a RA alsa 193 MC AIOE Sl CC PIO ceases none ogee cee see ass a oe ss 194 Port Selection A E oe PIPE PROT A A 195 Fakers ave len Si ii anemia dats 196 Reader COn SIA ada 201 Dispenser CO A do od stadans 202 Controlling Multiple Instruments Simultaneously oooooooonnoooonnnnnnnnnnnnnnnnnnnnnnoss 204 Dinos a 205 Optics a tcc cated A N 205 Sy Stem Lest la Misia sacs uli N
49. New Plate to prepare to read a plate Open an existing KC4 protocol or begin with an empty one 9 Select Protocol Reading to open or create a Clarity Protocol File and define its reading parameters See Parameter Input in Chapter 4 of the Clarity Operator s Manual to learn about the reading parameters 10 Return to KC4 s main view and define the data reduction parameters create the plate layout by defining and assigning well identifiers select curve parameters specify cut off and validation criteria create single and multi plate transformations define reporting and exporting options 11 Select Data Read Plate to read a microplate 12 Review the results 13 Select Data Print to print reports 14 Select Data Save to save the information or with KC4 Signature select Data Sign to electronically sign and then save the information 15 Select Display Curves to view the curve User s Guide to KC4 Getting Started e 39 Sample Files KC4 s installation program copies many sample protocol PRT and plate data files into a special SAMPLES folder inside the root KC4 folder and with KC4 Signature inside the KCShared database Examine these files to learn how to create KC4 protocols to support your assays The sample files cover a range of protocol options including single plate transformations multi plate transformations curve fitting parameters cutoff criteria and more Important The sample protocols
50. Push the carrier eject button to draw the plate back in The read will continue as scheduled Reader Dispense Injector model only Create and run a total of three protocols A Kinetic protocol to start the read Save results in a PLA file A Dispense only protocol to dispense the reagent A second Kinetic protocol to complete the kinetics after dispense Note Reading parameters in the second protocol must be identical to the first although kinetic settings can differ Gather the results in a single PLA file using KC4 s Append to Kinetic File feature Pathlength Correction le Pathlength Correction wavelengths i Bio Cell E Check Pathlength Correction to normalize absorbances to 1 cm This option is available for readers that support multiple wavelength reads up to 999 nm If Pathlength Correction is enabled KC4 1 Calculates the pathlength for each well Uptions 2 Calculates the corrected absorbance for each well 3 Reports the Corrected Data after Pathlength Correction is applied Pathlength Calculation KC4 calculates each well s pathlength according to the following formula A977 nm Agoo nm well A977 nm Agoo nm 1 0 cm water e The well is measured at 977 nm and again at 900 nm KC4 calculates the difference in measurements then divides that result by the absorbance of water at 1 cm Note 977 nm and 900 nm represent default Test and Reference Wavelengths respectiv
51. SPL2 will be included in the table 296 e PowerReports User s Guide to KC4 e Click the Data tab to customize the contents of the table Curve Parameter Table Properties Selection Data re ee De rR m gt C e me gt Ce m om gt Cancel Help KC4 presents a default set of Data items that will be included in the table although any available item can be included in the list To add an item to the list highlight the first empty Data field and click the left mouse button once A list of available data items will appear Click on the desired item To modify an item in the list highlight 1t and click the left mouse button once A list of available data items will appear Click on the desired item To delete an item from the list highlight the row then click the right mouse button and select Delete To insert a new item between two existing items highlight the row above which the new item will be inserted Click the right mouse button and select Insert For data sets that represent numerical information Click the corresponding Format field then select Decimal or Scientific For the Decimal format click the corresponding Precision field to set the number of decimal places 0 to 5 Click OK to close this dialog and insert the object into the document EA Microsoft Excel Worksheet in lt KC4 gt ee el oo eel a 7 User s Guide to KC4 P
52. SPL4 SPL4 SPL4 UE 50 000 50 000 50 000 50 000 S50 000 40 000 50 000 50 000 Fi p le 4 E z z L 2 F gt 50000 e Dilution values are defined as 10 000 20 000 30 000 40 000 50 000 and 60 000 e Replicates is set to 3 and both Auto Select options are enabled e Wells Al through F12 are highlighted to assign IDs and dilutions Plate Layout Well Settings 3 r Replicates Auto Select Type Sample Control 6 a Mb WE HIG Poe A In sPLCS le El MaE Aref vert E gt q Filling Dil E J Bl le How Vert a L Li a SPLCS SPLCE E SPH 1 0000 SPL2 doo SPL 000 1 SP4 aoo SS 1o E A 100 LL i i 2 i ae ae VA e Dilution values are defined as 1 000 10 000 100 000 and 1000 000 e Replicates is set to 1 and both Auto Select options are selected e Wells A2 through D2 are highlighted to assign the ID and dilutions then A4 through D4 and so on to A12 through D12 154 e Protocol Menu User s Guide to KC4 Defining and Assigning Concentrations If the plate layout contains Standard wells or if you want to generate curves based on control or sample wells e g for a titer assay the location and the expected concentrations of these wells must be defined and assigned in the plate layout KC4 uses the expected concentration values as the basis for calculating the curve they represent the x axis values The measurement values retrieved from the reader or the re
53. Scanning Horizontal Reading points i E Individual Well Auto Scaling Cancel Help e The Horizontal Reading Points setting represents the total number of points to be read across the center of each well Valid entries are odd integers from 1 to 31 e Individual Well Auto Scaling individually adjusts the min max x and min max y settings for each well to best display its scanning curve If enabled this feature overrides the settings of the min max x and min max y Note This feature only affects the scale of an individual zoomed well The scanning curves display showing all wells on the plate 1s not affected User s Guide to KC4 Protocol Menu e 115 With Area scanning Scanning Read Matra Size 3 3 M Individual Well Color Auto Scaling Shaking Shaking Cancel Help Read Matrix Size represents the number of measurements taken across and down each well If for example the Read Matrix Size is 5 x 5 a total of 25 measurements are taken Individual Well Color Auto Scaling determines how the color palette 1s built when the measurement points are graphically displayed On color monitors and printers blue shades represent the lowest measurement values purple shades represents mid range measurement values and red shades represent highest measurement values On black and white monitors and printers white represents the lowest measurement values increasing shades of gray represent increasing measurement v
54. Sensitivity 98 Top Probe Vertical Offset 101 SynergyHTTR 87 SynergyHTTR with Injectors Dispenser configuration 202 Dispenser control 247 Emptying the tip prime troughs 247 Initializing the dispenser 202 Priming the dispenser 247 Purging the dispenser 247 Read and dispense protocol 119 Reading parameters 88 Setting the injector position 202 Syringe setup 202 User s Guide to KC4 System Administrator Administrator requirements 8 Appointing 8 Initial password 19 System audit trail Exporting events 225 Local events 224 Shared events 224 System menu Diagnostics 205 Overview 193 Plate formats 240 Reader control 245 Readers 194 Setup 230 System requirements 9 System security 211 System test 205 T Table format reports 181 Table objects in PowerReports 279 286 Technical assistance center TAC 4 Technical support 4 Temperature Control 117 Temporary files Creation following database connection failure 233 Importing into shared database 228 Text separators for importing and exporting 231 Time formats for kinetics 231 Time is approximate indication 120 Time resolved fluorescence 117 Timeout session 216 Timing control 120 Tip prime troughs 247 Tip prime troughs 122 Toolbar 31 237 Buttons 237 User s Guide to KC4 Customizing 237 Top Probe Vertical Offset 101 TplRep_1 could not be found 20 Transformations Formula syntax 160 163 Multi Plate
55. Subtract instrument background option was not enabled KC4 imports the raw data values and the background value and then subtracts the background value from all wells User s Guide to KC4 Data Menu e 53 Signing Data Files KC4 Signature provides an option for users to permanently attach or sign their names to global data GLB files Notes While KC4 does not permit users to sign plate data PLA files if a plate data file and a protocol file are open at the same time selecting Data Sign will combine the two individual files into one signed GLB file KC4 only supports file signatures when the multi user identification feature is in use See Security in System Menu for more information To sign a global data file l Za 2 Open the desired file if it is not already open Select Data Sign or click the Sign toolbar icon The New Data Audit Trail Signature dialog will appear New Data Audit Trail Signature E Reason Authorship al Representative of User Identification User MAJ Full Marne M ary Ann Jones Click the Reason drop down arrow and select the reason to be associated with this signature Signature Reasons are site defined by the System Administrator under System Security Signature Reasons 54 e Data Menu User s Guide to KC4 4 Optional If two users are signing on behalf of a third KC4 user check the Representative of box and then choose a name from the drop
56. System Setup For an interval greater than 99 59 59 set the time format to HHH MM 56 e Data Menu User s Guide to KC4 Exporting Plate Data Export Export KC4 can export plate measurement data to a text file a Microsoft Excel spreadsheet or a Microsoft Word document This section contains instructions for exporting to a text file only using the KC4 export engine e See Export in Protocol Menu for more information on the available export engines e See PowerReports for more information on the Excel and Word exporting engines The export file format can be defined within a protocol under Protocol Export or after the plate is read by selecting Data Export The text delimiter used during the export is selected under System Setup To export data e When the plate read is complete select Data Export e Ifexport parameters are defined in the protocol KC4 requests the entry of a file name Enter the file name and then click Save to export the data Data exported to a text file can be used by any other program with the ability to read an ASCII file e If export parameters are not already defined in the current Protocol an Export template appears The example below shows the template used to export data to a text file Export gt Protocol Description e Assay Description p Available Data Exported Data Delta OD Corr Template Concentrations Delta OD Symbols Delta OD Corr Protocol Audit Trail Concen
57. The KC4 software package includes the Biograph module for curve viewing and manipulation Biograph can be used either as a stand alone product or in conjunction with KC4 See Biograph for more information File Signatures KC4 Signature users can electronically sign protocol files and global data files Signing a file is easy select Protocol Sign or Data Sign and KC4 will guide you through the process Electronic signatures are permanent and cannot be modified and they remain a part of the overall protocol or global data file for the life of that file Signatures can be viewed by selecting Data Audit Trail or Protocol Audit Trail Audit Trails KC4 automatically logs events for user actions and stores these events inside audit trails e KC4 v3 4 maintains a Data Audit Trail for each plate and global data file These audit trails contain events for Change and Mask actions applied to the measurement data e KC4 Signature maintains three types of audit trails Protocol Data and System These audit trails contain events for many user activities including login logout protocol and global data file creation and modification plate reading and more Main Screen KC4 s functions are controlled through the main screen which contains a Title Bar Menu Bar Toolbar and Status Bar Title Bar KCA L NADH glb created from Lactate NADH prt 26 KC4 L HADH pla Lactate NADH_prt The title bar shows the current open file s Its
58. Trail Events Esport audit trail events from fi 0 01 2002 to fi 03172002 A Delete events after exportation Save in E Archive Shared Events da August 002 tat September 2002 tut File name October tet Save as type Text files tet Cancel Help WE Enter a valid date range in mm dd yyyy format Select a destination folder for the text file Enter an identifying file name SY A Optional Check Delete events after exportation to permanently remove the events from the audit trail 8 Click OK to export the events If events were deleted after export the System Administrator should do the following to compact the database 1 Ensure that no KC4 users are currently logged into the shared database 2 Select System Setup Database 3 Inthe Shared Database or Local Database area as appropriate click Repair Compact and then click OK When the process is complete the database should be smaller in size User s Guide to KC4 System Menu e 225 Maintain Files This feature only applies to KC4 Signature KC4 Signature provides a Windows Explorer style file maintenance utility for working with files stored in the shared database Authorized users can create folders move files delete files and so on all within the secure KC4 environment Events associated with all actions are automatically logged in the System Audit Trail To access the file maintenance utility select System Maint
59. View Window Help sae oe Table Title Well Analysis Table Properties Coordinate Jas Default ID SPLE C4 SPLC3 0 5000 EU ml D3 SPL4 10 000 Cone Dil 10 000 D4 SPLC4 0 5000 EU ral E3 SPL5 10 000 E4 SPLC5 0 5000 EU AS SPL6 10 000 lt lt Remove e The Selected Wells list contains all wells that will be included in the table To append a well to the Selected Wells list enter the well s Coordinate in a format resembling Al or F11 then click the Add gt gt button To remove a well from the Selected Wells list highlight the well then click lt lt Remove User s Guide to KC4 PowerReports e 293 e Click the Data tab to customize the information to be included in the table for the selected wells KC4 presents a default list of Data sets to be included in the table although any available data set can be selected See Statistics for information on working with data sets Click OK to close this dialog and insert the object into the document taj Document in lt KC4 gt Microsoft Word Normal Tmes New Ronan 510 294 e PowerReports User s Guide to KC4 Validation Results A Validation Results table lists the validation conditions and their corresponding statuses To create a Validation Results table click the Tables button in the RSO toolbar then click on Validation Results The table object is automatically inserted into the document 239 Docu
60. and down arrow keys to move from well to well in the matrix When the down arrow is pressed from a well in the bottom row the cursor automatically jumps to the well in the top row of the next column e Place the cursor over the desired well and enter the OD or RFU RLU if fluorescence luminescence value e Click OK to end the editing process and calculate results e For multi wavelength reads enter data for the first wavelength and click OK For the remaining wavelengths select the appropriate data set and click Change to enter values e For kinetic reads enter data for Reading 1 and click OK For the remaining readings update the Reading field and click Change to enter values Note With kinetic plate data each reading number set must be incremented to mimic the kinetic plate reading cycle The time base will be taken from the kinetic reading parameters defined in the protocol 50 e Data Menu User s Guide to KC4 Reading Plate Data from a File KC4 can read endpoint or kinetic data from an ASCII Text file The file must be formatted properly and the data must be delimited with acceptable separators To import Endpoint or Kinetic data e Examine the text file to see what character is used to delimit the data Typical delimiters include semi colon comma and tab e InKC4 select System Setup Set the Import Export Separator so that it matches the character used within the text file e If desired create or open a
61. appearance depends on the current Preferred Data File Format setting selected via System Security Files When the Preferred Data File Format is set to GLB e The set of brackets embraces the Global Data File name extension GLB e Ifapreviously saved protocol was used to generate the GLB file the title bar includes the text created from lt protocol name gt prt When the Preferred Data File Format is set to PLA e The first set of brackets embraces the Plate Data File name extension PLA e The second set of brackets embraces the Protocol File name extension PRT e An asterisk next to any file name indicates that its contents have changed but the file has not yet been saved 30 e Getting Started User s Guide to KC4 Menu Bar Data Edit Protocol Display System About e The Data menu contains functions for acquiring and processing measurement data e The Edit menu provides tools for copying data from the screen in KC4 and pasting it into a spreadsheet word processor database manager or text editor Data cannot be pasted back into KC4 e The Protocol menu contains functions for creating modifying and saving assay protocols e The Display menu provides access to the Biograph module and also displays the results of the Validation criteria defined in the protocol e The System menu contains special functions for configuring readers maintaining user accounts configuring security parameters settin
62. are expected to yield a mid range RFU or RLU value High Wells are usually the highest standards or high positive controls and are expected to yield a high but not over range RFU or RLU value User s Guide to KC4 Protocol Menu e 91 e Specify the Base and High Values The Base Value is the fluorescence or luminescence value that the Low and Experimental Wells must exceed during sensitivity determination This ensures that the plate produces values that are large enough to be meaningful The High Value is the fluorescence or luminescence value that the High Well s should approach but not exceed The default setting is 90000 RFU RLU and the maximum value is 99999 for the FL600 and 99998 for the FLx800 and the Synergy HT Practically this value could be anywhere from 10000 to 90000 Note Ifthe Best signal to Noise Ratio cannot be calculated use Scale to High Wells Use First Filter Set Sensitivity If multiple filter sets are defined you can opt to use the sensitivity determined with filter set 1 for other filter sets by choosing Use First Filter Set Sensitivity i M Automatic Sensitivity Adjustment Scale to High wels Best Signal to Noise Ratio C Scale to Low wells Use first filter set sensitivity Bandwidth Verification Only applies to fluorescence luminescence readers KC4 evaluates each filter set against two criteria 1 Overlap Evaluation By definition the bandwidth rating on the fil
63. available Standards could not be calculated The data set specified for curve fit calculation may no longer exist Select Protocol Curves Curve Description If the Data to Interpolate field contains asterisks this is the case Choose another data set and recalculate results e Data to process lt gt not available for kinetic calculations The data set specified for kinetic analysis may no longer exist Select Protocol Kinetic Analysis If the Data to Process 1 or 2 field is empty this is the case Choose another data set and recalculate results e Log Axis cannot be used with a negative or null value KC4 was unable to calculate the curve because one or both Axes is set to calculate logarithmically and an input value such as a standard OD is negative or null Select Protocol Curves Curve Fit to view the current curve parameters e Not enough valid standards Each curve fit requires a minimum number of valid standards either not enough standards are defined in the plate layout or following results calculation too many standards were determined to be invalid e Some cutoffs could not be calculated Symbols could not be determined The cutoff criteria may reference a data set or well ID that no longer exists or a referenced well ID may contain an invalid value Select Protocol Cutoffs to examine the current criteria e The archived curve could not be loaded Path is probably wrong This message was likely preceded by a
64. become locked Only the System Administrator is permitted to reinstate locked user accounts Valid entry range 2 to 10 attempts 3 Specify the number of minutes that the KC4 session may remain idle before it becomes locked and users are required to re login A session is considered idle if the user is not operating the keyboard or mouse and if the software is not currently controlling an instrument Valid entry range 1 to 1440 minutes Note For security purposes KC4 prevents lower level users from logging in after a higher level user has been automatically logged out of a session if a user intentionally logs out this restriction does not apply For example if the Administrator is automatically logged out only the Administrator is permitted to re login If a Power User is automatically logged out only Power Users and the Administrator are permitted to re login If a Standard User is automatically logged out any valid user may re login 216 e System Menu User s Guide to KC4 Enable Force user to type ID to require the user to manually type in his or her User ID in the Login and Signature dialogs Disable this option to permit the user to select his or her User ID from a drop down list Click OK to save the changes and close this dialog To set Password parameters l 2 Enable the desired Password parameters by checking the appropriate boxes Specify the minimum number of alphanumeric characters required to define a
65. button ensure that the reader is connected to the computer and that the communication parameters are properly configured Data Read Plate Save a data file Plate data files raw data use the extension PLA Global data files raw data protocol parameters use the extension GLB Selection of the target directory is possible using the Browse facility Data Save Sign a data file In a multi user system global data files raw data protocol parameters can be electronically signed Data Sign Print the results report The appearance of the button depends on the Report Engine setting under Protocol Options Excel Word or KC4 Report templates are created under Protocol Report Data Print Preview the results report before printing The appearance of the button depends on the Report Engine setting under Protocol Options Excel Word or KC4 Report templates are created under Protocol Report Data Print Preview Export the plate data to a file The appearance of the button depends on the Export Engine setting under Protocol Options Excel Word or KC4 Export templates are created under Protocol Export Data Export Enter Sample IDs associated with the plate This can be done before a plate is read or after a plate read when results are displayed in KC4 Data Samples Define the text to replace the prompts in the Data Plate Description and Data Read Plate dialogs Protocol Plate Description Define or modify reading
66. consider inserting multiple Well Data Table objects Area Scanning protocols Well Data Table Properties Selection Data Wiel Jat First Matris Column fi Last Matrix Column js Cancel Help e Fach table reports the area scanning data for one well Specify the Well to be represented in the table User s Guide to KC4 PowerReports e 291 e Area scanning data is reported in a matrix format representing the measurement points taken across and down each well Specify the First and Last Matrix Column to include in this table object Note If Word is the reporting exporting engine each table object can contain a maximum of 15 columns so you may need to create multiple table objects to print all of the information for the well For example if the Read Matrix Size is 25 x 25 create one table object with First and Last Matrix Columns set to 1 and 15 then create another table object with First and Last Matrix Columns set to 16 and 25 All protocols e Click the Data tab to change and or customize the data set Well Data Table Properties Selection Data Meas OD Blank Meas OD Meas OD Corr Each Well Data table reports information for one data set If the current protocol parameters generate multiple data sets click on the Data field to open a drop down list of all available data sets Click the Format field then select Decimal or Scientific Click the Precision field to set the number of decimal pla
67. cutoffs that reference well identifiers ensure that all identifiers have been assigned under Protocol Plate Layout e Cutoffs must be entered in ascending order If entering formulas the calculated results of the formulas must be in ascending order to be considered valid For example if Data to Interpret is set to Concentrations and the cutoffs are defined as STD1 STD2 and STD3 then the calculated concentrations must be in ascending order such as 10 000 20 000 and 30 000 e Each cutoff can contain any of the following Numeric values 1 500 85000 2 0E 8 Well identifiers NC BLK STD1 Well coordinates Al H12 P24 A formula combining any of the above CTL1 CTL2 100 Cutoff Formulas The following table contains valid symbols and functions for use within cutoff formulas Symbol or Function Well coordinates MEAN lt ID gt SUD CV lt TD gt LOG lt x gt POW lt x gt lt y gt SQRT lt x gt MEN CS ey SZ i MAX lt gt PS yo p lt Zee 174 e Protocol Menu Description Addition Subtraction or negation Division Multiplication Represents inclusion The value of a specific well The mean of the specified well identifier The standard deviation of the specified well identifier The coefficient of variation of the specified well identifier Represents the LOG10 function The value of lt x gt raised to the power of lt y gt The square root of
68. define global parameters including special Word Excel reporting options A Select Data New Plate to prepare to read a plate Open an existing protocol or begin with an empty protocol Select Protocol Reading to define reading parameters such as wavelengths plate type and temperature 8 Return to the main view and define the data reduction parameters create the plate layout by defining and assigning well identifiers select curve parameters specify cut off and validation criteria create single and multi plate transformations define reporting and exporting options 9 Select Data Read Plate to read a microplate 10 Review the results 11 Select Data Print to print reports 12 Select Data Save to save the information or with KC4 Signature select Data Sign to electronically sign and then save the information 13 Select Display Curves to view the curve 38 e Getting Started User s Guide to KC4 Quick Start for Clarity Microplate Luminometer Users Clarity users will follow a slightly different sequence of events 1 Connect the luminometer to the computer if not already connected 2 Start the Clarity software and set up the communications port 3 Important Close the Clarity software 4 Start KC4 and log in if required 5 Select System Readers and set Clarity as the current reader 6 Select System Setup to define global parameters including special Word Excel reporting options 7 Select Data
69. dialog will default to this Protocols Folder The user is permitted to modify his or her Protocols Folder 8 Click OK to save the changes and return to the Security dialog Connecting Reader s to the Computer M KC4 v3 4 M KC4 Signature Before KC4 can be used to communicate with a microplate reader the reader must be properly connected to the computer on which KC4 is installed The following briefly describes how to connect the reader to the computer For more detailed information on reader setup refer to the reader s user manual 1 Attach the appropriate cable or adapter to an available serial or USB port on the back of the computer Plug the other end of the cable to the serial or USB port on the reader Connect the power cable to the back or side of the reader Plug all power cords into grounded electrical outlets Use of a surge protector is strongly recommended 5 Use KC4 to set and test the communication parameters as described under Reader Selection in the System Menu chapter User s Guide to KC4 Installation and Configuration e 25 Where to go from here M KC4 v3 4 M KC4 Signature KC4 is a powerful software program with many features and customizable options Learn more now by following these recommendations e Go to the Getting Started chapter and Review essential information on file types plate data acquisition data reduction and more Learn about KC4 s menu structure and h
70. down list Y Representative of Patrick Walter Barmes Representative 1 Representative 2 User ba Full Name Im ary Ann Jones Password fia 5 Enter or select your User ID If Representative Of is checked two users must enter or select their User IDs Enter your Password The characters will not be visible on the screen Click Sign amp Save If the file has not yet been saved a Save As dialog will appear Current Folder DE Wones Mary AAN TE E K i Modified DMA Ouarnt AN A Quant File Mame Phenal Type rca Data Protocol glb Cancel 8 Using the folder location tools provided select the storage location 9 Enter the desired File Name and click OK The signature is applied immediately and KC4 logs the event in the Data Audit Trail select Data Audit Trail to view Data Audit Trail Date l User l Action l Comment 0819 02 16 21 18 MAJ Protocol DB WJones Mary iMNucleic AcidPhenol prt 0819 02 16 21 18 selected De a2 16 21 32 MAJ Plate read started 081 9 02 16 21 33 MAJ Sample Plate successtully read 081902 16 22 05 MAJ File Created 031902 16 22 05 MA Signed for Authorship by Mary Ann Jones User s Guide to KC4 Data Menu e 55 Append to Kinetic File KC4 provides the option to build a kinetic file using individual endpoint read plates The currently open plate can be appended to the previously read plate This feature allows plates to be read on a discont
71. e Interval Defines the time interval between individual kinetic reads The maximum valid Interval is equal to the current Run Time The minimum valid Interval depends on the current reader type and certain protocol parameters such as the number of wavelengths specified the plate format the number of wells to be read and whether or not shaking 1s selected For certain readers a Minimum button is available under the Interval box When this button 1s clicked KC4 communicates with the reader to determine the minimum kinetic interval based on the currently selected Reading parameters For all readers when the OK button is clicked KC4 automatically checks the validity of the Interval value If it is insufficient for the currently defined Reading parameters a theoretical minimum value is displayed Note For readers that support more than one read mode speed the returned value is based on the current reading mode For some instruments the read mode is set through the instrument s front panel For others it is defined as part of the protocol e Reading Number Represents the total number of kinetic reads KC4 calculates this automatically based on the currently defined Run Time and Interval The maximum number of kinetic reads is 300 e Allow Well Zoom During Read If this feature 1s enabled you can click on one or more wells in the Results dialog to view the kinetic curve s as the plate is being read KC4 updates the graph and kineti
72. e PowerReports User s Guide to KC4 Well Data A Well Data table presents measurement values for kinetic spectrum or scanning protocols e For Kinetic protocols the table lists measurement values by time of kinetic reading e For Spectrum protocols the table lists measurement values by wavelength e For Linear Scanning protocols the table lists measurement values by reading position e For Area Scanning protocols each table presents one well s data in a matrix format To create a Well Data table click the Tables button in the RSO toolbar and then highlight Well Data to open a list of available data sets Click on the data set to be represented in the table the Well Data Table Properties dialog appears cay Document in lt KC4 gt Microsoft Word Eile Edit wiew Insert Format Tools Table Window Help Mormal Times New Roman 10 B Z U Fields Plates Tables Curves x Close Statistics Well IDs Well Data Well Analysis Results Fitting Results Kinetic Spectrum and Linear Scanning protocols Well Data Table Properties Selection Data Top Left well E BottormAight Well He Cancel Help i e Define a rectangle of wells to be included in this table Note This table reports information in columns one column per well You may wish to experiment with the maximum number of columns that can display legibly per table If you want to display information for many wells 1 e more than 10 12
73. filter s to be used Define a rectangle of wells to be read 1f reading a partial plate For a kinetic protocol click the Kinetic button to define the read time and interval A A A O For an endpoint linear scanning protocol click the Scanning button to define the horizontal reading points Optional Define Shaking parameters Optional Define Temperature Control parameters Optional Select Pre Read Blank Plate Optional Select Read Barcode to instruct the instrument to scan the barcode as the plate is drawn in to the reading chamber e a 10 Optional Select Monitor Wells and then click Options to define related parameters 11 Click OK to verify the parameters and close this dialog User s Guide to KC4 Protocol Menu e 79 FL600 Reading parameters Reading Type Reader End Point FL600 on COM2 C Absorbance Fluorescence Luminescence Kinetic metic Scanning Options Filters Filter Set C ie 2 Escitatian 485 20 a 485 20 Emission 530 25 530 25 Optics Position Bottom Y Top Y Sensitivity Auto i 00 Plate Options Options Temperature Control Type se WELL PLATE Size 8x12 C Yes l FreHeating First well Al Last wel H12 e Ho Temperature IE Reading Direction Horizontal Vertical Lag Time 00 00 00 Shaking Pre Read Blank Plate eee a E dl o Monitor wells Notions fe Before even reading Before
74. first reading coc eo SAA A A A 10 11 I2 13 14 Select a Detection Method of Absorbance Fluorescence or Luminescence Select a Reading Type of Endpoint or Kinetic For a fluorescence or luminescence protocol define the Filter Sets to be used For an absorbance protocol define the Wavelengths and Optics Position to be used Select a Plate Type and choose a Reading Direction For a kinetic protocol click the Kinetic button to define the read time and interval For an endpoint or kinetic scanning protocol enable the Scanning checkbox and then click the Scanning button to define the read matrix size Optional For a spectrum protocol click the Scales button to customize the graphical data display Optional Define General Options parameters Optional Define Shaking parameters Optional Define Temperature Control parameters Optional Select Pre Read Blank Plate Optional Select Monitor Wells and then click Options to define related parameters Click OK to verify the parameters and close this dialog 80 e Protocol Menu User s Guide to KC4 FLx800 Reading p Reading arameters Type Reader alla ueno on COME iz Fluorescence f Luminescence Kinetic Kinetic Scanning Update Filters Filters Filter Set Cc s Excitatiorr 485 20 485 20 485720 456 20 Emission 528 20 528 20 528 20 520 20 i Optics Position Bottom Bottom T
75. following 1034789489 Custom Format 1 126570 84830 25150 22860 102310 61740 2 3 34990 Bio Tek 12345 Highlight the entire line click the right mouse button and select Copy Switch to the open lt plate database gt txt file Scroll to the end and place the cursor on the first available blank line Click the right mouse button and select Paste The custom plate format should appear 9 Save the lt plate database gt txt file and close it Return to KC4 10 Within the System Plate Formats dialog click Import Locate the modified lt plate database gt txt file and click Open The Plate Formats list will update to show the contents of this file Important KC4 does not verify that the imported file has a valid structure or contains valid values It is the user s responsibility to verify the structure and content of the plate formats after they have been imported 11 Click OK to return to the main view Note KC4 version 3 02 introduced a new Plate Height dimension for plate formats This 1s the distance from the bottom mounting surface of the plate to the top face of the plate If you are importing a plate database from an earlier KC4 version be sure to measure the plate height and add it via the Modify button in the Plate Formats dialog User s Guide to KC4 System Menu e 243 To import the plate database from the earlier KC4 version option 2 1 Launch KC4 v3 4 KC4 Signature 2 Select System Plate Formats and click Im
76. for details e When all values have been entered click Send Filters The values will be downloaded to the instrument overwriting its existing wavelength table 200 e System Menu User s Guide to KC4 To retrieve filter wheel settings from the instrument e Click Get Filters The values will be uploaded to KC4 overwriting the values in this dialog Special filter position requirements for the Synergy HT with Injectors model e For Read amp Dispense protocols KC4 offers an option to block the light between measurements to inhibit photo bleaching Single filter set protocols If light blocking is enabled a blocking filter Plug must be placed in the excitation filter wheel in one of the two positions next to the excitation filter that is specified in the protocol Dual filter set protocols If light blocking is enabled two blocking filters Plugs must be placed next to each other in the excitation filter wheel Reader Configuration KC4 provides probe size information for some readers via the Reader Configuration dialog Clarity Microplate Luminometer users KC4 s Reader Configuration parameters do not apply to the luminometer Reader Configuration Absorbance probe 7 Probe size 1 4 mm z Cancel Fluorescence Lumnezcence probes Hep Top Probe size E mm F Y mm Get Probe Sizes Bottom Probe size The contents of this dialog vary depending on the currently selected reader The following par
77. format Formulas are entered in cells representing the wells within the microplate To define Single Plate Transformations e For readers that support multiple plate formats ensure the correct format is selected under Protocol Reading e Ensure that identifiers have been assigned under Protocol Plate Layout e Select Protocol Single Plate Transformations Single Plate Transformations ji Init All Transf Init ranst Transf fi C Copy Current Formula Edit Well Direction Dataln z j i ata In M 620 z C Diff Between Lines Data Out toxicity C Diff Between Cols 2 3 El E 8 0 lt BLK1 BLK1 X BLK2 P lt BLK2 lt BLK2 HY PSE est Fee ese esse BLK K BLK1 lt BLK2 X BLK2 lt BLK2 PXBLK1 K BLK1 D lt BLK2 X BLK2 X BLK2 PBLK1 BLK1 PX BLK2 PX BLK2 BLK2 PBLK1 K BLK1 RBLK2 BLK BLK2 P BLK1 X BLK1 lt BLK2 X B LK2 2 B LK2 P BLK1 X BLK1 lt BLK2 X BLK2 X BLK2 r Current Formula BLKIAETAL1 BLK1 100 Place the cursor in a well and enter the desired formula The formula is visible in the Current Formula box at the bottom of the dialog The Transf field indicates the current transformation level Level 1 transformations can be passed for further manipulation into Level 2 and Level 3 transforms for successive manipu
78. in the appropriate field Shared Local or Diagnostics 19 Click Start Upgrading to begin the process When prompted enter name s for the converted databases The original databases will remain unchanged Default names contains a date and time stamp as shown below Upgrade the Shared database az Save In a K C4 Signature Y 3 3 da jt KCLocal mdb sa KCShared MDB File name KCShared_Upgraded_030815_130528 MDB Save as type Databases Cancel 20 Click Save to complete the upgrade Z When the process is complete KC4 will ask if you would like to read the Readme file Question 5 7 Would you like to read the Readme file now 16 e Installation and Configuration User s Guide to KC4 21 Click Yes or No as desired If you click No or when you exit the text editor the installation process finishes and then the Setup Complete dialog appears Setup Complete Setup has finished copying files to your computer Before vou can use the program you must restart Windows or Your Computer No will restart my computer later Remowe any disks from their dives and then click Finish to complete setup is 22 Remove the CD from the drive and click Finish to restart the computer User s Guide to KC4 Installation and Configuration e 17 Starting KC4 M KC4 v3 4 M KC4 Signature To start KC4 1 Double click on the KC4 icon on the desktop or open the Start menu and select
79. in the blank wells is subtracted from the raw data values in all wells on the plate or from the corrected data values if a Blank Plate was read KC4 requires that the same identifier be used for all blank wells The maximum number of blank wells that can be defined is limited only by the number of available wells on the plate You cannot define concentrations or dilutions for blank wells The default blank well ID is BLK and its default color is white it will print in black on reports To change the ID text and or color or to define a descriptive name for the blank wells for use on reports click the box to the right of the ID field Blank Ed BLK Plate Background WA Cancel Help e Enter an ID using 1 to 10 characters The ID can contain letters numbers and underscores It cannot contain any spaces Blank Well is valid Blank Well is not The ID cannot be identical to a formula operand X MIN MAX LOG POW SQRT CV SD and MEAN are not valid IDs If an ID is currently used in the grid its text can be changed here The change will automatically be reflected in the grid e Optional Enter a descriptive Name using up to 20 alphanumeric characters This name can be included on reports e Click on the Color box to open a drop down list showing all of the available colors Click on the desired color 144 e Protocol Menu User s Guide to KC4 Assay Control Wells Well Settings Type A
80. lt pathname gt lt filename gt gph was not found message If this is the case the protocol references a stored curve in the location specified in that message Select Protocol Curves Curve Description Under Curve Origin if File is selected click Browse to locate the appropriate gph stored curve file e The curve fit method is not compatible with the standards The curve could not be calculated Every curve fit requires a minimum number of valid standards there may not be enough valid standards to support the current curve fit Select Protocol Plate Layout to add more standard wells or select Protocol Curves to choose another curve fit and then recalculate results e The curve is not strictly increasing nor strictly decreasing The curve is ambiguous more than one X value was calculated for a particular Y value ex more than one concentration was calculated for at least one OD FU LU User s Guide to KC4 Display Menu e 191 e There must be at least two valid standards The plate currently contains only one valid standard and at least two are required to calculate the curve Check to see if any of the standard wells is currently masked or contains an out of range or another non numeric notation e Values out of range Log Axis cannot be calculated KC4 was unable to calculate the curve because one or both Axes is set to calculate logarithmically and an input value such as a standard OD 1s out of range Select Protocol Cur
81. lt x gt The minimum of the defined variables The maximum of the defined variables Example CTL1 0 100 BLK 0 010 ETA Sun ag CALLAS TAO A1 B1 MEAN POS1 SD BLK CM ob LOG SPL10 POW STDTA33 SORT A1 B1 MIN EV STCL CV CTL2 MAX A1 B1 C1 User s Guide to KC4 Defining Symbols Symbols are assigned to wells after cutoff evaluation After plate reading open the Symbols results data set to see the assigned symbols as well as the calculated cutoff values The number of available Symbols fields exceeds the number of defined cutoffs by one During data reduction if the value of a well is less than the first cutoff the first symbol 1s assigned to the well If the value of a well is greater than or equal to the first cutoff but less than the second cutoff the second symbol is assigned to the well and so on See Results Symbols later in this manual for more information Each Symbol can contain up to 8 alphanumeric characters Common symbols include NEG EQV POS The appearance of each symbol can be customized by clicking 1ts corresponding Font button Symbols Font Font style iria A egular Allegra BT F Amer ype Md BT Aral Black FT Arial Narrow FT AvantGarde Bk BT T AvantGarde Md BT E frects M Strikeout F Underline J AaBbYyZz Color sal MM Back westen This i a TrueType font This same font will be used on both pour screen and your printer e
82. mas nms a aD a a ams m ATS mo 127 45 171 63 22290 ENE 24 576 55 376 66 491 78 302 6639 232550 85 117 86 144 27 128 16 694 29306 13 463 e Values outside the range of the standard curve are reported as lt lowest conc or as gt highest conc where the lowest conc and highest conc represent the minimum and maximum concentrations plotted on the standard curve respectively Note These two values may be different than the min max standard concentrations if an extrapolation factor is applied See System Menu Setup Format for information on using out of range values in statistical calculations e To calculate values outside the normal limits of the curve select Protocol Curves Axes to increase the pon Factor reason this may occur is 1f the value exceeds the limits of the standard curve 254 e Results User s Guide to KC4 e Select Display Curve s or click the Biograph toolbar button to view curves Biograph R BIOGRAPH Windows Application STANDARD CURVE File Edit View Graph Window About O ia Mal el e e ar BS STANDARD CURVE mu 4 Parameters y a di nic b d a 2 485 b 1 32b c 49 10 d 0 07268 A 0 9998 R 0 9996 er 0 01 452 User s Guide to KC4 Results e 255 Symbols If Cutoffs are defined in the protocol the specified set of data is evaluated against the cutoff criteria and each well is assigned a user defined Symbol To view the assigned symbols select the Symbols dat
83. may not be authorized to do so User s Guide to KC4 System Menu e 227 The Select a Folder dialog will appear Select a folder Folders eked va nucleic acid assay files foo och gt ECA V3 B Nucleic Acid Assay Files Drives E c Network J Delete files after extraction Select the target Drive and Folder either the local hard drive or a network drive 4 Optional Check Delete files after extraction to permanently remove files from the database after they are exported 5 Click OK to continue Files are exported in their proprietary formats Files can be opened by a KC4 program that does not use the shared database or they can be imported pasted from the clipboard into another shared database If files were deleted after export the System Administrator should do the following to compact the database 1 Ensure that no KC4 users are currently logged into the shared database 2 Select System Setup Database 3 Inthe Shared Database area click Repair Compact and then click OK When the process is complete the database should be smaller in size Importing Files Protocol plate data and global data files residing outside the shared database can be imported into the database through the Maintain Files dialog This includes any unsaved files that were automatically saved to a temporary location following a database connection failure To import a file 1 Select System Maintain Fi
84. measurements with up to six uniquely defined Filter Sets There are many applications for multiple filter sets including e Assays that have more than one fluorophore in the well e Comparison of results between the top and bottom optics positions e Comparison of results between rapid and static mode e Comparison of results with varying static mode settings e Comparison of results with varying sensitivity settings To define Filter Sets e Ensure the protocol is appropriately set for Absorbance Fluorescence or Luminescence detection method e Select the Filter Set radio button corresponding with the number of sets required e Define the wavelengths Absorbance detection method For each filter set use the drop down lists to select the desired wavelength Fluorescence detection method For each filter set use the drop down lists to select the Excitation and Emission wavelengths User s Guide to KC4 Protocol Menu e 95 Luminescence detection method For maximum sensitivity select Lum E to indicate an empty position in the filter wheel and a plug in position E in the excitation wheel For each filter set use the drop down lists to select the Emission wavelength e Note To prevent damage to the reader KC4 performs bandwidth verification as part of the validation within the Reading Parameters dialog e The instrument may be equipped with one or two optical probes positioned above Top and or below Bottom the
85. must be considered as examples provided for demonstration and guidance purposes only If you plan to use these protocols or similar ones in a real application it is your responsibility to validate the protocol parameters including the Excel sheet content if PowerReports is used before using them e The SAMPLES folder in the KC4 root folder also contains a Sample Protocol Descriptions doc file describing the protocols e Files with the extension PRT represent protocol files e Files with the extension PLA represent plate data files e Files with the extension GLB represent global data files containing plate data and protocol parameters e Open protocol and plate data files with similar names such as Quantitative prt and Quantitative pla Note Not every PRT file has a corresponding PLA file 40 e Getting Started User s Guide to KC4 Data Menu Overview The Data Menu contains functions for acquiring processing and storing plate data This chapter covers the following topics Creating opening saving and closing data files Reading plates Entering plate data manually Retrieving plate data from an external file Applying electronic signatures to global data files Appending a new set of plate data to an existing kinetic file Exporting plate data Printing and previewing reports Entering descriptive information for the plate Entering sample names Viewing the data file s audit trail User s Guide to KC4
86. name is entered KC4 will provide a warning to avoid an accidental overwrite Click Yes to continue with the overwrite or No to rename the file before saving to open it and view its contents Closing Protocol Files To close a protocol file select Protocol Close If the protocol is newly created or 1f modifications were made to an existing protocol KC4 provides an option to save DB Jones Man 4 Nuclerc Acids Olgo Quant prt already exists A Do you want to replace it e Click Yes to save the new or modified information and close the protocol e Click No to close the protocol without saving changes e Click Cancel to close this dialog and return to the open protocol The new or modified information is retained but not yet saved 72 e Protocol Menu User s Guide to KC4 Signing Protocol Files KC4 Signature provides an option for users to permanently attach or sign their names to protocol files Note KC4 only supports file signatures when the multi user identification feature is in use See Security in System Menu for more information To sign a protocol file 1 Open the desired file af it is not already open 2 Select Protocol Sign The New Protocol Signature dialog will appear New Protocol Signature Reason Authorship Representative of User Identification a a User MA Full Mame Ju ary Ann Jones Password SY Cancel Help 3 Click the Reason drop down arrow and s
87. of Bio Tek Instruments Inc Bio Cell is a trademark of Bio Tek Instruments and Bio Cell is patented under U S patent number 5 963 318 Microsoft is a registered trademark and Windows Windows 95 98 NT 2000 XP Word and Excel are trademarks of Microsoft Corporation All other trademarks are the property of their respective holders Restrictions and Liabilities Limited Software Warranty Information in this document is subject to change and does not represent a commitment by Bio Tek Instruments Inc Changes made to the information in this document will be incorporated in new editions of the publication No responsibility is assumed by Bio Tek for the use or reliability of software or equipment that is not supplied by Bio Tek or its affiliated dealers Bio Tek Instruments Inc Bio Tek warrants for a period of ninety 90 days after the date of delivery that all software manufactured and sold by Bio Tek the Software will conform as to all substantial operational features to Bio Tek s then current published specifications meaning those specifications in effect as of the date of purchase and will be free of defects which in Bio Tek s sole judgment substantially affect system performance This Warranty is limited to the original purchaser the Purchaser and cannot be assigned or transferred All claims under this Limited Warranty must be made in writing or fax to Bio Tek attention Service Department
88. op Costar 96 UW Costar 96 well dish Export Costar 96 well half area Costar 96 white opaque Donatech 96 Iramularn flat bos Import crea eeo The Plate Formats dialog lists the currently defined plate types To add a new plate click New To modify a plate highlight the plate name and click Modify Warning Modifying the dimensions of the installed plate types 1s not recommended consider adding a new plate type instead Please contact Bio Tek with any questions regarding the current dimensions To view the format information for any plate highlight the plate name and click View To delete a plate highlight the plate name and click Delete To export plate format information to a text file click Export The export function 1s useful if you wish to add custom plate formats from earlier KC4 versions See Exporting Importing Plate Formats for more information To import the contents of an external plate database file either a txt or a pld file click Import The import function 1s useful if you wish to work with custom plate formats created with an earlier KC4 version See Exporting Importing Plate Formats for more information 240 e System Menu User s Guide to KC4 Plate Description When creating or modifying a plate information is entered in the Plate Description dialog Plate Description x Name Coming E well Catalogue 420343 Manufacturer Coming 126570 um Top Left s
89. performed when a protocol specifies Pre Read Blank Plate Pathlength Correction with Bio Cell and the plate is configured with blank wells This example is for explanation only the typical application does not use all three raw data correction options Processing steps 1 The Blank Plate is read at every wavelength defined in the Reading parameters dialog 2 The Bio Cell is read at 977 nm and 900 nm 3 The Sample Plate is read at 977 nm and 900 nm 4 The Sample Plate is read at every wavelength defined in the Reading parameters dialog Calculation steps performed for each wavelength defined in the Reading parameters dialog 1 The basis of calculations begins with the raw Sample Plate readings 2 The Blank Plate readings are subtracted from the Sample Plate readings The result of this calculation becomes the basis for step 3 3 The mean of the blank wells is calculated and subtracted from every well on the plate The result of this calculation becomes the basis for step 4 4 The value in each well is multiplied by the difference in readings of the Bio Cell at 977 nm and 900 nm The result of this calculation becomes the basis for step 5 5 The value in each well is divided by the difference in readings of the Sample Plate at 977 nm and 900 nm The result of this calculation is the final corrected value 128 e Protocol Menu User s Guide to KC4 The image below shows the data sets available following a three wavelength read with
90. select the Emission wavelength e Note To prevent damage to the reader KC4 performs bandwidth verification as part of the validation within the Reading Parameters dialog User s Guide to KC4 Protocol Menu e 97 e The instrument may be equipped with one or two optical probes positioned above Top and or below Bottom the assay plate Specify the Optics Position to be used for each filter set Tips Evaluate the proper reading position by specifying two or more filter sets one each for the Top and Bottom optics positions The top position is advantageous for homogeneous solutions using solid black plates The bottom position is advantageous for cellular assays and assays requiring a covered plate to maintain sterility The recommended Optics Position for Luminescence readings 1s Top e Specify the Sensitivity setting for each individual filter set Valid settings are 0 and integers from 25 to 255 Auto indicates that the Auto Sensitivity Adjustment feature has been enabled Note With Dispense protocols if two filter sets are defined the field for the second filter set always shows Auto and will use the first filter set s sensitivity The signal from the range of fluorescence and luminescence assays can be very weak very strong or anywhere in between For each assay the sensitivity of the photomultiplier PMT should be adjusted to ensure that the signals from all wells fall within the appropriate dyna
91. smaller of the two bandwidths Ifthe Time Resolved option is enabled the determination of BW maximum and BW minimum must be made by comparing the emission bandwidth to twice the excitation bandwidth Once again this accounts for the triangular shape of the transmission profile when using the monochromator If the absolute value of the difference in the excitation and emission center wavelengths 1s greater than the sum of the maximum bandwidth and half the minimum bandwidth then no proximity condition exists If itis less than or equal to then a proximity condition does exist e When all of the reading parameters have been defined and the user attempts to validate them by clicking OK to KC4 evaluates each filter set pair against the proximity formula Ifthe result is such that there is no proximity condition for any of the defined filter set pairs and there are no other parameter definition violations KC4 will allow you to OK out of the Reading Parameters dialog Ifthe result is such that there is proximity condition for any filter set pair KC4 will display a message resembling the following Warning the bandpasses for filter set 2 are too close However KC4 does not prohibit you from saving and running protocols that produce proximity warning messages User s Guide to KC4 Protocol Menu e 93 Detection Method Detection method Absorbance Fluorescence C Luminescence Multithode Certain readers such as
92. such as Pathlengths and the resulting data set will be available from the Data drop down list To learn how to do this access KC4 s help system contents and select Results gt Data Sets gt Pathlengths Click on Click here for more information on reporting the pathlength for each well User s Guide to KC4 Results e 257 Kinetic Data KC4 provides several options for viewing kinetic data The default view is Kinetic Curves a RESULTS Data Kinetic CUES me 3 E 7 E g 10 11 12 Kinetic curs eee E SA EA A AF Coett 107 Mint 00 00 00 Min y 1 0000 Scales Calculations Mast 00 05 00 Mas y 4 9740 am 258 e Results Each well displays a graph of the measurement values taken over time Hold down the Ctrl key and click on up to 8 wells to overlay the kinetic curves in one display Click the Scales button to adjust the data points displayed for each well The current scaling parameters are shown in the lower left corner of the Kinetic Curves display Click the Calculations button to alter the kinetic calculation zone for all wells Click on a well to enlarge the view of its kinetic curve m Well El A8 well E1 A8 1000 1500 2000 2500 3000 Time s Selected Curve m 405 E1 gt Options Recalculate Max Y 148 00 mOD mn Delta t at Max 00 11 15 6 ees M 405 0 982 Coeff Reg 1 000 Lag Time 00 06 31 T 00 12 30 User s Guide to KC4 This dialog can display up to 8 overlaid
93. system requirements 9 2 Install KC4 on one computer and restart the computer 11 3 Start KC4 and log in as the System Administrator 18 6 e Installation and Configuration User s Guide to KC4 Special note for Clarity Microplate Luminometer users The Clarity PC software must also be installed on the PC s Install Clarity before performing the step Connect reader s to the computer and establish communication KC4 Signature Single PC with one or more users Step Task Page 21 5 Change the System Administrator s password 6 Copy KCShared mdb to a secure network location 22 7 Configure KC4 to use the KCShared mdb file on the network 23 8 Create one or more new user accounts 24 9 Connect reader s to the computer and establish communication 25 KC4 Signature Multiple PCs in a network environment Step Task Page l Appoint a System Administrator 8 2 Ensure that all computers meet the minimum system requirements 9 3 Install KC4 on one computer and restart the computer 11 4 Start KC4 and log in as the System Administrator Change the System Administrator s password 18 5 21 23 24 User s Guide to KC4 Installation and Configuration e 7 Appointing a System Administrator M KC4 Signature Before installing the software one individual at the site must be appointed as the KC4 System Administrator The System Administrator is typically responsible for the following Determining which computers will h
94. the Start and Stop values in the Protocol Reading Parameters dialog Formula From and To allow the specification of a range of read numbers upon which to perform calculations The default values correspond with the total number of wavelengths to be read based on the Start Stop and Step values in the Protocol Reading Parameters dialog Example Start Stop Step values set to 400 600 10 respectively The default From and To values are 1 and 21 indicating a calculation zone from 400 600 nm Setting From to 2 and To to 18 would change the calculation zone to 410 570 nm Mean Min Max specifies the number of successive wavelength readings to be averaged prior to the determination of the Minimum and Maximum OD values Check the Formula radio button to define a formula to structure calculations by a formula Enter the formula into the long field Example The formula W1 W2 W3 calculates the sum of the ODs for the readings at wavelengths 1 2 and 3 If the plate is read from 400 to 410 nm in 2 nm increments W1 will correspond with the OD at 400 nm W2 with the OD at 402 nm and W3 with the OD at 404 nm Enter a Result Name in which to store the formula results This name can be used as a Data In variable in Transformations Mean Min Max specifies the number of successive wavelength readings to be averaged prior to the determination of the Minimum and Maximum OD values 138 e Protocol Menu User s Guide to KC4 Scannin
95. the calculation type Select the data set for spectrum analysis as well as the calculation type Select the calculation type and related options for linear scanning analysis Configure the layout names and colors of blanks standards controls and samples Define expected concentrations or dilutions Define and apply formulas to one or more wells within the plate Define calculations between different sets of plate data Define curve fitting parameters Define the criteria used to assess the validity of the results or the suitability of calibrators for inclusion in subsequent calculations Define the conditions used to screen selected data into defined categories based on fixed or variable threshold limits Customize the contents and appearance of the report Customize the contents and format of the data export file View the contents of the Protocol Audit Trail Administrator and Power Users Specify which protocol and plate specific functions if any shall be protected from use by Standard Users Select automated functions Choose the reporting and exporting engines Enable the protocol audit trail function Select Advanced to access special protocol menu options Plate Description Raw Data Correction Multi Plate Transformations Validation and Cutoffs User s Guide to KC4 Display Menu The Display Menu provides access to the Biograph module and also displays the results of the Validation criteria define
96. the mean of the wells containing the identifier CTRL1 minus three Standard Deviations of these wells It is the equivalent of MEAN CTRL 3 SD CTRL X A5 A6 2 The result of this formula is the sum of the current well value and the mean of the A5 and A6 wells LOG X MEAN POS1 The result of this formula will log the ratio of the current well over the mean of POSI MAX A1 A2 A3 MIN B1 B2 B3 This formula calculates the difference between the maximum value of Al A2 A3 and minimum value of B1 B2 B3 SPLn BLKn CTRLn BLKn This formula can be used for toxicology assays to calculate toxicity percentages User s Guide to KC4 Protocol Menu e 161 Multi Plate Transformations TransF KC4 provides for up to three levels of Multi Plate Transformations The three different levels of transformations may be interdependent or completely independent of each other In addition the results of any Single Plate Transformations can be incorporated into a Multi Plate Transformation To define Multi Plate Transformations e Ensure that all protocol parameters that have an effect on data reduction have been defined e Select Protocol Multi Plate Transformations Multi Plate Transformation Transf H fi mu Kinetic Data In Xi Dataln Mt 405 fie Reading t Al H 42 Dataln M 2 630 fe Reading fan H eo Data Ir Eile Reading Data Out Formula x1 Ae Ex 2 41 443
97. the suitability of select wells If the criteria are not met KC4 generates a warning to indicate that results are suspect Protocol Validation Define Cutoffs to screen selected data into defined categories based on fixed or bs variable threshold limits During data reduction results are evaluated against the C Fs cutoff criteria and each well is assigned a user defined symbol Protocol Cutoffs els Walidati uko Create a report template The appearance of the button depends on the Report ag Engine setting under Protocol Options Excel Word or KC4 The template is stored with the protocol when it is saved Protocol Report Create an export template The appearance of the button depends on the Export Engine setting under Protocol Options Excel Word or KC4 The template is T stored with the protocol when it is saved Protocol Export Aid Define various protocol options including the report and export engines and hate automatic save append report and export procedures Protocol Options fa Specify which functions in the currently open protocol shall be protected from use So by unauthorized users Protocol Security Open the Maintain Files dialog to create folders move files delete files and so on within the shared database System Maintain Files Launch the Biograph application to view the calculated curve s This button 1s accessible only 1f results are displayed and a curve has been successfully g
98. to change it as soon as possible 7 Optional Specify a folder location from which the user will most often access protocol files When the user selects Data New Plate to initiate a plate read the protocol selection dialog will default to this Protocols Folder 8 Click OK to save the changes and return to the Security dialog User s Guide to KC4 System Menu e 213 Modifying User Accounts Standard and Power Users Standard Users and Power Users are authorized to modify the Password and Protocols Folder fields within their own accounts The ID Full Name Group Membership and Status parameters can only be modified by the System Administrator To modify your user account begin by logging into KC4 with your user ID and Password Once logged in select System Security highlight your name and click Edit The KC4 User Settings dialog will appear Security eee x Users Login Protected functions Audit Trail Signature Reasons Files M Mutter identification E Administrator System Administrator tl Mary Ann Jones KC4 User Settings E ID Ma Full Marne Mary Ann Jones e Group Membership Password Standard User l Current password Can not have access to Protected Functions Power User New password at hare access to iotectea Functions but ath hob aainttnstiate Users Administrator Hss complete access to allunctone Status a including Wee eomintetration 0 l ER ked cut z Protocols Fold
99. to include out of range values in statistics calculations Raw Data Report Export Calculation Weighting of Standard Deviation N C H T Use out of range values in statistics e Select an option for the Weighting of Standard Deviation N 1 Used in calculations where the data is assumed to represent a sample from a larger population N Used in calculations where the data is assumed to represent the total population from which the sample was taken e Choose whether or not to use out of range values in statistics Out of range values are those values reported as lt xxxx or as gt xxxx Example SPL1 has three replicates reported with the following concentrations well Al 10 230 well A2 lt 10 out of range value well A3 10 450 If the option is not selected the software will not use the concentration of well A2 in the Statistic calculations Therefore the mean standard deviation and CV of SPL1 will be 10 34 0 155 and 1 5 respectively If the option is selected concentration of well A2 is taken into account the value of 10 is used in the statistic calculations Therefore the mean standard deviation and CV of SPL1 will be reported as lt 10 22 0 225 and 2 2 respectively Important Exercise caution when enabling this option as it can lead to biased results particularly for standard deviation and CV As a warning statistic results arising out of the use of this option are displayed in italic in the r
100. to reinstate the checkmark 268 e Biograph User s Guide to KC4 Graph Menu Use the Graph Menu to view and or modify certain characteristics of the current curve Standards Use the Standards dialog to view enter or modify the x and y values of standards Note When Biograph is launched from KC4 these values cannot be modified Standards Ea Std Std std otd4 std St db std tdg 4 hask E E SD 1 E E 4 a 10 22025 0 143543 2 101 2 304 20 2069 0 203647 2213 1 325 a0 1 5575 0 0162635 1 543 1 526 40 0 8605 0 226951 0 7 1 021 50 0 408 0 177464 0 305 0 556 ED O2405 010530353 0 203 0 270 Cancel E s The abscissa values are listed in the x column These are typically the standard concentration values The ordinate values are listed in the y columns These are typically the measurement values OD RFU RLU You can enter up to 12 y values replicates per standard The M y column calculates the mean for each row of y values Note The curve is calculated based on the mean y values not on the individual replicate values The SD y column calculates the standard deviation for each row of y values To mask a y value check the Mask box then click on the desired y value It will temporarily change to To unmask a y value keep the Mask box checked and click on the desired It will change back to its original value Click OK to display the curve Axes Fit Interpolations
101. tu taniebesctalee asia 59 IPI ys Fahri eect a ace ct einen each at aera a acen ch anette aah charac aes secon ch aetna ace tn a uaneatines saan 59 PUMPS VIS Wy iscsi A i iG wears areas dee es teen ences 59 PENES Sl 2 EEE E E T E E EE E E INEOS 59 ii e Contents User s Guide to KC4 Plate Des capta 60 A A II E EA 61 AMO NUDE sao EE E EEEE R S 62 Mport Sample Name Sienn a E EN A ATA 62 Expor Sample NM cee cee 62 Printing Sample IDs ieor E E E E ee 62 Daa Aadi Trasses T TA A TS 63 EDIT MENU 0 ide 65 Edita TOOS ar rte re err N oO Te no 65 PROTOCOL MEN U ios adan aR ha coacalco tases 67 ONEIN o CIP TI ET er tors fern ne OARS re POR ae ee EP E 67 Workine With POCO COIS ssc cer earns tte E EEEIEE NONNE ial a aiken 68 Where and How Are Protocols Stored oooccccccccccccccncnnnncnnnnnnnnnnnnnnnnnnnnnnnnnnnnininin ss 68 Opening Existing Protocol les A RE 68 Creatine NG Wek GOtOC Ol aia 69 Modityine Protocol Param Cters AAA 71 Davin Protocol PIES essai bets e A A 71 Closino Protocol FES A A sade ie aia AS 72 O ProrocoL F IES iaa 73 AI E ean hence tensa 75 Detimine Reading Parameters siemens scons ekctucn i o e ed ed 76 e E E E E E E A tue se E E T E ele T gi ELISA epee e ee E E SO 78 E E N E ee 79 GOO os ars annie Sasi a a O a a a 80 A IA 81 Power Wave MPower Wy ave dd 82 Power Wave XAM nl O sor 83 TOWLE WV VS A M Sele linorna td 84 POWER WaVe Sala 85 VT y H eaa aS A 86 Syner M HAT TIME RESO Ce eaen E oad aacuaredsonti
102. valid password Valid entry range 2 to 10 characters Specify the number of days that a password may be used before it KC4 requires the user to change it If a user allows a password to expire his or her account will become locked Only the System Administrator is permitted to reinstate locked user accounts Valid entry range 1 to 10000 days Specify the number of days in advance of the password expiration date that KC4 should alert the user to the upcoming expiration Valid entry range 1 to the Password Expiration value or 30 whichever is smaller Specify the number of unique passwords that KC4 shall remember for each user s account to prevent that user from defining a recently used password Valid entry range 2 to 20 passwords Click OK to save the changes and close this dialog User s Guide to KC4 System Menu e 217 Protected Functions Select System Security Protected Functions to access a list of functions which the System Administrator can protect from use by unauthorized users Note The picture below shows all system level protectable functions it may or may not represent your present KC4 configuration KC4 Signature users The functions shown as protected below must be protected to ensure 21 CFR Part 11 compliance Security x Users Login Protected functions Audit Trail Signature Reasons Files Protected functions Le Functions only available to AdmiristratorPower Users Modify Reader Setting
103. 0 SPL1 SPL3 SPL5 SPL7 SPL9 B STD2420 000 SPELI GELS eer hoe SPL SELO C STD3 30 000 SPL2 SPL4 SPL6 SPL8 SPL10 D STD4 40 000 SPL2 SPL4 SPL6 SPL8 SPL10 Plate M 405 IZRA 56 A 0 006 2 818 0 967 4 044 2 925 2 399 B 0 751 4 480 4 114 3 733 0 871 4 295 C 1 532 2 568 1 520 0 075 0 457 1 822 D 2 737 0 829 4 943 2 228 0 595 0 023 Plate Concentrations peo Ss pay eo A 11 065 gt 41 500 21 774 gt 41 500 gt 41 500 37 731 B 19 367 gt 41 500 gt 41 500 gt 41 500 20 704 gt 41 500 C 28 070 39 614 27 936 11 834 16 091 31 302 D 41 498 20 236 gt 41 500 35 826 17 629 11 255 Plate Symbols AS AIO A NEG OUT EQV OUT OUT POS B EQV OUT OUT OUT EQV OUT C POS POS EQV NEG NEG POS D POS HOV OUT POS NEG NEG 58 e Data Menu User s Guide to KC4 Printing Reports After plate measurement data has been acquired and calculations are complete KC4 can generate a user customizable set of results reports Reporting options are selected and customized within each protocol by selecting Protocol Reports Print Select Data Print or click the appropriate toolbar icon to print the reports selected in the protocol Print Preview oat ae at i a Preview Preview Proview Select Data Print Preview to see what the report will look like before it is printed Print Setup To change the printer paper size or source or page orientation select Data Print Setup to access a standard Print Set
104. 33 e Tip With some operating systems KCShared mdb is installed in the Application Data folder under Documents and Settings All Users If you are looking for the Application Data folder on your computer and can t find it 1t is probably hidden To reveal this folder open Microsoft Explorer and select Tools Folder Options View Make sure that Show hidden files and folders is enabled To change the shared database Path 1 Click Browse A standard file open dialog will appear 2 Locate the desired shared database file and click Open Control will return to the Database dialog and the Path field will fill in with the new name 3 Click OK A confirmatory message will appear stating that the database change will not take effect until KC4 1s closed down and relaunched Modify system setup is a protectable function If you have tried without success to change the Path you may not be authorized to do so Contact your KC4 System Administrator 4 Click Yes to close KC4 5 Multi user sites Repeat these steps for all computers that use the shared database The Restore Connection button becomes active after a critical database error is detected if the database path did not change This button must be clicked after a critical database error occurs For example if the network connection drops while KC4 is open a critical database error can occur because the shared database is no longer accessible When the network connectio
105. 4 and so on e Click the Clear button to delete all values from all cells e When all of the dilution values have been entered click OK to return to the Plate Layout dialog Assigning Dilutions There are different techniques for assigning dilutions You may wish to experiment with the software to get comfortable with assigning Replicates choosing neither one or both Auto Select options and toggling the Horizontal Vertical filling options It might also be helpful to sketch your plate map on a piece of paper to use as a reference To assign dilutions with Well IDs e Select the necessary Well Settings Type and ID then choose the first dilution value to be assigned e Enter the number of dilution Replicates per control or sample e Choose the dilution replicate filling direction Horizontal or Vertical e To advance well IDs and or dilution values automatically choose Next ID if available and or Next Dil e Choose the dilution group filling direction Horizontal or Vertical e Highlight the well s to assign dilutions and well IDs Examples are provided on the following pages 152 e Protocol Menu User s Guide to KC4 Plate Layout Wall Settings Type assay Control ID Emi apl Dil kwa Jd CTRLA ETRLA 4 0000 8 0000 16 000 Plate Layout Auto Select F Nesii I Next Cil l e Filing C Horz e Wert NETAS Wall Settings Type Assay Control 2 ID CTRL2
106. Add gt gt e To remove an item from the list highlight the item then click lt lt Remove e Click OK to close the dialog and insert the object into the document 304 e PowerReports User s Guide to KC4 Ed Microsoft Excel Worksheet in lt KC4 gt File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves x Close KO Ms AAA AA ESOO EGSA ENE A Meas OD HG Meas OD H12 Wavelength mm 8 9 10 11 14 15 16 me 8 TI aa 20 Sheet1 Ready II A A Nu IS ES O KC4 generates temporary curve s using fictitious data After the plate is read KC4 rebuilds the curve s with real plate data Certain characteristics of this curve can be modified including colors and labels Double click anywhere on the graph to enter the editing mode a shaded box should appear around the graph When this box appears click the right mouse button once to open a menu of items that can be edited User s Guide to KC4 PowerReports e 305 Previewing and Printing the Report When plate reading and data reduction are complete the report 1s ready for formatting and then previewing and or printing To format the report that is to fill all of the objects in with current plate data select Data Print Preview or Data Print or click the appropriate toolbar button During the formatting process KC4 displays a progress bar as shown below Ls ECA Brev16_prt
107. C4 automatically updates the Data Audit Trail to reflect the action Data Audit Trail Date User Action Comment 052002 17 02 47 JonesR Plate read started 08 20 02 17 02 48 Jonesk Sample Plate successfully read 08 2002 17 02 59 Jonesk Change Yalue Well 41 M 405 Old 0 362 Mewe 0 000 adjusted value in blank well ss To mask a well within any data file Open the desired file 2 Within the Results window click the Data drop down arrow and select the desired data set Note When a well is masked KC4 excludes its raw measurement value from further calculations 3 Click the Mask box The System Administrator can protect the use of the Mask function If you are trying without success to use this function you may not be authorized to use it 4 Click on a well in the grid to mask it click again to unmask When the well s have been masked click OK to recalculate results User s Guide to KC4 Data Menu e 45 KC4 automatically updates the Data Audit Trail to reflect the action Data Audit Trail Date User Action Comment i 082002 170837 Jonesk Plate read started 08 2002 17 08 38 Jones Sample Plate successfully read 082002170857 Jones Mask Value Well El M 405 masked standard outlier STD4 1 seo To modify protocol parameters within a GLB file 1 Open the desired file 2 Use the Protocol menu to select a parameter category to modify such as Plate Layout Single
108. CFR Part 11 compliant sites setting this parameter to glb will help to streamline the file signing process 222 e System Menu User s Guide to KC4 Login LogOut During typical use with KC4 v3 4 individual users do not log into the software A System Administrator can optionally log into change the Administrator password specify protected functions and specify a preferred data file format During typical use with KC4 Signature users must log into KC4 to gain access Each user 1s identified by a short User ID a longer Full Name and a 2 to 10 character Password see User Accounts on page 212 for more information In addition the System Administrator logs in to set up and maintain user accounts and to customize security options To log into KC4 1 Select System LogIn or System Administrator LogIn The Login dialog will appear Please enter your login information User k Administrator Full Name System Administrator Password Quikca Help With KC4 Signature the Login dialog also appears each time KC4 is launched 2 Click the User drop down arrow and select your User ID Ifthe User field does not have a drop down arrow type the ID directly into the field Type your Password For security purposes it will appear as asterisks on the screen Click OK KC4 will determine whether or not the User ID Password combination is valid and will check to see if your account is locked for any reason If
109. Click on the Color box to open a drop down list showing all of the available colors Click on the desired color User s Guide to KC4 Protocol Menu e 145 Sample Wells Well Settings Type Sample ho H ID SPLi Dil None z m Sample wells contain the unknown sample solutions to be processed Dilutions or concentrations can be optionally assigned Unique IDs should be used to distinguish between the different sample groups The maximum number of unique sample well IDs that can be defined is limited only by the number of available wells on the plate The default ID prefix is SPL and the default IDs are SPL1 SPL2 SPL3 etc The default color for all IDs is black To change the ID prefix and or colors click the box to the right of the ID field Sample ID pretis JSPL e Enter an ID Prefix using 1 to 7 characters The prefix can contain letters numbers and underscores It cannot contain any spaces and it cannot end in a number If the prefix is currently used in the grid its text can be changed here The change will automatically be reflected in the grid e For each group number click on the Color Sequence box to open a drop down list showing all of the available colors Click on the desired color KC4 uses the first color for SPL1 then the second for SPL2 and so on It restarts with the first color when the whole list of colors has been used To remove the last color of the current se
110. Control T Blank W Sample T Sample Control Index al From fi to fro Cancel Help Select the Identifier s to be represented in the table If Sample and or Assay Control 1s selected Index is by default set to All meaning all sample and or assay control wells will be included in the table To include a subset of all sample and or assay control wells use the Index From and To fields to enter the limiting group numbers In the example above Sample groups 1 through 10 SPL1 to SPL10 will be represented in the table Click the Data tab to customize the contents of the table Ed Microsoft Excel OLE Stat Document in Worksheet in lt KC4 gt A File Edt View Window Help 4 23 2 Statistic Table Properties Selection Data bts Operator Format Precision 2 Ca w O e S Ce wo O 7 Cs mo ech a C Coneomatons oem O a we M2 B30 Corr Delta On Concentrations User s Guide to KC4 PowerReports e 287 KC4 presents some default Data sets for inclusion in the table although any available data set s can be selected To add a data set highlight the first empty Data field and click the left mouse button once A list of available data sets will appear click on the desired set to select it To change an existing data set highlight 1t and click the left mouse button once A list of available data sets will appear click on the desired data set to select it To delete an existing
111. Created 068 2802 15 2424 KEO Reading parameters changed Changed wavelength value from 405 to 340 nm Changed Plate Type to Corning 96 06 25 02 15 25 31 KSO Plate Layout changed Assigned samples in duplicate 06 25 02 15 26 11 KSO Curves changed Set x axis title to Lactate Conc ugin and y axis title to Absorbance 068 2802 15 26 50 KSO Report changed Added standard curve to report template 06 25 02 15 27 15 KSO Options changed Changed Report Engine to Word 06 25 02 15 2746 KSO Report changed Re created the report template in Word ses e Events are sorted by date time stamp and include the ID of the user logged in at the time the action taken and any comments that were entered by the user when the action was performed e The contents of the protocol audit trail can be copied to the Clipboard by highlighting the desired cells and pressing Ctrl C e The contents of the protocol audit trail cannot be edited or deleted User s Guide to KC4 Protocol Menu e 183 Security KC4 provides the ability for the Administrator to protect the use of certain protocol data functions by unauthorized users To view or modify the protected functions associated with a particular protocol select Protocol Security advanced function Security Protected functions je Functions only available to Administrator Power Users Re read plate ue Mask a value e Change a value Edt Protocol Parameters Edit Plate Information
112. Dil 2 0000 Y te Hor Plate Layout Well Settings Type assay Control 2 l ID Jeria Dil 2 0000 f Ao CTRL CTRL2 CTRL2 CTRL2 CTRL2 CTRL 2 0000 4 0000 5 0000 16 000 32 000 64 000 Er ET E Ar A F Hest IM Nest Dil Filling C Horz fe Vert E uto Select TF Hesi I Next Dil Filling C Horz fe Vert e Dilution values are defined as 2 000 4 000 8 000 16 000 32 000 and 64 000 e The Auto Select option Next Dilution is selected The Next ID option is not available for Assay Control groups they must be incremented manually e Wells Al through A6 are highlighted to assign CTRL1 and its dilutions CTRL2 is then selected and assigned to wells B1 through B6 User s Guide to KC4 Protocol Menu e 153 Plate Layout Well Settings n o aaa ae See Type Sample 1 a Hb 3 T W NextID I Next Dil ID sPL1 le Jus f Hor C Vert p Filing Dil 10 000 d d C Hor Wert A ICA OEI PEI CI CI IC A CI CI JC E SPL1 SPL1 SPL1 SPL2 SPL2 SPL SPL3 SPL3 SPL3 SPL4 SPL4 SPL4 10 000 10 000 10 000 10 000 10000 10 000 10 000 19 000 10 000 10 000 10 000 10 000 9 SPL4 2U WOU E SPL4 30 000 30 000 3 i a a 30 000 30 000 PL1 gt SPL2 SPL pa SPLS SPLS SPLA PL4 SPL4 SPLE f 40 000 40 000 40 000 40 000 40 000 0 000 40 000 40 000 40 000 40 000 i SPL1 SPL1 SPL2 SPL2 SPL2 SPL3 SPL3 SPL3
113. E e KC4 generates a temporary curve using fictitious data After the plate is read KC4 rebuilds the curve with real plate data e Certain characteristics of this curve can be modified including colors and labels Double click anywhere on the graph to enter the editing mode a shaded box should appear around the graph When this box appears click the right mouse button once to open a menu of items that can be edited 302 e PowerReports User s Guide to KC4 Dose Response Curve s Dose response curves are generated by a protocol with assay control sample or sample control wells with concentrations or increasing dilutions defined Up to 5 curves can be overlaid in one curve object To insert a Dose Response curve click on the Curves button in the RSO toolbar then click on Dose Response The Graph Properties dialog appears listing all available curves by Well ID Graph Properties E Available Curves Selected Curves SPL SFL SPL3 lt lt Remove e The Available Curves box lists all curves made available by the current protocol parameters e The Selected Curves box lists all curves that will be overlaid in a single curve object e To add a curve to the list highlight an available curve then click Add gt gt e To remove a curve from the list highlight the curve then click lt lt Remove e Click OK to close the dialog and insert the object into the document Ed Microsoft Excel Worksheet in lt KC4 gt
114. E Data In not available for Multi Plate Transformation 1 Some cutoffs could not be calculated Symbols could not be determined teete creer trece J ocerte preteen creer ceerr Peete rece PETE Warnings and their probable causes include e Atleast one validation condition failed The protocol defines validation conditions and one or more conditions has failed Select Display Validation Results to see the current status of each condition e Cutoffs are not in increasing order Symbols can not be determined The protocol defines cutoff criteria the results of which are not in increasing order and therefore Symbols Pos Neg etc cannot be determined For example if criteria 1s defined like this Symbols Cut Off and if CTL1 2 050 and CTL2 1 432 cutoffs are not in increasing order Select Protocol Cutoffs to view modify the criteria 190 e Display Menu User s Guide to KC4 e Data In not available for Multi Plate Transformation lt 1 2 or 3 gt A multi plate transformation references a data set that no longer exists Select Protocol Multi Plate Transformation to choose another data set and then recalculate results e Data In not available for Single Plate Transformation lt 1 2 or 3 gt A single plate transformation references a data set that no longer exists Select Protocol Single Plate Transformation to choose another data set and then recalculate results e Data to interpolate are not
115. Excel Worksheet in lt KC4 gt 1647 File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves E Close Statistics Well IDs Validation Results Protocol Audit Trail Data Audit Trail Calculation Log Report e This list contains all available categories of data based on the current protocol settings e Available categories can include Statistics Well IDs Well Data Well Analysis Results Validation Results Fitting Results Protocol Data Audit Trail and Calculation Log Report Statistics A Statistics table calculates the Mean Standard Deviation and Coefficient of Variation CV for the Standard Sample Control and or Blank groups defined in the protocol To create a Statistics table click the Tables button in the RSO toolbar then highlight Statistics A list of available data sets appears Ed Microsoft Excel Worksheet in lt KC4 gt 1687 File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves e Close Statistics Mal 405 Well IDs M2 630 Fitting Results M1 405 Corr M2 630 Corr Delta OD Concentrations Symbols e The list contains those data sets made available by the current protocol parameters e Select the data set to be represented in the table 286 e PowerReports User s Guide to KC4 The Statistic Table Properties dialog appears Statistic Table Properties Ea Selection Data Identifier W Standard Assay
116. File Storage and Format Options in System Menu for more information To backup archive files you can either copy the entire database to the desired medium or you can export files to a disk and then copy them to the desired medium See Maintain Files in System Menu to learn more about exporting and optionally deleting files from the shared database See also Audit Trail in System Menu to learn about exporting and optionally deleting system audit trail events to reduce the size of KCShared mdb Plate Data Acquisition KC4 provides three methods for acquiring plate data retrieving results from a microplate reader entering plate data manually and reading data in from an existing text file Refer to the following for more information e Reading Plates on page 48 e Entering Plate Data Manually on page 50 e Reading Plate Data from a File on page 51 Data Reduction and Analysis When the data has been retrieved and data reduction is complete KC4 displays the raw data associated with the first defined filter wavelength The availability of additional data sets depends on the current protocol parameters and can include raw data at other filters wavelengths blanked data corrected data concentrations symbols kinetic curves and more Refer to the following for more information e Results Overview on page 251 e Data Sets on page 253 User s Guide to KC4 Getting Started e 29 Biograph
117. ID SFL Dil 10 000 A When the Type and ID fields are set click the button to the right of the Dil Field The Concentrations Dilutions dialog appears Concentrations lutions RS e am e ow W Fact E Te aww Dilution Concentration ICON E Use this dialog to enter the dilution values required for the assay Dilutions are must be entered in ascending or descending order starting with Dilution 1 10 000 in the example above e Choose the Conc Dil Type option Dilution e Enter the starting dilution in cell 1 Subsequent dilutions can be entered manually or automatically Enter dilutions manually in cells 2 3 and so on Use the down arrow key to advance to the next cell or click the mouse in the desired cell To increase dilutions automatically choose an Auto option of Incr or Fact Enter the required Increment or Factor value then click on cell 2 to assign the next dilution Use the down arrow key to advance to the next cell or click the mouse in the desired cell User s Guide to KC4 Protocol Menu e 151 Incr Increments dilutions by the entered value In the example above the starting dilution is 10 and subsequent dilutions increase by 10 Enter a negative number e g 10 to decrement dilution values Fact Increase dilutions by an entered factor value If the starting dilution is 2 increasing by a factor of 2 subsequent dilutions become 4 8 16 32 6
118. Mame Delta OD caca t The Transf field indicates the current transformation level Level 1 transformations can be passed for further manipulation into Level 2 and Level 3 transforms for successive manipulation Data In represents the data set upon which the formulas will be applied The Data In choices will vary depending on the protocol type and reading parameters Within one Multi Plate Transformation you can select up to three discrete assay data sets for combined calculations You must select and position each data set in the appropriate location holder 1 e X1 X2 and X3 The data sets must be of similar type 162 e Protocol Menu User s Guide to KC4 You can select previously stored data files these files must be correctly formatted text files and only contain end point readings For kinetic protocols Reading can be set to All or from 1 to the total number of readings Use the location variables X1 X2 and X3 to construct the Formula The Name field assigns a name to the transformed data and saves the settings and formula The transformed data can then be used as the Data In for further calculations including Kinetic Analysis and other Multi Plate Transformations Syntax The following table contains symbols that can be used in Multi Plate Transformations Type of Symbol Description Example Algebraic symbols reyes Ta st Figures scientific notation 2 45E 08 Automatic Formula G
119. Parameters Edit Reading JEdit Plate Layout Edit Raw Data Correction jEdit Well Analysis JEdit Single Plate Transformations Edit Multi Plate Transformations jEdit Curves Edit Validation Iw Edit Cut ofts gt Mar n mMm Checked Cancel Help functions may not be performed by unauthorized users With KC4 v3 4 an unauthorized user 1s any user other than the System Administrator With KC4 Signature an unauthorized user is one who is designated as a Standard User The following list includes all protocol level protectable functions and describes the behavior the unauthorized user can expect from KC4 for each function when it is protected 184 e Protocol Re read plate The user cannot re read a plate if one is already open because re reading a plate deletes the initial values The user must first select Data New Plate and then Data Read Plate Mask a value Within the Results window the user cannot click the Mask box to exclude wells from calculations Change a value Within the Results window the user cannot click the Change box to change any measurement values Edit Protocol Parameters If this box is checked the user cannot edit any protocol parameters Edit lt individual protocol parameter gt The user cannot modify the particular protocol parameter For example if Edit Plate Layout 1s protected the user cannot define or assign new well IDs expected concentration values o
120. Plate Transformations Report and so on Select Protocol Advanced to see additional options including Cutoffs and Validation Note Reading parameters such as filters wavelengths and plate geometry cannot be modified after a plate has been read 3 Within the appropriate protocol parameter dialog make the desired change and click OK to recalculate results The System Administrator can protect some or all protocol parameters from being modified If you are trying without success to modify these parameters you may not be authorized to do so KC4 Signature automatically updates the Data Audit Trail to reflect the action Data Audit Trail Date User Action Comment l 08 2002 17 26 34 Administrator Protocol DB ones Mary Aucleic Acid260_250Ratio prt 06 2002 17 26 34 selected 08 20 02 172707 Administrator Plate read started 082002 172714 Administrator Blank Plate successtully read 0820 02 17 27 25 Adminiztrata Me Fiat Somes til read 08 2002 17 25 01 Admin Plate Layout changed Changed the SPL color from black to blue for reporting purposes only OS 2002 17 20 41 Th Report changed Designed the report template seo Saving Data Files After creating or modifying a data file save it by selecting Data Save or DatalSave As Save stores the data file under its current name If the file does not have a name KC4 will ask you to enter one Save As copies the contents of an existing data file to a new da
121. PowerWaveX Select 84 PowerWaveXS 85 SynergyHT 86 SynergyHTTR 87 SynergyHTTR with Injectors 88 uQuant 89 Reading parameters reference 90 Reading plate data from a file 51 Reading Type 113 Real time kinetic zoom 103 Records 29 Archival 29 Retention 29 Recovery percent calculation 155 Reference wells FL600 100 104 Refr Filter 99 Registering KC4 229 Reinstalling KC4 229 Report Engine 186 User s Guide to KC4 Reports Excel 186 KC4 177 186 Keep open after printing 186 Preview before printing 59 Printing 59 Protecting the document 186 Selecting the reporting engine 186 230 Word 186 Restoring connection to KCShared 234 Retaining records 29 Run KC4 in trial mode 18 Run Time 102 S Sample control wells 143 147 Sample name list report 180 Sample names 61 Sample protocol and plate data files 40 Sample wells 143 146 Saving data automatically 185 Scale to high wells 90 Scale to low wells 90 Scales Individual Well Auto Scaling 114 Kinetic reads 114 Spectrum reads 114 Scanning 262 Analysis 139 Area 115 Integral 141 Linear 115 139 SD function 160 Secured protocol and data file storage 222 Security 184 211 Send filters to reader 200 Send wavelengths to reader 196 199 Sensitivity 96 98 Session idle timer 216 Index e 321 Session timeout 216 Set Lamp Off during measurement 100 Setup 230 Formats 231 Protocol defaults 230 Shaking 116
122. Ratio prt 123 Nucleic Acid Protocols Pe 280 atic i B User Folders 3 Administrator Cut 3 Dligo Quant p Copy s OLIGO Quant Pose 3 Phenol glb Rename 29 Murdoch Sandra A 2 Phenol pri Delete 123 Barnes Robert G With the options available through this menu authorized users can e Export the highlighted item s to a user designated location outside the shared database e Delete Cut the highlighted item s and move them to the clipboard e Copy the highlighted item s to the clipboard without deleting them e Paste or import folders and files from the clipboard into the current folder e Rename the highlighted folder or file e Delete the highlighted item s without first moving them to the clipboard Exporting Files Protocol plate data and global data files residing inside the shared database can be exported out of the database through the Maintain Files dialog Important When files are exported out of KC4 s secured database they will reside on a disk or the network in a user designated location It 1s the site s responsibility to ensure the security of exported files To export a file 1 Select System Maintain Files Locate the desired file 2 Highlight the file click the right mouse button and select Export to Disk The System Administrator can protect the use of the Export and Delete Files After Extraction functions If you are trying without success to export and or delete files you
123. TARTED 0 lO a 27 ON NV PP E E E N E E NE 27 PSS titi all In ornati OM eenn n nA T AA 28 PS AN 28 A A 28 Record Retention A sein E tet ecruashas RA 29 Plate Data Acqua T eevee 29 User s Guide to KC4 Contents e i Data Keduction and Analy siS 4ncsniewcmaseie ee TA 29 A A EAN 30 PIE STATES di A ta 30 Audi Lal aa 30 E En A SEO E SET RIOR OT nem ose 30 TS Bat 30 Ml E 31 A E R E E A bodies a AE sion nse beta bs E N 31 Sas ws CAN EEE E EE A E it 31 Otek Retorenco GUMS iS 32 Data M Desene aii 32 A O AER AE A O AER E E 33 PHOLOC OW ME Menene na a EEEE A E E ali 33 Display Meinar al 35 SV Ste wt MEU de 35 Abou a eee eee eee 36 Navigating the Help SS a RE 3 A g A E TE A E E T O Een 38 Quick Start for Clarity Microplate Luminometer US rS occcccnnccnnccnnncnnnnnnnnnnnininininess 39 Sample E O O iia 40 DATA MENU opti 41 O 41 Working with Data Piles a is 42 Where and How is Plate Data Stored 00 0 0 ccececeeeeeeeeeeeeeeesseessesessesseesseeeeeeeeegs 42 Creatinora New Data ler daa 43 Openin Exin Data Els ri 44 Modify mne Data Fes 45 a e E E 46 Closmo Data FES etoen E ii 47 Ran Pte a 48 Unintentional Termination of a Kinetic Read sck entices 49 Pnterine Plate Data Manta lly roeie die a cdo do 50 Reading Prate Data Tom a Pienaar T E A A a 51 Simi Data leia tol 54 Append to Giant bl POPE A OP Pe S6 Export sian tata eee iaaeecd bimeein wate eames 57 PrAT MIM IE MOUS A aie taentindadateian Manteca abel taadactiebadateind Misia
124. The default Font is MS Sans Serif Regular 10 point or similar in Black KC4 presents for selection the fonts that are already installed on your PC typically in your Windows Fonts folder e Change the Font Font Style Size and color as desired Add Effects such as a Strikeout or an Underline Symbols will appear on the results display and on printed reports as they are defined within this dialog User s Guide to KC4 Protocol Menu e 175 Out of Range Symbols Out of range symbols jour El Concentration below minimum Jour T Concentration above maximum When Data to Interpret is set to Concentrations the Out of Range Symbols fields are available at the bottom of the Cutoffs dialog Following data reduction e Wells with concentrations less than the minimum standard concentration are assigned the Concentration below minimum symbol e Wells with concentrations greater than the maximum standard concentration are assigned the Concentration above maximum symbol e Note Use of the Extrapolation Factor may cause the upper and lower limits for concentration interpretation to be extended The default out of range symbols are OUT and OUT Each Symbol field can contain 0 to 8 alphanumeric characters The appearance of each symbol can be customized by clicking its corresponding Font button as described in the previous section Cutoffs Evaluation Cutoffs are evaluated during data reduction If the cutof
125. Well settings 143 Plate Layout 142 181 Plate mode FL600 103 SynergyHTTR with Injectors 119 Plate objects in PowerReports 279 283 Plate shaking 116 Plate Shaking Before First Every Reading 116 Duration 116 Intensity 116 Plates Signing electronically 54 PLD file 10 244 PLUG notation 197 200 Point to Point curve fit 164 167 Polynomial Regression 164 167 Port 195 POW function 161 Power users Group membership 213 Protocol level protected functions 184 Index e 319 System level protected functions 218 PowerReports Curve objects 279 302 Dollar sign character 280 Editing an object 280 Field objects 279 282 How it works 274 Overview 273 Plate objects 279 283 Results objects 279 RSO toolbar 279 System requirements 276 Table objects 279 286 PowerWave 82 196 PowerWaveHT 82 196 PowerWaveX 83 196 PowerWaveX Select 84 196 PowerWaveXS 85 Preferred data file format 222 Pre Heating 117 246 Pre read blank plate 111 Print a list of sample names 61 Printing data automatically 186 Printing reports 177 306 Probe orientation FL600 96 Probe orientation FLx800 SynergyHT H TTR 98 Probe size 194 201 Prompt 75 Prompt for name 185 Protected functions Protocol level 184 System level 218 Protocol Creating a new protocol 69 Defaults 230 Defined 28 67 Modify 71 Options 185 Protected functions 184 Save Save As 71 Security 184 Protocol audit trail Po
126. a set ie RESULTS Data Symbols Template M405 5 E T Concentrations s av eav OUT OUT OUT OUT NEG A av cor rav Fav Fl Fae EY OUT OUT OU 256 e Results E 3 10 11 le ERF ERF POS ERF fer Fay POS POS EGY fav EQU NEG Ss EEES co sOy Fav Fey Fay fay y Pr y a on On 5358 on ou u T T O d If the cutoffs are in increasing order symbol assignment is determined as follows Each well is evaluated against the first cutoff If the value of the well is less than the criteria then the first symbol is assigned to the well If the value of the well is greater than or equal to the first cutoff then it is evaluated against the next cutoff If the value of the well is less than the next criteria then the symbol immediately preceding the cutoff criteria is assigned to the well This process continues until the last cutoff criteria is encountered If the value of the well is less than the last criteria then the symbol immediately preceding the last cutoff criteria is assigned to the well If the value of the well 1s greater than or equal to the last criteria then the symbol immediately following the last cutoff criteria is assigned to the well If the cutoffs are not in increasing order the message Cutoffs are not in increasing order is not defined in the Plate Layout a well that is masked or a well that is out of range or has been failed by the attached micro
127. ach time you make a change e Ifyou are running KC4 with an external robotic application such as Bio Tek s Bio Stack PC Control software click Minimize KC4 when running in OLE mode When KC4 starts it will automatically minimize permitting access to the external application 232 e System Menu User s Guide to KC4 Database KC4 uses three different databases for storing information These databases are secure meaning that they can only be opened and updated through KC4 e KCShared mdb contains security and plate format information With KC4 Signature this database also contains user accounts protocol plate data and global data files and their associated audit trails and a system audit trail for shared events Following installation KCShared mdb is stored on the computer s hard drive but it can be moved to a network location for secure storage and multi user access This database may also be copied and renamed if for example your site requires a separate shared database for each project e KCLocal mdb contains all of the settings defined by the user under System Readers and System Setup With KC4 Signature this database also contains an audit trail for local events KCLocal mdb is stored on the computer s hard drive and it cannot be moved or renamed e CALIB mdb contains Optics and Universal Plate Test setup values and saved test results CALIB mdb is stored in the root KC4 folder It cannot be moved or renamed and t
128. ad step with Shake During Read enabled counts as two steps e The steps are listed in the order in which they will be executed The Description column provides key information about the step such as volume rate or intensity The Start column indicates the time the step will begin The End column indicates the time the step will complete The Comment column contains additional information about the step such as tip prime settings To edit a step highlight it and double click or click the right mouse button and select Edit To delete a step highlight it and press the Delete key or click the right mouse button and select Delete e The steps are also displayed graphically to show how they will be executed on the plate over time Dispense step The solid area represents the action on the first well Cross hatches represent the time necessary to process other wells on the plate Shake step The solid area represents the action on the entire plate Read step The solid area represents the action on the first well and for each kinetic interval if this is a kinetic protocol Cross hatches represent the time necessary to process other wells on the plate Time indicators A Process time indicates the time at which a particular step will begin relative to the start time of the very first step in the operation Process times are displayed in grey light text Kinetic protocols only A Kinetic time indicates the tim
129. add a new plate click Add User s Guide to KC4 System Menu e 207 The Edit Universal Plate dialog appears Edit Universal Plate Plate Type 7 filter universal plate PH 7260522 a Serial Number i 27824 Wavelength List 0 151 0 143 0 137 0 133 0 141 0 141 0 131 0 593 0 550 0 545 0 543 0 545 0 540 0 463 dl SEI dl aa do Teale 1 6284 1 693 1 676 1 365 2 145 1 902 1 3585 1 535 1 754 1 756 1 451 2 355 2 508 2 446 2 422 2 344 2 303 1 304 WaveLength of peak in the interval between 580 nm and 590 nm ses OF Cancel Sa 1 187 1 0935 1 095 1 081 1 030 1 080 0 922 If this is a new plate select the Plate Type then enter its Serial Number The top row of the wavelength grid shows eight unique wavelengths for use with the Universal Plate Test The default wavelengths correspond with the calibration test plate data sheet To modify one or more of these wavelengths click Wavelength List Refer to the calibration data sheet that came with the test plate to determine the expected OD for each test well at each wavelength For each Location C1 D4 E2 F5 G3 H6 enter the OD associated with that well at each wavelength For readers that support spectral scanning a Peak Wavelength value is required Enter the value in nanometers nm Click OK to save the changes and close the dialog or Cancel to close the dialog without saving changes To run the Universal Plate Test e Ensure that the reader is properly c
130. ading Type End Point C Kinetic Spectrum Select a Reading Type before defining additional Reading parameters The Reading Type options Endpoint and Kinetic are available for all readers supported by KC4 For certain readers the Spectrum reading type is also available e During an Endpoint Read every well on the plate is read with one two or multiple wavelengths If supported by the current reader the Scanning feature is available for endpoint protocols Click the Scanning button to define linear or area scanning parameters e During a Kinetic Read every well on the plate is read a user specified number of times with one two or multiple wavelengths Click the Kinetic button to define kinetic reading parameters e During a Spectrum Read multiple readings are taken across a wavelength range The objective is to plot a graph with absorbance or transmittance versus wavelength Click the Scales button to customize the graphical data displays optional e The Reading Type options for the Synergy HT with injectors contains an additional Dispense checkbox to enable the fluid dispense option User s Guide to KC4 Protocol Menu e 113 Scales for Kinetic Reads By default the Scales parameters automatically calculate an appropriate box size to display the kinetic reactions The default values can be manually overridden by deselecting one or both Auto checkboxes e Absorbance reads min t is automatically
131. ading Type Reader End Point Power ayer on COM2 Kinetic Kietie S Canning Spectrum Scales Wavelengths 405 E20 oOo Calibrate Plate Shaking First well E Last well 412 Intensity 0 Duration o z Temperature Control f Before every reading Before first reading G Yes I PreHeating Pre Readings CoNo Temperature 37 ale Blank Plate Lag Time joo 0 00 Pathlength Correction Wavelengths Fiotel pat Options Read Barcode Monitor wells Options AA A AA 10 11 12 coca 9 Select a Reading Type of Endpoint Kinetic or Spectrum Define the Wavelengths to be used Calibrate the wavelengths if necessary Define a rectangle of wells to be read if reading a partial plate For a kinetic protocol click the Kinetic button to define the read time and interval For an endpoint linear scanning protocol click the Scanning button to define the horizontal reading points Optional For a spectrum protocol click the Scales button to customize the graphical data display Optional Define Shaking parameters Optional Define Temperature Control parameters Optional Select and define Pre Reading options Optional Select Read Barcode to instruct the instrument to scan the barcode as the plate is drawn in to the reading chamber Optional Select Monitor Wells and then click Options to define related parameters Click OK to verify the parame
132. ain Files All files and folders currently stored in the shared database will be displayed Maintain Files DB i Type Modified S Endpoint ELISAs 3 DNA Quant Kinetic Assays ANA Quant of Miscellaneous Protocols 3 260_2BDRatio prt KC4 Protocol 11 21 02 16 33 42 13 Nucleic Acid Protocols 34320 prt KC4 Protocol 04 17 01 14 11 50 a 19 User Folders EDTA prt KC4 Protocol 04 11 01 17 03 06 23 Phenol prt E C4 Protocol 11421402 16 33 10 Si Administrator 239 Bames Robert G na Guest A Murdoch Sandra 4 ea Osborne Ky L oat Stolz Richard F e The Maintain Files dialog contains two frames The left frame displays the folder file tree The right frame displays the contents of the current folder All changes are made within the right frame e Click E e Click as to create a new folder e Click ona folder L e Highlight a folder or file and click to delete it J to move up one folder level to open it and view its contents e Click to update the view to show any folders files that may have been added by another user after you opened this dialog e Click the right mouse button to open an editing menu see Edit Options on the next page 226 e System Menu User s Guide to KC4 Edit Options Click the right mouse button to open an editing menu Maintain Files Curent Folder DB User Folders Jones Mary A 3 Endpoint ELISAs J ON4 Guant 9 Kinetic Assays 2 RNA Guant 9 Miscellaneous Protocols 280
133. allow quick access to KC4 functions Accessibility of a particular toolbar button depends on whether or not its function may be used in the current environment For example the Biograph button is accessible only if results are displayed and a curve has been generated When KC4 is installed the toolbar contains a set of default buttons These buttons can be customized by selecting System Toolbar Customize Toolbar Available toolbar buttons El Move Up m blove Down F e To add a button to the toolbar highlight the desired button under Available Toolbar Buttons then click Add e To remove a button from the toolbar highlight the button under Current Toolbar Buttons then click Remove e To change the position of a button in the Current toolbar list highlight it then click Move Up or Move Down e To restore the toolbar to its default buttons click Reset e To save the changes and update the toolbar click Close On most displays the toolbar can easily display up to 16 icons All possible icons are shown and described below and on the following pages Create a protocol using the Protocol Wizard This is offered as an alternative to using the Protocol Menu Protocol Wizard a A Wizard Initialize memory for a New data file Click this button before reading a plate Data New User s Guide to KC4 System Menu e 237 Read a plate or enter data manually or read data from a file Before clicking this
134. alues and black represents the highest measurement values Tip Ifthe plate 1s likely to yield values ranging from very low to very high enable Individual Well Color Auto Scaling to help with the visual differentiation between points within each well Intensity i Duration 30 E Before every reading C Before first reading If plate shaking is available with the currently selected reader the Shaking parameters are accessible Endpoint Spectrum protocols Shaking occurs immediately before the plate is read Kinetic protocols Shaking can occur before the first reading only or before every reading Intensity Acceptable values include 0 No Shaking 1 Low 2 Medium 3 High 4 Variable not supported by all readers that support shaking Duration Defines the absolute time period for shaking in seconds Before First Before Every Reading Defines when the shaking will occur For non kinetic reads shaking occurs before the first reading For kinetic reads shaking can occur before the first read or before every read 116 e Protocol Menu User s Guide to KC4 Temperature Control Temperature Control it Yes Ho Y Pre Heating Temperature 37 E Lag Time joo 0 00 For readers that support computer controlled incubation the Temperature Control parameters are accessible Select Yes to indicate plate incubation Select No to indicate no incubation default Check Pre Heating to pre heat t
135. ameters options may be available e The Absorbance Probe size field displays the diameter of the absorbance probe e The Top Probe size field displays the diameter of the top probe If the reader is an FL600 use the drop down list to choose the size e The Bottom Probe size field displays the diameter of the bottom probe If the reader is an FL600 use the drop down list to choose the size e If supported by the reader click the Get Probe Sizes button to retrieve the size s of the probe s currently installed in the attached reader User s Guide to KC4 System Menu e 201 Dispenser Configuration For the Synergy HT with injectors KC4 provides access to special configuration functions and settings Reader Configuration Reader Synergy HTTA welrjectors on COM Probe sizes Dispenser 1 Dispenser 2 Status Initialized Iritisize Calibration volumes Injector postion Emeeen i Next to Top Probe Above Bottom Probe Initializing the Dispenser If the dispenser module was connected before the reader was turned on or if a system test was run the dispenser should initialize automatically If for any reason the dispenser does not initialize automatically you can initialize it from KC4 This may be necessary if the module was connected to the reader after the reader was turned on or following the installation of a new syringe To initialize the dispenser e InKC4 select System Readers click the Conf
136. and the kinetic read was interrupted Data recelwed to this point has not been validated by the reader Do vou with to keep the data IF vou answer Mo the data willbe PERMANENTLY DISCARDED As the message states the read was interrupted and KC4 is providing the opportunity to save or discard the kinetic data received up to this point The data is unvalidated which means the reader did not or could not provide KC4 with the expected valid status at the end of the plate read The term unintentional termination applies to the following situations e The reader was turned off or the serial cable was disconnected during the read e KC4 received an error status string at the end of the kinetic read Note If the unvalidated data is saved after this error the last kinetic point for each well is deleted due to its proximity to the error status e KC4 received an unexpected response from the reader or the reader stopped operating during the read possibly due to a hardware error or an internal software error e MS Windows experienced a communication error in the low level COM processes If the message shown above appears in KC4 take a moment to try to determine its cause If the reader was inadvertently turned off or the serial cable was disconnected the reader may have performed normally up to the point of termination in which case the data would be valid If the termination appears to have been induced by the reader the data ma
137. and then try again Note If the current reader is set to Clarity the Configuration and Port buttons are dimmed and inaccessible because configuration parameters and port settings can only be accessed through the Clarity PC software The Filters Wavelengths button is dimmed and inaccessible because it does not apply to the luminometer Note If the computer has more than one COM port you can control more than one instrument at a time by running multiple instances of KC4 See Controlling Multiple Instruments Simultaneously at the end of this section for more information 194 e System Menu User s Guide to KC4 Port Selection Use the Port Selection dialog to select the COM port number 1 to 9 and to access additional communication parameters Clarity Microplate Luminometer users Port settings can only be accessed through the Clarity PC software See the Clarity Operator s Manual for more information Port Selection 3 Reader SpnergpHT COM port 2 Y Setup Cancel Help e Click the drop down arrow and select the number that represents the COM Port KC4 should use when communicating with the reader e Click Setup to define additional communication parameters Fort COM Fort Parameters Transmission Speed a00 Cancel Data Bits le H Parity No H Hep Stop Bits Ja H Important It is the user s responsibility to define the correct parameters otherwise communication err
138. are biotek com User s Guide to KC4 Appendix A Records Submission e 309 310 e Appendix A Records Submission User s Guide to KC4 Appendix B Windows File Caching Windows File Caching This topic applies to sites accessing a shared database stored on a network server By design most write and read database operations are intended to be performed synchronously by KC4 For example when a system audit trail event or a file is created by a KC4 instance it should be immediately visible in other instances using the same database However because of other factors beyond the control of KC4 like network and operating system delayed write optimizations there is no way to guarantee such behavior To improve synchronization it is recommended when KC4 1s used on several networked PCs connected to the same shared database to disable the network redirector file caching In addition while file caching 1s enabled there is a potential for data loss following a network connection failure According to Microsoft when the Windows redirector opens a file for read or read write access the redirector utilizes the Windows system cache Therefore when data is written to the file it is written to the cache and not immediately flushed to the redirector The cache manager flushes the data at a later time If an unrecoverable network error occurs while the data is being transferred to the remote server it may cause the write request to fail and an appl
139. articular wavelength this most likely means that the measurement value over ranged Spectrum calculations such as Min OD Max OD and W at Min OD Max OD associated with the currently selected plot are displayed Click on the down arrow associated with Selected Curve to choose a different curve Moving the mouse cursor over a data point in the currently selected curve will display coordinates for that point Data displayed can include Wavelength OD and Wavelength value in nanometers Click on an individual data point to temporarily exclude it from further calculations then click the Recalculate button to exclude the point from calculations Important Modifying data may adversely affect the final results To restore an excluded data point click on the point then click Recalculate Click the Options button to adjust the Spectrum Analysis parameters for the curves shown in this dialog User s Guide to KC4 Results e 261 Area Scan Data The default view for area scan results contains the graphical representation of the measurements taken down and across each well a RESULTS Data Scans M 405 e To view a different set of data click the Data drop down arrow and select Mean OD RFU RLU Std Dev OD RFU RLU or CV OD RFU RLU e Click on a well to zoom in on it E M 405 WELL Al E Data Mode Recalculate Close Mean OD 2 5558 Std Dev OD 1 4183 CY OD 55 492 Well Size 34 99 mm Probe Size 1
140. asked well KC4 surrounds the value with asterisks 0 234 9999 98999 e To denote biased values KC4 surrounds the value with exclamation points 0 068 123 456 Note Biased values may appear in a Statistics table if Use out of range values in statistics is enabled under System Setup If for example the concentration for one of three standard replicates is reported as gt 40 the Standard Deviation and CV values are considered suspect KC4 surrounds the values with exclamation points to indicate that it 1s the user s responsibility to determine whether or not the results are valid Statistics z Concentr weno cancion wen mans Focas Ne Mean Stapev cve STL 10 41 0 006 9 024 3 9 026 0 004 0 042 K 0 007 9 032 0 006 9 024 STD 20 5 1 235 E 3 18 955 1 229 1 231 l STD3 30 o 4 999 gt 41 500 3 32 350 E 2319 l 2 321 STD4 40 36 996 37 008 ee 012 Z 033 D2 p3 C 2age Fane 0202 User s Guide to KC4 PowerReports e 307 Performing the Data Export When plate reading and data reduction are complete the export document is ready for formatting and saving To format the export document that is to fill all of the objects in with current plate data select Data Export or click the appropriate toolbar button When formatting completes KC4 displays the Save As dialog SS E 1x Ea em QX C m To E File name Dose Response xls E Save Save as type Microsoft Excel Workbook xls
141. assay plate Specify the Optics Position to be used for each filter set Tips Evaluate the proper reading position by specifying two or more filter sets one each for the Top and Bottom optics positions The top position is advantageous for homogeneous solutions using solid black plates The bottom position is advantageous for cellular assays and assays requiring a covered plate to maintain sterility The recommended Optics Position for Absorbance readings is Bottom KC4 provides a warning if Top is selected although this selection will not be prohibited The recommended Optics Position for Luminescence readings 1s Top e Fluorescence or Luminescence method only Specify the Sensitivity setting for each individual filter set Valid settings are 0 and integers from 25 to 255 Auto indicates that the Auto Sensitivity Adjustment feature has been enabled The signal from the range of fluorescence and luminescence assays can be very weak very strong or anywhere in between For each assay the sensitivity of the photomultiplier PMT should be adjusted to ensure that the signals from all wells fall within the appropriate dynamic range of 0 to 99999 relative fluorescence or luminescence units RFU or RLU Tips Typical assays using 96 or fewer well plates require sensitivities between 75 and 190 Assays using 384 well or other higher density plates will likely require much higher sensitivities due to narrower o
142. atistics box then click the Add button STATIS TES Ml 405 Identif Name Conc Dil Well Std Dew CW SPL1 SMPO 1 000 A 3 606E 3 1 5215 SPL Sel PODIO 1 000 3 215E 3 0 2145 SPL3 Se PODOS 1 000 2501 E 3 0 2045 Se PODOA 1 000 0 1423 127 45 User s Guide to KC4 Protocol Menu e 181 Export Export With KC4 s standard exporting function you can customize the contents of export files All KC4 users have access to this standard function Exporting through Microsoft Excel 2000 and Word 2000 is also supported with KC4 s PowerReports function This section only discusses the standard exporting function the KC4 report engine The export engine 1s selected under Protocol Options Select Protocol Export to customize the export file for the open protocol Export E Protocol Description TT Assay Description Available Data Exported Data Template MM 405 Concentrations Symbols Protocol Audit Trail Data Audit Trail Calculation Log Report Interpolation Sample names Cut Offs Sample name list Plate Template 405 Concentrations Format Plate 7 Table Statistics Controle Standards Samples Kinetic readings From Tot lt Remove k Insert gt gt it Cancel Help e Check Protocol Description and Assay Description to include this information in the file e The Available Data window displays the data and information available for exportin
143. ats to store information e Protocol PRT files contain instructions for controlling the attached microplate reader Protocols also contain information required by KC4 to analyze plate measurement data e Plate data PLA files contain raw measurement values plate description information prompts comments date and time of reading and sample names e Global data GLB files contain plate data and protocol information The GLB file format is offered as an alternative to maintaining two separate protocol and plate data files and is the required data file format for electronic signature support In addition Clarity Microplate Luminometer users will work with Clarity protocol files which use a BPF extension These files are referenced by KC4 s PRT and GLB files and they contain the reading parameters required to control the luminometer File Storage File storage options vary depending on site requirements and the current KC4 configuration e Files may be stored individually on the computer s hard drive on a CD or diskette or on a network to which the computer is attached This is the storage method that users of KC4 version 3 0x and earlier are familiar with Windows Explorer or a similar application can be used to view the file names and their locations and to move copy rename and delete files e With KC4 Signature PRT PLA and GLB files may also be stored inside a secure shared access database This database named
144. ave As copies the contents of an existing protocol to a new protocol with a different name Note The layout of the Save or Save As dialog varies slightly depending on whether or not the Secured Protocol and Data File Storage option is enabled but the functionality is the same In the following example this option is enabled Current Folder be ES Mary AN ucleic Acid K i Modified DNA Quant AL RNA Guant 3 260_280R atio prt 146E KC4 Protocal 04 11 01 15 31 10 1654320 prt T4KB KC4 Protocol 0417401 13 11 50 16 Phenol prt 14KB KC4 Protocol 051001 15 23 22 File Name Dligo G Want prt Type C4 Protocol prt Cancel e The Current Folder field or the Save In field you will see one or the other shows your current location within the overall folder structure Ifthe drive is represented as DB this means that files are currently being stored in KC4 s secure shared access database If the drive is represented by a single letter such as C or D this means that files are stored individually on the computer s hard drive or on a network to which the computer is attached See Where and How Are Protocols Stored for more information User s Guide to KC4 Protocol Menu e 71 e The default file name is NewProtocol prt e Enter a new File Name and click OK to save it e Click J to move up one folder level e Click Ef to create a new folder e Click on a folder L e Ifan existing file
145. ave KC4 installed on them Determining which users will have access to certain KC4 functions Installing KC4 Creating and maintaining user accounts Customizing security settings Reviewing records Establishing a procedure and schedule for record retention and archival Administrator Requirements The FDA s Electronic Signatures Rule 21 CFR Part 11 contains requirements that sites must meet in order to be in compliance The KC4 Signature System Administrator should be cognizant of the following Administrator The site shall select an Administrator whom will be the person responsible for all high level administration of the program This person will control access to the program by adding new users structuring the individual users authority levels and reporting to management as appropriate on any unauthorized use of the program Personnel Qualifications Personnel that develop maintain or use electronic record electronic signatures shall have the education training and experience necessary to perform their assigned tasks Written Policies There shall be written policies which hold individuals accountable and responsible for action initiated under their electronic signatures in order to deter record and signature falsification There shall be written revision and change control procedures to ensure that the program is administered in compliance with the FDA s requirements Record Archiving and Deletion The site is respons
146. ax V then click Calculation Options Kinetic Analysis Max se Calculation one i Fromt 00 00 00 Tot 02 00 00 C From fi To j3 Y calculation on 3 paints Unit i m D C OD Per Min Cancel Help Calculation Zone Typically the Calculation Zone is adjusted to ignore erroneous data point s generated at the beginning of a kinetic assay due to assay noise e Fromt Tot Defines a calculation zone based on time The default values span the defined kinetic Run Time e From To Defines a calculation zone based on number of reads The default values include all reading points User s Guide to KC4 Protocol Menu e 133 V calculation on n points e This option defines the number of successive points to be included in the calculation of the maximum slope e The minimum number of points is 2 The maximum number of points is the total number of points within the calculation zone e Only the points defined within the calculation zone are used e mOD or OD dependent on kinetic dynamic range e Per sec or Per sec2 dependent on rate order e Per min or Per min2 dependent on rate order e Per hour or Per hour2 dependent on rate order Mean Min Max The Mean Min OD is the mean minimum OD based on n points The Mean Max OD is the mean maximum OD based on n points KC4 calculates the Mean Min and Mean Max ODs as follows e Starting at the first point in the calculation zone KC4 evaluates
147. ble for Multi Plate Transtonmmation 1 The 4 parameter fit is not convergent tis probably notthe most adequate fit Same cutoffs could not ke calculated Symbols could not be determined 178 e Protocol Menu User s Guide to KC4 Kinetic Curves Report The Kinetic Curves report shows the kinetic curve in every well in a plate format For example Kinetic Curves TAVA VAVAVAVAVAVAVA Well Zoom Report Each Well Zoom report contains the kinetic curve for one well e To specify a Well Zoom report highlight Well Zoom in the Available Data box enter the desired Wells for kinetic zoom then click the Add button e Upto 8 kinetic wells can be overlaid in a single graph Select the Wells for kinetic zoom then click Overlay Wells Zoom on well 41 61 Well 4161 Mi 405 Ad Mean 1234 0 mOD ma Coeff Reg 0 9132 hl 405 B1 Mean 1343 4 mOD ma Coeff Reg 0 9944 User s Guide to KC4 Protocol Menu e 179 Validation Report The Validation report contains every validation condition defined for the protocol and its resulting Status e The possible status values are Verified Not Verified and Could Not Be Evaluated VALIDATION Data type M405 Condition Status 1 0 000 lt BLE 0 200 VERIFIED Data type MM 405 Corr Condition Status 1 O 000 lt NEG1 lt 0 500 VERIFIED 2 2500 lt POS lt 3 000 NOT VERIFIED Cutoffs Report The Cutoffs report lists the defined cutoffs and their calculated results
148. c calculations as each measurement value is retrieved from the reader Important When the Allow Well Zoom During Read box is checked a warning message appears If the computer speed is less than 100 MHz or if there is insufficient memory this feature should not be used Kinetic Parameters FL600 The Kinetic dialog contains special Mode parameters for the FL600 Mode ie Plate i Well P Eject between well Cancel Reading Run Time 00 10 00 Interval 00 01 00 Help Reading number 11 e Allow Well Zoom during read Scales miri E 00 00 00 maz t 00 10 00 e Auto min y o mas pr 59999 M Auto M Individual well Auto Scaling e In Plate Mode the FL600 repeatedly reads a whole or partial plate for a specified number of times and for up to 6 filter sets User s Guide to KC4 Protocol Menu e 103 e In Well Mode the FL600 repeatedly reads a well for a specified number of times for up to 2 filter sets This mode is typically used for fast kinetic reactions Check Eject between well to eject the assay plate between well readings This feature is useful for example for accommodating reagent addition Measurement Options Filter Set E scitatiors E mission Optics Position Top Sensitivity 75 125 Options Options To define Measurement Options such as Sampling or Automatic Sensitivity Adjustment for a particular filter set start by clicking its Options button Note If the curr
149. catez mp f Hoz f Vert Choose the Auto Select option Next ID This automatically increments sample groups SPL1 SPL2 SPL3 and so on Auto Select I NetID F ext i Select the Filling option Vertical to assign groups vertically Filling C Horz s Vert Finally go to the grid and click the mouse once in the box above the A and to the left of the 1 This fills all wells with samples ee es ee oe oe a SFE SPL1 PL1 SEIS SPLS SPLS S5PL17 e SPL2 SPL2 SPL2 SPL10 SPL10 SPL10 SPL16 SPL3 SPL SPL m During data reduction KC4 will automatically calculate and display the Mean Standard Deviation and CV for each sample group click the Statistics button from within the Data display to view this information Sir Lig SIAC SAC PL11 Ss es ey eT STATISTICS 150 e Protocol Menu User s Guide to KC4 Defining and Assigning Dilutions KC4 provides the ability to define dilutions for Assay Controls Samples and Sample Controls Defining Dilutions Dilution values can be assigned to wells when the well IDs are assigned or they can be assigned to existing well IDs by clicking the Dilutions Only button This section discusses assigning dilutions and well IDs at the same time When defining dilutions start by selecting the desired well type The dilution values will remain associated with this well type Customize the well IDs if necessary Well Settings Type Sample sh
150. ce of the plate during the read The valid range for the offset parameter 1s determined on a plate by plate basis The calculation is based on the plate height and the maximum travel of the top probe User s Guide to KC4 Protocol Menu e 101 Top Probe Height Z where Lyga amm E min 15 0 mm Height In order to be considered valid the vertical offset value must satisfy the following conditions e 1 0mm lt offset lt 32 5 plate height e offset gt 15 0 mm plate height Plate heights are defined via System Plate Formats Kinetic Parameters If the Kinetic reading type is selected in the Reading dialog click the Kinetic button to define related parameters Reading Aun Time 00 20 00 Interval 00 01 00 Reading number 2 Minimum T Allow Well Zoom during read Scalez mint 00 00 00 mar E 00 20 00 wW Auto min y 1 0000 mar y 5 0000 m Suto Individual Well Auto Scaling e Ifthe FL600 is the current reader the Kinetic dialog contains some additional parameters See the next section for more information e The default time format for kinetics is HH MM SS To change the time format select System Setup Specify the total kinetic Run Time and the Kinetic Interval KC4 automatically generates the Reading Number based on these settings e Run Time Defines the duration of the complete kinetic run The maximum run time is 48 hours 102 e Protocol Menu User s Guide to KC4
151. ceptable Intensity values include 1 Low 2 Medium 3 High 4 Variable e Click OK to save the settings and add the Read step to the list Well Mode Options The Dispense and Read steps are available with Well Mode The Timing Control feature is not available Read amp Dispense Read mode C Plate Description lid DISPENSE Syringe 25pl at 250 pltzec i DISPENSE Syringe 25pl at 250 pl sec ld READ during 2 000 Interval 0 100 Format 5555 sss Dispenser 41 PA ul l Dispenser 2 250 pls Read Steps o 000 0 17 D 0 340 0 540 00740 0 940 0 000 0 200 0 400 0 600 Comment Tip Prime 5 pl before the process Tip Prime 5 pl before the process 21 Reads Time scale 0 000 to 2 610 oom 1 1 aja 11540 1 740 1 940 2 140 1 200 1 400 1 600 1 800 Scales Cancel Help time is approximate and reader configuration dependent e Click the Dispense and Read buttons to create new steps Up to nine 9 steps may be defined Shaking is not permitted in Well Mode Endpoint protocols require one Read step 124 e Protocol Menu User s Guide to KC4 Kinetic protocols require at least one Read step Note If the protocol contains two or more Read steps the Kinetic Interval 1s represented as Uneven on reports Dispense steps cannot overlap Only one dispenser can be in use at a time A Read step can overlap a Dispense step For example the dispense may occur during a kinetic r
152. ces 0 to 5 Click OK to close this dialog and insert the object into the document taj Document in lt KC4 gt Microsoft Word Eile Edit View Insert Format Tools Table Window Help Header Times NewRoman 10 B Z U BE Fields Plates Tables Curves x Close b El 4 Page 1 Sec 1 11 fit T Col 1 REG ki ExT O 292 e PowerReports User s Guide to KC4 Well Analysis Results A Well Analysis Results table lists various statistics for kinetic spectrum or scanning protocols e For Kinetic protocols the table lists the kinetic analysis calculations e g Max V Delta t at Max V Lag Time etc determined for each well e For Spectrum protocols the table lists the Minimum and Maximum measurements and their corresponding wavelength values for each well e For Area and Linear Scanning protocols the table lists the Mean OD Standard Deviation and Coefficient of Variation calculated for each well To create a Well Analysis Results table click the Tables button in the RSO toolbar and then click on Well Analysis Results The Well Analysis Table Properties dialog appears iy Document in lt KC4 gt Microsoft Word File Edit View Insert Format Tools Table Window Help Normal Times Mew Roman 10 B Z E Fields Plates Tables Curves x Close Statistics Well IDs Well Data Well Analysis Results Fitting Results taj Document in lt KC4 gt Microsoft Word File Edit
153. cesecceeeeeceeeees 117 Top Probe Verica OA EE diia 117 NA e A 118 Creatine a Read Dispense Protocol vets naire se A 119 Plate Mode ODIOS lata li da 120 Well Mode OPON aire a a Di 124 Raw Daa Orie A a 128 Blank Plate Subac Oisen nan i a nE A A EA SE EE 129 Blank Wells SuUDi ACH OM lt a tos 130 Pathlen dii OTC CON 130 KMEC ANAIY I Seis ed ee 131 Dee a E E E A E E E T A A A E A 131 Meam Vs aa 132 ON 132 Mean MA aa 134 Onset OW Das tU e o 135 O O Meer OMe ae RTE SME RE See 136 No Calena Oec e A 136 A O O E 136 iv e Contents User s Guide to KC4 SPecleum ANALYSIS cee SA nae ae eee 137 pte arc A E o nue dana aiaaeiaacauialan tana taaaynics Mamuunenaats 137 E AA A EE EO E E 137 FOMU liae A 138 Scanno ANa yS Serea O 139 Meam EDENO AA II II 139 Mean Mia Massana 140 A A A EEA EE EEA tence 141 EA AAA e O 142 Well Typesand does 143 Selecting and Customizing Well Types amp 1DS ooooonnnnnononacananananonanoooooorro nono nonnonnos 144 ASNO WED od 149 Defining and Assigning Dilutions cccccccnnnnnnnccnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnneniness 151 Defining and Assigning Concentrations cccccccccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 155 Sine le Plate Trans ormatlOnS saroian a E 159 Formula da is 160 Formula Eximp Col AA A A 161 Malti Plate Trans ornati OMS ia 162 SVAR O NT 163 Automatic Formula SAO tdi 163 CU lios 164 CURVY ES e 164 CUA oa 165 CURVE F a O O tias 166 CURVE TINCT OL ATOM 5 ta 169
154. ck OK to open it to open it and view its contents e Click to update the view to show any folders files that may have been added by another user after you opened this dialog 44 e Data Menu User s Guide to KC4 Modifying Data Files After data has been retrieved from the reader or entered manually or read in from a file KC4 permits authorized users to change or mask raw measurement values KC4 automatically logs an event to the file s Data Audit Trail any time a change or mask action occurs Note Data Audit Trail behavior differs between KC4 v3 4 and KC4 Signature Turn to page 63 to learn more Important Changing or masking wells may significantly alter or even invalidate results e Only raw measurement values can be changed Raw data set names resemble M 405 M 3 490 and M 485 528 e When a well is masked KC4 excludes its raw measurement value from further calculations To change a raw measurement value within any data file 1 Open the desired file 2 Within the Results window click the Data drop down arrow and select a data set that contains raw measurement values e g M 405 or M 2 485 528 3 Click the Change box The System Administrator can protect the use of the Change function If you are trying without success to use this function you may not be authorized to use it 4 Click on a well in the grid and change its value When the changes have been made click OK to recalculate results K
155. ck the Reader drop down arrow To view a list of tests for a specific operator click the Operator drop down arrow To view test results highlight the desired date time from the list then click View To permanently delete test results from the database highlight the desired date time from the list then click Delete System Test Clarity The System Test for the Clarity Microplate Luminometer must be performed using the Clarity PC software Refer to the Clarity Operator s Manual for instructions Optics Test FL600 To perform an Optics Test with the FL600 e Ensure that the reader is properly connected to the computer and that all communication parameters are set correctly e Select System Diagnostics Optics Test e The FL600 Reader Diagnostics dialog will appear Click Start to run the test e As each individual test completes its pass fail status appears on the display e When the tests are complete the results will resemble the following 206 e System Menu User s Guide to KC4 Diagnostic of the Bro tek FL600 Reader FL600 on COM Model Number 210 Serial number 4 EEPROM version les Instrument status O Date of manutacture 17 78 95 Quarter 4 Voltages General Memory Pass Reset PASS Temperature PASS 28 1 C Y Axis Top PASS 15 PASS 143W Y Asis Bottom PASS 15 PASS 15 5 W Transport PASS 24 PASS 237 v Filter Ex Pass HT Fail PASS 4 Probe Change Ex PASS Fi
156. computer s must meet or exceed the following requirements Pentium IT processor 300 MHz minimum Microsoft Windows 98 NT 2000 XP operating system 128 MB RAM recommended Microsoft Office 2000 9 0 3821 SR 1 or newer Office XP Office 2003 User s Guide to KC4 Installation and Configuration e 9 Upgrading from an earlier version of KC4 M KC4 v3 4 M KC4 Signature Upgrade Notes If you are upgrading from KC4 version 3 1 3 2 or 3 3 and you plan to uninstall your existing version we recommend you do so after successfully installing version 3 4 This will allow the 3 4 installation program to recognize your current registration information You are not required to uninstall an earlier KC4 version If you choose to uninstall a pre 3 1 version of KC4 however be sure to preserve the KC4_32 pld file found in the root KC4 folder This file may contain custom plate formats that must be imported into the new plate database to support any protocols that reference custom plate formats See Plate Format in System Menu to learn more If you are upgrading from KC4 version 3 1 3 2 or 3 3 and you plan to reuse existing Local Shared and or Diagnostic databases make a note of the names and current locations of these databases They will have to be upgraded for use with version 3 4 During the version 3 4 installation KC4 will automatically run a Database Upgrade Utility to do this KC4 will search all fixed non removable drives e
157. d e Assigning the well identifiers to the appropriate locations on a row x column grid To perform these tasks select Protocol Plate Layout or click the Layout toolbar icon The Plate Layout dialog appears in the row x column format of the plate type selected under Protocol Reading Plate Layout Wall Settings Replicatez Auto Select Type Sample aaf mp FP NestlD I Next Dil Im SPLI f Horz C Yer Filling Dil None El C Hoer Vert ee A STD 57101 57101 SPL1 SPL1 SPL1 SPLY SPL7 SPLY SPL13 SPL13 SPL13 10 000 10 000 10 000 17 0000 5 0000 10000 10000 5 0000 10000 1 0000 5 0000 10 000 STO STO STO SPL SPL SPL SPL SPL SPL SPL 4 SPL1 4 SPL14 20 000 20 000 20 000 1 0000 S0000 10000 10000 5 0000 10000 1 0000 5 0000 10 000 c S TOS S5TO3 S5TO3 SPL3 SPL3 SPL3 SPL3 SPL SPL SPL15 SPL15 SPL15 40 000 40000 40 000 17 0000 50000 10000 10000 50000 10000 1 0000 50000 10 000 357104 57104 57104 SPL4 SPL4 SPL4 SPL10 SPL10 SPLIO SPLI6 SPLI6 SPL16 BO 000 80 000 80 000 1 0000 5 0000 10 000 1 0000 5 0000 10 000 1 0000 5 0000 10 000 STDS STDS STDS SPLS SPLS SPLS SPL11 SPL11 SPL11 SPL1F7 SPLIT SPL1AF 160 00 160 00 160 00 1 0000 5 0000 10 000 1 0000 5 0000 10 000 1 0000 5 0000 10 000 F STD STD STD PLE PLE PLE SPL12 SPL12 SPL12 SPL16 SPL18 SPL1B 320 00 32000 320 00 1 0000 5 0000 10 000 1 0000 5 0000 10 000 1 0000 5 0000 10 000 G Pos Pos
158. d blank well 4 1 0822002 14 09 56 MAJ Report changed Designed report template 08 2202 141015 MAJ Signed for Authorship by Mary Ann Jones seo 74 e Protocol Menu User s Guide to KC4 Plate Description Prompts The Plate Description dialog appears just prior to each plate read to provide the optional entry of plate specific information Plate Reading x Date 29 Dec 00 Time 09 59 47 Plate Description Name J rthur Knight Lot Code 1 736 002 395 Tithe Tech Il Water Lot 18b Site EACH Test 2 of 10 Comments Enter Manually Cancel Read From File Help This dialog gathers information to be stored in the plate file along with the measurement data KC4 allows you to define up to six free text fields to appear in this dialog they are shown above as Name Title Site Lot Code Water Lot and Test Select Protocol Plate Description to customize the names of these fields Plate Description E Frompt 1 Name Prompt 4 Lat Code Prompt 2 Title Prompt 5 water Lot Frompt 4 Site Prompt 6 Test Cancel Help e Text entered here substitutes the Prompt text seen in the Data Plate Description and Data Plate Read dialogs e To customize a prompt simply overwrite the default text in the Prompt field e Enter up to 15 alphanumeric characters in each field e Ifa field is left empty the prompt will be kept out of the Plate Description dialog User
159. d in the protocol feKC4 NewPlate Data Edit Protocol Display System Curve s Calculation Log Report Validation results ow Toolbar Curve s Launch Biograph and view the curve s Calculation View calculation warnings associated with the current data Log Report Validation Check the status of every Validation condition defined in the protocol Results Toolbar Show or hide the toolbar System Menu The System Menu contains special functions for configuring readers configuring system security setting global parameters performing reader diagnostics and more Data Edit Protocol Display System Readers Siow e Diagnostics Fun Optics Test Optics Test History Security Logln LogUut Define Universal Plates Audit Trail Aun Universal Plate Test Maintain Files Universal Plate Test History me Registration Upgrade Setup Toolbar Plate Formats Reader Control Readers Change or configure the current reader Diagnostics Run an Optics Test or Universal Plate Test on the attached reader Define characteristics for a Universal Test Plate View historical test results User s Guide to KC4 Getting Started e 35 Security Administrator LogIn LogOut LogIn LogOut Audit Trail Registration Upgrade Setup Toolbar Plate Formats Configure system security parameters and maintain user accounts Log into KC4 as the System Administrator single user system Log into KC4 using a uni
160. d of Absorbance Fluorescence Luminescence or Multi Mode Select a Reading Type of Endpoint Kinetic or Spectrum Optional For a fluorescence protocol enable the Time Resolved option Se YS For an absorbance protocol define the Wavelengths and Read Mode to be used Calibrate the wavelengths if necessary 5 Fora fluorescence luminescence or multi mode protocol define the Filter Sets to be used Select a Plate Type and define the First and Last Wells For a kinetic protocol click the Kinetic button to define the read time and interval For an endpoint area scanning protocol enable the Scanning checkbox and then click the Scanning button to define the read matrix size 9 Optional For a spectrum protocol click the Scales button to customize the graphical data display 10 Optional Define Shaking parameters 11 Optional Define General Options parameters 12 Optional Define Temperature Control parameters 13 Optional Select and define Pre Reading options 14 Click OK to verify the parameters and close this dialog User s Guide to KC4 Protocol Menu e 87 Synergy HT Time Resolved Injectors Reading Type End Point C Kinetic Spec SHUT IV Dispense Read amp Dispense Scales l Time Resolved Reader Synergy HTTA weInjectors on COM2 Detection method C Absorbance Fluorescence Luminescence Multi Mode Filters Filter Set Mode Emission
161. d with your KC4 package 14 Click the Registration Card button to verify the number and open a registration form Registration card Main Registration Information Customer License Number license number are Sah U2GH TH3D 37 a Last Marne Jones Upgraded product O RES Aerdlock protected Hardlock SMH ER Company Bio T ek Instruments Inc Salutation Dr O KEJ Sotware protected Department ne License Number Gee Address 1 Highland Park Keuna A iors Address 2 Box 998 SNE heck City Winooski System Information E A Moo FC Brandi Compaq Zip Postal Code 05404 Processor Pentium 4 1693 MHz Country USA OS verion windows 2000 5 00 Phone 1802 655 4040 OS Add Info Service Pack 2 E mail Jioness biotek com Beeder SynerqyHTTR Fax re02 655 3849 Send by fas Send by e mail Close 14 e Installation and Configuration User s Guide to KC4 15 Enter the required information and click Send by Fax to print a copy of this form for later faxing or Send by E mail to send the information to Bio Tek via email Bio Tek will respond within two business days with your password Ifyou are upgrading from a previous KC4 version or from KCjunior enter the requested KC4 hardlock serial number KC4 software license number or KCjunior serial number as appropriate Click Check to verify the number If you are reinstalling KC4 you must submit a separate Reins
162. dard Users PJS added and Peter power 1014 02 13 39 40 Administrator Login settings changed Changed Lock user account from 5 to 4 failed login attempts 1041402 13 40 17 Administrator Logout Computer Y W2K BRITTINK Session 22607518 1041402 13 40 19 PJS Failed login attempt Computer VV2K BRITTINK Session 2657315 1014 02 13 40 25 PJS Failed login attempt Computer 2K BRITTURK Se ion 426071310 1041402 13 40 27 FPJS Failed login attempt Computer 2K BRITURK Se ion 2607518 104 402 13 40 35 PJS Failed login attempt Computer VV2K BRITTINK Session 2657315 1041 4 02 13 40 45 Administrator Login Computer Yv2K BRITTINE Session 226073181 10401402 13 41 06 Administrator Users PJS unlocked Unlocked Peter Smith s account tt became locked after his 4 tailed login attempts 109402 13 41 29 Administrator Logout Computer VW2K BRITTINK Session 2667315 104402 13 41 35 PJS Login Computer Vv2k BRITTINK Session 2687318 The System Audit Trail displays Shared and Local events recorded for the following activities e User login and logout e System security updates user accounts login and password parameters protected functions audit trail notifications and file location and format e Plate format modifications e Universal test plate modifications e Reader settings changes reader type probe selection communication parameters and filters wavelengths e System setup changes protocol de
163. data set highlight the entire row then click the right mouse button and select Delete To insert a new data set between two existing sets highlight the row above which the new data set will be inserted Click the right mouse button and select Insert Selection Data JO operator Format Precision a w O S S o m al Decimal Decimal For data sets that represent numerical information an Operator can be applied to perform a specific calculation Nb displays the number of wells in the group Mean calculates the average measurement for each well group Std Dev calculates the Standard Deviation for each well group CV calculates the Coefficient of Variation for each well group as Std Dev Mean x 100 Click OK to close this dialog and insert the object into the document Ed Microsoft Excel Worksheet in lt KC4 gt 687 File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves x Close Mean Std Dev CV Symbols PII Sheet1 Ready AA A A A A Es 288 e PowerReports User s Guide to KC4 Well IDs A Well IDs Table lists well identifiers and their names To create a Well IDs table click the Tables button in the RSO toolbar and then click on Well IDs The Statistic Table Properties dialog appears 149 Document in lt KC4 gt Microsoft Word Eile Edit View Insert Format Tools Table Window Help Normal gt Times New Roman 10 B Y U Fields Plat
164. default setting of 00 00 00 allows the well s to be read as quickly as possible Multi Mode Only applies to the Synergy HT line of instruments Detection method C Absorbance Fluorescence C Luminescence Multi Mode Synergy HT instruments with basecode versions of 2 10 or higher support KC4 s Multi Mode detection method option When Multi Mode is selected more than one detection method can be defined within a single protocol When Multi Mode is enabled the following Reading parameters are supported End Point reading type with up to 6 wavelengths or filter sets Kinetic reading type with up to 6 wavelengths or filter sets Time Resolved Fluorescence HTTR Pre Read Blank Plate Monitor Wells Automatic Sensitivity Adjustment The following Reading parameters are not supported Rapid and Sweep read modes absorbance reads are always performed in Normal mode Spectrum reading type Area scanning Pathlength correction Absorbance wavelength calibration via the Reading Parameters dialog and before performing the read Eject between filter set reads Read amp Dispense 108 e Protocol Menu User s Guide to KC4 If dispensing 1s required during a kinetic read here are two options Manual Dispense Create a protocol with a long kinetic interval During an interval when the reader is idle Push the carrier eject button on the front of the reader to eject the plate Perform the dispense using a pipette
165. dialog and insert the Field Object into the document The document shown on the next page contains six Field Objects Each object is represented by a 1 row x 3 column table Notice that Field objects do not have to be separated by a blank line or row See Object Components for more information 282 e PowerReports User s Guide to KC4 cay Document in lt EC4 gt Microsoft Word Eile Edit wiew Insert Format Tools Table Window Help Normal Times Mew Roman 10 B F E ES Fields Plates Tables Y Close a E Technician Site Reading Date Reading Time Protocol File Name o Data File Name g B El 4 Page 1 Sec 1 111 E En Col 1 RES RR EXT Inserting Plate Objects A Plate Object presents information in a matrix corresponding with the plate type selected in the protocol For example if the protocol specifies a 96 well plate the plate object contains 8 rows and 12 columns of data To insert a Plate Object click the Plate button in the RSO toolbar A drop down list appears containing all of the data sets available for inclusion in the Plate Object taj Document in lt KC4 gt Microsoft Word File Edit View Insert Format Tools Table Window Help Normal Times Mew Roman 10 HB Y U Fields Plates Tables Curves J Close Mati 405 X M 2 630 M 1 405 Corr MZ 6350 Corr Delta OD Concentrations Symbols e Data set availability is based on t
166. ding Points in the protocol is 13 The default From x and To x settings are 6 and 6 respectively To change the calculation zone to include only the three points to the left and right of center 0 set From x to 3 and To x to 3 6 3 0 3 6 Points in calculation zone All scanned points 13 total Mean Min Max Select Mean Min Max to determine and report the Mean Minimum and Mean Maximum measurement values based on n points By default the calculation zone includes all of the Horizontal Reading Points defined in the protocol and the n value is 1 To change the calculation zone and or the n value click Calculation Options Scanning Analysis Mean Min Max Ea Calculation Zane f From E Tox le e From f Tot 13 Mean Minhas on i points Cancel Help e Currently only the From x To x method for determining the calculation zone 1s supported See Mean Std Dev CV for more information e The default number of points n used to calculate the Mean Minimum and Mean Maximum values is 1 KC4 calculates the Mean Minimum and Mean Maximum values as follows Starting at the first point in the calculation zone KC4 evaluates n points and calculates the mean KC4 repeats the operation starting at the second point in the calculation zone then repeats again starting at the third point and so on KC4 then compares all mean values to determine the minimum and maximum values 140 e Protocol Men
167. dministrator To permanently delete a user account 1 Login as the System Administrator 2 Select System Audit Trail to see if the user is currently logged into KC4 You can still delete the account but it will not take effect until the user logs out and tries to log back in or tries to sign a file 3 Select System Security Highlight the desired account name and then click Delete A confirmatory message will appear click Yes to confirm or No to cancel the process Note The System Administrator s account cannot be deleted Changing an Account s Status Administrator cc oocohO AA The Status box within the KC4 User Settings dialog shows whether or not that user s account is currently locked and allows the System Administrator to lock or unlock the account When a user s account is locked the user cannot log into KC4 and cannot sign files A user s account may become locked through one of three ways e Intentional lock by the Administrator through this dialog e Automatic lock if the user has exceeded the defined number of successive failed login attempts e Automatic lock if the user s password has expired To unlock an account uncheck the Status box and click OK to return to the Security dialog Click OK again to return to the main view The change is effective immediately the unlocked user is now permitted log into KC4 and to sign files To lock an account check the Status box and click OK to return to t
168. e Click OK to display the new curve Closing a Curve Select File Close to close the current curve If the current curve is one generated dynamically using data from KC4 clicking Close will close both the curve and the Biograph application If the current curve is a stored gph file clicking Close will close the file but leave Biograph open Saving a Curve Graph File Select File Save or File Save As to save the current curve as a graph file Graph files of the type GPH contain calculated curve fitting values A stored GPH file can be used as the basis for calculating concentrations in a protocol that does not contain standard wells Tip You can use refer to this curve in KC4 as the basis for calculating concentrations in a protocol that contains no standards To do this select Protocol Curves in KC4 then enter the name of this GPH file in the Curve Origin File field 266 e Biograph User s Guide to KC4 Opening a Graph File Select File Open to retrieve a stored graphic file with the extension GPH and manipulate it in the same manner as newly created graphs Look in S Samples 7 ella El Standard Curve O1 gph File name Frequency gph Files of type GRAPH gph Cancel e To open a file highlight its name then click Open e To look for a file in a different folder click the drop down arrow next to the Look In field or click the previous folder button Printing Curves Select File P
169. e remaining KC4 root folder was moved renamed or deleted This error message occurs because one of the files used for building PowerReports cannot be found The error may occur e When starting KC4 if Excel is the default reporting or exporting engine and the Preferred file format is GLB e When creating a new protocol or GLB file if Excel is the default reporting or exporting engine Follow these steps to solve the problem 1 Close KC4 2 Launch Microsoft Excel The TplRep_1 xla could not be found message will appear 3 Click OK 4 In Excel select Tools Add Ins The TplRep_1 xla Add In will be selected 5 Unselect the TplRep_1 xla Add In Excel will display an error message indicating that the file could not be found and prompts you to delete this Add In Click Yes to delete the Add In Quit Excel 8 Restart KC4 The TplRep_1 xla could not be found message should no longer appear 20 e Installation and Configuration User s Guide to KC4 Changing the System Administrator s Password M KC4 v3 4 M KC4 Signature We recommend that you change the System Administrator s password immediately following software installation to ensure a secure operating environment To change the System Administrator s password 1 Log in as the System Administrator if you have not already done so KC4 v3 4 Select System Administrator Login KC4 Signature Select System LogIn LogOut 2 When the Login dialog ap
170. e CVs although selecting a large number of samples per well typically results in only marginal improvement Consider a setting that represents the optimal combination of sensitivity and speed The recommended setting to achieve a balance between speed and sensitivity is 10 measurements per well Note The reader takes approximately 10 milliseconds to perform each measurement Delay before sampling is the pause time of the optical probe above or below the microwell before the measurement is taken Valid options are 0 10 0 35 0 50 and 2 00 seconds The recommended setting for fluorescence assays to ensure the probe 1s stopped for measurement but for the minimum amount of time is 0 35 seconds For luminescence reads that allow long reading times a delay of 2 0 seconds will provide lower CVs This delay of 2 0 seconds is not recommended for fluorescence reads Delay between samples is the time delay between measurements taken in each well The valid range is from 0 to 2550 milliseconds The recommended setting is 10 milliseconds Longer delays between samples may result in better CVs between replicates Measurement Mode FLx800 Synergy HT Measurement Mode Nb Samples per well li O Delay before sampling 350 msec Delay between samples li msec To define the measurement mode parameters for the FLx800 and Synergy HT Nb Samples is the number of measurements taken per well The valid range 1s from to 255
171. e Open dialog will appear Open Look in a EC4 Shared Area d jE R C5hared MOE File name KCShared MDB Files of type Databases 2 2 Use the file tree to locate the KCShared mdb file on the network Highlight the file and click Open The Database dialog will reappear and the Path field will update to show the new location 3 Click OK to close the Database dialog Note With KC4 Signature an Audit Trail dialog will appear to log this event Enter an appropriate comment and click Close Audit Trail E Date 103102 16 03 55 User Administrator E vents H System Database changed from C Documents and Settings 4ll Users4pplication D atas Bio Tek l Comment Reconfigured K C4 to point to EShared mdb on the network ou must quit and restart KC4 before the new settings will take effect Do you really want to quit KC4 5 Restart KC4 User s Guide to KC4 Installation and Configuration e 23 Creating New User Accounts M KC4 Signature KC4 Signature supports the creation of individual user accounts to ensure that only authorized users can gain access to the system and to any protected functions To create a new user account 1 Select System Security Users 2 Click New The KC4 User Settings dialog will appear Security E E x Users Login Frotected functions Audit Trail Signature Reasons Files W Multi User identification Administrator System Administrator KC4 User S
172. e at which a reading begins relative to the start time of the very first read in the operation which is always 00 00 00 Kinetic times are displayed underneath the Process times in black dark text Note that in a kinetic protocol each read step will have a Process time and a Kinetic time associated with it User s Guide to KC4 Protocol Menu e 121 Dispense Step Parameters Use this dialog to create or modify a Dispense step Dispense step Dispenser li E Tip Prime Priming before the process a Wolume 5 pl Dispense Start time 00 00 00 Shortest after SHAKE 10 s Intensity 1 Wolume 100 pl Rate 300 E Wy Sec Cancel Help time le approximate and reader configuration dependent e Select the number of the Dispenser to be used for this step The numbers available for selection correspond to the numbers on the dispense box e Select a Tip Prime option Tip priming is performed in a small removable priming trough located in the left rear The purpose of tip priming is to compensate for any fluid loss at the dispense tip due to evaporation since the last dispense The trough holds up to 1 ml of liquid and must be periodically emptied and cleaned by the user KC4 must then be told that the trough has been emptied via the System gt Reader Control dialog If the trough overflows refer to the Synergy HT Operator s Manual for decontamination instructions None No prime Before the
173. e available colors Click on the desired color 148 e Protocol Menu User s Guide to KC4 Assigning Well IDs After defining the Well Settings assign the Well ID s in the grid area of the Plate Layout dialog Here is a very simple example for assigning blank wells to the plate e Select the Blank well type from the Type drop down menu e The ID field shows the default identifier BLK Well Settings Type Blank ha ID ELK e Assuming you wish to use the BLK identifier highlight the well s in the template that should contain blanks then release the mouse button e The average of the measurement values in wells Al and A2 will be subtracted from every well on the plate KC4 provides options for defining replicates and automatically updating the Well ID numbers The following example shows you how to define Replicates and use the Auto Select and Filling features to assign samples in triplicate e Select the Sample well type from the Type drop down menu Wall Settings Type Sample w Empty Blank Assay Control Sample al onto ha 3 E stendara Ma m e The ID field shows the first ID in the sequence SPL1 Wall Settings Type Sample g ID SFL zj Dil None d User s Guide to KC4 Protocol Menu e 149 Set the number of Replicates to 3 then select the Horizontal filling option Sample replicates will be assigned horizontally Al A2 A3 B1 B2 B3 and so on Repli
174. e is from 1 to 255 measurements The value reported for each well represents the average of its measurements Usually the more samples per well the better the CVs although selecting a large number of samples per well typically results in only marginal improvement Consider a setting that represents the optimal combination of sensitivity and speed The recommended setting to achieve a balance between speed and sensitivity is 10 measurements per well e Delay before sampling is the pause time of the optical probe above or below the microwell before the first flash The valid range is from 10 to 2550 milliseconds The recommended setting to ensure the probe 1s stopped for measurement but for the minimum amount of time is 350 milliseconds e Delay between samples is the time delay between the end of data collection and the beginning of the next flash in each well The valid range is from 0 to 255 milliseconds The recommended setting is 5 milliseconds Longer delays between samples may result in better CVs between replicates e Note for Injector models The following values are fixed for Dispense protocols Plate Mode Well Mode Nb Samples per well 10 Nb Samples per well 1 Delay before sampling 350 Delay before sampling 10 Delay between samples 24 Delay between samples 0 If the Time Resolved option in the Reading Parameters dialog is checked specify the Time Resolved Fluorescence options e Delay is time delay between the flas
175. e one ENE T AANE I o STE AA ee ae eo Os 264 BIOGRAPH ais 265 DS A A 265 Ed a e o AA e o o ete ers bea 266 AA A tested state eae eae 266 TGs fin Ore UI RARA 266 Savino a O FE organ er errr sr ne ND er ee 266 Oe Iii Oa Grape coo 267 EATS ce gree ae ade ee de ee ee ee ede es 267 EXT O co aa 268 MI ae enano AE ie nes adas a oN dla TU Da 268 User s Guide to KC4 Contents e vii TO A nescence mse se ie st ls aoa a 268 THE Status Ba 268 OA Menta tata beapetencesebaad teaniates 269 SP O 269 Axess Pit Interpola OS aa scada 269 Overa yO TAPAS aaie 270 TV WALON ss sk A ele aaa eet 271 POWERREPOR TS dic 273 Heh Powered Report 215 HOWIE INOT K Senee ten ce ters ate et cane eats AE ican eats E E canes meet 274 System REGUS ISIS orra steal ates cene E OE E 276 Preparino KCA fort POW CEC PO ed 276 Semino Global OPUS iaa ata 276 Seino Protocol Spec iic O OS 271 Detinine the Protocol Paola 278 Creating Edining De Ud 278 W rkme wiih Results Dd 279 The Results Objects TODA 219 Object COMPONENTS serie cect no ee ne 280 Ob eet E a e OS o 281 Desi nino he Docena ria rot 282 Inserte Fier Obs oa 282 Insertine plate Objects idas 283 Insertino Pablo Di cis pia 286 SMS CU e dl 302 Prevyiewine and Printing the Report usan 306 Preview IO 10 1810 1 Reena E A ae Pie ese E ee Peer 306 Special NOTAM ONS a 307 Pertormins the Data EXPO A A donee ens 308 TIE CUE Ort tO Excite 308 APPENDIX A RECORDS SUBMISSION cccccssssceeeeeeeeeeeeeeeeee
176. e sample was taken e Choose whether or not to use out of range values in statistics Out of range values are those values reported as lt xxxx or as gt xxxx Example SPL1 has three replicates reported with the following concentrations well Al 10 230 well A2 lt 10 out of range value well A3 10 450 If the option is not selected the software will not use the concentration of well A2 in the statistic calculations Therefore the mean standard deviation and CV of SPL1 will be 10 34 0 155 and 1 5 respectively Ifthe option is selected concentration of well A2 is taken into account the value of 10 is used in the statistic calculations Therefore the mean standard deviation and CV of SPL1 will be reported as lt 10 22 0 225 and 2 2 respectively Important Exercise caution when enabling this option as 1t can lead to biased results particularly for standard deviation and CV As a warning statistic results arising out of the use of this option are displayed in italic in the report Formats Select System Setup Formats to choose an import export separator and a time format for kinetics Protocol Defaults Formats Start up Database Import Esport Separator L C Tab Other Time Formats for Kinetics HH MM 55 C HHH MM 5555 sss seconds milliseconds C 55555555 seconds sssssszs milliseconds Cancel Help User s Guide to KC4 System Menu e 231 e Select an Import Expo
177. e the tip with fluid before dispensing to each well e If tip priming will be performed enter the Volume in microliters The valid range is 5 to 20 e Set the Start Time for the step Enter the time manually SSSSS sss or check Shortest and select the step immediately after which this Dispense step shall be performed e Enter the dispense Volume in microliters The valid range is 5 to 1000 e Select the dispense Rate in microliters per second The default rate 1s the maximum available for the attached instrument e Click OK to save the settings and add the Dispense step to the list 126 e Protocol Menu User s Guide to KC4 Read Step Parameters Use this dialog to create or edit a Read step Read step Start time 0 340 Run time 2 000 z Interval 0 100 z Reading number 21 T Block light between reads e Enter the Start Time in seconds sssss sss e Kinetic protocols only Specify the Run Time This is the length of time from the start of the first kinetic read to the start of the last kinetic read Specify the time Interval for repeat reading of the plate section of wells or individual well The maximum valid Interval is equal to the current Run Time KC4 automatically calculates the Reading Number by dividing the Run Time by the Interval and adding 1 e Optional For non TR Fluorescence reads check Block light between reads to block the light between measurements to inhibit photo bleaching
178. ead to show the rate of change immediately following the injection of fluid This can also be accomplished by creating two Read steps one immediately before and one immediately after the Dispense step e The steps are listed in the order in which they will be executed The Description column provides key information about the step such as volume rate or intensity The Start column indicates the time the step will begin The End column indicates the time the step will complete The Comment column contains additional information about the step if needed To edit a step highlight it and double click or click the right mouse button and select Edit To delete a step highlight it and press the Delete key or click the right mouse button and select Delete e The steps and are also displayed graphically to show how they will be executed on each well over time Dispenser 1 Dispenser 2 and Read steps are shown in parallel Dispense step The solid area represents the execution of the Dispense for one well Read step The solid area represents the execution of the Read step for one well Time indicators A Process time indicates the time at which a particular step will begin relative to the start time of the very first step in the operation Process times are displayed in grey light text Kinetic protocols only A Kinetic time indicates the time at which a reading begins relative to the start time of the very
179. eading To create edit reference a Clarity protocol file within a KC4 protocol make sure that Clarity is set as the current reader System Readers and then select Protocol Reading The Reading dialog will appear as shown below Reader Clarity Clarity Protocol Path C Program Files BioT ek Clarity protocols 6 TF bpt Create New Edit Browse kmete scales crest o e To create a new Clarity protocol file click Create New The Create Protocols dialog will appear Select a protocol type and click Create to advance to the parameter input series of dialogs e To search for and open an existing Clarity protocol file click Browse The file s extension must be BPF Highlight the file and click Open The file s name and location will appear in the Path field e To edit reading parameters for the currently selected file click Edit The Protocol Settings dialog will appear Make the necessary changes and then click Save to store the settings using the current file name or Save As to store the settings using a new file name e Optional For Fast Kinetic protocols click the Kinetic Scales button to adjust the min max t and min max y scales for curve displays e When finished click OK to save changes or Cancel to discard them You can now define data reduction parameters via the Protocol Menu Plate Layout Transformations Cutoffs Reports etc or run the protocol by selecting Data Read Plate User s Gu
180. ected reader You can for example import a data file containing raw luminescence data and then create a KC4 protocol to apply transformations calculate curves generate reports and so on The following requirements must be met to ensure a successful import e The Clarity file must have a bti extension e Before the bti file is saved in the Clarity PC software the Displayed digits after period field under Options Visual Settings must be set to 0 e Ifyou wish to import a bdf file open it in the Clarity PC software select File Save As and select the bti file type e Batch Protocols must contain a maximum of 300 reads and have a total measurement time of less than 48 hours e Ifthe file has a measurement comment associated with it Measurement Measurement Comment the comment cannot contain any empty lines For example the following comment would cause the file import to fail because the second line is empty 1st line Measurement performed by Brian Smith 2nd line lt empty gt 3rd line Results approved by Sharon Jones Notes on background measurement values If the Clarity file specified a background measurement and the Subtract instrument background option was enabled KC4 imports the subtracted values as well as the background value for informational purposes This value can be viewed in KC4 by selecting Protocol Reading when the data file is open If the Clarity file specified a background measurement and the
181. eeesnseessssssseeeeeeeeeees 309 Records UDS Oi 309 APPENDIX B WINDOWS FILE CACHING cccsscscseeeeeeeeeeeeeeeeeeesesssssssssseeeeeeeeeees 311 Windows Els CAC ING op cece 2s cant acetal ce cane ohetnaulicecaisial N acsteateeecevendeect 311 INDE ees ser E O 313 viii e Contents User s Guide to KC4 Introduction KC4 Software Bio Tek s KC4 software provides rapid flexible and powerful assay analysis with a multitude of applications The user interface is simple and uncluttered and provides a quick step by step setup and read process for even the most complex assays Applications include Research DNA quantitation Fluorescence assays Bio Industrial Pharmaceutical Development Interfaces with most Bio Tek autoreaders Clarity FLx800 PowerWavexX 340 EL800 PowerWave PowerWaveX Select ELx800 PowerWave 340 PowerWave XS EL808 PowerWave HT Synergy HT ELx808 PowerWave HT 340 uQuantiM FL600 PowerWaveX Extensive and complete analysis of your laboratory applications Fluorescence and absorbance ELISA Spectral scanning from 200 999 nm with the wQuant and all PowerWave PowerWaveX and Synergy HT models Endpoint assays Kinetic assays Endotoxin analysis Cytotoxicity and titer assays User s Guide to KC4 Introduction e 1 Defines and controls all reading protocols Selection of filters wavelengths Shaking parameters Incubation Flexible kinetic options Read Dispense o
182. elect the reason to be associated with this signature Signature Reasons are site defined by the System Administrator under System Security Signature Reasons 4 Optional If two users are signing on behalf of a third KC4 user check the Representative of box and then choose a name from the drop down list Representative of Patrick Walter Barmes Representative 1 Representative 2 User Mii Full Name z an Ann Jones 5 Enter or select your User ID If Representative Of is checked two users must enter or select their User IDs Enter your Password The characters will not be visible on the screen Click Sign amp Save User s Guide to KC4 Protocol Menu e 73 If the file has not yet been saved a Save As dialog will appear Current Folder De Wones Mary ANN ucleic Acid 4 K l Modified 0MN4 Quart q ANA Quant 3 260_280Ratio prt 14 EB KC4Protocol 04 11 01 15 31 10 E 34320 pri 14EB EC4 Protocol 04 7 7 07 13 11 50 1 Oligo Quant prt TERE ELA Protocal 0417201 130228 File Name Phenol Type kca Protocal prt Cancel 8 Using the folder location tools provided select the storage location 9 Enter the desired File Name and click OK The signature is applied immediately and KC4 logs the event in the Protocol Audit Trail select Protocol Audit Trail to view A oaa E User Action Comment 08 2202 14 08 55 File Created 08 2292 14 09 35 MAJ Plate Layout changed Adde
183. ely These wavelengths can be changed by clicking the Options button User s Guide to KC4 Protocol Menu e 109 e KC4 uses a default value of 0 18 for the absorbance of water at 1 cm This value can be changed by clicking Options and defining a different Absorbance of Solution at 1 cm Alternatively click Bio Cell to calculate the absorbance of water at 1 cm value using Bio Tek s Bio Cell plate Use the drop down menu to select the location of the cuvette in the plate Corrected Absorbance Calculation KC4 calculates the corrected absorbance in each well according to the following formula Awavelength well pathlength of well e Ifa Blank Plate is pre read and or if blank subtraction is performed select the advanced protocol option Raw Data Correction to control the data used in this formula Pathlength Correction Options Click the Options button to change the Test or Reference Wavelength values or to change the pathlength constant PathLength Correction Options Test Wavelength nm 977 Reference Wavelength nm 300 Lancel Absorbance of solution at 1 cm 018 Help e The valid range for the Test and Reference Wavelength fields is 200 to 999 nm e The default Absorbance of solution at 1 cm value is 0 18 To use a different constant value when calculating pathlengths simply enter the desired value Reporting the Pathlength Values KC4 automatically reports the Corrected Data after Pathlength Correction i
184. enance Moving a Syringe to its Maintenance Position If a syringe needs to be installed or replaced it must first be moved to its Maintenance Position To do this using KC4 e InKC4 select System Readers click the Configuration button and then click the appropriate Dispenser tab 1 or 2 e Click Move Syringe to its Maintenance Position e The syringe plunger will move to its furthest from home position The syringe can then be disconnected form the drive bracket and unscrewed from the valve see the Synergy HT Operator s Manual for detailed instructions e Click OK to close the dialog User s Guide to KC4 System Menu e 203 Controlling Multiple Instruments Simultaneously If the computer has more than one COM port more than one instrument can be controlled at a time by running multiple instances of KC4 Important Please review the items under Technical Issues before attempting to control multiple instruments simultaneously To control multiple instruments simultaneously Connect the instruments to the computer one per COM port Launch a single instance of KC4 Open System Readers Select a reader model define and test its Port settings then set up its Filter Wavelengths table Repeat for each attached reader Once communication is established for all attached readers launch additional instances of KC4 one per reader Create open and run protocols within each KC4 instance Technical Issues
185. enerated Biograph Display Curve tt Control the attached reader s door carrier movement or incubation This button is E accessible only if these functions are supported by the current reader Control System Reader Control User s Guide to KC4 System Menu e 239 Plate Format This dialog lists all of the plate types currently stored in the shared database For each plate type the database contains formatting information such as width length height and the number of rows and columns When a plate read is initiated KC4 checks to see which plate type is selected in the Reading Parameters dialog and then sends the appropriate formatting information to the reader The reader uses this information to precisely position the plate when taking measurements Not all readers support all of the plate formats in this list Consult your reader s operator s manual for a list of supported plate sizes KC4 comes with format information for more than forty industry standard microplates You can add your own microplates by selecting System Plate Formats Plate Formats x Bio Tek PCA high adapter Cliniplate 96 flat bottom Cliniplate 96 flat bottom blac Cliniplate 96 flat bottom whit Corning 6 well Corning 96 well flat bottom Costar 1 8 8 flat bottom Costar 12 well Costar 4 well Costar 48 well Costar 6 well Costar 96 black opaque Costar 96 clear bottom black Costar 96 DMA BIND Costar 96 flat bottom Costar 96 half area black
186. eneration For readers with multiple wavelength gt 2 reading capability a default Multi Plate Transformation is automatically created when exactly 2 wavelengths or filter sets are specified These default parameters can be changed e The Protocol menu switches to Advanced Mode e The Multi Plate Transformation dialog contains X1 lt Meas OD 1 gt X2 lt Meas OD 2 gt Formula X1 X2 Data Out Delta OD e In addition if the protocol is Kinetic The Kinetic checkbox becomes enabled The Reading fields for both X1 and X2 are set to All Note Once the Reading Parameters dialog is saved and closed with 2 filter sets wavelengths specified the Delta OD Multi Plate Transformation will not automatically clear if the protocol is then changed to or 3 6 filter sets wavelengths The user must open the Multi Plate Transformation dialog and manually empty modify the fields User s Guide to KC4 Protocol Menu e 163 Select Protocol Curves to define various curve fitting parameters including the data set to interpolate and the fitting method Interpolations are defined here as well Curve Description Select Protocol Curves to select the data set to be used for curve calculations and to specify the origin of the curve Curves Curve Description Axes Curve Fit Interpolation Data To Interpolate as 405 Corr Brose e The Data to Interpolate setting represents the type of data to be used for c
187. ent reader is the FL600 and the reading type is Kinetic click the Options button associated with Filter Set 1 Options selected here are applied to all defined filter sets Measurement Mode FL600 Measurement Mode C Rapid Mode f Static Nb Samples per well 10 al Delay before sampling 0 35 Sec Delay between samples i 0 nsec To define the measurement mode parameters for the FL600 e Choose between Rapid and Static mode Rapid Mode reads each well while the assay plate is in motion This provides for faster reading than with Static mode but the CVs might be lower The FL600 is equipped with fluorescent plastic compounds called reference wells which help provide the best results when using Rapid Mode to read the assay plate See General Options FL600 for more information Static Mode performs a specified number of reads in each well with a specified delay between each read The reading performs more slowly than with Rapid Mode but the CVs are usually better Nb Samples Delay before sampling and Delay between samples pertain specifically to Static Mode 104 e Protocol Menu User s Guide to KC4 If Static Mode was selected specify the Sampling options Nb Samples is the number of measurements taken per well Valid options are 1 2 3 4 10 20 50 and 100 measurements The value reported for each well represents the average of its measurements Usually the more samples per well the better th
188. enter the temperature in the Requested field and then check On to begin pre heating On enables the incubation unit the temperature will rise or fall to the Requested temperature as appropriate Off disables the incubation unit the temperature will fall to ambient e The Actual field reports the current temperature Celsius of the incubation unit e For the FL600 the Ambient field reports the current temperature of the outside environment via a sensor located within the reader refer to the FL600 user manual for sensor location information e The Total Time field reports the total time elapsed since the On box was checked Associated with the Total Time field the Beep after checkbox and field can be enabled to request that KC4 beep continuously after a specified duration The duration begins when the incubation unit is turned on e The Time since reached field reports the total time elapsed since the incubation temperature has reached the Requested temperature Associated with the Time since reached field the Beep after checkbox and field can be enabled to request that KC4 beep continuously after a specified duration The duration begins when the incubation temperature reaches the Requested temperature e The REACHED indication appears when the incubation temperature has reached the Requested temperature Note As the temperature of the incubation unit approaches the Requested temperature 1t may take a few minutes to settle wit
189. eport 188 e Protocol Menu User s Guide to KC4 Display Menu Overview The Display menu allows you to check the verification status of your data and or to activate the Biograph module to view adjust and otherwise interact with test results as displayed on a curve or set of curves Curve s Select Display Curves to load Biograph and view the curve or curves generated using the current plate data This option is available only if the protocol defines a standard curve or dilution curves fifa BIOGRAPH Windows Application STANDARD CURYE E File Edt View Graph Window About Haa S em 4 Parameters y a d 1 4 s c b d a 0 02575 b 1 345 c 2717 d 4 933 A 0 9998 A 0 9997 er 0 02578 e When Biograph is loaded every curve calculated for the current plate appears in a window See Biograph for information on changing the curve fitting method deleting standard points printing the graph and more e To quit Biograph and return to KC4 select File Exit User s Guide to KC4 Display Menu e 189 Calculation Warnings Select Display Calculation Warnings to view a list of data reduction calculation warnings issued by KC4 against the current set of data If this list is empty KC4 found no errors with the calculations ie KC4 NewPlate created from Phenol prt Data Edit Protocol Display System Zetting Layout Aralere lis RESULTS Data Symbols Audit Trail ie Calculation warnings Be
190. eport document Ero export document Tip If you don t see these buttons in KC4 s toolbar you can add them by selecting System Toolbar 278 e PowerReports User s Guide to KC4 Working with Results Objects A Results Object is a placeholder of information in an Excel or Word document that is filled in with results data during the print preview print or export function The Results Objects Toolbar When KC4 launches Excel or Word it automatically adds a Result Objects toolbar to the standard Excel Word toolbar Ed Microsoft Excel Worksheet in lt KC4 gt Window Help x cose gt Remember that a results object is a placeholder of information which will be filled in by KC4 after the plate is read Each object specifies which item of information to use where the information will be located in the document and how it will be presented font style color size etc Fields Plates Tables UVES Close eee EEE Insert a Field Object Each object contains a single piece of information such as a cut off value a file name or a curve coefficient Clicking the Fields button opens a dialog with lists of available categories and related items to choose from Categories include Curve Fit Cut Offs File Information Plate Description and Reading Parameters Insert a Plate Object to present data in a row x column format matching the plate type defined in the protocol Clicking the Plates button
191. er DE ProtocolsiMary Jones Browse le Cancel Help To set a new password 1 Enter your Current password if one exists 2 Enter a New password using up to ten alphanumeric characters no spaces Note If the New password field is inaccessible the correct Current password must first be entered 3 Enter the new password again to Confirm it 4 Click OK to save the password and close the dialog Ifthe message Password minimum length is not respected appears this means that the System Administrator has set a minimum password length between 2 and 10 characters and the password you entered is too short Important The System Administrator cannot retrieve your password if you forget it If you do forget your password contact the System Administrator to request a temporary one and then change it immediately 214 e System Menu User s Guide to KC4 To specify a Protocols Folder 1 Using the Browse button select the folder location from which you will most often access protocol files When you select Data New Plate to initiate a plate read the protocol selection dialog will default to the folder defined here If no folder is specified KC4 will default to the most recently accessed folder 2 Click OK to save the change and close the dialog Specifying a Protocols Folder is optional and is only available if the Secured Protocol and Data File Storage feature is in use Deleting a User Account A
192. es Tables pl Close Statistics Well IDs Statistic Table Properties Selection Data Identifier Standard TO Assay Control M Blank M Sample M Sample Control e Select the Identifier s to be represented in the table If Sample and or Assay Control is selected Index is by default set to All meaning all sample and or assay control wells will be included in the table To include a subset of all sample and or assay control wells use the Index From and To fields to enter the limiting group numbers In the example above Sample groups 1 through 10 SPL1 to SPL10 will be included in the table User s Guide to KC4 PowerReports e 289 Click the Data tab to customize the contents of the table tay Document in lt KC4 gt Microsoft Word File Edit View Window Help slee 2 a Table Title Statistic Table Properties a operator Format Presieion A a a ane 3 M 405 Corr N E a7 Imaos M 405 Corr For the Well IDs table KC4 automatically inserts the Well ID and Name data sets Any available data set can be included in the list See Statistics for information on working with data sets Click OK to close this dialog and insert the object into the document iy Document in lt KC4 gt Microsoft Word Eile Edit View Insert Format Tools Table Window Help Normal Times Mew Roman 10 BE 7 U Fields Plates Tables lt Close 290
193. eter of any well e Height is the distance from the bottom mounting surface of the plate to the top face of the plate User s Guide to KC4 System Menu e 241 Microplate Measurement When creating or updating plate dimensions it is essential that precise measurements be taken Use calipers with precision to 0 01 microns to gather the following measurements A standard ruler is not precise enough Important Dimension values are used by the software to calculate reading positions Any inaccuracies in these dimensions could significantly affect your results The following diagram demonstrates the proper method for taking measurements for a standard 96 well plate Top Left X Diameter Top Left Y gt MIDA CA A Aaa ft A O Width Bottom Right Y F G y i p Bottom Right X gt lt Length gt Exporting Importing Plate Formats If the following message appears when opening a protocol created with a 3 0x 2 x version of KC4 the protocol references a custom plate format not currently found in KC4 KC4 xX This file refers to a plate definition D 1034 7252771 which le not in the plate database If you wish to work with 3 0x 2 x protocols that reference custom plate formats you have two options 1 Export the current plate format information into a text file manually add the custom plate format s to this file
194. ettings ID Ma Full Hame Man Ann Jones Group Membership Password Standard User Current password Can not have access to Protected Functions Power User New password Can have access to Protected Functions but can not administrate Users 9 Administrator Hae complete access to al tunctiaone Status eluding weer administratia mo Protocols Folder DB Protocolstlones Man Ann Browse Cancel Help 3 Enter a unique ID using to 16 alphanumeric characters The user will enter or select this ID when logging into KC4 and when signing files 4 Enter the user s Full Name This name will be associated with events logged by this user s actions and with signatures applied by this user 5 Choose a Group Membership Standard Users are denied access to protected functions The System Administrator determines which functions shall be protected from use by Standard Users Power Users are permitted access to protected functions Power Users cannot administer user accounts or modify Security settings 6 Enter a temporary password for the user in the New and Confirm fields Share the password with the user with instructions to change it as soon as possible 24 e Installation and Configuration User s Guide to KC4 7 Optional Specify a folder location from which the user will most often access protocol files When the user selects Data New Plate to initiate a plate read the protocol selection
195. f a particular identifier 172 e Protocol Menu User s Guide to KC4 Cutoffs Cutoff Cutoffs screen selected data into defined categories based on fixed or variable threshold limits During data reduction KC4 evaluates results against the cutoff criteria assigns each well a Symbol Select Protocol Cutoffs with the Protocol Menu in Advanced mode to define Cutoffs and Symbols Cut Offs Data To Interpret Concentrations Symbols Cut Ofts A Cte CO2 ETL snee A An C 0 6 LOr COG m Out of range symbols our F Concentration below minimum ours F Concentration above masimum Cancel Help e After plate reading open the Symbols data set to see the assigned symbols and the calculated cutoff values Selecting the Data to Interpret The Data to Interpret setting represents the set of data to which the cutoffs will be applied For example if Data to Interpret is set to Concentrations the cutoffs will be applied to the calculated concentrations KC4 presents for selection the applicable data sets that are made available by the current protocol definition User s Guide to KC4 Protocol Menu e 173 Defining Cutoffs KC4 supports the definition of up to 40 cutoffs These are the criteria against which the plate data will be evaluated e Enter cutoff values or formulas in the C O fields Click ll or LES to see the next or previous 8 fields e Before defining
196. faults format settings start up options and database settings e User customizable application settings such as toolbar and window characteristics A note on event sorting under the Shared tab The Shared tab displays events recorded by all computers on the network The date of an event is supplied by the computer on which the event occurs Because the clocks on different computers are not synchronized consecutive events on different computers may not be recorded with consecutive dates Therefore events under the Shared tab are displayed in the order of their recording in the shared database not in the order of the event dates 224 e System Menu User s Guide to KC4 Exporting Audit Trail Events System audit trail events can be exported to a text file and optionally deleted from the System Audit Trail This feature may be used as part of a site s procedure for archiving past events Deleting events after export helps to reduce the size of the KCShared and or KCLocal databases Important When audit trail events are exported to a text file the text file does not reside within KC4 s secure database it resides as a single file in a user designated location It is the site s responsibility to ensure the security of exported system audit trail event files To export audit trail events 1 Select System Audit Trail 2 Click the Shared or Local tab 3 Click Export The Export Audit Trail Events dialog will appear Export Audit
197. ficulties or defects traceable to Purchaser s errors or system changes shall be billed to Purchaser at Bio Tek s standard time and materials charge Licensing Agreement Document Revision LICENSE Single Use You have the non exclusive right to use the enclosed KC4 program This program can only be used on a single computer unless a site license or additional network passwords have been purchased through Bio Tek Instruments You may transfer the program from one computer to another provided that the program is used with only one computer at a time You may not electronically transfer the program from one computer to another You may not distribute copies of the program s or documentation without the prior written consent of Bio Tek Instruments Inc or as stipulated in a sub licensing agreement Network Use You have the non exclusive right to use the enclosed program This program can only be used on a single file server The number of concurrent users on the network at any time may not exceed the number of licensed copies of this software You may transfer the program from one server to another provided that the program is used with only one server at a time You may not distribute copies of the program s or documentation without the prior written consent of Bio Tek Instruments Inc or as stipulated in a sub licensing agreement You may not use copy modify loan lease or transfer the program s or documentation or any copy exce
198. fined criteria Once the criteria are met the plate read begins Click the Options button to define the criteria Monitor wets O O OOOO O Data to monitor Mi 405 Wells to monitor at B2 Stop Monitoring when E least one well 0 500 si Delay between readings Joo 001 O Cancel Help e Select the Data to Monitor representing the filter or wavelength value to be used during well monitoring The available options depend on the current selections in the Reading dialog Only measured raw absorbance fluorescence or luminescence values are presented for selection User s Guide to KC4 Protocol Menu e 107 Enter the well or Wells to Monitor Multiple wells must be contiguous and entered as a range e g Al A2 or G10 H12 Define the Stop Monitoring When criteria to tell the instrument when to conclude the monitoring process Choose a conditional option At least one well in the range must meet the criteria the Average of the monitored wells must meet the criteria or All wells in the range must meet the criteria Choose an operator gt gt lt or lt Enter the measurement value to be met For Absorbance protocols the valid range is 1 000 to 5 000 For Fluorescence and Luminescence protocols the valid range 1s 0 to 99998 FLx800 and Synergy HT or 99999 FL600 If desired enter a Delay Between Readings of the monitored well s in HH MM SS format The
199. first read in the operation which is always 00 00 00 Kinetic times are displayed underneath the Process times in black dark text Note that in a kinetic protocol each read step will have a Process time and a Kinetic time associated with it User s Guide to KC4 Protocol Menu e 125 Dispense Step Parameters Use this dialog to create or modify a Dispense step Dispense step Dispenser li E Tip Prime Friming before the process Volume 5 pl Dispense Start time 0 000 5 Volume 25 ul Rate 250 Wy Sec Cancel Help time le approximate and reader configuration dependent e Select the number of the Dispenser to be used for this step The numbers available for selection correspond to the numbers on the dispense box e Select a Tip Prime option Tip priming is performed in a small removable priming trough located in the left rear of the carrier The purpose of tip priming is to compensate for any fluid loss at the dispense tip due to evaporation since the last dispense The trough holds up to 1 ml of liquid and must be periodically emptied and cleaned by the user KC4 must then be told that the trough has been emptied via the System gt Reader Control dialog If the trough overflows refer to the Synergy HT Operator s Manual for decontamination instructions None No prime Before the process Prime the tip with fluid once before the entire process begins Before each well Prim
200. fr Filter select the reference filter for a dual wavelength read First Well Last Well Most readers support reading a subset of the entire plate based on contiguous microplate wells First well 41 Last well H12 To specify the range of wells to be read If applicable select the appropriate Plate Type The default Last Well field value will automatically update to match the selected plate type Set the First Well and Last Well fields to indicate the range of wells to be read For example if First Well is set to A1 and Last Well is set to B3 KC4 reads a rectangle of wells Al A2 A3 B1 B2 and B3 General Options FL600 KC4 provides options for controlling certain reader functions preceding and during plate reads such as lamp control and plate ejection Click the Options button from within the Reading Parameters dialog to open the General Options dialog The available recommended settings vary depending on the reading mode General Options T Eject between Filter Set Read W Skip reference wells Cancel Lamp Control Help W Check Lamp before measurement T Set Lamp Off during measurement User s Guide to KC4 Protocol Menu e 99 Absorbance mode Enable Eject between Filter Set Read to perform an initial reading of a blank plate followed by the assay plate s at some later time provide the ability to add reagents to the same plate between readings or enable reading up to 6 different plates w
201. fs are in increasing order symbol assignment is determined as follows e Each well is evaluated against the first cutoff If the value of the well is less than the criteria then the first symbol is assigned to the well e Ifthe value of the well is greater than or equal to the first cutoff then it is evaluated against the next cutoff Ifthe value of the well is less than the next criteria then the symbol immediately preceding the cutoff criteria is assigned to the well e This process continues until the last cutoff criteria is encountered If the value of the well is less than the last criteria then the symbol immediately preceding the last cutoff criteria is assigned to the well If the value of the well is greater than or equal to the last criteria then the symbol immediately following the last cutoff criteria is assigned to the well 176 e Protocol Menu User s Guide to KC4 Reports T Report Report With KC4 s standard reporting function you can customize the contents and to some extent the appearance of printed reports All KC4 users have access to this standard function Reporting through Microsoft Excel 2000 and Word 2000 is also supported with KC4 s PowerReports feature This section only discusses the standard reporting function the KC4 report engine The report engine is selected under Protocol Options Select Protocol Reports to customize the report for the open protocol Report I Header KC4
202. g Available Data selectable items are dependent upon the type of protocol selected as well as the defined Reading Parameters Select an individual item by clicking it with the left mouse button To select groups of like items hold the Ctrl key down while highlighting them Click the Add gt gt button to transfer the item s to the bottom of the Selected Data list Click the Insert gt gt button to insert item s into the Selected Data list Highlight the item in the Report field above which the inserted item should appear e See Formats in System Menu Setup for information on choosing the text delimiter 182 e Protocol Menu User s Guide to KC4 Protocol Audit Trail This feature only applies to KC4 Signature Every protocol file has a Protocol Audit Trail associated with it This audit trail automatically records events performed on the file since the file was created first saved Each event records an action such as Plate Layout changed or Reading parameters changed and includes a date and time stamp the ID of the user logged in at the time and any comments that were entered by the user when the action was performed The audit trail cannot be edited and it remains a permanent part of the file for the life of that file that is if the file is deleted the audit trail is also deleted To view a protocol s audit trail select Protocol Audit Trail Date User Action Comment 0520 02 15 24 11 KSO File
203. g diskette ZIP JAZ and network drives are not automatically searched for the following files KCLocal mdb KC mdb and Calib mdb You will have an opportunity to specify additional databases for upgrade during the installation You can also run the utility after KC4 is installed via the System Setup Database menu option Note When KC4 prepares to upgrade a database it makes and saves a copy of the original The original database will remain unchanged and it can still be used with the earlier KC4 version The upgraded databases are not backwards compatible That is a KC4 version 3 4 database will not be recognized by KC4 version 3 1 10 e Installation and Configuration User s Guide to KC4 Installing KC4 M KC4 v3 4 M KC4 Signature Follow these steps to install KC4 1 Start Windows Note If you are prompted with a Windows password entry dialog enter a valid password this helps to ensure a complete installation If you were not prompted with the Windows password entry disregard this note Close any unnecessary Windows programs Place the CD in the CD ROM drive Click the Windows Start button and select Run When the Run dialog appears type lt drive gt Setup exe where lt drive gt represents the CD rom drive oe Alternatively use the Browse function to access the drive and then click on setup exe Click OK After a brief pause the Welcome dialog appears Click Next to continue An informational d
204. g Analysis If linear scanning is selected in an endpoint protocol select Protocol Scanning Analysis to specify data reduction options Scanning Analysis Calculation Type Calculated Data Mean OD i Mean Std Dev CY Std Dey OD CY OD Mean Min Max Integral Calculation Options i Cancel Help e Select the Calculation Type to be performed on each scanned well e Click the Calculation Options button to define the calculation zone By default the calculation zone includes all of the Horizontal Reading Points defined in the protocol Mean Std Dev CV Select Mean Std Dev CV to calculate and report the Mean Standard Deviation and Coefficient of Variation for all points in the calculation zone By default the calculation zone includes all of the Horizontal Reading Points defined in the protocol To change the calculation zone click Calculation Options Scanning Analysis Mean Std Der CY Calculation Zane ie From x Tox le oo From 1 Tot ra caos 00 Currently only the From x To x method for determining the calculation zone is supported By default From x represents the outermost scanned point to the left of center 0 and To x represents the outermost scanned point to the right of center These values can be changed to base calculations on a subset of all scanned points User s Guide to KC4 Protocol Menu e 139 In the example below the number of Horizontal Rea
205. g global parameters performing reader diagnostics and more e On the far right of the menu bar the About menu provides access to the Help system Bio Tek s home page and the About KC4 screen Toolbar el of Layout Patra Wiehe sf Options The Toolbar contains buttons that when clicked provide quick access to KC4 functions Accessibility of a particular toolbar button depends on whether or not its function applies to the current environment For example the Biograph button is accessible only if results are displayed and a curve has been generated When KC4 1s installed the toolbar contains a set of default buttons These buttons can be customized by selecting System Toolbar Status Bar The Status Bar at the bottom of the main screen displays the current function reader and COM port the name of the person currently logged in if applicable and any enabled keyboard functions User s Guide to KC4 Getting Started e 31 Quick Reference Guides This section briefly describes the contents of each menu in KC4 Data Menu The Data Menu contains functions for acquiring and processing plate data ie KC4 NewPlate Protocal Data Edit New Plate Open Close Save Save As Read Plate Sign Export Display System Cirit Ctrl 0 Chrl 5 Chri F New Plate Clear memory to prepare to read a new plate Open Open an existing plate PLA or global GLB data file Close Close the ope
206. gnal This feature is deselected by default Luminescence mode Enable Eject between Filter Set Read to perform an initial reading of a blank plate followed by the assay plate s at some later time provide the ability to add reagents to the same plate between readings or enable reading up to 6 different plates with one protocol and plate file This feature is available for Endpoint reads only Skip reference wells is deselected for luminescence readings and cannot be changed Check Lamp before measurement is deselected by default The lamp does not need to be checked before measurement Set Lamp Off during measurement is selected for luminescence readings and cannot be changed The lamp is turned off during measurement to avoid damage to the photomultiplier 100 e Protocol Menu User s Guide to KC4 Reading the Reference Wells It is strongly recommended that you read the reference wells if Rapid Mode is enabled If however reading the Reference Wells must be skipped consider the following e During an Endpoint read when prompted to Insert plate and press OK do so within 60 seconds e Ifa Lag Time is specified for an Endpoint or Kinetic read the maximum recommended time is 60 seconds If a longer lag time is required read the reference wells first or use Static Mode to read the plate e During a Kinetic read the maximum time interval is 60 seconds If a longer time interval is required read the reference wells fir
207. h and the beginning of data collection Itis also referred to as the delay before integration Valid values are 0 usec and 20 to 16000 usec The default value is 20 usec Note A delay of 0 usec is not the same as a fluorescence read with the time resolved option turned off They differ in two ways 1 the excitation wavelength is generated by the monochromator and 2 the user can specify the data collection time which 1s not an available parameter for normal fluorescence reads 106 e Protocol Menu User s Guide to KC4 e Data Collection Time is the amount of time for which readings are collected after the delay before integration time has expired The valid range is from 20 to 16000 usec The default value is 100 usec The following is a simplified schematic of the measurement mode parameters Delay before sampling Time y Delay between samples before collection integration 3 Dela before collection integration before collection between integration samples Plate carrier stops moving Flash 1 Flash 2 Flash Nb samples gt D Nb Samples per well Delay before sampling Delay between samples Delay a b O N Data Collection Time Monitor Wells If the currently selected reader supports well monitoring the Monitor Wells option is available The feature enables the instrument to repeatedly read a single well or a group of wells and evaluate the measurements against pre de
208. he Security dialog Click OK again to return to the main view The change is effective immediately the locked user is no longer permitted to log into KC4 or to sign files e Note Ifthe user is presently logged into another KC4 session he or she is prevented from signing files but is not automatically logged out User s Guide to KC4 System Menu e 215 Login and Password Parameters This feature only applies to KC4 Signature Select System Security Login to access Login and Password parameters Only the System Administrator is authorized to modify these parameters Users Login Protected functions Audit Trail Signature Reasons Files Login Lock user account after E pa failed login attempts IY Lock session after 20 minutes of idle T Force user to type ID Password M Minimum password length at least 5 a characters e Password expiration 30 days Advise User 5 pa days in advance IW Password reuse Remember last 5 me passwords Cancel Help Note The options shown as enabled above must be enabled to ensure 21 CFR Part 11 compliance The options values however are site definable To set Login parameters 1 Enable the desired Login parameters by checking the appropriate boxes 2 Specify the number of successive failed login attempts a user may make before becoming locked out of KC4 This feature only applies to Standard and Power User accounts the System Administrator s account cannot
209. he current protocol settings e Each object can contain from 1 to 20 data sets For example an object can contain the Well ID the corrected data value and the concentration value for every well Or 1t can contain just the concentrations e To proceed select one data set even if you want to include multiple data sets in the same object User s Guide to KC4 PowerReports e 283 The Plate Properties dialog appears Plate Properties Selection Data Top Left well E Bottorm Atght tell H12 Readingk The Top Left Well and Bottom Right Well set the boundaries for the plate object The default settings coordinate with the plate type defined in the protocol The Reading field is available for kinetic protocols to specify which kinetic read to include in the object Each plate object can display information for one kinetic read e Click the Data tab to select the data sets to include in the plate object KC4 presents one or more default Data sets for inclusion in the object although any available data set s can be selected Plate Properties EZ Selection Data OO J e nat Precision 7 A e oeoo om O s 7 hi2 630 11 405 Corr hie 630 Corr META Delta OD Concentrations To add a data set highlight the first empty Data field and click the left mouse button once A list of available data sets will appear click on the desired set to select it To change an existing data set highl
210. he currently selected reader and COM port are displayed at the top of this dialog If you need to change the COM port click Cancel to exit this dialog now and then click the Port button KC4 s currently defined wavelength values are displayed in fields 1 through 6 e To change download and calibrate wavelength values entered the desired values in these fields and then click Send Wavelengths The values will be downloaded to the instrument overwriting its existing wavelength table e To retrieve wavelength values from the instrument click Get Wavelengths The values will be uploaded to KC4 overwriting the values in this dialog User s Guide to KC4 System Menu e 199 Synergy HT Fluorescence Luminescence The Synergy HT instrument has one four position excitation wheel and one four position emission filter wheel Each filter wheel can contain any combination of filters plugs and empty locations depending on assay requirements Use this dialog to specify the contents of each filter wheel The settings here will be made available as default selections within the Reading Parameters dialog Important It is essential that you conform to the specific Bio Tek instrument procedures for altering the filter wheel configuration The Synergy HT does not automatically detect which filters 1f any are installed Therefore it is your responsibility to ensure that KC4 s wavelength table exactly matches the contents of each filter wheel F
211. he incubator before the plate carrier is drawn in Enter the desired set point Temperature in C Define the Lag Time to elapse before the reader initiates the first reading cycle The reader draws the plate carrier into the reading chamber waits for the Lag Time to elapse then reads the plate Lag Time is available for all reader models including those that do not support incubation The valid entry range for most readers is 00 00 00 to 00 59 59 HH MM SS For the FL600 the range is 00 00 00 to 08 00 00 Time Resolved Fluorescence Synergy HT Time Rezolved If supported by the reader the Time Resolved option is available when the selected detection method is Fluorescence Ifthis option 1s enabled additional measurement mode parameters become available in the Measurement Options dialog accessed by clicking the Option button located in the Filters box to specify the length of time to delay before collecting readings and the length of time during which readings will be taken Top Probe Vertical Offset See General Options Synergy HT User s Guide to KC4 Protocol Menu e 117 Wavelengths For readers equipped with a monochromator e g PowerWave PowerWaveX uQuant M Synergy in Absorbance mode the Wavelengths set of parameters is available within the Reading parameters dialog For Endpoint and Kinetic reads Calibrate e Click the radio button associated with the desired number
212. he read time and interval For an endpoint area scanning protocol enable the Scanning checkbox and then click the Scanning button to define the read matrix size Optional For a spectrum protocol click the Scales button to customize the graphical data display Optional Define Shaking parameters Optional Define General Options parameters Optional Define Temperature Control parameters Optional Select and define Pre Reading options Click OK to verify the parameters and close this dialog 88 e Protocol Menu User s Guide to KC4 uQuant Reading Type Reader C End Point Quant on COM2 f Kinetic kinetic AOS caninas Spectrum Wavelengths Stark 400 nm Step fro nm Shop B00 an Plate W Calibrate Pre Readings Type 48 WELL PLATE Size bee I Blank Plate First well 5 Last well Fs Monitor ells Options q A A A E cuca Hee Select a Reading Type of Endpoint Kinetic or Spectrum Define the Wavelengths to be used Calibrate the wavelengths if necessary Select a Plate Type and define the First and Last Wells For a kinetic protocol click the Kinetic button to define the read time and interval For an endpoint area scanning protocol enable the Scanning checkbox and then click the Scanning button to define the read matrix size Optional For a spectrum protocol click the Scales button to customize the graphical data display Optional Select a
213. here are no special functions associated with 1t Select System Setup Database to access information and functions related to KC4 s shared and local databases Shared Database The Shared Database area contains the current location and name of the shared database a Restore Connection button and a Repair Compact button Protocol Defaults Formats Startup Database Shared database Thiz database mainly includes Security and Plate Format information It may also include Protocol and Data files See Security Settings lt can be shared by several networked PCs Path METEXCHANGESKC4KCShared mdb Browse Restore connection RepairComparct The Path setting must be changed 1f the location and or the name of the KCShared mdb file changes For example following KC4 installation the System Administrator is advised to make a copy of the installed KCShared mdb file and store the copy on a shared location on the network To gain access to this shared file the Path setting must change to reference the network location e Before changing the Path a KCShared mdb file must already exist in the new location this feature does not create a new KCShared mdb file e The shared database name is not required to be KCShared mdb If your site requires the use of multiple shared databases one per project for example the file can be renamed via Windows Explorer The mdb extension IS required User s Guide to KC4 System Menu e 2
214. hin an acceptable tolerance 0 5 C During this settling period the REACHED indication may appear for a few seconds disappear then reappear moments later The Time since reached field will automatically reset to 00 00 00 each time the REACHED indication appears If the Beep after checkbox associated with Time since reached field is enabled and set to for example 10 minutes KC4 will beep 10 minutes after the incubation temperature has consistently settled within 0 5 C of the Requested temperature 246 e System Menu User s Guide to KC4 Dispenser Synergy HT with injectors Use the Dispenser dialog to perform dispenser specific functions such as prime and purge Reader Control Information Door Carrier Pre Heating Dispenser Dispenser f Status Primed Prime Purge Volume i OOO yl Rate 225 pisec Tip Prime Wessel O pl remaining Dump all Tip Prime Tubs Priming the Dispenser Before running a Read amp Dispense protocol the syringe s and tubing must be primed with the fluid to be used To prime the dispenser e Fill the supply bottle with a sufficient volume of the fluid to be used for the prime e Important Place the priming plate on the carrier Bio Tek PN 7142002 e InKC4 select System Reader Control and click the Dispenser tab e Select the Dispenser 1 or 2 associated with the supply bottle e Enter the volume to be used for the prime from 5 to 5000 ul e Select a prime Rate
215. ial file maintenance utility for viewing the file names and their locations and for moving copying renaming deleting importing and exporting files 42 e Data Menu User s Guide to KC4 Creating a New Data File Before a plate can be read and before plate data can be entered manually or read in from a file a new data file must be created To create a new data file 1 Select System Readers and set the Current Reader to the appropriate target reader model 2 Select Data New Plate or click the toolbar icon shown above to clear memory to prepare to read a new plate 3 IfKC4 is currently configured to create plate data PLA files by default do one of the following Select Protocol New to create a new protocol Select Protocol Open to open an existing protocol Select DatajRead Plate to initiate the plate read If KC4 is currently configured to create global data GLB files by default selecting Data New Plate opens the New Data File dialog Hew Data File Ed Select a Protocol A320 DAA Quant Oligo Quant Phenal ae ANA4 Quant Path DB Jones Mary 64260 280R atio prt Cancel Browse This dialog allows you to choose which protocol to open and use for the plate reading Click the Browse button to search through the folder structure Highlight the desired protocol and click OK To start with an empty protocol and customize its parameters highlight Empty Protocol and clic
216. ialog appears Welcome to the KC4 Setup program This program will install RC4 on pour computer Ik is strongly recommended that you exit all Windows programs before running this Setup program Click Cancel to quit Setup and then close any programe you have running Click Hest to continue with the Setup program WARNING This program 12 protected by copyright law ard International treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and Will be prosecubed to the maximum extent possible under law Cancel User s Guide to KC4 Installation and Configuration e 11 8 Click Next to continue The Software License Agreement dialog appears Software License Agreement Fa Please read the following License Agreement Press the PAGE DOWN key to see the rest of the agreement LICENSE You have the non exclusive right to use the enclosed KC4 program This program can only be used on a single computer unless a site license or additional network licenses have been purchased through Bio Tek Instruments ou may not transher the program from one computer to another ou may not electronically transter the program from one computer to another ou may not distribute copies of the programs or documentation without the prior written consent of Bio Tek Instruments Inc or as stipulated in a sub licensing agreement fou map not use copy modify or
217. ible to ensure that archiving or other file management techniques are suitable such that electronic records generated by the system shall be accurate and readily retrievable Records may be removed by deleting the entire shared database or individual files within the database This action may be conducted by the Administrator or by any Power User User Identity Any person that will be authorized to use an electronic signature will have their identity confirmed by the Administrator prior to granting them program access Only the genuine owner of the electronic signature is allowed to access the program through their ID password combination System users should be informed that accessing the program using someone else s logon is a violation of the FDA rule 8 e Installation and Configuration User s Guide to KC4 Password Expiration Recall All passwords must be checked recalled or revised at an interval appropriate with the security needs of the organization Personnel whom no longer work at the establishment shall have their program access capabilities deactivated in a timely manner Certification to FDA The site must certify to FDA that the electronic signatures utilized on records are intended to be as legally binding as handwritten signatures prior to or at the time of record submission to FDA The FDA rule should be consulted for details and method to be used Notification of Attempted Security Breaches The software utilizes an erro
218. ication error to occur Here is an example scenario The network connection failed because of a problem on the server The KC4 user unaware of the failure chose to save a GLB file to the shared database just seconds after the failure Because file caching was enabled the file actually went into temporary storage in the Windows system cache to await redirection to the shared database on the network KC4 gave the appearance that the file was saved successfully no error message appeared and the asterisk disappeared from the title bar After a moment Windows detected the failure and displayed an appropriate error message and the user decided to quit KC4 Upon closing KC4 did not present an option to save the file because KC4 thought that the file was already saved When the network connection was re established the user launched KC4 but could not locate the GLB file because it never reached the shared database For more information visit http support microsoft com and search for current articles on file caching or write caching User s Guide to KC4 Appendix B Windows File Caching e 311 312 Appendix B Windows File Caching User s Guide to KC4 Index indication in statistics 307 indication in statistics 307 256 4 Parameter logistic fit 164 168 A Absorb checkbox 197 Absorbance read method 112 Administrator Appointing a System Administrator 8 Initial password 19 System Admi
219. ide to KC4 Protocol Menu e 77 ELx800 Reading Type Reader End Point ELxS00 on COM Kinetic Kinetic Seenning Filters Meas Filter 405 Pre Read Blank Plate Refr Filter W Monitor wels Options Plate Type se WELL PLATE Size 8x12 First well 5 Last well h12 Cancel Help Select a Reading Type of Endpoint or Kinetic Select the filter s to be used Select a Plate Type and define the First and Last Wells For a kinetic protocol click the Kinetic button to define the read time and interval oo a gt a fo For an endpoint area scanning protocol enable the Scanning checkbox and then click the Scanning button to define the read matrix size Optional Select Pre Read Blank Plate Optional Select Monitor Wells and then click Options to define related parameters Click OK to verify the parameters and close this dialog 78 e Protocol Menu User s Guide to KC4 ELx808 Reading Type Reader End Point ELxs08 1 on COM C Kinetic Kimete Scanning Filters Temperature Control f Meas Filter 405 Yes M Pre Heating C Mo Temperature 37 oe f Refr Filter fezo Lag Time 00 00 00 Shaking Intensity li a fro PF Pre Read Blank Plate Before even reading 4 Read Barcode Before first reading T Monitor welle UO 2tone Plate First well 5 Last well 412 coc 0 Select a Reading Type of Endpoint or Kinetic Select the
220. if the databases reside on a drive that is not automatically searched click the Stop button to advance to the Database Upgrade Utility dialog see next step When the search is complete if KC4 has detected database files created by former KC4 versions an upgrade option message box appears E C4 detected database files created by former C4 versions Do you want to upgrade and use them in this new version Note the Database Upgrade Utility may be executed later via the System S etup Database menu 18 Click Yes to upgrade the database s now or No to skip the upgrade at this time The Database Upgrade Utility can be run manually through KC4 via System Setup Database User s Guide to KC4 Installation and Configuration e 15 If you click Yes the Database Upgrade Utility dialog appears Database Upgrade Utility Please select the database file s to upgrade and to use on this PL Shared database eso ocuments and Settings All Users Application Datas Bio Tek Instruments AECA Sig Local databaze Continue to use current file no upgrade Diagnostics database lt Continue to use current file no upgrade Browse Stark upgrading Cancel Click the drop down arrows to see which databases KC4 found Click on the desired database name to select it for upgrading Click the Browse button to search for a database When the database is located highlight it and click Open KC4 will insert the name
221. ight it and click the left mouse button once A list of available data sets will appear click on the desired set to select it To delete an existing data set highlight its entire row then click the right mouse button and select Delete 284 e PowerReports User s Guide to KC4 To insert a new data set between two existing sets highlight the row above which the new data set will be inserted Click the right mouse button and select Insert Selection Data C romat Pression CO E For data sets that represent numerical information click the corresponding Format field then select Decimal or Scientific For the Decimal format click the corresponding Precision field to set the number of decimal places 0 to 5 Click OK to save the settings and insert the new Plate object into the document as demonstrated below ij Document in lt KC4 gt Microsoft Word File Edit Yiew Insert Format Tools Table Window Help Normal Arial 9 B Z Z HDE ARAY s BelsH Fields Plates Tables Curves v x Close hh i i EX a EX oo E en E oe a E Plate Title E D S Page 1 Sec 1 1 1 ae Un Col 1 REG MRE ExT OVR ujoj 4 N User s Guide to KC4 PowerReports e 285 Inserting Table Objects A Table Object presents results data in a columnar format To insert a Table object click the Table button in the RSO toolbar A drop down list of available data categories appears Et Microsoft
222. ignore erroneous data point s generated at the beginning of a kinetic assay due to assay noise e Fromt Tot Defines a calculation zone based on time The default values span the defined kinetic Run Time e From To Defines a calculation zone based on number of reads The default values include all reading points e mOD or OD dependent on kinetic dynamic range e Per sec or Per sec2 dependent on rate order e Per min or Per min2 dependent on rate order e Per hour or Per hour2 dependent on rate order Max V With Max V the calculated value is the maximum slope KC4 calculates the maximum slope as follows e Starting at the first point in the calculation zone KC4 evaluates n points and calculates the slope among these n points e Next KC4 repeats the operation starting at the second point in the calculation zone and repeats again starting at the third point and so on e KC4 then compares all calculated slopes to determine the maximum slope e KC4 will register the Delta t time in the middle of the point where the V max is calculated 132 e Protocol Menu User s Guide to KC4 KC4 also calculates the kinetic Lag Time which is the time intercept between the line of maximum slope of the propagation phase and the absorbance baseline at time 0 as illustrated below Paint of max slope Absorbance Time Min By default the calculation zone starts at time zero To change the calculation zone select M
223. iguration button and then click the appropriate Dispenser tab 1 or 2 e Click the Initialize button The syringe drive will move to its home position and its sensors will be verified Upon successful completion the Status should show Initialized 202 e System Menu User s Guide to KC4 Setting the Injector Position The instrument dispenses fluid using two injectors The injector heads can be positioned to dispense fluid from three possible locations two adjacent to the top probe and one above the bottom probe The default positioning is one adjacent to the top and one above the bottom Moving the injector head from one position to another must be done manually see the Synergy HT Operator s Manual for instructions After making an adjustment the new position s must be configured in KC4 for recognition by the reader To configure the injector positions in KC4 e InKC4 select System Readers click the Configuration button and then click the appropriate Dispenser tab 1 or 2 e Click the radio button corresponding with the current physical injector head position Next to Top Probe or Above Bottom Probe e Click OK to save the change and close the dialog Syringe Setup KC4 provides access to special syringe setup functions for maintenance and calibration purposes Important Do not change the syringe positions or calibrate the dispensers unless instructed to do so as part of installation upgrade or maint
224. iles Select a Preferred Data File format Enable or disable the Secured Protocol and Data File Storage feature and select a Preferred Data File format User s Guide to KC4 System Menu e 211 User Accounts This feature only applies to KC4 Signature A significant component of a secure software environment is the ability to create individual user accounts to ensure that only authorized users can gain access to the system and to any restricted functions KC4 Signature s Multi User Identification feature allows a site designated System Administrator to create and maintain user accounts Users Login Protected f M Multi User identification Multi User Identification is automatically enabled upon KC4 Signature software installation and it must remain enabled to ensure 21 CFR Part 11 compliance If however you wish to disable this feature select System Security and uncheck the Multi User Identification box This action will cause all user accounts except the Administrator s to be permanently deleted When Multi User Identification is disabled users are not required to login to KC4 but the Administrator can still log in and perform the following tasks e Change the Administrator s password e Define system level protected functions e Set audit trail notification options e Enable or disable the Secured Protocol and Data File Storage feature e Select a Preferred Data File Format 212 e System Menu User s Guide to KC4
225. in ul second e Click Prime to start the process e When the process is complete carefully remove the priming plate from the carrier and dispose of the fluid Purging the Dispenser KC4 provides a special utility to purge fluid from the dispense tubing and syringes by pumping the fluid in reverse back into the supply bottle To purge the dispenser e InKC4 select System Reader Control and click the Dispenser tab e Select the Dispenser 1 or 2 associated with the supply bottle e Enter the desired purge Volume from 5 to 5000 ul e Select a prime Rate in ul second e Click Purge to start the process User s Guide to KC4 System Menu e 247 Emptying the Tip Prime Trough Read amp Dispense protocols can specify a tip prime to compensate for any fluid loss at the dispense tip due to evaporation since the last dispense Tip priming is performed in a small removable priming trough located in the left rear of the carrier The prime trough holds up to 1 ml of liquid and must be periodically emptied and cleaned by the user The trough must be emptied periodically and KC4 must be told that the trough has been emptied To empty the tip prime trough e Carefully remove the trough from the carrier and empty it e Clean the trough as required by your site and then put it back on the carrier Refer to the Synergy HT Operator s Manual for decontamination instructions e InKC4 select System Reader Control and click the Dispenser
226. inetic Analysis Plate Layout and so on Select Advanced to see additional options including Cutoffs and Validation The major headings in the remainder of this chapter are aligned with the Protocol menu options refer to these sections for further instruction You may not need to use all of the options but you must at a minimum define the Reading parameters For more information see Defining Reading Parameters later in this chapter Protocol Wizard mt i Wizard As an alternative to using the Protocol Menu to create a new protocol KC4 provides a Wizard to guide you through the process Protocol parameters are conveniently categorized for easy selection and intuitive setup Click the Wizard toolbar button or select Protocol Wizard to begin Wizard Plate Settings Basic Analysis Advanced Analysis Outputs HES COHOHON Y Allows the definition of assay prompts that will appear each time a plate is read E Aeading Parameters Yio Allows the definition of reading parameters these include filters shaking Al temperature control lag time before reading kinetic time parameters and scaling of kinetic graphical display options available are reader dependent favo Allows to define positions of samples controls standards Options allow automatic or manual filling replication and assigning of dilutior s and concentrations where appropriate Back Next gt oo Help e Click on a but
227. inuous time base unlike pure kinetic readings e Alternatively it is possible to append several kinetic readings to one file If a kinetic reading sequence is halted ex to inject reagents then 1t is possible to resume the kinetic reading and append the resumed data onto an existing file e The Append feature can be set to perform automatically after a plate read See Protocol Options in Protocol Menu for more information To append a set of plate data to a kinetic file e When the plate read is complete select Data JAppend to Kinetic File The Append to Kinetic File dialog appears Append to Kinetic File Ea Append to existing file jc SECA Kinetic pla Cancel Hew Help The last opened plate file name appears as the default Change the path and or file name if desired Click New if this set of data represents the starting kinetic read 1 e if subsequent reads will be appended to this one If no file name has been entered KC4 will produce the standard Save As dialog Click Open to search through the directory structure to locate the target file Click OK to continue A dialog will appear requesting entry of a Reading Time relative to the first reading Append To Kinetic File ES Reading Time relative to first reading no 00 35 Cancel Help The default value represents the elapsed time since the last plate read enter a new value if required Note The time format is defined under
228. ion 127 BPF file 77 Clarity protocol files 28 Creating new 77 Opening existing 77 BTI file 53 Importing 53 Buttons in toolbar 237 C Calculate recovery percent 155 Calculation log report 178 301 Calculation options 188 Calculation warnings 190 CALIB mdb database 233 Calibrate Endpoint reads 118 Kinetic reads 118 Spectrum reads 118 Calibration plate test 205 207 Calibrators 143 Changing measurement values How to 45 Results window 251 Check Lamp before measurement 99 100 Clarity 77 Background measurement value 53 File importation failures 53 Heating 250 314 e Index Importing BTI files 53 Priming 249 Protocol definition 77 Protocol files 28 Reader control 249 Reading parameters 77 Coefficient of variation 160 Coefficient of Variation 264 Communication port 195 Compact database After exporting and deleting audit trail events 225 After exporting and deleting files 227 Local database 235 Shared database 233 Computer requirements 9 Concentrations Defining in plate layout 155 Contacting Bio Tek Instruments Inc 4 Control wells 143 Controlling multiple instruments simultaneously 194 Converting databases 15 236 Copy Current Formula 160 Corrected data 128 Critical database error Restoring connection to KCShared 234 Current folder 44 47 68 71 Current plate 164 Current reader 194 Curve axes 165 Curve description 164 Curve fitting 164 166 Curve in
229. ispense El Shake Read fee st 100 pl fe Mi WY YY 5 avy Y Y fees We lf te Wh lA Me 00 00 00 00 04 30 00 02 30 00 04 30 00 06 30 00 03 30 00 10 30 00 13 30 00 00 00 00 03 00 00 04 00 00 06 00 00 03 00 00 10 00 Timing control is OM Incubation time it exactly the same for each well Scales Ok Cancel Help O O athe same apaec time i approximate and reader configuration dependent e Timing Control is enabled by default To disable Timing Control check the box When Timing Control is enabled all steps are processed in the same way with regard to timing Incubation time is exactly the same for each well Dispense and Read steps are performed at the same pace When Timing Control is disabled all steps are performed as quickly as possible Incubation time may vary across the microplate Dispense and Read steps may be performed at different speeds 120 e Protocol Menu User s Guide to KC4 e Click the Dispense Shake and Read buttons to create new steps Up to nine 9 steps may be defined Steps cannot overlap They must be performed one after another With Endpoint protocols only one Read step may be defined Endpoint protocols do not require a Read step the protocol can be Dispense only Kinetic protocols require at least one Read step Note Ifthe protocol contains two or more Read steps the Kinetic Interval is represented as Uneven on reports A Kinetic Re
230. ith one protocol and plate file This feature is available for Endpoint reads only Skip reference wells is selected for absorbance readings and cannot be changed Enable Check Lamp before measurement to check lamp output prior to measurement This feature is selected by default Deselecting this feature speeds up the reading process however a lamp output failure may not be detected Enable Set Lamp Off during measurement to allow for the reading of an unexcited signal This feature is deselected by default Fluorescence mode Enable Eject between Filter Set Read to perform an initial reading of a blank plate followed by the assay plate s at some later time provide the ability to add reagents to the same plate between readings or enable reading up to 6 different plates with one protocol and plate file This feature is available for Endpoint reads only Skip reference wells is deselected by default The FL600 is equipped with fluorescent plastic compounds call reference wells which help provide the best results when using Rapid Mode to read the assay plate Skip reference wells should be deselected for use with Rapid Mode reads Enable Check Lamp before measurement to check lamp output prior to measurement This feature is selected by default Deselecting this feature speeds up the reading process however a lamp output failure may not be detected Enable Set Lamp Off during measurement to allow for the reading of an unexcited si
231. k OK The Preferred Data File Format is selected via System Security Files User s Guide to KC4 Data Menu e 43 Opening Existing Data Files Select Data Open to open a previously saved plate or global data file Note The layout of the Open dialog varies slightly depending on whether or not the Secured Protocol and Data File Storage option 1s enabled but the functionality is the same In the example below this option is enabled J ONA Guant ANA Quant 6 KB EC4 Data 08 19 02 14 37 32 2 EB EC4 Data 08 19 02 14 35 11 amp OLIGO Quant pla r EB EC4 Data 05 31700 19 19 28 File Name 260_280Fatio pla Type kca Data pla Cancel e The Current Folder field or the Look In field you will see one or the other shows your current location within the overall folder structure Ifthe drive is represented as DB this means that files are currently being stored in KC4 s secure shared access database If the drive is represented by a single letter such as C or D this means that files are stored individually on the computer s hard drive or on a network to which the computer is attached See Where and How is Plate Data Stored for more information e Click LE to move up one folder level e Click ona folder L e Click the Type drop down arrow to change the type of files being viewed Options include e KC4 Data PLA KC4 Data Protocol GLB All Files e Click on a file and cli
232. kage 3 Click the Registration Card to verify the license number and then open a form Enter the required information and send the form to Bio Tek via fax or email 4 Bio Tek will respond with a password via fax or email When you receive your password from Bio Tek enter it in the Password field and click OK KC4 Reinstallation If you are reinstalling KC4 you must fill out and submit a Reinstallation Form to obtain another password Reinstallation is defined as e Re installing a KC4 version on the same PC for example after a hard disk replacement e Un installing a KC4 version from one PC and installing the same version on another PC A Reinstallation Form doc and txt formats complete with instructions can be found in the root KC4 folder User s Guide to KC4 System Menu e 229 Setup Select System Setup to set various global parameters including reporting and exporting engines import export separator and startup options Protocol Defaults Select System Setup Protocol Defaults to specify several default settings for new protocols Setup Protocol Defaults Formats Start up Database Reporting Engine KC4 amp Microsoft Ww ord C Microsoft E xcel Document Formatting Standard Faster without font customization Y Exporting Engine C KC4 C Microsoft W ord Microsoft E xcel Document Formatting Enhanced slower with font customization Weighting of Standard Deviation i H Cc N W Use out of
233. kinetic curves Kinetic calculations such as Max V Coeff Reg Delta t at Max V and Lag Time associated with the currently selected plot are displayed Click on the down arrow associated with Selected Curve to choose a different curve Moving the mouse cursor over a kinetic data point in the currently selected curve will display coordinates for that point Data displayed can include Read OD and Reading Time Click on an individual data point to temporarily exclude it from further calculations then click the Recalculate button to exclude the point from kinetic calculations Important Modifying data may adversely affect the final results To restore an excluded data point click on the point and then click Recalculate Click the Options button to adjust the Kinetic Analysis parameters for the curves shown in this dialog Refer to Kinetic Analysis under Protocol Reading Parameters in the Protocol Menu section of this guide for more information Dispense During Kinetic Read The Synergy HT with injectors model provides the ability to dispense fluid into microwells as part of a fluorescence or luminescence Read amp Dispense protocol When the protocol specifies a dispense operation during a kinetic read the Kinetic Curves display will represent the dispense operation in a box with cross hatches on the Time axis as shown below No readings are taken during a dispense operation 100000 30000 0000 0000 60000 50000 40000
234. l KC4 refers to these settings to know which engine s to engage These default settings can be overridden within a protocol explained in the next section Setup Protocol Defaults Formats Start up Database Reporting Engine Co KC4 Microsoft ord C Microsoft E scel Document Formatting Standard Fazster without font customization Exporting Engine C KCa C Microsoft ford Document Formatting Enhanced slower with font customization e Select the default Reporting Engine and Exporting Engine the ones you plan to use most frequently 276 e PowerReports User s Guide to KC4 e Ifyou have selected Microsoft Word for either or both engines choose a Word Document Formatting option of Standard or Enhanced 4 With Standard formatting the print preview and print functions perform quickly but any custom Plate Layout or Cut Offs fonts are not applied With Enhanced formatting the print preview and print functions may take longer but all of your custom Plate Layout and Cut Offs fonts will be applied e Click the Start up tab to customize additional options Protocol Defaults Formats Start up Database Start Microsoft ord when launching KC4 M Start Microsoft E scel when launching KC4 Optional Check Start when launching KC4 if you want Word and or Excel to open automatically when KC4 is launched With the program open in the background document opening and formatting will perform more quickly
235. lable if Scanning is enabled The Blank Plate data is subtracted from the actual microplate data if the Blank Plate Subtraction option is selected in the Raw Data Corrections dialog an advanced protocol option Read Barcode Read Barcode If the attached reader is equipped with an internal plate barcode scanner use the Read Barcode feature to read the code when the plate 1s read If for any reason the barcode cannot be read you will be prompted to manually enter the plate barcode See Protocol Options at the end of this chapter to find out how to use the scanned or entered barcode as the data file name User s Guide to KC4 Protocol Menu e 111 Read amp Dispense 4 Dispense Read Dispense The Synergy HT with Injectors provides the ability to dispense fluid into microwells as part of a Read amp Dispense protocol To enable this feature click the Dispense checkbox To define the Dispense Shake and or Read steps click Read amp Dispense See Creating a Read amp Dispense Protocol starting on page 119 for detailed instructions Read Mode Read Mode Rapid a Some reader models support multiple Read Modes for absorbance protocols The selection of one read mode over another generally depends on the desired outcome of the assay better precision versus faster plate reading The available read modes are e Normal mode is the slowest of the three available modes After positioning the well over
236. lation Data In represents the data set upon which the formulas will be applied The Data In choices will vary depending on the defined protocol parameters User s Guide to KC4 Protocol Menu e 159 For kinetic protocols a Kinetic checkbox is provided If this box is checked the Data In options represent plate data such as M 1 405 or M 2 630 If this box is not checked the Data In options represent the results of kinetic calculations such as Max V and Coeff Reg Transf fi W Kinetic Data In Mi 405 Data Out Mi 630 he The Data Out field assigns a name to the transformed data The transformed data can then be used for further calculations including Multi Plate Transformations Select Copy Current Formula to copy the current formula into one or more wells Clicking on the title row or column auto fills the wells selected with the current formula Clicking on the top left corner auto fills the whole plate Select Edit Well to edit the formula in a particular well Select Diff Between Lines to perform subtraction between rows then select A B or B A Select Diff Between Cols to perform subtraction between columns then select 1 2 or 2 1 Click Init All Transf to reinitialize all Single Plate Transformations to their most recently saved settings Click gt to expand the width of the columns to view and access long formulas The maximum expansion shows a single column of well
237. lculations User s Guide to KC4 Results e 263 Statistics From within any Results window click the Statistics button to view the statistical data associated with the results SAMPLES Well ID SPL1 264 e Results STATISTICS Hame Conc Dil Well M 620 Hb Mean Std Dev CU oh os 0 ee y y ewes E os000 o E Z iaa ba o300 2 ot osc ee oo o o 2 osso o wow oo a ee Controls standards Tip To view statistics for a different data set go back to the main Results window choose the desired data set and then click Statistics To view statistics for a different set of identifiers click Samples Controls or Standards The available columns are protocol dependent Name The name of the sample associated with the current well Conc Dil Concentration or dilution of the current well Well Coordinates of the current well Nb Represents the number of replicates that were used to calculate statistics Mean Mean of the replicate group Std Dev Standard deviation of the replicate group CV Coefficient of variation of the replicate group 1 e the standard deviation expressed as a percentage of the mean User s Guide to KC4 Biograph Overview Biograph exe The KC4 software package includes the Biograph module for curve viewing and manipulation Biograph can be used either as a stand alone product or in conjunction with KC4 The Biograph functions are desc
238. le s audit trail cannot be viewed from within KC4 but it can be added to report and export templates e With KC4 Signature Change Mask and many other actions may be logged to the audit trail A file s audit trail can be added to report and export templates and can also be viewed by selecting Data Audit Trail Data Audit Trail Date User l Action Comment 0816 02 14 55 28 JonesR Protocol DB Synergy Absorbance prt 0516 02 14 58 25 selected 0816 02 14 55 46 Jones Plate Layout changed Added blank wels 41 and 22 0816 02 14 59 01 Jones Report changed Added M405 Corr to report template 0841 5 02 14 59 21 Jonesk Options changed Changed weighting of standard deviation to M 1 0816 02 14 59 35 Jonesk Plate read started 0816 02 15 00 03 Jones Sample Plate successtully read 0841602 15 0217 JonesR File Created 05 1602 15 02 17 Jonesk Signed for Authorship by Robert Jones 061602 15 03 16 Administrator Signed for Approval by System Administrator Events are sorted by date time stamp and include the ID of the user logged in at the time the action taken and any comments that were entered by the user when the action was performed The contents of the data audit trail can be copied to the Clipboard by highlighting the desired cells and pressing Ctrl C The contents of the data audit trail cannot be edited or deleted User s Guide to KC4 Data Menu e 63 64 e Data Menu User s Guide to KC4 Edit Menu Edi
239. le contains no time indicators then the time parameters defined via Protocol Reading parameters will be used as defaults The following example represents the kinetic data set generated from 5 readings in the 24 well format to ensure a successful import of all five reads shown in this example create a protocol and set the kinetic parameters so that the Reading Number is 5 Time 00 00 00 0 000 0 007 0 013 0 020 0 026 0 033 0 00470 01170 01770 02470 03070 037 0 008 0 014 0 021 0 028 0 034 0 041 0 012 0 018 0 025 0 032 0 038 0 045 Time 00 00 30 0 000 0 026 0 053 0 079 0 105 0 132 0 016 0 042 0 068 0 095 0 121 0 147 0 03210 0580 0840 11070 1377 0 163 00AT 0 2 07450 10050 126 0 15330 179 Time 00 01 00 O200070 0597 0 11870 1783 0123170296 00360095505 1394072135032 127033 1 US 0714013070189024 030870367 0 1077 0 1667 022257 0 2847 0 343 0 402 Time 00 01 30 0 000 0 099 0 197 0 248 0 284 0 356 005070 195250 194 029032270381 O LO eee 2 else ser 39040 420 Ost 6770 21 67 0 2 O 170 4035 0 499 Time 00 02 00 0 00070 17670 251 0 299 0 37870 446 0n LITA 0 Dz OS De oO 0739TA 0AA 0 257 0 275 0 331 0 388 0 438 0 482 0 239 0 296 0 369 0 402 0 492 0 557 52 e Data Menu User s Guide to KC4 Importing Clarity Luminescence Data Files Applies to Clarity Microplate Luminometer users only KC4 supports the importing of Clarity results files bti through its Read from File feature when Clarity 1s the currently sel
240. les Locate and make current the folder in which the imported file will reside 2 Launch Windows Explorer and locate the desired file Highlight the file click the right mouse button and select Copy 4 Switch back to KC4 Place the cursor inside the Maintain Files dialog click the right mouse button and select Paste The System Administrator can protect the use of the Import function If you are trying without success to import files you may not be authorized to do so 228 e System Menu User s Guide to KC4 Registration Upgrade Twenty one 21 days after installation when the Trial mode is over your KC4 software will switch to Demonstration mode unless you enter a valid password To apply for and enter a password 1 Select System Registration The Registration dialog will appear Registration Current License settings Instructions Running mode Network Mode E C4 is delivered with a 21 days Trial license In order to get a permanent license you need to License number LOSM KMVKM Registration Upgrade 1 Type the License Number provided with the product Liesmes number 2 Fil the Registration Card and send it to Bio Tek E mail passwordt biotek com Fas 1 802 655 38 49 System ID U2 lt 7JH3F 00 Registration Card 3 Type the password received from Bio Tek and click OF Password 2 Ifthe License Number field is empty enter the license number provided with your KC4 software pac
241. llar sign will be replaced by actual information during the print print preview or export process In the example above the dollar sign will be replaced by the system date recorded when the plate was read By default the font color of this character is set to Auto The color will be whatever the color is in KC4 The color and font type and size can be overridden in the template using the application s font customization tools Do not delete or move a dollar sign without deleting or moving the entire object e A gray editing button as shown in cell D2 To change the contents of an existing object double click the gray editing button The subsequent dialog depends on the object type 280 e PowerReports User s Guide to KC4 Object Fulfillment After a plate is read and the operator initiates the print preview print or export function KC4 begins the formatting process and transfers data into the prepared document A Formatting Office document message lets you know that something is happening Ls KC4 Brev16_prt Brev_pla Editing report Data Edit Protocol Display System Settings Layout AT eSTe E z Options 3 a E g 3 10 11 006 0063 0 561 0 695 O415 0 670 045 0566 0814 0636 0 071 Hod 073 0664 0913 0608 064 03934 0041 When formatting is complete the document can be printed viewed or exported depending on the current process User s Guide to KC4 PowerRep
242. lowing information e The KC4 software version and revision numbers such as Version 3 4 Rev x found under the About menu e The license type v3 4 or Signature e The product serial number e The specific steps which reproduce your problem e Any error messages displayed on the screen e A daytime phone number and fax number if available e Your name and company information Phone Support Contact the Technical Assistance Center between 8 30 AM and 5 30 PM Eastern Standard Time EST and or Customer Service between 8 00 AM and 5 30 PM EST Monday through Friday excluding holidays Bio Tek Instruments Main Number Technical Assistance Center European Coordination Center Facsimile Support 802 655 4040 800 242 4685 toll free in the U S 802 655 4740 outside the U S 49 0 7136 9680 Send a fax with your questions or requests for help 24 hours a day to Technical Assistance Center European Coordination Center Written Communication Send your written comments to Bio Tek Instruments Inc Technical Assistance Center P O Box 998 Highland Park Winooski Vermont USA 05404 0998 Electronic Communication Send E Mail or find us on the Web E Mail tac biotek com 802 655 3399 49 0 7136 968 111 Bio Tek Instruments GmbH Kocherwaldstrasse 34 D 74177 Bad Friedrichshall Germany Web http www biotek com To visit the our website from within KC4 select
243. lter Em PASS Lamp Monitor PASS W243 Probe Change Ern PASS Ex Position PASS 2 97 W LS PASS Em Position Pass 1 12 Bias Bass View Report Click View Report to see a complete listing of commands exchanged between KC4 and the FL600 during the diagnostic procedures This report can be printed and faxed to Bio Tek for help with technical support Click Close to close the dialog and return to the main display Test results are automatically saved in a database in the KC4 folder Universal Plate Test The Universal Plate Test uses Bio Tek s Universal Calibration Test Plate to validate and document the consistency of an absorbance reader s alignment accuracy reproducibility and linearity Every Bio Tek Universal Test Plate comes with a Calibration Test Plate data sheet The data sheet lists the expected absorbance reading for each location in the test plate at several different wavelengths This data must be entered into KC4 before the Universal Test Plate can be used To define Universal Test Plates e Select Setup Diagnostics Define Universal Plates The Define Universal Test Plates dialog appears listing the currently defined test plates by serial number Define Universal Test Plates Ea 21239326 Universal Test plate 129874 7 Filter Universal Test plate Delete Add oe ses AE gait To modify an existing plate highlight the desired serial number then click Modify or to
244. lue 92 High wells 90 91 HOLE notation 197 200 Horizontal Reading Points linear scanning 115 I Identifiers 181 Importing a plate database 242 Importing files into the shared database 228 Importing plate data Clarity BTI files 53 Importing plate data 51 Expected file formats 51 Importing sample names 61 Selecting a text separator 231 Importing sample IDs 61 User s Guide to KC4 INCORRECT READER MODEL CONNECTED message 69 Incubation Pre heating 117 246 Temperature control 117 Individual Well Auto Scaling 114 115 Individual Well Color Auto Scaling 116 Init All Transf 160 Installing the software 11 Instruments controlling more than one at a time 194 Integral 136 Kinetics 136 Scanning 141 Intensity plate shaking 116 Interpolation 169 Examples 169 170 Formula syntax 170 Interpolation results 298 K KC4 32 pld file 10 244 KCLocal database Compacting 235 Explained 233 Location 235 Repairing 235 KCShared database Compacting 233 Configuring KC4 22 Explained 233 Location 233 Renaming 233 Repairing 233 Restoring connection to 233 Used for file storage 222 Keep open after exporting 187 Keep open after printing 186 Kinetic analysis 131 163 259 Data to process 131 Formula 136 Index e 317 Integral 136 Lag time 133 Max V 132 Mean Min Max 134 Mean V 132 No calculation 136 Onset OD 135 Kinetic curves Overlaying 258 Repo
245. luminescence assays to prevent ambient light from entering the measurement chamber HOLE indicates an empty location in the filter wheel An empty location in the Emission filter wheel is typical for luminescence assays e For each Center wavelength value enter its accompanying Bandwidth Note This information is printed on the side of each filter See the FLx800 operator s manual for details Bandwidth is the filter bandpass width in nanometers of the installed filter For example the Center Bandwidth combination of 485 20 will transmit light from 475 to 495 nm 10 nm on either side of the center e When all values have been entered click Send Filters The values will be downloaded to the instrument overwriting its existing wavelength table To retrieve filter wheel settings from the instrument e Click Get Filters The values will be uploaded to KC4 overwriting the values in this dialog 198 e System Menu User s Guide to KC4 Synergy HT Absorbance The Synergy HT performs absorbance reads in the range of 200 to 999 nm Use this dialog to specify and calibrate six wavelengths to be made available as default selections within the Reading Parameters dialog Wavelengths E Reader SynergyH T I on COM Absorbance Fluorescence Luminescence Wavelengths 1 405 G gt Send Wavelengths Note To exchange wavelength information between KC4 and the instrument the two must be communicating T
246. ly depending on whether or not the Secured Protocol and Data File Storage option 1s enabled but the functionality is the same In the example below this option is enabled Current Folder be Wones Mary 4Nucleio Acid K i Modified OMA Cluarit 11 BN4 Quant 6 260_280R atio prt E C4 Protocol 041101 15 31 10 1434320 pri E C4 Protocol 04 17 01 13 11 50 3 Dligo Quant prt E C4 Protocol 04 17 01 13 02 28 EEE Phenol prt E C4 Protocal 05710701 16 29 22 File Mame Phenol prt Type KC4 Protocal prt Cancel e The Current Folder field or the Look In field you will see one or the other shows your current location within the overall folder structure Ifthe drive is represented as DB this means that files are currently being stored in KC4 s secure shared access database If the drive is represented by a single letter such as C or D this means that files are stored individually on the computer s hard drive or on a network to which the computer is attached See Where and How Are Protocols Stored for more information 68 e Protocol Menu User s Guide to KC4 e Click J to move up one folder level e Click on a folder J to open it and view its contents e Click ona file and click OK to open it e Click to update the view to show any folders files that may have been added by another user after you opened this dialog Upon opening a protocol KC4 evaluates it to determine 1 if it was c
247. m within KC4 To perform an Optics Test for all readers except the Clarity and the FL600 Ensure that the reader is properly connected to the computer and that all communication parameters are set correctly Select System Diagnostics Run Optics Test The Run Optics Test dialog appears a AA pQuanton COMI Reader Serial Number 123583 y Reader response Enter the reader serial number then click Start to run the test When the test is complete the Identification dialog appears Identification of the optics test Fram Ik Brittirig Company Bio T ek Instruments Telephone 655 4040 Fas Number Date 101498 15 29 36 User s Comment October maintenance check Cancel Help Enter the From Company Telephone Fax Number and Comments as appropriate Click OK to continue KC4 displays the results and then saves them in a database in the KC4 folder User s Guide to KC4 System Menu e 205 Optics Test History Following each Optics Test KC4 saves the test results in a database CALIB MDB located in the KC4 folder To view historical Optics Test results e Select Setup Diagnostics Optics Test History The Optic Test History dialog appears Optics Test History Ea Selection Reader Operator 1045498 11 31 11 AM 10 5 98 11 49 55 AM 10 5 98 4 07 13 PM 1045 98 6 04 32 PM 1010 00 3479 01 04 4 View Delete Help To view a list of tests for a specific reader cli
248. measurements The value reported for each well represents the average of its measurements Usually the more samples per well the better the CVs although selecting a large number of samples per well typically results in only marginal improvement Consider a setting that represents the optimal combination of sensitivity and speed The recommended setting to achieve a balance between speed and sensitivity is 10 measurements per well Note The reader takes approximately 10 milliseconds to perform each measurement Delay before sampling is the pause time of the optical probe above or below the microwell before the measurement is taken The valid range is from 10 to 2550 milliseconds The recommended setting to ensure the probe is stopped for measurement but for the minimum amount of time is 350 milliseconds Delay between samples is the time delay between measurements taken in each well The valid range is from 0 to 255 milliseconds The recommended setting is 1 millisecond Longer delays between samples may result in better CVs between replicates User s Guide to KC4 Protocol Menu e 105 Measurement Mode Synergy HT Time Resolved Measurement Mode Nb Samples per well i O Delay before sampling 350 msec Delay between samples li nsec Time Rezolved Fluorescence Delay 20 Sec Data Collection ime 100 Sec Specify the Sampling options e Nb Samples is the number of measurements taken per well The valid rang
249. ment in lt KC4 gt Microsoft Word File Edit View Insert Format Tools Table Window Help Header Times Mew Roman 10 B 7 es Fields Plates Tables Close L TENE Statistics Well IDs Validation Results Header Times Mew Roman B Fields Plates Tables Y Close Table Title T User s Guide to KC4 PowerReports e 295 Fitting Results A Fitting Results table lists coefficient values for all curves in the assay To create a Fitting Results table click the Tables button in the RSO toolbar and then click on Fitting Results The Curve Parameter Table Properties dialog appears Ed Microsoft Excel Worksheet in lt KC4 gt File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves x Close A1 Statistics A Well IDs a ees Fitting Results Curve Parameter Table Properties se Selection Data Identifier Standard Assay Control T Blank e Sample TT Sample Control Index Cal i From I to fro Cancel Help e Select the Identifier s to be represented in the table 4 If Sample and or Assay Control 1s selected Index is by default set to All meaning all sample and or assay control wells will be included in the table To include a subset of all sample and or assay control wells use the Index From and To fields to enter the limiting group numbers In the example above Sample groups l and 2 SPLI and
250. mic range of 0 to 99998 relative fluorescence or luminescence units RFU or RLU Tips Typical assays using 96 or fewer well plates require sensitivities between 35 and 150 Assays using 384 well or other higher density plates will likely require much higher sensitivities due to narrower optical probes Luminescent reactions may require sensitivities up to 255 To determine the optimum sensitivity for an assay use Scale to High Wells or Scale to Low Wells Scale to High Wells is used for fluorescence and bright luminescence assays Scale to Low Wells is used for weakly luminescent reactions See Automatic Sensitivity Adjustment for more information As the sensitivity setting increases so will the fluorescence values If many wells result in OVRFLW indicating RFU or RLU values greater than 99998 the sensitivity setting is probably too high If each well contains more than one fluorophore one of which might give brighter or weaker results assign each filter set its own sensitivity Ifthe field shows Auto the Automatic Sensitivity Adjustment feature is enabled e Click the Options button to specify the Measurement Options for each individual filter Set 98 e Protocol Menu User s Guide to KC4 Filters Filters Meas Filter 405 i Refr Filter ezo The Filters parameters include e Meas Filter select the measurement filter for a single or dual wavelength read e Re
251. n absolute value or a relative value compared with a reference OD the Basis Value You can either manually enter this value or have KC4 calculate it mean of the n first points By default n 1 thus the Basis Value 1s the OD of the first point e KC4 returns the time for each well to pass the Onset OD value e Note For fluorescence and luminescence reads KC4 performs calculations based on fluorescence units Onset RFU and luminescence units Onset RLU respectively To set the calculation zone select Onset OD or Onset OD then click Calculation Options Kinetic Analysis Onset OD Basis Value Calculation f Mean of the E first points C OD o Cancel Help Basis Value Calculation e Mean of the n first points For relative determination of the Basis Value e OD For absolute determination of the Basis Value Examples Onset OD 0 5 Basis Value Mean of first 2 points KC4 calculates the time that 1s required for the OD to increase by 0 5 OD compared with the mean of the 2 first points of the kinetics Onset OD 15 Basis Value Mean of the first point KC4 calculates the time that 1s required for the OD to increase by 15 compared with the first point of the kinetics Onset OD 1 6 Basis Value 0 KC4 calculates the time that 1s required for the OD to reach the value of 1 6 Basis Value Onset OD User s Guide to KC4 Protocol Menu e 135 Formula Select Formula to define a formula to calcula
252. n data file If the file has not yet been saved KC4 provides the option to save before closing Save Save the current plate data PLA or global data GLB file Save As Save the open file under a new name Read Plate Read a plate using the attached reader enter plate data manually or read plate data in from a file Sign Electronically sign the current GLB data file Export Export data to a text file to an Excel spreadsheet or to a Word document Append to Append the current set of plate data to an existing file Kinetic File Print Print a report according to the Protocol Report settings Print Preview Print Setup Plate Description Preview the report before printing Define the print setup before printing Enter information about the current plate using up to six free text fields plus a large comments field Samples Define the names of samples Audit Trail View the contents of the Data Audit Trail Exit Exit KC4 32 e Getting Started User s Guide to KC4 Edit Menu The Edit Menu contains editing functions commonly found in Windows fe KC4 NewPlate Data Edit Protocol Display System Cut Ctrlass AR Copy Che poe iar Paste t Cut Deletes selected data from the document and copies it to the Clipboard Copy Copies selected data to the Clipboard Paste Pastes data from the Clipboard into the document Protocol Menu The Protocol Menu contains functions for creating modifying
253. n is restored click Restore Connection to reconnect KC4 with the database It may be necessary to click the Restore Connection button several times to successfully restore the database connection If this message appears Restore Database Connection X Unrecoverable database error please retry IF the problem persists itis recommended to quit EEA E Cancel e Click Retry to try to restore the database connection again e Click Cancel to abort the process and regain control of KC4 e Click Quit KC4 Unsaved files will be saved as temporary files on the computer s hard drive the pathname will be provided These files can be renamed and moved using Windows Explorer and optionally imported into the shared database The Repair Compact button should be used following these events e After reconnecting to the shared database following a database error in case the database has been corrupted e KC4 Signature After System Audit Trail events are exported and deleted to reduce the size of the database 234 e System Menu User s Guide to KC4 e KC4 Signature After files are exported and deleted from the Maintain Files dialog to reduce the size of the database e Note Repair Compact may only be performed if no other KC4 session 1s using this file When Repair Compact is clicked KC4 performs a safety backup of the database in its present location KCShared yymmdd hhmmss mdb before beginning the repair compact procedu
254. n or to the right to make rows or columns larger Well Types and IDentifiers Select and customize Well Types and Well IDentifiers within the Well Settings area of the Plate Layout dialog then assign them to one or more locations within the grid area of the dialog The Well Type tells KC4 which function to perform using the measurement value in the well KC4 supports five different Well Types Blank used during full plate blank subtraction Assay Control used during assay validation or as calibrators Sample representing the unknown samples to be processed Sample Control representing the control well s that are associated with a specific sample Standard used to generate a standard curve on which data will be interpolated The Well ID is the descriptive link between the measurement value retrieved for the well and the Well Type assigned to it For example The average of the values in the wells of type Blank and labeled with the Well ID BLK will be subtracted from every well on the plate as a part of KC4 s blank subtraction function User s Guide to KC4 Protocol Menu e 143 Selecting and Customizing Well Types amp IDs The customization features vary among the different Well Types Each Well Type is addressed individually starting below and continuing on the following pages Blank Wells Well Settings Type Blank ID BLK El During data reduction the average of the raw data values
255. n points and calculates the mean among these n points e Next KC4 repeats the operation starting at the second point in the calculation zone and repeats again starting at the third point and so on e KC4 then compares all of the calculated means to determine the minimum and maximum of these values e KC4 also calculates the Delta t at Min Max OD e Note For fluorescence and luminescence reads KC4 performs calculations based on fluorescence units Mean Min Max RFU and luminescence units Mean Min Max RLU respectively To set the calculation zone select Mean Min Max OD then click Calculation Options Kinetic Analysis Mean Min Max OD Calculation Zone e Frome 00 00 00 Tot 00 30 00 B From 1 Tot ra Mean Min Mas on f points Cancel Help 134 e Protocol Menu User s Guide to KC4 Calculation Zone Typically the Calculation Zone is adjusted to ignore erroneous data point s generated at the beginning of a kinetic assay due to assay noise e Fromt Tot Defines a calculation zone based on time The default values span the defined kinetic Run Time e From To Defines a calculation zone based on number of reads The default values include all reading points Mean Min Max on n points e Selects the number of points to be included in the average calculation Onset OD Onset OD The calculated value 1s the time when the OD reaches a set decrease increase Onset OD that can be defined by a
256. n the Protocol Reading Parameters dialog e To change download and calibrate wavelength values entered the desired values in these fields and then click Send Wavelengths The values will be downloaded to the instrument overwriting its existing wavelength table e To retrieve wavelength values from the instrument click Get Wavelengths The values will be uploaded to KC4 overwriting the values in this dialog 196 e System Menu User s Guide to KC4 FL600 wavelengths Reader FLEO0 on COM Excitation Emission Cancel Center Bandwidth Center Bandwidth Absorb Lum Help KC4 cannot automatically retrieve the wavelength information from the FL600 therefore this information must be entered manually Consult the Order Entry Form that was shipped with the unit to determine which filters were originally installed If this information cannot be found note the reader serial number then contact Bio Tek Instruments Tip If the exact filter setup is unknown you can remove the FL600 s shroud to examine the filters in the Excitation and Emission filter wheels The filters should be imprinted with the wavelength and bandwidth Refer to the FL600 Hardware Guide for instructions A F represent the 6 filter positions on the Excitation and Emission filter wheels Excitation refers to the light directed at the sample in the well Emission refers to the light emitted by the sample in the well Center is the center wavelength in
257. nabled The Repair Compact button should be used following a KCLocal mdb database error When the button is clicked KC4 performs a safety backup of the database in its present location KCLocal_ yymmdd hhmmss mdb before beginning the repair compact procedure User s Guide to KC4 System Menu e 235 Database Upgrade Utility Use the Database Upgrade Utility to update Shared Local and Calibration databases created with earlier versions of KC4 so that they will be compatible with the current version of KC4 For example if you are upgrading from KC4 version 3 1 to 3 4 your version 3 1 databases must be converted before they can be used by KC4 version 3 4 This utility runs automatically during installation It can also be run manually from the System Setup Database dialog To run the Database Upgrade Utility l Zz 3 If possible identify the locations and names of all databases to be upgraded Close all other instances of KC4 running on the PC If upgrading a database located on a network drive make sure that no other KC4 users are currently accessing that database Close any open unsaved KC4 files Select System Setup and click the Database tab Click the Database Upgrade Utility button KC4 automatically begins a search of the fixed non removable drives e g diskette ZIP JAZ and network drives are not automatically searched KC4 searches for the following files KCLocal mdb KC mdb and Calib mdb Note This aut
258. nanometers of the installed filter Bandwidth is the filter bandpass width in nanometers nm of the installed filter does not apply to absorbance filters Example The Excitation Center setting of 485 20 refers to a center wavelength of 485 nm with a the total bandpass of 20 nm This combination will transmit light from 475 to 495 nm 10 nm on either side of the center A check in the Absorb column informs the software that the corresponding filter is an absorbance filter If at least one Absorb box 1s checked the HOLE notation appears in the Center field for Excitation position F Note It is not advised to check Absorb for fluorescence filters because the bandpass is not fixed at 8 to 10 nm but can be as wide as 50 nm or more Some fluorescence filters with narrower bandpasses however might be able to function as absorbance filters A check in the Lum column informs the software that the corresponding filter will be used for luminescence reading If at least one Lum box is checked the PLUG notation appears in the Center field for the Excitation position E Note When a Lum box is checked its corresponding Bandwidth field is not disabled although the bandwidth is not used during luminescence plate reading User s Guide to KC4 System Menu e 197 FLx800 The FLx800 has one four position excitation wheel and one four position emission filter wheel Each filter wheel can contain any combination of filters
259. ncated and the character V is added at the beginning of the name to indicate that the name has been truncated For example LE001 LE002 etc e Sample IDs can be entered using a barcode scanner attached to the computer To do this place the cursor in cell 1 or any empty cell Scan the item with the barcode scanner The first 12 characters of the ID will appear in KC4 Move the cursor down to the next empty cell then scan the next item User s Guide to KC4 Data Menu e 61 Auto Numbering To automatically number the sample IDs sequentially e Check the Auto Numbering box e Jn cell 1 type the identifying name and starting number e g Smp1 for Sample group 1 Leave the cursor in cell 1 e Press the down arrow through as many cells as there are sample groups on the plate KC4 will automatically increment the suffix number Smp2 Smp3 and so on Import Sample Names The Import option allows you to import a sample name list from a text file Click Import and then locate and select the file to import e The import file must contain one name label per line e The number of IDs in the file should equal the number of samples as defined under Protocol Plate Layout If there are more IDs in the text file than samples defined on the plate the extra IDs at the end of the file will remain unused e Ifa Sample ID in the text file exceeds 12 characters only the first 12 characters will be imported Export Sam
260. nd define Pre Reading options Optional Select Monitor Wells and then click Options to define related parameters Click OK to verify the parameters and close this dialog User s Guide to KC4 Protocol Menu e 89 Reading Parameters Reference All possible reading parameters and certain related topics are discussed on the following pages Information 1s organized alphabetically by parameter name or topic Clarity Microplate Luminometer users Refer to Chapter 4 of the Clarity Operator s Manual for information on reading parameters Automatic Sensitivity Adjustment Enable Automatic Sensitivity Adjustment to let KC4 automatically select the most appropriate sensitivity value for a particular filter set There are three methods to choose from Scale to High Wells Scale to Low Wells and Best Signal to Noise Ratio The best method depends on your application some experimentation may be necessary Note for Synergy HT w Injector models With Dispense protocols if two filter sets are defined and Automatic Sensitivity Adjustment is enabled for the first filter set Use first filter set sensitivity is the required selection for the second filter set Scale to High Wells Scale to High Wells searches for two consecutive gain values where the measured values are lower and higher than the defined high well values E i Automatic Sensitivity Adjustment a f Scale to High wels Best Signal to Noise Ratio Scale to Low wells U
261. ndards fall within the linear area of the curve one either side of the inflection point 168 e Protocol Menu User s Guide to KC4 Curve Interpolation Select Protocol Curves Curve Interpolation to define Y values and or formulas for which a concentration will be calculated Curve Description AMES Curve Fit Interpolation A Y Formula Che Os CTL2 1 015 4 50 6 50 Cancel KC4 supports the definition of up to 25 Interpolation values and formulas e Before defining interpolations that reference well identifiers ensure that all identifiers have been assigned under Protocol Plate Layout e ach interpolation can contain any of the following Numeric values such as 1 500 85000 or 2 0E 8 Well identifiers such as NC BLK or STD1 Well coordinates such as Al H12 or P24 A formula combining any or all of the above such as CTL1 CTL2 100 e Click the Biograph button after a plate is read to view the Interpolation Results e Interpolation results can be included on reports and in export files e Interpolation formula syntax is described on the next page User s Guide to KC4 Protocol Menu e 169 Interpolation Formulas The following table contains valid symbols and functions for use within Interpolation formulas Symbol or Function Well coordinates MEAN lt ID gt SD lt ID gt CV lt ID gt LOG lt x gt POW lt x gt lt y gt SQRT lt x gt MIN lt x eS AZS i
262. ng parameters and then click OK to continue The Plate Reading dialog appears Plate Reading Date 14 Feb 01 Time 16 18 29 PI Description mmmn aaa Name Sandra Allen Lot Code Title Tech Il Water Lot Site Winooski Test Comments Enter Manually Read From File 48 e Data Menu 970 385 275 16 2 of 5 Cancel Help User s Guide to KC4 The Plate Reading dialog allows optional information and comments to be added to the plate This information is filed along with the data for future reference Optional Enter data in the Plate Description fields and enter Comments Tip The data entry field names can be customized via Protocol Plate Description Click Start Reading to initiate communication with the attached microplate reader Following a successful plate read the measurement values will be transferred to KC4 and displayed in the Results window Click Enter Manually to enter plate data using the keyboard See Entering Plate Data Manually on page 50 for more information Click Read From File to read plate data from a stored file See Reading Plate Data From a File on page 51 for more information Unintentional Termination of a Kinetic Read If a kinetic read is unintentionally terminated or if at the end of the read KC4 receives an error status from the reader KC4 presents the following message An error in communication to the reader has occurred
263. ngs including report and export engine and the weighting standard deviation e Set global parameters including the import export text separator and the time format for kinetics e Customize the arrangement of the toolbar buttons e Create and maintain microplate measurement information User s Guide to KC4 System Menu e 193 Reader Selection Select System Readers to select or change the current reader Important Be sure to set and test communication parameters before attempting to read a microplate Reader Selection E Current Reader uGuant on COM Cloze Available Readers Help Quant on COME Clarity EL 800 HE on COM EL 808 1 on COM FLEO0 on COM FL 800 on COM 2 Configuration Port Fiters Wavelenaths e Choose a reader from the Available Readers list then define the Configuration Port and Filters Wavelengths parameters e Click Port to define communication parameters including Port number Transmission Speed Data Bits Stop Bits and Parity e Click Filters Wavelengths to align KC4 s wavelengths table with the reader s internal table e If applicable click Configuration to define the optical probe size so that it matches the current hardware configuration e When all parameters are set click the Current Reader button KC4 will attempt to communicate with the attached reader If communication fails KC4 provides information for solving the problem Follow the instructions
264. nistrator requirements 8 Administrator 8 Allow well zoom during read 103 Anti Log 160 Append to kinetic file 56 185 Application Data folder not visible in Explorer 22 234 235 Archiving records 29 Area scanning 115 262 Area under the curve 136 Assay control wells 143 145 Assay description 177 182 Assigning names to sample groups 61 Audit trail 63 Data audit trail 63 Notification dialog 220 User s Guide to KC4 PowerReports 300 Protocol audit trail 183 System audit trail 224 Automatic append to kinetic file after reading 185 Automatic export after reading 187 Automatic print after reading 186 Automatic save after reading 185 Automatic sensitivity adjustment Best signal to noise ratio 91 Scale to high wells 90 Scale to low wells 90 Selecting in a protocol 96 98 Available readers 194 Averaging in formulas 160 Axes 165 B Bandwidth 197 200 Bandwidth verification 92 Barcode Enabling the option to read 111 Using as file name 185 Base value 92 Basis value calculation 135 BDF file 53 Importing 53 Best signal to noise ratio 91 Bias value 96 98 Biograph 189 265 Index e 313 Bio Stack Minimizing KC4 when running in OLE mode 232 Sample IDs 61 Bio Tek contacting for technical assistance 4 Blank plate subtraction 111 128 Blank wells 143 144 Blank wells subtraction 128 Block light between reads Filter position requirements 201 Read step opt
265. nt 186 e Protocol Menu User s Guide to KC4 Export Select Protocol Options Export to select the export engine and define related parameters Ram Data Report Export Calculation Engine KC C Microsoft W ord E MicrosoftB Excel Export Director ESKEA Data Files Browse Formatting File Format Microsott E scel Format g W Automatic Export after reading Keep Open after exporting Protect e Select the Engine for designing export templates KC4 This is the standard method for designing export files that KC4 has always provided The contents are customizable but the appearance 1s completely controlled by KC4 you cannot change the font style or size for example Word If your computer has Microsoft Word 2000 installed on it you can design export file templates using Word and take advantage of all of 1ts formatting features Excel If your computer has Microsoft Excel 2000 installed on it you can design export file templates using Excel and take advantage of all of its formatting features In addition you can create custom Excel formulas that reference KC4 data e Ifyou chose Word as the engine choose a Document Formatting option of Standard or Enhanced If Standard is selected document formatting is faster because KC4 does not apply any custom font characteristics from the Plate Layout or Cutoffs dialogs Well IDs and cutoff symbols are printed using just the default font cha
266. o dispense fluid into microwells as part of a Read amp Dispense protocol To enable this feature begin by clicking the Dispense checkbox within the Reading parameters dialog Reading parameters Reading Type f End Point a Detection method Kinetic C Absorbance Fluorescence C Luminescence O Multi Mode Spectrum Reader Synergy HITA welnjectors on COM M Dispense Read amp Dispense Filters Filter Set e Note The Dispense option is not available if Multi Mode is selected e Define or select additional Reading parameters within this dialog including Reading Type Filter Set s Plate Type and Temperature Control e Click the Read amp Dispense button to open the Read amp Dispense dialog Use this dialog to define the Dispense Shake and or Read steps for this protocol Read mode ell Comment Format HH Mk4 55 Time scale 00 00 00 to 00 76 30 oo0m 1 1 ajal A Dispense Shake Read Steps JE le Timing control is ON Incubation time it exactly the same for each well Cancel Help Dispensing and read steps are performed at the same speed time if approximate and reader configuration dependent e Select a Read Mode If Plate mode is selected each step in the sequence is performed on all wells of the plate step after step For example if the step sequence is Dispense Read fluid is dispensed into all wells of the plate and then the wells are read Timings a
267. o help protect the system and its data from intentional or inadvertent harm or loss Select System Security to view or edit these options Security Ea Users Login Protected functions Audit Trail Signature Reasons Files j W Multi User identification Administrator System Administrator Mew TAM Alan Ww Thompson BEN Ben E Franklin Edit MEW Martha E Washington G SLJ Susan L Jennings Delete Up to six feature tabs are available within the Security dialog Availability depends on the current KC4 license and whether or not the System Administrator 1s logged in KC4 v3 4 KC4 Signature Users Edit the Administrator s password Edit the Administrator s password Establish and edit individual user accounts and enable or disable Multi User Identification Login does not apply Enable and define various Login and Password parameters for application in a multi user environment Protected Determine which KC4 functions Determine which KC4 functions shall be protected Functions shall be protected from use when from use by Standard Users the Administrator is not logged in Audit Trail does not apply Enable or disable the appearance of an Audit Trail Notification dialog each time an event is logged to a Data or Protocol Audit Trail or to the System Audit Trail Signature does not apply Define a list of terms for users to choose from when Reasons signing files to explain why they are signing F
268. o the list e Note Shaking can also be defined as a part of the Read step if repeated shaking is required between each kinetic reading interval Read Step Parameters Use this dialog to create or modify a Read step Read step E E Start time 00 02 30 Shortest after SHAKE 10 s Intensity 1 Run time Joo 0 00 Interval 00 02 00 Reading number 6 Shake during read Intensity i Duration o E Cancel Help time le approximate and reader configuration dependent e Set the Start Time for the step Enter the time manually HH MM SS or check Shortest and select the step immediately after which this Read step shall be performed e Kinetic protocols only Specify the Run Time This is the length of time from the start of the first kinetic read to the start of the last kinetic read User s Guide to KC4 Protocol Menu e 123 Specify the time Interval for repeat reading of the plate section of wells or individual well The maximum valid Interval is equal to the current Run Time KC4 automatically calculates the Reading Number by dividing the Run Time by the Interval and adding 1 If Shaking before each kinetic read is required specify the shake Intensity and then specify the shake Duration in seconds a Duration of 0 means no shaking will occur Note Shaking can also be defined as an individual step outside of the Read step using the Shake button in the Read amp Dispense dialog Ac
269. of wavelengths e Click Mto open a list of available wavelengths then click on a wavelength to select it If the desired wavelength is missing from the list type the value directly into the wavelength field e Optional If the reader is connected clicking Calibrate will update KC4 s wavelength table and the reader s internal wavelength table If KC4 s wavelength table already matches the reader s wavelength table calibration is not necessary If calibration is necessary but the reader is not connected right now calibration will perform automatically when a plate read begins For Spectrum reads Step li 0 nm W Calibrate e Define the wavelength range by entering the Start and Stop values in nanometers e Define the reading interval by entering the Step value in nanometers For example if the Start Stop and Step values are 300 600 and 50 nm respectively the plate will be read at 300 350 400 450 500 550 and 600 nm e Optional Select Calibrate If Calibrate is selected the reader will always perform calibration at the wavelengths specified in the protocol just prior to plate reading If Calibrate is not selected the reader will calibrate at only those wavelengths specified in the protocol that have not yet been calibrated since the reader was turned on 118 e Protocol Menu User s Guide to KC4 Creating a Read amp Dispense Protocol The Synergy HT with injectors provides the ability t
270. ol enable the Scanning checkbox and then click the Scanning button to define the read matrix size Optional For a spectrum protocol click the Scales button to customize the graphical data display Optional Define Shaking parameters Optional Define General Options parameters Optional Define Temperature Control parameters Optional Select and define Pre Reading options Click OK to verify the parameters and close this dialog 86 e Protocol Menu User s Guide to KC4 Synergy HT Time Resolved Reading Type Reader SynergyHTTA I on COM2 End Point Detection method C Kinet C Absorbance Fluorescence Luminescence Multi Mode metic Spectrum 5 a Kinetic Scales 5 Y Time Resolved Filters Filter Set 9 Mode Fluo E Excitation 485 10 Emission 528 20 Optics Position Top v Sensitivity f uto Options Plate Temperature Control Type 96 WELL PLATE y Size 8x 12 Yes E PreHeating First well 61 Last well 1H12 Q No Temperature f z Lag Time 00 00 00 Shaking Intensity o Duration o Pre reading Pre Read Blank Plate te Before ever readina Before first reading General Options Eject between Filter Set Read Ps Bio tel far g UF Top Probe Vertical Offset fi mm Monitor Wells BGS Cancel Help O Pathlenath Eorection tayelenaths MaS Select a Detection Metho
271. omatic search will not find Shared database files if their names do not begin with KC Ifyou know the names and locations of the databases you wish to upgrade and or if the databases reside on a drive that is not automatically searched click the Stop button When the search is complete or when the Stop button is clicked the Database Upgrade Utility dialog appears Database Upgrade Utility Please select the database file s to upgrade and to use on this PL Shared database cap ocuments and Settings All Users Application Data Bio Tek Instruments 3 15KC4 Sig Local database lt Continue to use current file no upgrade Diagnostics database lt Continue to use current file no upgrade Browse Start upgrading Cancel Click the drop down arrows to see which databases KC4 found Click on the desired database name to select it for upgrading Click the Browse button to search for a database When the database is located highlight it and click Open KC4 will insert the name in the appropriate field Shared Local or Diagnostics Click Start Upgrading to begin the process When prompted enter the name s for the converted databases The original databases will remain unchanged When the process is complete restart KC4 The upgraded database s will be in use 236 e System Menu User s Guide to KC4 Toolbar Sakkings Layout The Toolbar contains buttons that when clicked
272. on to enable this feature and then enter the Y axis intercept value Enter 0 the default value to force the curve through the origin User s Guide to KC4 Protocol Menu e 167 The value entered here determines the coefficient a of the polynomial equation y gtx fx ex dex c x b x ta Curve Fit Options Ea e Y axis intercept o Cancel Note By forcing the curve through the origin the R squared value may be decreased because the equation may not describe the data of the standards as well It is the user s responsibility to check the validity of the data when using this feature Spline with Smoothing Spline fitting with a smoothing factor The curve will appear smoother as the smoothing factor is increased The zero factor will force the curve fit through all data points The minimal number of standards for the spline fit is 4 Note The unknown concentrations are calculated using an approximate calculation method linear interpolation between 200 points evenly spaced on the x axis 4 Parameter logistic fit a d y d lt ich a theoretical response at concentration 0 b measure of slope of curve at its inflection point c value of x at inflection point d theoretical response at infinite concentration x concentration y response OD The minimum number of standards is 4 It is recommended that e at least one standard is not far from each asymptote e at least 2 sta
273. onents you do not want to install Components lv Sam ples PLA PAT Destination Folder CARCA 3 0 Rev 22 Browse Space Required 16355 K E Space Available 2096832 K _Disk Space Cancel 11 Check the components you want to install and click Next The Select Program Folder dialog appears Select Program Folder x Setup will add program icons to the Program Folder listed below Tou may type a new folder name or select one from the existing Folders list Click Mest to continue Program Folders kca 3 1 Fev es Existing Folders E Cjunior Microsoft Office Tools Mouse are Network Associates PYCS Tracker lt Back Cancel 12 Accept the default Program Folder or enter a new one Click Next to continue with the installation process User s Guide to KC4 Installation and Configuration e 13 After a few moments the Registration dialog appears Registration El Thank you for purchasing EC KC4 is delivered with a 21 days Trial license In order to get a permanent license you need to 1 Type the License Number provided with the product 2 Fill the Registration Card and send it to Bio Tek E mail passwordlibiotek corn ae Fax 1 802 655 38 49 Registration Card Tou will then receive a password from Bio T ek Later on you will need to complete F C4 registration by entering this password Into EC Cancel Help 13 Enter the License Number provide
274. onnected to the computer and that all communication parameters are set correctly e The reader should be equipped with the necessary filters to perform the test Refer to the calibration data table that came with the test plate to determine the required wavelengths e Select System Diagnostics Run Universal Plate Test 208 e System Menu User s Guide to KC4 The Run Universal Plate Test dialog will appear Run Universal Plate Test Ea Test setup Reader uLuart Serial Number i 39139 Universal plate jie 259 7 Filter Universal Test plat W Peak absorbance search Wavelength 405 450 430 550 620 630 Operator Ik Britting Comments ia maintenance check Run Test Close Help Enter the Serial Number of the reader under test or click the drop down arrow to choose an existing number Click the Universal Plate drop down arrow to select the test plate to be used If the desired test plate is not in the list select Setup Diagnostics Define Universal Plate to define a new test plate If supported by the reader the Peak Absorbance Search option is available If the box is checked the Peak Absorbance Search is performed in addition to the standard Universal Plate Test Choose the wavelength s to be included in the test by clicking the required cells in the Test Yes No column Click in a cell to toggle Yes No Note If N A appears for a wavelength that you want to test update the
275. ontrol is available under the System menu This feature can be used to view information about the attached reader control the reader door or carrier and to control incubation Information Before contacting Bio Tek for technical assistance use the Information dialog to retrieve the serial number part number software version and basecode checksum from the attached reader Reader Control x Serial Number 128730 Basecode Part number 260207 Version 1 12 Checksum EE3D Door Carrier The Door Carrier functions are reader dependent The attached reader must have robotic capability such as the ELx808 or FL600 Reader Control x Plate In Plate Out e Select Plate In to move the plate carrier into the plate reading chamber and close the door 1f applicable e Select Plate Out to move the plate carrier out of the plate reading chamber and open the door if applicable User s Guide to KC4 System Menu e 245 Pre Heating Use the Pre Heating dialog to set the reading chamber temperature turn the incubation unit on or off or to set a timer to beep after a specified duration or when the requested temperature has been reached Reader Control xl Information Door Carrier Pre Heating Temperature Requested 370 C I On Off Actual ern E Ambient E Total Time 00 01 06 Beep after 0 mn Time since reached 00 00 00 Beep after 0 mr e To set the reading chamber temperature
276. op Top r 50 00 Sensitivity 50 i O0 1 Plate Type First well Shaking Options Options Options Options Temperature Control 48 WELL PLATE Size 6x8 Yes E Ee HEt Mo Temperature E Al Last well Fs Lag Time 00 00 00 FT Pre Read Blank Plate Intensity o Diuratiorr o 5 Mental Mita fe Before evem reading Before first esca M Eject between Filter Set Read AA a ae 10 INIA 12 ES Help Select a Detection Method of Fluorescence or Luminescence Select a Reading Type of Endpoint or Kinetic Optional Click Update Filters to retrieve current filter values from the instrument Define the Filter Sets to be used Select a Plate Type and define the First and Last Wells For a kinetic protocol click the Kinetic button to define the read time and interval For an endpoint area scanning protocol enable the Scanning checkbox and then click the Scanning button to define the read matrix size Optional Define Shaking parameters Optional Define Temperature Control parameters Optional Select Pre Read Blank Plate Optional Select Monitor Wells and then click Options to define related parameters Optional Select Eject Between Filter Set Read Click OK to verify the parameters and close this dialog User s Guide to KC4 Protocol Menu e 81 PowerWave PowerWave HT Reading Type End Point O Kinetic Spectrum Wavelengths fe 1 405 Calibra
277. op selected items between the two lists The Form Feed option inserts a page break Audit Trail Reports If Protocol Audit Trail is selected a list of all changes made to the protocol is included on the report Protocol Audit Trail 0845 02 14 2316 MAJ FileCreated 3 3 3 3 SS 08 15 02 14 38 12 WAJ Multi Plate Transformation changed Adjusted Delta OD transformation O84 502 14 46 07 Report changed Designed the report template 08115102 15 11 23 Signed for Authorship by Mary Ann Jones 08 16 02 09 35 28 Signed for Review by Robert Michael Barnes If Data Audit Trail is selected a list of all changes made to the data file is included on the report Data Audit Trail MAJ Protocol DB Wones Mary AiNucleic SO AcidiPhenolprt 06 7102 10 21 15 08 19 02 16 23 32 MAJ Platereadstarted 08 19 02 16 24 27 MAJ Sample Plate successfully read 08 19 0216 26 53 MAJ File Created CT 08 19 02 16 26 53 MAJ Signed for Authorship by Mary Ann Jones S O 081 9 02 16 48 22 WAJ Options changed Changed the weighting of 08 20 03 09 25 38 RMB Signed for Review by Robert Michael Barnes Calculation Log Report If Calculation Log Report is selected warning s appear on reports if a special event occurs during the calculations Events can include a failure during the calculation of a transformation the standard curve or cut offs Calculation Log Report Calculation Warnings Data In not availa
278. opens a list of available items to include in the plate such as Well ID 405 405 Corr and Concentrations Insert a Table Object to present data in a columnar format Clicking the Tables button opens a list of available items to present in this format including Statistics Well Data Analysis and Fitting Results Insert a Curve Object such as a standard curve a set of dose response curves or a kinetic scanning or spectrum well zoom Delete an object The cursor must be placed inside the object when this button is clicked Close minimize Excel or Word and return to KC4 The document is automatically saved as a part of the protocol Get Help on PowerReports User s Guide to KC4 PowerReports e 279 Object Components Objects are inserted into the document using the Result Objects toolbar See Field Objects Plate Objects Table Objects or Curve Objects for specific instructions for inserting the different types of objects The example below shows a Field Object in an Excel document EA Microsoft Excel Worksheet in lt KC4 gt Ready A e A z In the report or export document any object contains 3 unique items e An identifying title as shown in cell B2 The text and appearance of this title can be modified To change the text simply overwrite the existing title To change the appearance use the application s font customization tools e A dollar sign character as shown in cell C2 The do
279. option is disabled The Eject Between Filter Sets option is disabled The Top Probe Vertical Offset option is disabled The top probe height will be automatically set to 1 mm above the top of the plate Note The Dispense option is not available when Multi Mode is selected 94 e Protocol Menu User s Guide to KC4 Eject Between Filter Set Read This feature is available for fluorescence and luminescence protocols if you are defining an endpoint read with two or more filter sets Enable Eject between Filter Set Read to e Perform an initial reading of a blank plate followed by the assay plate s at some later time e Provide the ability to add reagents to the same plate between readings e Enable reading up to 6 different plates with one protocol and plate file If this feature is enabled KC4 ejects the plate after one filter set read then displays a message to click OK to start the next filter set read See also General Options FL600 or General Options Synergy HT Excitation Emission Filter Sets FL600 Filters Ee hep yet c ca gt ad CN 75 Excitation 485 20 465 20 Pe 485 20 485 20 485 20 485 20 Emission 590 35 590 35 590 35 590 35 590 35 590 35 6 Optics Position Bottom Bottom Bottom Top Top a Top 75 125 175 Options Options Options Options Options Options Sensitivity f 25 li r5 The FL600 can take
280. or equal to then an overlap condition does exist e When all of the reading parameters have been defined and the user attempts to validate them by clicking OK to KC4 evaluates each filter set pair against the overlap formula If the result is such that there is no overlap condition for any of the defined filter set pairs and there are no other parameter definition violations KC4 will allow you to OK out of the Reading Parameters dialog Ifthe result is such that there is an overlap condition for any filter set pair KC4 will display a message resembling the following Please select alternative filters for filter set 1 The bandpasses overlap KC4 will not allow you to OK out of the Reading Parameters dialog until there are no overlapping filter set pairs 2 Proximity Evaluation Although the transmission profile is quite vertical it does flair out at very low transmission levels indicating an increase in the bandwidth In order to account for the bandwidth at these lower transmission levels KC4 performs a proximity check on each filter set pair The formula used to evaluate each pair of filter sets to determine if they are too close to each other is as follows ICW excitation CW emission gt BW maximum BW minimum 2 where CW excitation is the center wavelength of the excitation filter CW emission is the center wavelength of the emission filter BW maximum is the larger of the two bandwidths BW minimum is the
281. or specific instructions on changing filters see the Synergy HT Operator s Manual Note To exchange wavelength information between KC4 and the instrument the two must be communicating The currently selected reader and COM port are displayed at the top of this dialog If you need to change the COM port click Cancel to exit this dialog now and then click the Port button Wavelengths Reader SynergyH T on COM Absorbance Fluorescence Luminescence Excitation Emission Center Bandwidth Center Bandwidth Get Filters 360 H jad Send Filters ES 20 Cancel Help To change the current settings and download them to the instrument e Enter wavelength values in the Center fields or use the drop down boxes to select PLUG or HOLE PLUG indicates a solid plug in the filter wheel A plug in the Excitation filter wheel 1s typical for luminescence assays to prevent ambient light from entering the measurement chamber HOLE indicates an empty location in the filter wheel An empty location in the Emission filter wheel is typical for luminescence assays e For each Center wavelength value enter its accompanying Bandwidth This is the filter bandpass width in nanometers of the installed filter For example the Center Bandwidth combination of 485 20 will transmit light from 475 to 495 nm 10 nm on either side of the center The bandwidth is printed on the side of each filter See the SynergyHT Operator s Manual
282. ors will likely occur Ensure the communication settings match those defined on the reader Transmission speed Values commonly accepted by readers are between 2400 and 9600 bps Data Bits Options range from 4 8 bits Parity Options include No Odd Even and Mark Stop bits Options include 1 1 5 and 2 bits User s Guide to KC4 System Menu e 195 Filters Wavelengths Use the Wavelengths dialog to ensure that KC4 s wavelengths table is aligned with the reader s internal table Note It is essential that you conform to the specific Bio Tek reader procedures for altering the configuration When using filter based readers it is your responsibility to ensure that correct filters are allocated to their respective positions otherwise incorrect data will result Clarity Microplate Luminometer users KC4 s Filters Wavelengths options do not apply to the luminometer ELx Series PowerWave Series yQuant Reader Cancel Help Get Wavelengths Send Wavelenaths Note To exchange wavelength information between KC4 and the instrument the two must be communicating The currently selected reader and COM port are displayed at the top of this dialog If you need to change the COM port click Cancel now to exit this dialog and then click the Port button KC4 s currently defined Wavelength values are displayed in nanometers in fields 1 through 6 The settings here will be made available as default selections withi
283. orts e 281 Designing the Document This section explains how to work with Field Plate Table and Curve Objects Inserting Field Objects A Field Object represents a single item of information such as the name of the protocol file the calculated value of a cut off or the r squared value of a curve To insert a Field Object click the Fields button in the RSO toolbar The Insert Field Object dialog appears Insert Field Object Identification Category Curve Fit Cancel Name Ft v Help Format Hotatior Decimal Hb Dec E Select the Category of information that represents this Field Object Available options include Curve Fit Cut Offs File Information Plate Description Reading Parameters and System Information Select the Name of the field within that category Ifthe only available item in the list is the current protocol parameters do not generate any fields for this category For example 1f the protocol does not specify cut off criteria there will be no items available under the Cut Offs category Tip To learn more about the different fields and to see some examples click on the Insert Field Object Help button then click on a Category link If the field will contain a numeric value set the Notation to Decimal ex 0 003 or Scientific ex 3E 03 If Decimal notation is selected specify the number of decimal places Nb Dec to be displayed 0 to 5 Click OK to close this
284. otocol s Plate Layout the logged event is simply Plate Layout changed If the Protocol Audit Trail notification option is enabled however the user can provide additional text that will be logged with the event such as Added blank wells Al and A2 Here is a sample Audit Trail Notification dialog Audit Trail El Date 08 07 02 16 06 03 Everts Plate Layout changed Added blank wells 417 amp 42 Date A Action Comment TEP 05 0702 141322 TRE File Created 08 0702 16 06 03 TRE Plate Layout changed Added blank wels 41 4 42 se 220 e System Menu User s Guide to KC4 Signature Reasons This feature only applies to KC4 Signature Select System Security Signature Reasons to define a list of terms for users to select from when signing protocols and data files to explain why they are signing Only the System Administrator is authorized to modify these terms Users Login Protected functions Audit Trail Signature Reasons Files Signature reasons to display in the Data Sign dialog Authorship Review Approval Cancel Help Here is a sample New Data Signature dialog showing the Reason drop down list Hew Data Signature Reazon Review ki a Represent Modification Been ADD h dE User TRE Full Name Tom A Barmes Password Cancel Help User s Guide to KC4 System Menu e 221 File Storage and Format Options Select System Security Files to enable or di
285. otocol KC4 subtracts the value of each well of the blank plate from the corresponding well of the sample plate Kinetic read For each wavelength defined in the protocol within each kinetic read KC4 subtracts the value of each well of the blank plate from the corresponding wells of the sample plate Spectrum read At each wavelength read within the defined spectrum range KC4 subtracts the value of each well of the blank plate from the corresponding well and wavelength of the sample plate User s Guide to KC4 Protocol Menu e 129 Blank Wells Subtraction Within any protocol if one or more wells are defined as Blank under Plate Layout the Blank Wells Subtraction Raw Data Correction option automatically becomes enabled When this option is enabled Endpoint read For each wavelength defined in the protocol the average of the blank well s is subtracted from every well on the plate Note For readers that do not accept multiple gt 2 wavelengths if a dual wavelength read is defined KC4 automatically calculates the Delta OD first The average of the Delta OD values in the blank wells is then subtracted from every well on the plate Kinetic read For each wavelength defined in the protocol within each kinetic read the average of the blank wells is subtracted from every well on the plate Note For readers that do not accept multiple gt 2 wavelengths if a dual wavelength read is defined KC4 automatically calculates
286. ous engine and retain the current document if one exists e See Protocol Options at the end of Protocol Menu for more information on the different Report and Result Export options Defining the Protocol Parameters It is a good idea to define all or at least the majority of the protocol parameters before creating a report or export document KC4 customizes its lists of options that can be inserted into the document based on the current protocol parameters For example e Define Standard wells in the Plate Layout dialog and set the curve fitting parameters if the report will contain information about the standard curve such as a picture of the curve the curve equation and the R R values e Define Sample wells in the Plate Layout dialog and specify cutoff criteria if the report will contain custom formulas to determine the frequency of symbols assigned to samples and then generate a pie chart showing the percentages e Define the wavelengths in the Reading parameters dialog and assign blank wells in the Plate Layout dialog if the export file will contain raw and blanked measurement data at 405 nm and 490 nm Creating Editing Documents When you are ready to create or edit a document select Protocol Report or Protocol Export or click the appropriate toolbar button Create edit an Excel export document Create edit an Excel report document rot El cee Report Export Create edit a Word Create edit a Word r
287. ow to navigate the Help system Get started with the Quick Start Examine sample protocol and plate data files and read the corresponding assay descriptions e Go to the Data Menu chapter and read Working with Data Files to learn how to access generate modify and save plate and global data files e Goto the Protocol Menu chapter and read Working with Protocols to learn how to access create modify and save protocol files e Go to the System Menu chapter and read Security to understand all of the security options available to the System Administrator Protected Functions to learn how to protect certain KC4 functions from use by unauthorized users Database to learn about the different databases used by KC4 Exporting Importing Plate Formats to update the plate format database to support pre version 3 1 protocols that reference custom plate formats Importing Files to transfer your pre version 3 4 protocol and data files into the shared access database KC4 Signature users only 26 e Installation and Configuration User s Guide to KC4 Getting Started Overview Welcome to KC4 Getting Started introduces some important concepts for KC4 users including e A description of KC4 s key features under Essential Information e A tour of KC4 s Main Screen e An overview of each menu s contents under Quick Reference Guides e Tips on Navigating the Help System e A typical
288. owerReports e 297 Interpolation Results An Interpolation Results table lists the calculated X values for the Y values and formulas entered under Protocol Curves Interpolations To create an Interpolation Results table click the Tables button in the RSO toolbar and then click on Interpolation Results The Curve Parameter Table Properties dialog appears Ed Microsoft Excel Worksheet in lt KC4 gt 157 File Edit View Insert Format Tools Data Window Help Fields Plates Tables Curves X Close Statistics Well IDs Fitting Results Interpolation Results Protocol Audit Trail Data Audit Trail Calculation Log Report Curve Parameter Table Properties Selection Data Identifier le Standard Pe Assa Eont Blank Sample M Sample tonto Inder EA Comf to i Cancel Help e Standard is the only Identifier that can be included in an Interpolation Results report e Click the Data tab to customize the contents of the Interpolation Results table 298 e PowerReports User s Guide to KC4 Curve Parameter Table Properties Ea Selection Data Decimal KC4 presents a default set of Data items that will be included in the table although any available item can be included in the list See the previous topic Fitting Results for information on working with Data items Click OK to close this dialog and insert the object into the document Ed Microsoft Excel Worksheet in lt KC4 gt
289. parameter settings If this button is inaccessible select Protocol New Protocol to select a protocol type then click Settings to define the reading parameters Protocol Reading Configure the plate Layout by assigning a meaningful identifier to each well Typical identifiers include BLK STD and SPL Standard concentrations and dilutions are also defined here Protocol Plate Layout For kinetic spectrum or scanning protocols define related Analysis parameters for data reduction Protocol lt type gt Analysis Define or modify Single Plate Transformations to perform calculations within the same plate data set Typical uses include using specific wells as local blanks for other wells and calculating ratios between wells Protocol Single Plate Transformations Define or modify Multi Plate Transformations to perform calculations utilizing more than one plate data set For example create a multi plate transformation to subtract the 630 nm measurement values from the 405 nm measurement values in all wells Protocol Multi Plate Transformations 238 e System Menu User s Guide to KC4 Define or modify Curve fitting parameters Configurable parameters include Data to Interpolate Curve Fit Method and Interpolation values This button is available if well identifiers with concentrations or dilutions are defined in the Plate Layout Protocol Curve Define up to five sets of Validation criteria to assess
290. pears enter the password admin and click OK xl Please enter pour login information User Administrator Full Marne E patem Administrator 3 Users Protected functions Files E HMultUser mentiteastion Administrator System Administrator Change Password E Edit Current password Delete New password Ez Cancel Help OF Cancel Help 4 Enter a new password confirm the password and then click OK Important Do not forget the Administrator s password If you do you will have to reinstall KC4 User s Guide to KC4 Installation and Configuration e 21 Copying KCShared mdb to a Secure Network Location M KC4 v3 4 optional M KC4 Signature KCShared mdb is a database file installed by KC4 e With KC4 v3 4 KCShared contains security and plate format information e With KC4 Signature KCShared contains security and plate format information user accounts protocol plate data and global data files and their associated audit trails and a system audit trail for shared events Following installation KCShared mdb is stored on the computer s hard drive but a copy of it can be stored in a network location for secure record storage and for multi user access Note It is important to keep either the original KCShared mdb file or a copy of the network file on each user s hard drive in case the user needs to revert to it following an extended network connection failure
291. plate reader If a well contains OUT or OUT cutoffs are based on concentrations and the well fell below or above the lowest or greatest X value of the calculated standard curve User s Guide to KC4 e Click the Cutoffs button to see the cutoff criteria and the calculated cutoff value s Cut Offs Y alues H Y alue Formulae 0 05200 NC 2 5010 LPC 2 8760 HPC Cutoffs Values The contents of the Formulae in this dialog correspond with the Cutoffs defined within the protocol The calculated value of each cutoff is displayed in the Value column e A cutoff formula references a well ID that is not defined in the Plate Layout e A cutoff formula references a well that is masked e A cutoff formula references a well that is out of range or has been failed by the attached microplate reader Pathlengths Pathlength Correction can be enabled to normalize absorbances to 1 cm Its use is controlled within the Protocol Reading parameters dialog If Pathlength Correction is enabled KC4 first calculates the pathlength for each well The absorbance in each well is then divided by the pathlength resulting in the corrected absorbance When calculations are complete KC4 automatically reports the corrected values available by selecting M 405 Corr M 630 Corr etc from the Data drop down list You can easily create a Multi Plate Transformation to report the pathlength of each well You can give the transformation a descriptive name
292. ple Names The Export option allows you to export the generated identification labels to a text file for future use Enter the Sample IDs as required and then click Export to specify the export file name The export file will contain one name label per line Printing Sample IDs The Print options allow you to print the Sample IDs in a plate or list format e Click Print As Plate to print the Sample IDs in a matrix format corresponding with the plate format selected in the protocol The plate must be configured via Protocol Plate Layout prior to using this feature e Click Print As List to print the Sample IDs in a list format One Sample ID will be printed on each line 62 e Data Menu User s Guide to KC4 Data Audit Trail Every data file plate and global has a Data Audit Trail associated with it This audit trail automatically records events performed on the file since the file was created Each event records an action such as Sample Plate read or Mask Value lt well ID gt and includes a date and time stamp the ID of the user logged in at the time if applicable and any comments that were entered by the user when the action was performed The audit trail cannot be edited and it remains a permanent part of the file for the life of that file that is if the file is deleted the audit trail is also deleted e With KC4 v3 4 only Change Mask Read and Re Read delete initial values actions are logged in the audit trail A fi
293. port The Select plate database file to import dialog will appear Tip If the Plate Formats option is not available in the System Menu this means that the currently selected reader does not support customizable plate formats Select System Readers and choose a more appropriate reader Select plate database file to import Look in y KC4 3 0 Rev 29 a E BACKUP Office OLE CLIENT SAMPLES 12 Kc4_32 pld File name Kc4_32 pld Files of type kca 3 0 2 Plate Database pld Cancel A Set Files of Type to KC4 3 0x 2 x Plate Database pld 4 Use the file tree to locate the KC4 32 pld file from the previous KC4 version and then click Open The Plate Formats list will update to show the contents of this file Important KC4 does not verify that the imported file has a valid structure or contains valid values It is the user s responsibility to verify the structure and content of the plate formats after they have been imported 5 Click OK to return to the main view Note KC4 version 3 02 introduced a new Plate Height dimension for plate formats This is the distance from the bottom mounting surface of the plate to the top face of the plate If you are importing a plate database from an earlier KC4 version be sure to measure the plate height and add it via the Modify button in the Plate Formats dialog 244 e System Menu User s Guide to KC4 Reader Control If supported by the current reader Reader C
294. process Prime the tip with fluid once before the entire process begins Before the dispense Prime the tip with fluid before each dispense to a well e If tip priming will be performed enter the Volume in microliters The valid range is 5 to 20 The dispense rate will be the same as that selected for the well dispense e Set the Start Time for the step Enter the time manually HH MM SS or check Shortest and select the step immediately after which this Dispense step shall be performed e Enter the dispense Volume in microliters The valid range is 5 to 1000 e Select the dispense Rate in microliters per second The default rate 1s the maximum available for the attached instrument e Click OK to save the settings and add the Dispense step to the list 122 e Protocol Menu User s Guide to KC4 Shake Step Parameters Use this dialog to create or modify a Shake step 2 Shake step Stark time o0 01 30 Shortest after DISPENSE Syringel 100p at 3 Intensity i Duration i O amp Cancel Help time le approximate and reader configuration dependent e Set the Start Time for the step Enter the time manually HH MM SS or check Shortest and select the step immediately after which this Shake step shall be performed e Specify the shake Intensity Acceptable values include 1 Low 2 Medium 3 High 4 Variable e Specify the shake Duration in seconds e Click OK to save the settings and add the Shake step t
295. protocol e Select Data Read Plate Read from File then select the desired file If the file is formatted properly KC4 will read the data then calculate results according to the protocol parameters Import File Format for an Endpoint Read The file block size must correspond with the plate layout defined in the protocol For example a 96 well plate has 8 rows and 12 columns and a 24 well plate has 4 rows and 6 columns The following example represents the 96 well plate format 003 1 409 0 483 2 022 1 463 1 200 0 876 2 240 2 057 1 867 0 435 2 147 776128470 760700377022970 91170 368704157247171 11470 2987 0 012 ALA A AZ Oe As adas ALO DOS Lil Oseas LAS ks dad 95072200 77130070 TO A O Fle OL P23 9082 LA LS O O Lo US O 156 0 524 1 949 2 109 2 492 2 499 1 529 0 981 0 666 0 743 2 100 0 059 940 0 232 1 693 0 141 0 022 2 297 0 690 0 682 1 470 1 728 2 094 1 816 21270513718597 1 17171 14572 37371 86170 27171 4987 0 96371 83871 522 431 0 903 0 379 0 563 1 063 2 007 1 293 2 475 1 879 0 864 0 422 1 643 e e ON FF OF O User s Guide to KC4 Data Menu e 51 Import File Format for a Kinetic Read Before importing a kinetic text file create a protocol that specifies the exact number of kinetic reads that will be imported The first line in the text file can optionally contain a time indicator This value will be stored in the protocol overwriting protocol settings if they are different and used for any kinetic plotting If the fi
296. pt as expressly provided in this Agreement COPYRIGHT The KC4 program and its documentation are copyrighted You may not copy any program or the documentation except as for loading the program into the computer server as part of executing that program and for back up purposes respectively You must reproduce and include the copyright notice on the back up copy TERMS This license is effective until terminated You may terminate it by destroying the programs and documentation and all copies thereof This license will also terminate if you fail to comply with any term or condition of this Agreement You agree upon such termination to destroy all copies of the programs and documentation Rev Date Changes A March 2001 First Issue B December 2002 Updated entire manual to correlate with new licensing options KC4 v3 1 and KC4 Signature and their related new and modified features C September 2003 Changed KC4 v3 1 to v3 3 Added instructions for creating Read amp Dispense protocols for SynergyHTTR w Injectors Added instructions for configuring and controlling the Dispense Module Added instructions for using the Database Upgrade Utility D July 2004 E March 2005 Changed KC4 v3 3 to v3 4 Added instructions for using KC4 to control the new Clarity Microplate Luminometer Added instructions for using the new Multi Mode detection method for the Synergy HT line of instruments
297. ptical probes Luminescent reactions may require sensitivities up to 255 To determine the optimum sensitivity for an assay use Scale to High Wells or Scale to Low Wells Scale to High Wells is used for fluorescence and bright luminescence assays Scale to Low Wells is used for weakly luminescent reactions See Automatic Sensitivity Adjustment for more information As the sensitivity setting increases so will the fluorescence values If many wells result in OVRFLW indicating RFU or RLU values greater than 99999 the sensitivity setting is probably too high If each well contains more than one fluorophore one of which might give brighter or weaker results assign each filter set its own sensitivity Ifthe field shows Auto the Automatic Sensitivity Adjustment feature is enabled e Click the Options button to specify the Measurement Options for each individual filter set Endpoint Readings or once for all filter sets Kinetic Readings 96 e Protocol Menu User s Guide to KC4 Excitation Emission Filter Sets FLx800 Synergy HT Filters Filter Set a se Excitation 485 20 485 20 485 20 485 20 485 20 485 20 Emission 590 35 590 35 590 35 590 35 590 35 590 35 e 5 fe E Top C4 Optics Position Bottom Bottom Bottom Top Top Sensitivity 75 ji 25 li 5 75 li 25 li r5 Options Options Options Options Options
298. ptions Optional manual entry of data Reader diagnostics testing and archiving Customized assay development Graphical input for template creation with auto replication and fill and customized well ID text and colors Quick entry of standard concentrations and sample dilutions with auto increment and auto factor Control and assay validations may be created for each assay Multiple sample curves displayed and analyzed simultaneously including interpolations Powerful multi level transformation formulas allow manipulation of raw data transformed data curve fit results or statistical calculations at any point in the analysis KC4 s zoom feature allows detailed information observed for each well including the ability to highlight specific kinetic reaction wells absorbance and fluorescence Sample IDs may be entered manually or imported from a file Extensive curve fitting capabilities including Linear or Log axis definition Point to Point Linear Polynomial Cubic Spline 4 P and interpolation and graphic editing features Customized display and printed reports Quick access to all available results data including raw measurement values transformed values kinetic calculations concentrations plate layout and statistical data on controls standards and samples Masking and recalculation capability for authorized users Kinetic options Real time display of kinetic reactions individual well zoom Mean V Max V time at Ma
299. que ID and password combination multi user system View the contents of the System Audit Trail Enter a license number and password to ensure continuous use of KC4 after the trial period is over Set up global parameters including the import export text separator time format for kinetics weighting of standard deviation and Word Excel options Tell KC4 whether or not to use out of range values in statistics Customize the appearance of the toolbar by adding removing icons Create and modify plate formats and specify a default format for new protocols Reader Control Send a command to the reader to open or close the door insert or eject the plate and to control the temperature About Menu The About Menu provides access to the Help system Bio Tek and the About KC4 screen About f Help Topics Using Help Bio Tek Home Page About RCA Help Topics Open the Help window with its Contents Index and Find functions Using Help Tips on using the Help system Bio Tek Automatically launch the browser and go to Bio Tek s home page Home Page www biotek com About KC4 Check the software version and revision numbers view current system 36 e Getting Started performance parameters and find telephone and fax numbers for technical assistance User s Guide to KC4 Navigating the Help System Most KC4 dialogs contain a Help button that when clicked opens an informational Help dialog In addition to
300. quence set it to End of Sequence the last item in the list 146 e Protocol Menu User s Guide to KC4 Sample Control Wells wel Settings Type Sample Control 1 ID sPLO1 Y Dil 500 00 J Sample Controls are the controls that are associated with specific samples For example SPLC1 is associated with SPL1 assuming the default names are used This well type can be used to represent sample blanks or spikes Unique IDs should be used to distinguish between the different sample control groups The maximum number of unique sample control well IDs that can be defined 1s limited only by the number of available wells on the plate The default ID prefix is SPLC and the default IDs are SPLC1 SPLC2 SPLC3 etc The default color for all IDs is dark purple To change the ID prefix and or colors click the box to the right of the ID field Sample Control ID prefix SPLC Toon Sequence m A e Enter an ID Prefix using 1 to 7 characters The prefix can contain letters numbers and underscores It cannot contain any spaces and it cannot end in a number If the prefix is currently used in the grid its text can be changed here The change will automatically be reflected in the grid e For each group number click on the Color Sequence box to open a drop down list showing all of the available colors Click on the desired color KC4 uses the first color for SPLC1 then the second for SPLC2 and so on
301. r dilution values The user can however select Protocol Plate Layout to view the layout Menu User s Guide to KC4 Protocol Options le Options Select Protocol Options to customize various options for this protocol including automatic file saving report printing and data exporting Reporting exporting and calculation options can also be modified here if the desired options are different than the default ones specified under System Setup Raw Data Select Protocol Options Raw Data to specify automatic save and append options for raw data Raw Data Report Esport Calculation Save Directory EMKE Data Files Browse M Automatic Save after reading C Prompt for name Auto name C Use barcode as filename Automatic Append to Kinetic File after reading Cancel Help e Enter the Save Directory in which to store plate data files pla and glb associated with this protocol Click Browse to search for and select the desired location Note If files will not be saved automatically see below this location can be changed manually by selecting Plate Save or Plate Save As e Check Automatic Save after reading to automatically initiate the save plate data file function when the read is complete Select Prompt for Name to prompt the operator to manually enter the file name after the plate read or select Auto Name to let KC4 automatically name the file using the system date and a sequence n
302. r log system to notify the Administrator of log in failure incidents that exceed the limits they have established The administrator is responsible to immediately and urgently notify the appropriate personnel at the site if the activity appears to be an attempt to breach security Signature Representations The administrator should be made aware that while electronic signatures representations cannot be excised or added they can be copied by screen copy techniques and pasted into other documents These modified documents cannot be resaved in the secure program but may be printed out as 1s Verifying Minimum System Requirements M KC4 v3 4 M KC4 Signature Before installing KC4 verify that the computer s meet or exceed the following requirements Pentium processor 100 MHz minimum Microsoft Windows 95 98 NT 2000 XP operating system 64 MB of RAM minimum 30 MB of available hard drive space for the KC4 program files Additional available hard drive space for storing protocol and plate data files CD ROM drive Super VGA color monitor Keyboard and mouse ports Serial port for autoreader connection USB port with USB to serial adapter 1f required For users with Microsoft Word Excel 2000 or greater and Windows 98 NT 2000 XP PowerReports links KC4 with Excel and Word providing a powerful format for creating custom publication quality report and export documents For the PowerReports feature to run successfully the
303. racteristics If Enhanced is selected document formatting is slower because KC4 does apply custom font characteristics to Well IDs and cutoff symbols e Enter the Export Directory in which to store exported files associated with this protocol Click Browse to search for and select the desired location Note Select the export text file delimiter under System Setup e Select Automatic Export after reading to automatically export plate data to a file when the read 1s complete If Automatic Save after reading is selected the export file name will match the manually entered file name the automatically generated file name or the barcode depending on the automatic save parameters and will have a txt extension If Automatic Save after reading is not selected the export file name will be NONAME TXT e Ifthe export engine is Excel choose a File Format of Excel Excel CSV or Excel Text User s Guide to KC4 Protocol Menu e 187 e Select Keep Open After Exporting to keep the Word or Excel application open after the preview or after exporting to allow for temporary formula and or formatting adjustments To make permanent changes modify the export file via Protocol Export IfKeep Open After Exporting is enabled the Protect option can be enabled to prevent unauthorized changes to the document Calculation Select Protocol Options Calculation to select a weighting of standard deviation and tell KC4 whether or not
304. range values in statistics Cancel Help e Select the default Reporting and Exporting Engines With KC4 s standard reporting and exporting engines you can customize the contents and to some extent the appearance of printed reports and exported plate data 4 1 KC4 users have access to these standard functions Reporting and exporting with user designed Microsoft Excel 2000 and Word 2000 documents is supported with KC4 s PowerReports feature For more information on the Word Excel reporting options see PowerReports near the end of this manual If Word was chosen as the reporting and or exporting engine choose a Document Formatting option of Standard or Enhanced to tell KC4 how to prepare documents for printing and print previewing The default selection 1s Standard If Standard is selected document formatting is faster because KC4 does not apply any custom font characteristics from the Plate Layout or Cutoffs dialogs Well IDs and cutoff symbols are printed using just the default font characteristics If Enhanced is selected document formatting is slower because KC4 does apply custom font characteristics to Well IDs and cutoff symbols 230 e System Menu User s Guide to KC4 e Select the Weighting of Standard Deviation N 1 Used in calculations where the data is assumed to represent a sample from a larger population N Used in calculations where the data is assumed to represent the total population from which th
305. rations automatically after entering the starting concentration choose an Auto option of Incr or Fact then define the required Increment or Factor value Click on cell 2 to assign the next concentration Use the down arrow key to advance to the next cell or click the mouse in the desired cell Incr Increments concentrations by the defined value If the starting concentration is 10 000 with an Increment of 10 subsequent concentrations become 20 000 30 000 and so on Enter a negative number e g 5 to decrement concentration values Fact Increases concentrations by the defined value If the starting concentration is 1 000 with a Factor of 2 subsequent concentrations become 2 000 4 000 8 000 16 000 and so on e Optional Enter the Unit of measure for the X axis values on the curve using up to five alphanumeric characters e Click the Clear button to delete all values from all cells e For Sample Controls KC4 provides the Calculate Recovery option If this feature is enabled KC4 automatically generates a Recovery results data set containing the results of the following formula Conc SPLCn Conc SPLn ExpectedConc SPLCn x 100 e When all of the concentration values have been entered click OK to return to the Plate Layout dialog 156 e Protocol Menu User s Guide to KC4 Assigning Concentrations To assign concentrations with well IDs e Select the necessary Well Settings Type and ID then choo
306. re Local Database The Local Database area of the Setup Database dialog contains the current location of KCLocal mdb and a Repair Compact button Protocol Defaults Formats Startup Database Shared database This database mainly includes Secunty and Plate Format information It may also include Protocol and Data files See Security Settings lt can be shared by several networked PCs Path METEXCHANGESKC4KOS hared mdb Browse Restore connection HepaimEompach Local database This database mainly includes Setup and Readers information lk is used by all KC4 Y 3 3 instances run on this PC Path C Documents and Settings 4ll Users Application DataBio Tek Instruments Repair Compact Database Upgrade Utility Cancel Help The Path setting for KCLocal mdb cannot be changed If the pathname is larger than the width of the field click once in the field and press the right arrow button on your keyboard to scroll to the right Knowing where KCLocal mdb is stored is useful if your site requires a periodic back up of this file e Tip With some operating systems KCLocal mdb is installed in the Application Data folder under Documents and Settings All Users If you are looking for the Application Data folder on your computer and can t find it 1t is probably hidden To reveal this folder open Microsoft Explorer and select Tools Folder Options View Make sure that Show hidden files and folders is e
307. re generally long User s Guide to KC4 Protocol Menu e 119 If Well mode is selected the full sequence of steps is performed on each well well after well For example if the step sequence is Dispense Read fluid is dispensed into well A1 and the well is read fluid is dispensed into well A2 and the well is read and so on Timings are generally short fast e The Time is approximate indication at the bottom of this dialog means that Start and End times displayed with an asterisk were calculated using appromixation logic KC4 uses approximation logic when lt A step is added with Shortest after selected The reader is not communicating with KC4 The reader is unable to validate the Read amp Dispense settings In addition the asterisk will appear if the reader provided a time that can vary based on other reader settings that may change before the protocol is executed e g injector position Plate Mode Options The Dispense Shake and Read steps are all available with Plate Mode In addition the Timing Control feature is available Read amp Dispense Read mode Well Mew Comment Dispense E DISPENSE Syringel 100W at 300 plsec 00 00 00 00 00 59 Tip Prime 5 pl before the process Shak SHAKE 10 s Intensity 1 00 07 30 00 07 40 ore Wb READ during 00 10 00 Interval 00 02 00 00 02 30 0001329 6 Reads Read Format HH Mh4 55 Time scale 00 00 00 to 00 14 49 oom 31 1 aja D
308. reader s filter wavelength table to include the wavelength Enter the Operator name or click the drop down arrow to choose an existing name Enter Comments pertaining to this plate test optional Click Run Test to begin Follow the directions provided for loading and turning the test plate When the test is complete KC4 displays the results and then saves them in the CALIB MDB database in the KC4 folder User s Guide to KC4 System Menu e 209 Universal Plate Test History Following each Universal Plate Test KC4 saves the test results in a database CALIB MDB located in the KC4 folder To view historical Universal Plate Test results e Select Setup Diagnostics Universal Plate Test History The Universal Plate Test History dialog will appear Universal Plate Test History Ea Selection Reader Operator 10698 10 52 53 AM PASS 101698 3 39 77 PM PASS 10 20 98 12 11 02 PM PASS 10423298 4 17 55 PM PASS View Delete Help To view a list of tests for a specific reader click the Reader drop down arrow To view a list of tests for a specific operator click the Operator drop down arrow To view test results highlight the desired date time from the list then click View To permanently delete test results from the database highlight the desired date time from the list then click Delete 210 e System Menu User s Guide to KC4 Security KC4 provides several security options t
309. reated with an earlier version of KC4 and 2 which reader model it was created for e Ifthe protocol was created using an earlier version of KC4 the following message appears This file has been created with a former version of KC4 It is going to be converted As a precaution please verify after conversion that all of its parameters have been properly recovered Then save the converted file KC4 will not automatically save the converted file it is your responsibility to verify and then save the file either under its existing name or a new one e Ifthe protocol s reader model does not match the one currently selected under System Readers the following message appears Protocol file is not compatible with the current reader The current reader will be changed Click OK to open the protocol and allow KC4 to update the current reader selection under System Readers or click Cancel to return to the main view without opening the protocol If you click OK KC4 will set the current reader to the reader that was selected when the protocol was created If a different reader is then selected from the System menu KC4 will delete the reading parameters of the open protocol but will retain the data reduction steps of the protocol It will also set the protocol name to New Protocol prt The reading parameters must be re entered and the data reduction steps i e data in for curve fits transformations etc checked for correct inpu
310. ribed in the following sections e To start Biograph from within KC4 and view the current curve s select Display Curves or click the Biograph toolbar button e To launch Biograph in stand alone mode select Biograph exe from within the KC4 folder Both KC4 and Biograph allow you to select the mathematical curve that best fits the standards and this curve is used for calculating unknowns Each curve 1s displayed with its formula and the calculated coefficients and correlation parameters User s Guide to KC4 Biograph e 265 File Menu Use the File Menu to work with curves and graph gph files Creating a New Curve Select File New to define a new curve by manually entering the coordinates of the standard points Enter the abscissa values in the x column These are typically the standard concentration values Enter the ordinate values in the y columns These are typically the measurement values OD RFU RLU You can enter up to 12 y values replicates per standard The M y column calculates the mean for each row of y values Note The curve is calculated based on the mean y values not the individual replicate values The SD y column calculates the standard deviation for each row of y values To mask a y value check the Mask box then click on the desired y value It will temporarily change to To unmask a y value keep the Mask box checked and click on the desired It will change back to its original valu
311. rint to print the current curve File Print Preview to preview the printout or File Print Setup to adjust the printer settings The report contains a picture of the curve the name of the curve fit method the equation and the calculated coefficients 4 Parameters y a di c b d a 2 209 b 4 585 c 35 68 d 0 05681 R 1 000 A 1 000 er 0 002461 User s Guide to KC4 Biograph e 267 Exiting Biograph Select File Exit to close Biograph and return to KC4 or Windows wherever Biograph was originally launched from e Upon returning to KC4 all changes are lost and the following message appears BIO Windows Application Es AN changes will be lost If pou want them to be permanent please change the protocol i settings accordingly View Menu Use the View Menu to customize Biograph by hiding displaying the status bar and the tool bar The Toolbar By default the Toolbar is displayed below the menu options I BIOGRAPH Windows Application STANDARD CURYE File Edit View Graph Window About Haaa eel 17 e To hide the Toolbar select View Toolbar to remove the checkmark e To redisplay the Toolbar select View Toolbar to reinstate the checkmark The Status Bar By default the Status Bar is displayed at the bottom of the screen Save the active document with a new name CAP NUM D e To hide the Status Bar select View Status Bar to remove the checkmark e To redisplay the Status Bar select View Status Bar
312. rst having to create an export template User s Guide to KC4 PowerReports e 273 How it Works PowerReports uses Object Linking and Embedding OLE technology to interact with Microsoft Excel and Word providing you with total control over all report formatting using results objects created by KC4 KC4 sends information about the current protocol parameters to an Excel or Word document You design the document by selecting the pieces of information to include on the report or in the export file defining the locations for the different pieces of information and customizing the appearance of the document The document then becomes a part of the protocol and is available for editing any time the protocol is open Microsoft Excel or Word Document Inserts a Results Object toolbar Generates custom drop down menus EC4 Software User Creates a protocol based on current protocel parameters Chooses areporvexport engine Tells EC4 to launch Excel or Word Inserts objects into document using toolbar and custom menus Customizes document s appearance Launches Excel or Word Closes document to return to EC4 Sends current protocol information 0 Eca Embeds document in protocol User Initiates a plate read Previews report or export document Prints report or exports data 274 e PowerReports User s Guide to KC4 User s Guide to KC4 KC4 v3 0 Highland Pak Box 925 Winooski Vermon
313. rt 179 Viewing results 258 Kinetic interval 103 Uneven indication on reports 121 125 Kinetic read 131 Choosing the time format 231 Read was interrupted 49 Real time kinetic zoom 103 Selecting in a protocol 113 Kinetic reading parameters 102 103 Allow well zoom during read 103 Eject between well 104 FL600 103 Interval 103 Minimum kinetic interval 103 Plate Mode 103 Reading Number 103 136 Run Time 102 Scales 114 Well Mode 104 L Lag time Delay before reading 101 Kinetics 133 259 Lamp control 99 Last well 99 Lin axis 165 Linear regression 166 Linear Regression 164 167 Linear scanning 115 139 Local database Compacting 235 Explained 233 318 e Index Location 235 Repairing 235 Log axis 165 LOG function 160 Log report 301 Logging in to and out of KC4 223 Login parameters 216 Low wells 90 91 Lum checkbox 197 Luminescence 197 Emission filter wheel 200 Read method 112 M Maintain files 226 Manual data entry 50 Masking wells How to 45 Results window 252 MAX function 161 Max V 132 MEAN function 160 Mean Min Max 134 138 Mean V 132 Meas Filter 99 Measurement Options 104 Microplates measuring 242 MIN function 161 Min Max spectrum analysis 137 Minimize KC4 232 Minimum kinetic interval 103 Minimum password length 217 Monitor wells 107 Multi plate transformations 162 Multi Plate Transformations Formula syntax 163 Ove
314. rt Separator delimiter for use in export file formats Choose semi colon comma or tab or specify a custom separator such as in the Other field Before importing a plate data file ensure that the selected Import Export Separator matches what is used in the file e KC4 offers the following Time Formats for Kinetics HH MM SS Average duration kinetics HHH MM Long duration kinetics SSSS sss Average duration kinetics reported in seconds and milliseconds SSSSSSSS Average duration kinetics reported in seconds SSSSSSSSS Fast kinetics reported in milliseconds When involved in calculations such as Transformations Cutoffs and Validation conditions time values are always expressed in milliseconds regardless of the time formats chosen in the System Setup menu If you need to use other units define a single plate transformation to make the conversion When time values are used on the Y axis of a Standard Curve they are always expressed in seconds Start Up Select System Setup Start Up specify behavior each time KC4 is launched Setup Protocol Defaults Formats Startup Database T Start Microsoft ord when launching KC4 e Start MicrosoftBExcel when launching KC4 Minimize KC4 when running in OLE mode Cancel Help e Specify whether or not to start Word or Excel when launching KC4 Keeping these applications open saves time when creating new report and export templates you won t have to wait for them to load e
315. rview 162 Multi user identification Pal YA Multi mode 94 108 Multiple instruments controlling simultaneously 194 Multiple shared databases using 233 User s Guide to KC4 Multiple wavelength reads 109 118 N Naming samples 61 No Calculation 136 NO READER RESPONSE TIMEOUT message 69 Normal Mode 112 Normalizing absorbances to 1 cm 109 O OLE mode 232 OLE mode minimizing KC4 232 Onset OD FU LU 135 Optics Position 96 98 Optics test 205 206 OUT indication 256 Out of range wells 256 OVRFLW 252 P Parity 195 Password parameters 216 Passwords 213 Changing 214 Expiration 217 Minimum length 217 Unique 217 Pathlength Correction 130 Enabling 109 Raw data correction 128 Pathlengths Calculating 109 Reporting the pathlength for each well 257 Peak OD test 208 Picture mode 262 PLA files Plate data files 28 42 Plate data files 28 42 Plate database Exporting 242 Importing 242 Plate definition not found 242 User s Guide to KC4 Viewing contents of 240 Plate definition not found in plate database 242 Plate description 60 75 Plate format reports 181 Plate formats Custom 242 Exporting 242 Importing 242 List of 240 Plate height 241 Plate In Plate Out 245 Plate layout Assay control wells 145 Blank wells 144 Dilutions 151 Sample control wells 147 Sample wells 146 Standard wells 148 Undo the last action 143 Well IDs 143 144
316. s IM Modify Plate Formats 0 modify System Setup WwlRepair Compact databases Define Universal Plates e Delete Optics tests Universal Plate tests from history e Delete System Audit Trail events after export Create Folder In secured file storage Rename folder file in secured file storage e Delete Overte folder hle from secured file storage Move folder hle in secured file storage import file to secured file storage Esport file from secured file storage Checked functions may not be performed by unauthorized users e With KC4 v3 4 an unauthorized user is anyone other than the System Administrator e With KC4 Signature an unauthorized user is one who is designated as a Standard User 218 e System Menu User s Guide to KC4 The following list includes all system level protectable functions and describes the behavior the unauthorized user can expect from KC4 for each function when it is protected Modify Reader Settings Under System Readers the user can change the current reader but cannot alter the Configuration Port or Filter Wavelengths settings The user will be prevented from changing the current reader or from reading a plate if the action requires an update to KC4 s filters wavelengths table Modify Plate Formats Under System Plate Formats the user can view the dimensions of any plate The user cannot create modify or delete plates Modify System Setup Under System Setup
317. s The upper limit denoted by the last standard is 150 units The dynamic range therefore is 150 50 100 units Imposing the 20 extension means that the new range will be 100 x 1 2 equals 120 units This represents an increase in the dynamic of 10 units at the lower limit and 10 units at the upper limit Therefore the new lower limit will be 50 10 equals 40 units The new upper limit will be 150 10 equals 160 units e Notes Sample values that fall into the new limits will be calculated Those that fall outside these limits will be assigned gt than or lt than respectively Extrapolation cannot be used for the Spline routine nor the Point to Point fit These algorithms cannot calculate values outside the upper and lower defined standard points Curve Fit Use the Protocol Curves Curve Fit dialog to select the method by which the standard curve will be generated The default setting is Linear Regression Curve Description dyes Curve Fit Interpolation Curve Fit Point to Point C Linear Regression f Polpnomial regression Degree E Spline Smoothing factor o C 4 parameters Mptione Cancel Heb 166 e Protocol Menu User s Guide to KC4 It is important that the appropriate curve fit is applied to the data otherwise the results may be invalid Whichever curve fitting routine is used it is essential to check the shape of the curve prior to evaluating data The curve fit methods are described below
318. s Guide to KC4 Protocol Menu e 75 Defining Reading Parameters Sckkings The configuration of the Reading parameters controls plate reading and measurement data collection The availability of Reading parameters varies depending on the reader type currently selected under System Readers To define Reading parameters select Protocol Reading or click the Settings toolbar button to open the Reading dialog The following pages are organized by reader model locate your model to find the basic instructions for defining Reading parameters To learn more about a particular parameter search through the Reading Parameters Reference which is organized alphabetically 76 e Protocol Menu User s Guide to KC4 Clarity When KC4 is used to control the Clarity Microplate Luminometer two protocols are required a Clarity protocol file to specify the reading parameters and a KC4 protocol to specify the data reduction parameters The KC4 protocol references the Clarity protocol file Clarity protocol files have a BPF extension and they are typically but not necessarily stored on the PC in the C Program Files BioTek Clarity protocols folder BPF files cannot be stored in KC4 s shared database Clarity protocol files can be created and edited through either the Clarity PC Software or KC4 In either case information on Clarity s reading parameters can be found in Chapter 4 of the Clarity Operator s Manual under the Parameter Input h
319. s applied You can easily create a Multi Plate Transformation to report the Pathlength for each well on the plate Look in the Help system contents under Results Data Sets Pathlengths for more information 110 e Protocol Menu User s Guide to KC4 Plate Type Plate Type E WELL PLATE Size ax 12 Important Be sure to select the appropriate Plate Type Just prior to plate reading KC4 sends the plate s dimensions to the instrument This information guides the optical probe s to the correct reading positions relative to the microplate Choosing an inappropriate plate type could adversely affect results e Select a Plate Type from the drop down menu The items presented for selection come from the plate database which is maintained under System Plate Formats e The First Well and Last Well fields default to include the entire plate You can change one or both settings to read a partial plate Pre Read Blank Plate W Pre Read Blank Plate Check Pre Read Blank Plate to perform two reads at each defined wavelength The first read is performed on an empty or blank plate and the second on the sample plate The Blank Plate can represent a dry empty plate or a plate filled with solution e For Endpoint and Kinetic reads the blank plate is read at each wavelength or filter set defined in the protocol e For Spectrum reads the blank plate is scanned using the wavelength range defined in the protocol e This option is not avai
320. s in full screen Click a to contract the width of the columns Formula Syntax The following tables contain symbols and functions that can be used in Single Plate Transformations Type of Symbol Description Example Algebraic symbols ls eN Figures scientific notation 2 45E 08 X or X Represents the current well value coordinates of the well Represents the value of a particular well such as A2 or H12 160 e Protocol Menu User s Guide to KC4 Functions Applied to the Identifier MEAN POS1 SD NEG1 CV BLK Advanced Mathematical Functions LOG x POW x y POW 10 x SQRT x MIN x1 x2 x3 MAX x1 x2 x3 Formula Examples Description Example Represents the mean of identifier POS POSI by itself also represents the mean of all POS1 wells on the plate Represents the standard deviation of all valid wells in group NEGI Represents the coefficient of variation of all valid wells in group BLK Description Example Represents the log10 function Raises x to the power of y Calculates the Anti Log of the current well Square root of x Minimum value of defined variables Example MIN A1 B1 C1 Maximum value of defined variables Example MAX CV CTRLI CV CTRL2 See also the installed sample protocol files for both Single and Multi Plate Transformation examples these can be found in the SAMPLES folder in your main KC4 folder CTRL1 3 SD CTRL1 The result of this formula is
321. saatadatayei 206 Optics Test FE O00 did id iii 206 Universal Plite cic mee nen enter ae ee ee ee ee a re eerie aCe ciate ance enn 207 SECO en er ed Oe eee eee ee ge eee 211 MT SCT AC COU a e e 212 Essen and Password Para ti 216 orar A 218 Audit Trail Notification Options ti 220 A U O A E T OE cca he 221 Fil Storage and Format OPtiloiSennssnsnrsar a EE A RRT 222 For WEO Oaa dis 223 EOR AA A A O A AE E AAA T A AAT AAT 224 Exportine Audit Trail EYEN AN E 225 a O ee ee fod 226 FG ONS caldeado lll noseanae 221 Ex porine TOSS aa RO RU Ot OR Ve ee 227 vi e Contents User s Guide to KC4 Wn porto Ple a 228 Recistraton OTA iii deidad 229 O A E AE T T T T aan E ES ET E 230 PLOT o AAE Cn A EANA A 230 A A ET 231 A IA A O A 232 A ete eee Oe te IE eee RN Oe ein ne OTE eee NET ees mn ne ee 233 A A IN AA II TSO RUE Seren OEA 237 dE 240 Plate Des cap it ciis 241 Microplate Mica urea A AT 242 Exporting Importing Plate Formats A E 242 Reader om A O CON 245 ARO TVA OU AEE EEE AEE ETTE EEEE AEE een Todo en een ale ETELE EEE 245 A E NE E NAE E AN 245 Pre A II A TE A O N E 246 PA IN 247 Clarity Cl ios 249 RESUE TS ic adicas 251 O A A A ARA 251 Di II II DNR DRT ET SUNS PEO EE EA 253 CONCA OTA Sie ce as creases cea gh AA 254 A 256 A seal eal as eth aaa eon denoue nauiet unsaiiyaeub eeu uo etdunae naan 237 Ie A O AA AAT 258 Spectra Scan Data 260 A A E ee one ae PROS Oe eee we eR eee oe eee Ee eee 262 PANG ATS CATE aio ete A E EE 263 O ee
322. sable the Secured Protocol and Data File Storage feature and to select a Preferred Data File Format Only the System Administrator is authorized to modify these parameters Users Login Protected functions Audit Trail Signature Reasons Files W Secured Protocol and Data file storage Preferred data file format ke4 Data Prtcl GLB Secured Protocol and Data File Storage KC4 Signature only If this option is enabled all protocol plate data and global data files are stored inside a secure shared access database named KCShared mdb by default e These files can only be accessed from within KC4 they cannot be viewed or otherwise manipulated using separate file management software such as Windows Explorer e Important To ensure 21 CFR Part 11 compliance Secured Protocol and Data File Storage must be enabled e Clarity Microplate Luminometer users Clarity protocol files with a BPF extension cannot be stored in KC4 s Shared Database See page 77 for more information Preferred Data File Format When a user chooses to read a plate the default data file format will be PLA raw data or GLB raw data with protocol parameters whichever is selected as the Preferred Data File Format in this dialog e For sites unconcerned with 21 CFR Part 11 requirements the selection made here is merely a matter of preference pre version 3 1 KC4 users may be more comfortable with setting this parameter to pla e For 21
323. se first filter set sensitivity High Wells D1 D2 High Value soooo Starting Sensitrvity 100 e The High Wells represent those wells on which the calibration measurements will be taken They are typically wells with the highest expected signal measurement value on the plate such as the high standard wells e The High Value represents the upper limit of the expected range of values for the entire microplate The default value for the FL600 is 90000 RFU RLU with a valid range of 0 to 99999 The default value for the FLx800 and the Synergy HT is 80000 RFU LFU with a valid range of 0 to 99998 The recommended high value for all of these readers is 50 000 to 70 000 RFU RLU e The default Starting Sensitivity is 100 The valid range is from 25 to 255 The recommended starting sensitivity is 25 Scale to Low Wells Scale to Low Wells searches for two consecutive gain values where the measured values are lower and higher than the defined low value wells Scale to Low Wells is recommended for weakly luminescent reactions 90 e Protocol Menu User s Guide to KC4 T ue Automatic Sensitivity Adjustment a Scale to High wels Best Signal to Noise Ratio f Scale to Low wels Use first filter set sensitivity Low Wells A1 B2 Low Value 25 Starting Sensitrity i p e The Low Wells represent those wells on which the calibration measurements will be taken They are typically wells with the lowest expec
324. se the first concentration value to be assigned e Enter the number of standard sample or control Replicates e Choose the replicate filling direction Horizontal or Vertical e If you want well IDs and or concentration values to advance automatically choose Next ID if available and or Next Dil e Choose the group filling direction Horizontal or Vertical e Highlight the well s to assign the concentrations and well IDs Examples are provided below and on the next page Plate Layout Well Settings 3 r Replicates gt p uto Select Type _ Standard i Hb 2 al TP esti I Next Conc ID Prefix STD A GJ Hor Vert 4 Alles Cone 10 000 a Ho C Vet 10 000 i n e 0 000 30 000 TD4 TD4 40 000 40 000 STES 50 000 TDG 60 000 e Concentrations are defined as 10 20 30 40 50 and 60 e The Auto Select option Next Concentration is selected The Next ID option is not available for Standard groups they must be incremented manually e Wells Al through F2 are highlighted to assign the concentrations and IDs User s Guide to KC4 Protocol Menu e 157 Plate Layout E Well Settings i gt Auto Select Type Sample i miil l W NestiID M Next Conc ID SFL ale E 1 Filling Cone 7 0000 al Hore Yer e E E ee A E A el ae ele A ES PL1 SPL1 PL2 SPL2 SPL3 SPL3 PL4 PL4 SPLS SPLS SPLE PLE 1 0000 1 0000 1 0000 1 0000 1 0000 1 0000 1 0000 1 0000 T 0000
325. sequence of events under Quick Start e A Quick Start just for Clarity Microplate Luminometer users e Instructions for locating and working with Sample Files User s Guide to KC4 Getting Started e 27 Essential Information KC4 allows you to define instructions for controlling a microplate reader acquiring measurement data from that reader for reducing and analyzing the retrieved data and for printing exporting and saving the results The minimum instructions required for reader control and plate data acquisition are called reading parameters Reading parameters varying according to reader model but generally include reading type endpoint kinetic spectrum reading method absorbance fluorescence luminescence filters wavelengths shaking and incubation instructions well monitoring instructions plate geometry and various pre reading options including pathlength correction Reading parameters can be combined with data reduction instructions in a protocol In addition to storing the reading parameters a protocol can store plate layout information single plate transformations multi plate transformations curve fitting parameters cut off and validation criteria report and export templates security settings and more The KC4 software package also contains the Biograph module for viewing and manipulating curves Biograph can be used either alone or in conjunction with KC4 File Types KC4 uses several proprietary file form
326. set resembling M 1 405 Blank represents the measurement values for the Blank Plate at this wavelength A data set resembling M 1 405 Corr represents the corrected values after Blank Plate Subtraction Blank Wells Subtraction and or Pathlength Correction The Delta OD data set represents the difference in measurement values between two wavelengths The Concentrations data set represents the calculated concentration values Launch Biograph to see the curve The Conc Dil data set contains values calculated by multiplying the interpolated concentrations by any dilution factor imposed on the concentration The Symbols data set contains the cut off symbols assigned in accordance with the cut off criteria defined in the protocol The Kinetic Curves and Scanning Curves data sets contain the graphical representation of the measurement values in each well The results of any single or multi plate transformation can be identified by the name defined within the Protocol Single Plate Transformation dialog or the Protocol Multi Plate Transformation dialog For example if the name of a single plate transformation is toxicity select the toxicity data set to view the results User s Guide to KC4 Results e 253 Concentrations Concentrations are reported if standards are defined in the Plate Layout and the curve generated successfully fs RESULTS Data Concentrations Sot ase sm ze on nor a a fem ae amam ms cus na az se
327. set to a zero time value min y is set to 1 0000 OD max t is set to the Run Time value and max y is set to 5 0000 OD e Fluorescence and luminescence reads mint and min y are automatically set to zero values max t is set to the Run Time value and max y 1s set to 99999 RFU RLU FL600 or to 99998 RFU RLU FLx800 and Synergy HT e Individual Well Auto Scaling individually adjusts the min max t and min max y settings for each well to best display its curve It overrides the settings of the min max t and min max y Note This setting only affects the scale of an individual zoomed well The kinetic curves display showing all wells on the plate 1s not affected Scales for Spectrum Reads Scales By default the Scales parameters automatically calculate an appropriate box size to display the spectrum curves The default values can be overridden manually to change the scaling of the x and y axes If the Spectrum reading type is selected in the Reading dialog click the Scales button to open the Scales dialog Scales blin w 300 blan 400 Min p E 0000 Max y 5 0000 W Individual Well 4uto Scaling Cancel e The Scales parameter defaults are set as follows min w is set to the Start Wavelength max w is set to the Stop Wavelength min y is set to 1 0000 OD max y is set to 5 0000 OD The default values can be manually overridden by deselecting the Auto checkboxes e Individual Well Auto Scaling individ
328. ssay Control 1 ID fer Y Dil None Assay control wells are typically used to validate the assay or they can be used as calibrators Unique IDs must be used to distinguish between the different control groups e g NC for negative controls PC for the positive controls The maximum number of unique assay control well IDs that can be defined is limited only by the number of available wells on the plate Concentrations and dilutions can be optionally assigned The default IDs are CTL1 CTL2 CTL3 etc each with a different color To change the IDs and or colors or to define descriptive names for use on reports click the box to the right of the ID field Assay Control E HPS Hydrobtycin 1E4 gm Hydrokiycin 182 gm Distilled Water Cancel Help e Enter an ID using 1 to 10 characters The ID can contain letters numbers and underscores It cannot contain any spaces High POS is valid High POS is not The ID cannot be identical to a formula operand X MIN MAX LOG POW SQRT CV SD and MEAN are not valid IDs If an ID is currently used in the grid its text can be changed here The change will automatically be reflected in the grid If an ID is not currently used in the grid it can be deleted from this list by deleting the contents of the ID field e Optional Enter a descriptive Name using up to 20 alphanumeric characters This name can be included on reports e
329. st or use Static Mode to read the plate If the Reference Wells are skipped and the above time limits are exceeded the FL600 can provide unexpectedly high fluorescence values in the first well that is read on the assay plate Usually the first well is Al but since the software provides the ability to change the starting well the first well can be anywhere on the plate General Options Synergy HT KC4 provides options for controlling certain reader functions preceding and during plate reads such plate ejection and top probe positioning Measurement options e Eject between Filter Set Read Top Probe Vertical Offset 1 2 rim If you are defining an endpoint read with two or more filter sets and are utilizing either the fluorescence or luminescence detection method enable Eject between Filter Set Read to e Perform an initial reading of a blank plate followed by the assay plate s at some later time e Provide the ability to add reagents to the same plate between readings e Enable reading up to 6 different plates with one protocol and plate file If this feature is enabled KC4 ejects the plate after one filter set read then displays a message to click OK to start the next filter set read The Top Probe Vertical Offset parameter is available for fluorescence or luminescence reads when at least one of the filter sets uses the top optics position It allows you to define how far the top probe is positioned from the top surfa
330. sults of transformations applied to them represent the y axis values KC4 provides the ability to define concentrations for Assay Controls Samples Sample Controls and Standards Defining Concentrations When defining concentrations start by selecting the desired well type The concentration values will remain associated with this well type Customize the well IDs if necessary Wall Settings Type Standard ID Prefix STD is When the Type and ID fields are set click the button to the right of the Conc field for well types other than Standard this field will initially be labeled Dil The Concentrations or Concentrations Dilutions dialog will appear depending on the well type Concentrations 1 soes al Csm o a ae OS sma 20000 MECA PESCA Fact 2 PC E ot 00 Concentration METI Unit EU mL i 1 1 E E Unit Ju ral kt V Calculate Recovery Cancel Use this dialog to enter the expected concentration values required for the assay Values must be entered in ascending or descending order starting with concentration 1 1 000 and 5 000E 3 in the examples above e Enter the starting concentration in cell 1 Subsequent concentrations can be entered manually or automatically When entering concentrations manually use the down arrow key to advance to the next cell or click the mouse in the desired cell User s Guide to KC4 Protocol Menu e 155 To increase concent
331. t Single plate transformations Multi plate transformations Curve fitting parameters Validation criteria Cutoff criteria Reporting and exporting options User s Guide to KC4 Protocol Menu e 67 Working With Protocols This section provides instructions for creating modifying saving and accessing protocols Where and How Are Protocols Stored KC4 stores protocols using a proprietary file format and the PRT extension File storage options vary depending on site requirements and the current KC4 configuration e Files may be stored individually on the computer s hard drive on a CD or diskette or on a network to which the computer 1s attached This is the storage method that users of KC4 version 3 0x and earlier are familiar with Windows Explorer or a similar application can be used to view the file names and their locations and to move copy rename and delete files e KC4 Signature Files may also be stored inside a secure shared access database This database named KCShared mdb can be stored on a user s computer or on a shared access computer KC4 provides a special file maintenance window for viewing the file names and their locations and to move copy rename and delete files Opening Existing Protocol Files Select Protocol Open to open a previously saved protocol file If the Open option is not available in the Protocol menu close the plate data file Note The layout of the Open dialog varies slight
332. t The protocol can then be saved under a similar or new name Note If the attached reader does not match the one KC4 is expecting the messages INCORRECT READER MODEL CONNECTED or NO READER RESPONSE TIMEOUT and then Error when getting wavelength table from the reader may appear KC4 will proceed and change the reader under System Readers but you will have to attach the correct reader before running the protocol When the protocol is open KC4 s title bar changes to show the file name Use the Protocol menu or the toolbar icons to modify or view the protocol s parameters Creating New Protocols Important Before creating a new protocol select System Readers and set the Current Reader to the appropriate target reader model The reader does not have to be connected to the computer when you are creating or modifying protocols Clarity Microplate Luminometer users See page 77 for information on creating protocols To create a new protocol begin by doing one of the following to enable the options under the Protocol menu e Close any open plate data files and then select Protocol New e Ifthe Preferred Data File Format is set to GLB select Data New and then choose Empty Protocol User s Guide to KC4 Protocol Menu e 69 The next step is to define the protocol parameters required by your assay to control the reader and perform data reduction Do this by opening the Protocol menu and selecting each option in turn Reading K
333. t 05404 USA AFF 32 254 Date PowerReparts 1 glk 991 205 402 Test Validation 1431 2001 PowerReports e 275 System Requirements To put the power in PowerReports your computer must meet or exceed the following requirements e Pentium II processor or equivalent 300 MHz or faster e 64 MB RAM 128 recommended e Microsoft Office 2000 9 0 3821 SR 1 or newer Office XP Office 2003 To check your Office 2000 version launch Word or Excel and select Help About Microsoft Word or Excel e Windows 98 2000 NT XP Important If your computer does not currently meet all of these requirements you can still print reports and export data With KC4 s standard reporting and exporting functions you can customize the contents and to some extent the appearance of printed reports and exported plate data All KC4 users have access to these standard functions Preparing KC4 for PowerReporting KC4 offers three reporting exporting drivers or engines KC4 Microsoft Excel and Microsoft Word KC4 needs to know which engine s to you want to engage for editing previewing and printing reports and for editing and performing exports The following topics explain how to select default global reporting and exporting engines and how to override them when necessary Setting Global Options Under System Setup Protocol Defaults KC4 allows you to select a default Reporting Engine and a default Exporting Engine When you create a new protoco
334. ta file with a different name Note The layout of the Save or Save As dialog varies slightly depending on whether or not the Secured Protocol and Data File Storage option is enabled but the functionality is the same In the following example this option is enabled 46 e Data Menu User s Guide to KC4 Current Folder De Wones Man A4Nucleic Acid i Modified DNA Quarnt 3 ANA Guant J 260 280A atic pla E KB EC4 Data 08719702 14 37 32 J A320 pla KB ELA Data 081902 14 35 11 OLIGO Quant pla FEB ELA Data 05731700 79 13 28 File Name 4 40 Type kca Data pla Cancel e The Current Folder field or the Save In field you will see one or the other shows your current location within the overall folder structure Ifthe drive is represented as DB this means that files are currently being stored in KC4 s secure shared access database If the drive is represented by a single letter such as C or D this means that files are stored individually on the computer s hard drive or on a network to which the computer is attached See Where and How is Plate Data Stored for more information e The default file name is NewPlate e Entera new File Name and click OK to save it e Click EJ to move up one folder level e Click Ef to create a new folder e Click on a folder e Click the Type drop down arrow to change the type of files being viewed Options include KC4 Data PLA
335. tab The Tip Prime Vessel value shows KC4 s estimate of how much fluid is in the trough e Click Dump all Tip Prime Troughs The Tip Prime Vessel value will reset to 0 Note Ifthe tip priming trough overflows refer to the Synergy HT Operator s Manual for decontamination instructions Note When running a Read amp Dispense protocol KC4 may prompt you to empty the tip prime trough In this case KC4 will automatically open the System Reader Control Dispenser dialog 248 e System Menu User s Guide to KC4 Clarity Control Clarity Microplate Luminometer Use the Clarity Control dialog to control the microplate holder prime the reagent lines and injectors and control plate heating Prime Heating Close Help Microplate Holder Control The Clarity s microplate holder can be opened and closed only through software control e To extend the microplate holder click Plate Out e To retract the microplate holder into the reading and heating chamber click Plate In Note Do not forcibly push in or pull out the microplate holder If for any reason you cannot control the holder using the software use the Allan key supplied with the instrument to extend or retract the holder Refer to the Clarity Operator s Manual for more information Prime The reagent lines and injectors should be primed with the dispensing fluid before running the protocol Click the Prime button to open the Priming Parameters dialog
336. tallation Form to obtain another password Reinstallation is defined as 1 re installing a KC4 version on the same PC for example after a hard disk replacement or 2 un installing a KC4 version from one PC and installing the same version on another PC A Reinstallation Form doc and txt formats complete with instructions can be found in the root KC4 folder when the installation process is complete 16 Click Close to return to the Registration window and then click OK to continue KC4 will automatically run the Database Upgrade Utility This utility is used to update Shared Local and Calibration databases created with earlier versions of KC4 so that they will be compatible with the current version of KC4 For example if you are upgrading from KC4 version 3 1 to 3 4 your version 3 1 databases must be converted before they can be used by KC4 version 3 4 Database Upgrade Utility K C4 is searching for databases used by former versions Scanning LA Documents and SettingatAll Users4pplication DataBio T ek Instruments KC4 automatically begins a search of the fixed non removable drives e g diskette ZIP JAZ and network drives are not automatically searched KC4 searches for the following files KCLocal mdb KC mdb and Calib mdb Note This automatic search will not find Shared database files if their names do not begin with KC 17 If you know the names and locations of the databases you wish to upgrade and or
337. tatistical calculations For the FL600 a well will contain OVRFLW if the absorbance value for a well can be calculated but it is greater than 4 0 O D absorbance reads only or if the RFU RLU value exceeds 99999 fluorescence and luminescence reads only Tip If many or most of the wells contain OVRFLW the sensitivity setting in the protocol may be too high Note Wells containing OVRFLW are excluded from transformation equations and statistical calculations User s Guide to KC4 Data Sets The Results data sets presented for viewing depend on the current protocol parameters Click the Data menu drop down arrow to choose from a list of available data sets fea RESULTS Data M 405 MHI 405 Blank MHZ 630 Blank MHI 405 Corr 0 693 MEZEJO Cor bg Delta EEF ll 0 073 1 237 2 400 0 123 0 199 0 5044 The following describes just a subset of the many different Results data sets Template displays the identifier and any concentration or dilution value assigned to each well under the Protocol Plate Layout dialog M 977 SP and M 900 SP contain the measurement values of the Sample Plate at 977 nm and 900 nm respectively necessary for pathlength correction calculation Measurement numbers M 1 M 2 M 6 correspond with the wavelength selection order in the Reading parameters dialog A data set resembling M 1 405 represents the raw measurement values for the Sample Plate at this wavelength A data
338. te Read Mode Rapid id Flate Shaking Type 384 WELL PLATE Size 16 x 24 Intensity O Duration jo g First well at Last well P24 Before everreadina Before first reading Temperature Control Pre Readings Yes Pre Heating T Blank Plate f No Temperature E I Pathlength Correction Wavelengths p Lag Time 00 00 00 W Bio Cell 5 Options Read Barcode Monitor wele Options coca toe Select a Reading Type of Endpoint Kinetic or Spectrum 2 Define the Wavelengths and Read Mode to be used Calibrate the wavelengths 1f necessary 3 Select a Plate Type and define the First and Last Wells 4 Fora kinetic protocol click the Kinetic button to define the read time and interval For an endpoint linear scanning protocol click the Scanning button to define the horizontal reading points 6 Optional For a spectrum protocol click the Scales button to customize the graphical data display Optional Define Shaking parameters Optional Define Temperature Control parameters 9 Optional Select and define Pre Reading options 10 Optional Select Read Barcode to instruct the instrument to scan the barcode as the plate is drawn in to the reading chamber 11 Optional Select Monitor Wells and then click Options to define related parameters 12 Click OK to verify the parameters and close this dialog 82 e Protocol Menu User s Guide to KC4 PowerWaveX Re
339. te a value using data from individual reading points from the same well Designate an individual reading point using the identifier Rn where n represents the reading number then enter a Result Name in which to store the formula results This name can be used as a Data In setting in Transformations Formula examples e The formula R10 R5 calculates the difference in readings between data points 10 and 5 e The formula R e g R1 extracts a single reading from each well for endpoint data reduction No Calculation This option if selected overrides any calculations on the kinetic data and allows the display of kinetic graphics without data reduction e No Calculation is typically used to display overlaid curves Integral Select Integral to calculate the area under the curve according to the trapezoidal method shown below i n 1 Integral Y yt yn 2 Gu i Where y measurement value and x read point value By default the calculation zone spans the full Run Time and includes all of the reading points defined in the protocol Click Calculation Options to change the calculation zone Kinetic Analysis Integral Calculation Zone Fromt 0 000 sec Tot 120 000 SEC C From f Tot E e Fromt Tot Defines a calculation zone based on time The default values span the defined kinetic Run Time e From To Defines a calculation zone based on number of reads The default values include all reading points
340. ted signal measurement value on the plate such as blank wells negative controls or the zero standard wells e The Low Value represents the lower limit of the expected range of values for the entire microplate The default value is 25 RFU RLU and the valid range is from 0 to 99999 for the FL600 0 to 99998 for the FLx800 and the Synergy HT The recommended low value for all of these readers is 100 to 200 RFU RLU e The default Starting Sensitivity is 100 The valid range is from 25 to 255 The recommended starting sensitivity 1s 25 Best Signal to Noise Ratio With the Best Signal to Noise Ratio method KC4 instructs the reader to perform several pre readings on the plate to determine the optimal sensitivity The optimum sensitivity is based on the best signal to noise ratio obtained during these pre readings r M Automatic Sensitivity Adjustment a EE EEN Scale to High wells Best Signal to Noise Ratio Scale to Low wells Use first filter set sensitivity Low Wells at Ad Base Value 1100 Experimental wells C1 C4 High wells D1 D4 High alte fao000 e Designate certain wells on the plate as Low Experimental and High Wells Enter a single well location ex Al or a rectangle ex B1 C2 of well locations Low Wells are usually blank wells buffer wells or negative controls and are expected to yield a low RFU or RLU value Experimental Wells are usually low standards or low positive controls and
341. terpolation 169 Curve objects in PowerReports 279 302 Curve through origin 168 Curves Printing 189 Stored 266 Viewing 189 Custom plate formats 240 242 User s Guide to KC4 Cutoffs Defining 173 Evaluation method 176 Evaluation of results 256 Examples 174 Formula examples 174 Report 180 Symbols 173 Viewing calculated values 23 Viewing results 256 CV function 160 264 D Data audit trail Data menu 63 PowerReports 300 Data bits 195 Data files 42 Changing measurement values 45 Contents 28 Exporting 57 Masking wells 45 Modifying protocol parameters 45 Plate description 60 75 Signing electronically 54 Storage options 28 Data mode 262 Data to interpolate 164 Data to interpret 173 Data to monitor 107 Data to process 131 137 Database connection error Restoring connection to KCShared 234 Database upgrade utility 15 236 Databases KCLocal mdb 233 235 KCShared mdb 233 DB database drive 44 47 68 71 Default reporting and exporting engines 230 Delay before sampling 105 Delay between readings 108 Delay between samples 105 Delimiters 231 User s Guide to KC4 Delta OD 130 163 181 Detection method 94 108 112 Diagnostics CALIB database 233 Optics test 205 206 Universal plate test 205 207 Dilutions 151 Direct export to Excel 308 Dispense protocol 94 119 Dispenser Configuration 202 Control 247 Display grid 165 Document formatting
342. ters and close this dialog 84 e Protocol Menu User s Guide to KC4 PowerWave XS Reading parameters Reading Type End Point C Kinetic Spectrum Read Mode Rapid 96 WELL PLATE Size ax 12 First well 5 Last well H1 2 Temperature Control Yes D PreHeating Type Calibrate Shaking Intensity Duration fro ES Before even treading Before first reading Pre reading W Pre Read Blank Plate FP Pathlength Correction Wavelengths 10 11 User s Guide to KC4 i Ho Temperature E Lag Time 00 00 00 D BioCel 181 1 pan F Monitor Wells Cancel Help Select a Reading Type of Endpoint Kinetic or Spectrum Bptions Define the Wavelengths and Read Mode to be used Calibrate the wavelengths 1f necessary Select a Plate Type and define the First and Last Wells For a kinetic protocol click the Kinetic button to define the read time and interval For an endpoint area scanning protocol enable the Scanning checkbox and then click the Scanning button to define the read matrix size Optional For a spectrum protocol click the Scales button to customize the graphical data display Optional Define Shaking parameters Optional Define Temperature Control parameters Optional Select and define Pre Reading options Optional Select Monitor Wells and then click Options to define related parameters Click OK to verify the parameters and close
343. ters and close this dialog User s Guide to KC4 Protocol Menu e 83 PowerWaveX Select Reading Type Reader EF End Point Fome ave Select on COM Kinetic Kimetic Scanning Spectrum Calibrate Plate Shaking First well 5 Last well P24 Before even eadna D Before first reading Temperature Control Pre Aeadings Yes P Pie Heati o TT Blank Plate No Temperature HE Y Pathlength Correction Wavelengths E Lag Time 00 00 00 W Bio Cell 5 Options Read Barcode Monitor Welle Upton coca e Select a Reading Type of Endpoint Kinetic or Spectrum Define the Wavelengths to be used Calibrate the wavelengths if necessary Select a Plate Type and define the First and Last Wells For a kinetic protocol click the Kinetic button to define the read time and interval a A E For an endpoint linear scanning protocol click the Scanning button to define the horizontal reading points 6 Optional For a spectrum protocol click the Scales button to customize the graphical data display 7 Optional Define Shaking parameters Optional Define Temperature Control parameters Optional Select and define Pre Reading options 10 Optional Select Read Barcode to instruct the instrument to scan the barcode as the plate is drawn in to the reading chamber 11 Optional Select Monitor Wells and then click Options to define related parameters 12 Click OK to verify the parame
344. ters used for standard fluorescence reads represents the bandwidth at the 50 transmission level The transmission profile of these filters is essentially rectangular and therefore the bandwidth does not vary very much when the transmission levels change When KC4 checks for overlap 1t ensures that the excitation and emission filter center wavelengths are far enough apart that there is no overlap of their bandwidths The formula used to evaluate each pair of filter sets for overlap is as follows ICW excitation CW emission gt BW excitation BW emission 2 where CW excitation is the center wavelength of the excitation filter CW emission is the center wavelength of the emission filter BW excitation is the bandwidth of the excitation filter BW emission is the bandwidth of the emission filter Ifthe Time Resolved option is enabled the value of BW excitation must be multiplied by 2 prior to substituting it into the formula to account for the triangular shape of the transmission profile when using the monochromator Specifically although the monochromator has a bandwidth of 10 nm at 50 transmission levels it approximately doubles by the time the transmission levels approach zero 92 e Protocol Menu User s Guide to KC4 If the absolute value of the difference in the excitation and emission center wavelengths is greater than half the sum of the excitation and emission bandwidths then no overlap condition exists If it is less than
345. the Delta OD first Within each kinetic read the average of the Delta OD values in the blank wells is then subtracted from every well on the plate Spectrum read A blank average is calculated for each wavelength in the spectrum reading range The blank average for each wavelength 1s then subtracted from the absorbance read at the corresponding wavelength in each well Pathlength Correction With readers that support multiple wavelength reads up to 999 nm the Pathlength Correction option is available within the Reading parameters dialog If this option is selected absorbances are normalized to 1 cm If Pathlength Correction is enabled KC4 first calculates the pathlength for each well The absorbance in each well is then divided by the pathlength resulting in the corrected absorbance See Pathlength Correction in the Reading Parameters Reference section to learn more 130 e Protocol Menu User s Guide to KC4 Kinetic Analysis Use Kinetic Analysis to select pre data reduction parameters for kinetic reads This option becomes available within the Protocol menu when the Kinetic read method is selected Kinetic Analysis Calculated Data Data to process 1 l MHI 405 Man H1 Coeff Reg 1 Calculation Type Delta t at Max El C Meany a Coett Reg 2 Max Y Delta t at Max y H2 Mean Minhas OL Lag Time 2 C Onset OD a C Dreset OD ze C Fomula E EY Result Mame E No Calculation Calculation Options
346. the FL600 FLx800 and Synergy HT have multi detection read capability Before defining additional Reading parameters click the radio button associated with the detection method required for the assay Multi Mode is only available for the Synergy HT line of instruments When Multi Mode is selected more than one detection method can be defined within a single protocol When a Detection Method is selected KC4 customizes the Reading Parameters dialog so that only the options supported by the Detection Method are available for selection The Detection Method selection also instructs KC4 to display measurement values as OD optical density RFU relative fluorescence unit or RLU relative luminescence unit Dispense Option Reading parameters Reading Type i End Point Reader Synergy HITA welnjectors on COM 2 SOA Detection method Kinetic i C Absorbance 0 Fluorescence C Luminescence O Multi Mode O Spectun M Dispense Read amp Dispense Filters Filter Set The Synergy HT with Injectors provides the ability to dispense fluid into microwells as part of a Read amp Dispense protocol When the Dispense option is enabled the following limitations are applied Area scanning is not supported 1 or 2 filter sets may be defined The Shaking options are disabled within the main Reading Parameters dialog A Shake step may be added to Plate Mode readings in the Read amp Dispense dialog The Lag Time
347. the combination is valid and the account is not locked KC4 s main view will appear The system may be configured to automatically lock user accounts after a certain number of successive failed login attempts If your account is locked contact the System Administrator To log out of KC4 v3 4 1 Select System Administrator LogOut Control will return to KC4 s main view and all unprotected functions will become accessible To log out of KC4 Signature 1 Select System LogOut The Login dialog will appear KC4 s functions will remain inaccessible until the Administrator or another user logs back in Recommendation To maintain a secure environment log out any time you are away from the computer The Login dialog will remain on the screen and you or another user can log in at any time The System Administrator may also set KC4 to automatically log out after a set number of minutes if the system 1s idle User s Guide to KC4 System Menu e 223 Audit Trail This feature only applies to KC4 Signature Select System Audit Trail to view a list of all system level events stored in the shared and local databases currently in use to see which databases are in use select System Setup Database Audit Trail System shared Local Date User Action Comment 104 402 13 37 16 Administrator Login Computer W2K BRITTINE Session 2607318 1041402133903 Administrator Users MAJ added Added accounts tor Mary stan
348. the existing text e The Extrapolation Factor enables you to extend the abscissa range of the curve fitting routine beyond the limits of the standard data points generated from the assay See Defining Extrapolation below for more information Exercise caution when using extrapolation as the implied assumption is that the relationship of the x and y variables is valid outside the range defined by the standards This type of extrapolation should only be applied when prior knowledge of the relationship is known Typically this feature is used in assays where linear relationships are known Inappropriate use of the Extrapolation Factor feature may invalidate results generated from data falling into the extrapolation zone e By default the Min and Max y axis values for display and reporting purposes are determined automatically by KC4 These values can be overridden by deselecting Auto and manually entering the Min and Max values User s Guide to KC4 Protocol Menu e 165 Defining Extrapolation The extrapolation factor range must be between 1 and 3 Using a factor of 1 implies that the defined upper and lower standards are the limits for concentration interpretation However given some assays the upper and lower limits may be extended in this case tentative extension of the range may be imposed e Example Assuming a linear x axis we wish to extend the upper and lower limit by 20 The lower limit denoted by the first standard 1s 50 unit
349. the user can view the current Protocol Defaults Formats Start Up and Database settings The user cannot modify any of these settings Repair Compact Databases Under System Setup Database tab the user cannot access the Repair Compact buttons for the KCShared and KCLocal databases Define Universal Plates Under System Diagnostics Define Universal Test Plates the user can view the test plate settings but cannot add modify or delete a test plate The user can run a Universal Plate Test Delete Optics tests Universal Plate tests from history Under System Diagnostics Optics Test History and Universal Plate Test History the user can view historical test results but cannot delete any of them The remaining functions are only found in KC4 Signature Delete System Audit Trail events after export Via the Export button within the System Audit Trail dialog the user can export audit trail events to a text file but cannot select the Delete events after export option Create Folder in secured file storage When Secured Protocol and Data File Storage is enabled the user cannot create new storage folders within the System Maintain Files Protocol Save As and Data Save As dialogs Rename Folder File in secured file storage When Secured Protocol and Data File Storage is enabled the user cannot rename folders or files within the System Maintain Files Protocol Save As and Data Save As dialogs Delete Overwrite Folder File
350. this context sensitive help KC4 provides access to the entire Help system Select Help Index from the About menu to open the Help Topics dialog Help Topics KC4 Help System 3 E 21x Contents Index Find Click a topic and then click Display Or click another tab such as Index Introduction Ce Installation and Setup es Getting Started Ce Data Menu Edit Menu t Protocol Meru Overview MA Working With Protocols Working With Protocols Phere and How Are Protocols Stored Opening Existing Protocol Files e Creating New Protocols Modifying Protocol Parameters Saving Protocol Files Closing Protocol Files Print Cancel Double click on a book Y to see a topic s sub topics e Double click on a page Li to view the text for a topic e Highlight a topic then click Open to view the text for the topic e Click the Index tab to perform a keyword search e Click the Find tab to search the entire help system for a specific word or phrase e Click Print to print the text for a topic e Click Cancel to close the Help Topics dialog and return to KC4 User s Guide to KC4 Getting Started e 37 Quick Start When using KC4 you will typically follow some version of the steps shown below 1 Connect a microplate reader to the computer if not already connected 2 Start KC4 and log in if required 3 Select System Readers to define reader setup parameters and establish communication 4 Select System Setup to
351. this dialog Protocol Menu e 85 Synergy HT Reading Type Reader SynergyHT on COM t End Point Detection method ee C Absorbance Fluorescence Luminescence Multi Mode Kinetic 5 Spectrum ae P REE E E Filters Filter Set Mode Excitation Emission Flua a Fluo E 485 20 465 20 528420 e 7526 20 Optics Position Top Top Sensitivity Plate fo 100 Options Options Temperature Control Type 96 WELL PLATE Size Oe 12 Yes Pre Hee First well Shaking Intensity te Betore evemnteading O Before firstreadina General Options Al Last well H12 No Temperature E Lag Time 00 00 00 o Duration o z Pre reading FT Pre Read Blank Plate Pathlenoth Correction avelenathe e Eject between Filter Set Read Ja Biel fan Ef Ee Top Probe Vertical Offset i mm I Monitor Wells Lane 10 11 12 13 Cancel Help Select a Detection Method of Absorbance Fluorescence Luminescence or Multi Mode Select a Reading Type of Endpoint Kinetic or Spectrum For an absorbance protocol define the Wavelengths and Read Mode to be used Calibrate the wavelengths if necessary For a fluorescence luminescence or multi mode protocol define the Filter Sets to be used Select a Plate Type and define the First and Last Wells For a kinetic protocol click the Kinetic button to define the read time and interval For an endpoint area scanning protoc
352. ting Tools KC4 provides some editing tools for working with data in KC4 The two most commonly used tools are Copy to copy selected data to the clipboard and Undo to remove the most recent unsaved change Availability of editing tools depends on the current working environment for example the Cut and Paste options are not available within any measurement data grids or audit trail tables but they are available within data entry fields such as formulas Editing tools are available through the Edit menu by clicking the right mouse button or by pressing the shortcut key combination and they can include Cut Ctrl X Deletes the selected data from KC4 and moves it to the clipboard This function is not available within measurement data grids or audit trail tables Copy Ctrl C Copies the selected data from KC4 to the clipboard without deleting it This function is available within measurement data grids audit trail tables and data entry fields Note The values copied to the clipboard are separated by the character selected under System Setup Formats Paste Ctrl V Pastes data from the clipboard into the current KC4 location wherever the cursor is placed This function is not available within measurement data grids or audit trail tables Delete Delete key Deletes the selected data from KC4 without moving it to the clipboard This function is not available within measurement data grids or audit trail tables Select All Selects
353. ton e g Settings to define related parameters KC4 displays a check mark Y next to each category after it is accessed You do not have to define the parameters associated with every button Ata minimum however you should define the Reading Parameters under the Plate Settings tab e Click on a tab e g Basic Analysis to see more buttons e Click lt Back or gt Next to switch between tabs e Click OK to close the Wizard 70 e Protocol Menu User s Guide to KC4 Modifying Protocol Parameters To modify protocol parameters within an existing protocol prt or global data glb file begin by opening the desired file Once the file is open use the Protocol menu or the toolbar buttons to select a parameter category to modify such as Reading Plate Layout Single Plate Transformations and so on Notes Select Protocol Advanced to see additional menu options including Multi Plate Transformations Cutoffs and Validation When a file is being modified an asterisk appears next to its name in the title bar as a reminder to save the changes Oligo Quant prt With KC4 Signature KC4 automatically logs an event to the file s audit trail any time a change is made Saving Protocol Files After creating or modifying a protocol file save the information by selecting Protocol Save or Protocol Save As Save stores the protocol under its current name If it does not have a name KC4 will ask you to enter one S
354. transfer the program or documentation except as expressly provided in this License Agreement Copyright The programla and the documentation are copyrighted fou may not copy any program or the documentation except as for loading the program into the computer as part of executing that program and for back up purposes respectively You must reproduce and include the copyright notice on the back up copy la Do vou accept all the terme of the preceding License Agreement IF you choose No Setup Will close To install AC pou must accept this agreement lt Back 9 If you accept these terms click Yes to continue Otherwise click No to discontinue the software installation If you click Yes the Choose Destination Location dialog appears Choose Destination Location Setup will install AC4 in the following folder To install to this folder click Next To install to a different folder click Browse and select another folder fou can choose not to install LC4 by clicking Cancel to exit Setup Destination Folder CARLA 3 7 Revs Back Next gt Cancel 12 e Installation and Configuration User s Guide to KC4 10 The default location for the software is displayed Click Next to accept the default location or click Browse to specify a different location After a location is specified the Select Components dialog appears Select Components E Select the componente you want to install clear the comp
355. trations Data Audit Trail Symbols Calculation Log Report Sample names Sample names Cut Offs Sample name list Format Kinetic readings Plate ramet py lt lt Remove K O Table E Insert gt gt Statistics OF f controle c ance 7 Standards Samples Help e Select the desired data sets and then click OK to begin the export KC4 requests the entry of a file name Enter the file name then click Save to export the data Data exported to a text file can be used by any other program with the ability to read an ASCII file Regardless of the Secured Protocol and Data File Storage setting data is exported as an individual txt file outside the shared access database See Export in Protocol Menu for information on customizing the export template User s Guide to KC4 Data Menu e 57 Sample Export Text File ProtocolDescr Name C KC4 3 4 Sample Export prt Modified No ReadingParameters PlateSize 4 6 PlateType 24 WELL PLATE Wells A1 D6 ReadBlankPlate No PathlengthCorrection No ReadBioCell NO MonitorWell No Filter1 405 Calibrate No DataDescription Name C KC4 3 4 Sample Export pla Barcode 123 ABC 7890 ReadingDate 03 14 01 13 19 16 ReadingType Reader Temperature PromptTitles Prompt 1 Prompt 2 Prompt 3 Prompt 4 Prompt 5 Prompt 6 Prompts iir Comment This is a sample ReportTime 03 14 01 13 21 41 Plate Template livre A 6 A STD1 10 00
356. u User s Guide to KC4 Integral Use the Integral calculation option to provide the area under the curve according to the trapezoidal method shown below i n 1 Integral gt Yit Vier 2 x xm x i Where y absorbance and x scan point value Note that in linear scanning x x is equal to 1 and thus the calculation formula is reduced to i n Integral Yi y n 2 i By default the calculation zone includes all of the Horizontal Reading Points defined in the protocol To change the calculation zone click the Calculation Options button in the Scanning Analysis dialog Scanning Analysis Integral Calculation Zone Froms 6 Tox le pe From 1 Tot E e Currently only the From x To x method for determining the calculation zone is supported See Mean Std Dev CV for more information User s Guide to KC4 Protocol Menu e 141 Plate Layout KC4 allows you to describe the layout of the microplate s that will be associated with a protocol You are not required to do this To perform blank well subtraction generate curves assign dilution values or define formulas that reference well identifiers however you must create a Plate Layout The process involves e Selecting a well type Blank Assay Control Sample Sample Control or Standard e Defining well identifier s to be associated with the well type e Defining dilution or concentration values if require
357. ually adjusts the min max w and min max y settings for each well to best display each well s curve It overrides the settings of the min max w and min max y 114 e Protocol Menu User s Guide to KC4 Scanning J Scanning If supported by the current reader the Scanning feature is available for Endpoint reads Some readers support Linear scanning others support Area scanning e When performing a Linear Scan the reader takes multiple measurements in a line across the center of each well Linear scanning allows you to observe a pattern that may be present in the well bottom such as an agglutination pattern Readers that support Linear scanning include the ELx808 and all PowerWave models Note for PowerWaveX Select Linear scanning is supported for the 96 well plate type only e When performing an Area Scan the reader takes multiple measurements down and across each well in a matrix format Area scanning more accurately assesses cellular assays than reading once in the center of the well Readers that support area scanning include the ELx800 uQuant M FLx800 and Synergy HT e Note Ifthe Scanning button is dimmed and inaccessible well scanning cannot be performed with the currently defined plate type This may be due to a hardware limitation or an unacceptable combination of optic probe size and well diameter e Click the Scanning button to define the measurement points With Linear scanning
358. uine 87 Synergy HT Time Resolved Injector Scrios aasa 88 NA PP O 89 Reading Parameters RElerene AA A 90 Automatic Sensitivity A a 90 Bandwidth A innit ins AR 92 DE coa MA ts as ee 94 Dispense OPA Mali 94 Becet Between Filter Set Reade ahead T eee 95 Excitation Emission Filter Sets FLGOOO dos 95 User s Guide to KC4 Contents e iii Excitation Emission Filter Sets FLx800 Synergy HD occccccccnccnnnnnnnnnnnnnnnnnnnnnnnos 97 A ie te eue dee 99 IESE Wy Clie CIS tW E nennen ii ii eni A A aa 99 Said A A A 99 General Options Sy Mere O estatal ia 101 Kocic Sino o eo NP O E o OO TI eaten aes ee 102 mete Parameters Pl O00 od 103 Measurement cae ae ha aca ae a 104 Measurement Mode GF L600 ia ali 104 Measurement Mode FLx800 Synergy HD oooonnnnnnnninininanononanaoananananano nono nn ono nonononnos 105 Measurement Mode Synergy HT Time Resolved cccceseessessessseeseseeeens 106 Monor VN et ae orca A E ae te ease et tee ee etait A 107 MUA Ode ii 108 PACA o e 109 A 111 Pre Read Blank Plat A dos 111 RETA DAI id 111 A E an ree ee er ena OER ee eee eR onan A 112 Read Mod id 112 Reader Read SAO a A A e aca 112 Rd 113 Read no Dodo ito id 113 DG ALES FOR a AAA ee esata ee eae 114 Scales Tor Spectiuin INCAS ines coe ase cee eee 114 A a a a assassin oda es gdh adage nasa hd aA 115 O O IO A 116 Temperature Comos 117 Time Resolved Fluorescence Synergy HT cccccccsescecssscsecsccseccc
359. umber The use of Auto Name ensures a unique file name each time e Ifthe reader is equipped with a barcode scanner and Read Barcode is selected under Protocol Reading the Use barcode as filename option is available Select this option to automatically save the data file using the barcode name User s Guide to KC4 Protocol Menu e 185 Report Select Automatic Append to Kinetic File after reading to append the current data to an existing data file This feature allows you to perform several endpoint reads on one plate You can temporarily remove the plate to incubate it externally or perhaps to add reagents Enter the target directory and file name in the Kinetic File Name field Click the Browse button to search for and select the desired file Note Automatic Append to Kinetic File is not available if the protocol specifies pathlength correction or the pre reading of a blank plate Select Protocol Options Report to select the reporting engine and to set printing options Raw Data Report Esport Calculation Engine C KC4 Mictosoft yord C Microsoft Excel Formatting Standard Faster without font customization a Automatic Print after reading M Keep Open after printing Protect Select the Engine for designing report templates KC4 This is the standard method for designing reports that KC4 has always provided The contents are customizable but the appearance 1s completely controlled by KC4
360. up dialog User s Guide to KC4 Data Menu e 59 Plate Description The Plate Description dialog appears just prior to each plate read to provide the optional entry of plate specific information Plate Description Fa Reading Date 14 Feb 01 Reading Time 14 28 09 ae Plate Description Mame Sandra Allen Lot Code 570 385 275 Title Tech I Water Lot f E Site Winooski Test 2 of 5 Comments Cancel Help e The Reading Date and Time are automatically logged when the plate 1s read Enter additional data as desired and then click OK to continue with the plate read Data entry field names can be customized by selecting Protocol Plate Description e The information contained within this dialog is automatically stored in the data file when Data Save is selected 60 e Data Menu User s Guide to KC4 Samples Sample ID Select Data Samples to assign names to the sample groups previously defined under Protocol Plate Layout Export Print 4 Plate Print A List Each sample ID can be entered manually or the Auto Numbering feature can be used to automatically increment ID suffix numbers e The ID entered in cell 1 will be assigned to every replicate in Sample group one the ID in cell 2 will be assigned to every replicate in Sample group two and so on e Up to 20 alphanumeric characters can be used for each ID Note Ifa sample name cannot be printed in its entirety it is tru
361. urve fitting e Select Current Plate if the standard curve will be generated using data from the current plate e Select File if the standard curve will be based on a previously stored curve The stored curve will be used as the basis for calculating concentrations for the current plate Click Browse to search for the desired file The file must have a GPH extension Refer to the Biograph module for information on storing curves If a stored curve is used and the plate layout contains standard identifiers then any use of STDn in formulas will refer to the standard well s on the current plate and not to the standards in the stored curve 164 e Protocol Menu User s Guide to KC4 Curve Axes By default the x and y Axis data is transformed linearly and the Extrapolation Factor is set to 1 1 These options can be modified by selecting Protocol Curves Axes Curve Description ses Curve Fit Interpolation wt As Colin Log e Display Grid Title Concentrations Extrapolation Factor 2 ES te Lin Loc W Display Grid Tithe Delta OD Min o Max li W Auto Graph Title Standard Cure e Select either Lin or Log for the transformation of x axis or y axis data e If Display Grid is checked the graph will be displayed and printed with gridlines e The text in the x Axis and y Axis Title fields appears on standard curve displays and reports To modify a title e g toxicity or Delta OD overwrite
362. ves Curve Fit to view the current curve parameters Validation Results Selecting Display Validation Results shows the current status of each of the Validation conditions defined in the protocol Validation Ea Validation Set 1 Data lippe M 405 Condition 0 000 BLK 0 500 VERIFIED 2 790 CTL 3 250 MOT VERIFIED 0 000 CTL 0 500 COULD NOT BE EVALUATED e The Condition column lists the Validation criteria defined in the protocol e The Status column tells you whether or not each Condition has been met VERIFIED The condition has been met NOT VERIFIED The condition has not been met and results are therefore suspect COULD NOT BE EVALUATED The condition could not be evaluated This may occur 1f for example a condition refers to an identifier with masked replicate values e Important Ifa validation condition receives the status NOT VERIFIED then the results are suspect The responsibility for any masking or editing of the data lies with the user 192 e Display Menu User s Guide to KC4 System Menu Overview The options available under the System menu allow you to e Select the current reader and then set and test communication parameters e Run an optics test or a universal plate test on the attached microplate reader and view print store results e Configure security settings set up user accounts and define protected functions e Log into and out of KC4 e Set protocol default setti
363. werReports 300 320 e Index Protocol menu 183 Protocol defaults 230 Protocol description 177 182 Protocol file not compatible with current reader 69 Protocol files Contents 28 Creating modifying etc 68 Signing electronically 73 Storage options 28 Protocol options Calculation 188 Export 187 Raw data 185 Report 186 Protocol reading parameters Automatic sensitivity adjustment 96 98 Endpoint read 113 Filter Sets 95 97 Kinetic read 113 131 Measurement Options 104 Optics Position 96 98 Plate shaking 116 Read Mode 112 Reading Type 113 Sensitivity 96 98 Spectrum read 113 Temperature Control 117 Protocols folder in user account 213 PRT files Protocol files 68 Protocol Files 28 Signing electronically 73 Q Quick Start 38 For Clarity luminometer users 39 For other instrument users 38 R Rapid Mode 100 104 112 Raw data Options 185 Raw data correction 128 User s Guide to KC4 Read and dispense protocol 94 119 Read from file 51 Read Matrix Size area scanning 116 Read method 94 108 112 Read Mode Defining in protocol 112 Reader configuration 194 Reader Configuration 201 Reader control 245 Reader model 112 Readers controlling more than one at a time 194 Reading direction 113 Reading Number 103 136 Reading parameters Clarity 77 ELx800 78 ELx808 79 FL600 80 FLx800 81 PowerWave 82 PowerWaveHT 82 PowerWaveX 83
364. word length password aging and idle session time out Support for multiple users Each user 1s identified by a unique combination of User ID and encrypted password Users must log into KC4 with these identifiers to gain access Time stamped audit trails Activities such as user login logout protocol and data file creation and modification and plate reading are permanently logged in a secure database Embedded signatures Authorized users can electronically sign protocol and data files Electronic signatures are permanent and remain a part of the overall data record for the life of that record Secure record storage KC4 proprietary files prt pla and glb are stored in a secure shared access database Activities performed on files within this database such as rename move copy overwrite and delete are performed within the KC4 environment and every change is tracked in an audit trail In addition the System Administrator can configure the system so that these activities may only be performed by Power Users Protected functions The System Administrator can protect a variety of functions from use by Standard Users These functions include the deletion rename modification and overwrite of various record types User s Guide to KC4 Introduction e 3 Technical Support KC4 is backed by a superior support staff If the software fails to function perfectly please contact the Technical Assistance Center TAC and provide the fol
365. x File importation failure possible causes 53 Files File types 28 Maintenance 226 Signing 30 Storage options 222 Filter Sets 95 97 103 Filter wheels 197 200 Filters Wavelengths 194 196 Filter sets for SynergyHT HTTR 97 Filter sets for the FLx800 97 KC4 wavelength table setup 196 First well 99 Fitting results 296 FL600 80 Absorbance mode 112 Eject between well 104 Filter Sets 95 103 Fluorescence mode 112 General Options 99 Kinetic reading parameters 103 Luminescence read method mode 112 Measurement Options 104 Optics Position 96 Plate Mode 103 Rapid Static Mode 100 104 Reader Configuration 201 Sensitivity 96 Well Mode 104 Fluorescence Read method 112 Time resolved 117 Fluorophore 95 97 FLx800 81 Fluorescence mode 112 Luminescence read method 112 Measurement Options 104 Sensitivity 98 Wavelengths table 198 Force user to type ID 217 Formats 231 User s Guide to KC4 Formula examples 40 159 162 170 174 Formula for kinetic analysis 136 Formula for spectrum analysis 137 138 Formula syntax 160 Multi plate transformations 163 Single plate transformations 160 G General Options FL600 99 General Options SynergyHT 101 Get filters from reader 201 Get wavelengths from reader 196 199 GLB files Global data files 28 42 Signing electronically 54 Global data files 28 42 GPH files 266 Graph files 266 Group membership 213 H High va
366. x 251 all pun Bottom Right i 02310 pm Nb Columns 3 34990 pm Length Well Diameter B4530 um 22560 pm 61740 um width Top Lett or Bottom Right r Hb Fonz 2 Height LT Cancel e Name stores descriptive unique text appropriate for the microplate This name appears in the Plate Type selection list within the Reading Parameters dialog e Manufacturer stores the name of the microplate manufacturer e Catalogue stores the microplate identification number e Length is the longest dimension of the plasticware the x axis e Width is the shorter dimension of the plasticware the y axis e Top Left X is the distance from the left side of the plate to the center of well A1 e Top Left Y is the distance from the top of the plate to the center of well Al e Bottom Right X is the distance from the left side of the plate to the center of the last well on the plate In the case of a 96 well plate this would be H12 e Bottom Right Y is the distance from the top of the plate to the center of the last well on the plate In the case of a 96 well plate this would be H12 e Nb Columns is the number of vertical columns of wells when viewing the plate in its normal orientation In the case of a 96 well plate this would be 12 e Nb Rows is the number of horizontal rows of wells when viewing the plate in its normal orientation In the case of a 96 well plate this would be 8 e Well Diameter is the diam
367. x V Min Max OD Integral area under the curve and Onset calculations Screening options Definition of cutoffs by algebraic formulae and positive negative etc range calculations Validation options Formula to validate the Mean Standard Deviation or CV of controls Printed reports are completely customizable and can include results data sets in matrix or column format standard kinetic or dilution curves sample list audit trail protocol description custom header and page breaks 2 e Introduction User s Guide to KC4 For users with Microsoft Word Excel 2000 or greater and Windows 98 NT 2000 XP PowerReports links KC4 with Excel and Word providing a powerful format for creating custom publication quality report and export documents Reports can be previewed before printing Data exporting Data may be exported automatically for use with other programs including Excel Lotus and Paradox for further manipulation or report merging Exporting is not limited to just raw data all calculated results may be exported KC4 Signature complies with FDA s Electronic Signatures rule 21 CFR Part 11 System Administrator A site designated System Administrator creates and maintains Standard User low level and Power User high level accounts and specifies which KC4 functions shall be protected from use by Standard Users The System Administrator sets special password and login characteristics including minimum pass
368. y not be valid If a 4 digit error code is also supplied consult the reader s Operator s Manual Within the message box e If NO is selected all data collected up to this point is permanently deleted and cannot be retrieved e If YES is selected all data collected up to this point will be saved It is strongly advised that the data be analyzed to determine its validity User s Guide to KC4 Data Menu e 49 KC4 Signature users If Yes is selected an event is logged to the Data Audit Trail as demonstrated below Data Audit Trail Date User Action Comment l 06 23 04 09 42 21 Administrator Plate read started 06 23 04 09 42 54 Administrator Error 0200 detected refer to reader user s manual Running an Optics Te ecycling power on the instemmen 06 2304 09 47 58 Administrator Kinetic read was interrupted because of an error during communication with the reader User decided to not discard the received data 06 23 04 09 47 58 Administrator j ate T Running an Optics Test or recycling power on the instrument may clear the error 06 23 04 09 45 02 Administrator Read operation aborted Entering Plate Data Manually KC4 provides the option to enter Endpoint and Kinetic plate data manually via the keyboard Typically this information comes from other instruments or from previously acquired data s RESULTS Data M 405 Y J Change Mask Cancel Help e Use the up

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