AssayFit user manual version 1.6 research use only
2014 AssayFit 1.6 user manual AssayFit version 1.6 research use only user manual Curve fitting in excel 2007, 2010 and 2013 using the excel solver 1 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual AssayFit the flexible curve fitting software in Microsoft Excel. AssayFit is a software program for curve fitting and regression. It is specially designed for research and development of assays like ELISA tests, Luminex assays, other immunoassays or enzyme assays, however it can be used for a variety of other purposes. It is for research use only. Linear, 4PL (four parameter logistic), 5PL (5 parameter logistic), spline, point to point and second and third order polynomial regressions are included in the software. Users have the ability to add 10 custom fit functions. A calibration curve is fitted and unknown values are read from this calibration curve. The software works as an Add-In in Microsoft excel 2007, 2010 and 2013. A trial version is available. A video how the software works can be viewed on youtube: http://youtu.be/QztYGyK5I-A?hd=1 The IVDtools website is located at http://www.IVDtools.com 2 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Installing AssayFit Before installing AssayFit make sure that the excel solver is installed. AssayFit will not work without the solver. The trial and full version of the software from IVDtools come with an installer. Run the installer, then it will install the software in a directory. It is advised to install it in the directory the installer indicates. You must have read and write permission on the installation directory. If you cannot write, this will result in errors. You may need administrative rights to run the installer however in most cases this will not be required. appearance of the installed Add-Ins Then on the same sheet click on the Go button A form is shown with all already installed Add-Ins 3 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 You can see here that the Solver Add-in is installed. 4 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 The AssayFit menu There are two options how to use the AssayFit plugin. One is to use the special AssayFit tab in the excel ribbon. The other is through the AssayFit menu in the Add-Ins tab. If you click the AssayFit tab the AssayFit buttons appear. Clicking them will run the different scripts. Under the Add-ins tab a menu is located with the same options. 5 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Adding a new curve fitting sheet You can add a new curve fitting sheet by selecting clicking the ribbon button named “Add fit sheet” or the menu option: Add new empty IVDtools sheet. As alternative this option can be activated by the key combination: ctl, shift and N. Or use the menu option A new IVDtools sheet will appear with the default settings (in this case a 4PL regression) and default calibrators. These settings can be modified and saved in order to obtain your own personal sheet when adding a new IVDtools sheet. 6 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 7 2014 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual Entering calibrator values You can enter calibrator ID, dose and response values in the corresponding fields. Also the layout of the fields including the number of digits can be modified. The data points will be added to the graph, but the curve fit does not update until the menu option is selected to perform a curve fitting. When adding calibrators the field %, meaning the % of the maximum will be updated. 8 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Performing a curve fit The curve fit will be performed after selecting the menu option run AssayFit (formulas), or the key combination, clt-shift-f. The program will then perform a curve fit adding formulas in the fields. or As an alternative also the menu option Run AssayFit(values) can be selected. This also performs a curve fit, but the results of the fit will be entered as values. The sheet will then be locked. This option is preferable if you would like to lock the data or send the data to a person that does not have the AssayFit software installed. or A sheet can also be locked afterwards with the “Lock values” menu option. It can be unlocked by the “Unlock – formulas option” 9 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 10 2014 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Reading unknown values from the fitted curve Once a curve fitting has been performed, unknown response values can be used to read the corresponding dose value from the calibration curve. The response is entered and the software calculates the corresponding dose from the fitted curve. The response can be entered starting from row 24. 11 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 When using ELISA 96 well data the data can be entered in the corresponding fields on the right of the spreadsheet. One block can contain the ID values, one block the response values. In the last block the calculated dose values are displayed. 12 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual Selecting a new curve fit function The menu item “Fit function select” has a submenu with all the possible fit functions.: The ribbon has three submenu’s showing the same functions: 4PL1, 4PL2 Two four parameter logistic functions 5PL1 A Five parameter logistic function PtoP Point to point curve fitting, a straight line from one calibrator to the next Spline A spline function: a smooth curve through the data points LIN: Linear fit 2nd Poly: Second order polynomial regression 3rd Poly: Third order polynomial regression Additional to these standard functions 10 custom functions can be entered and selected through this menu. After selecting a new function the program will automatically start a new fit and display the fit function. In the example below the 4Pl2 function is selected 13 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 AssayFit will start fitting the data points with the new function. The fit function is entered into the Fitmodel field. The backfitmodel is used to backcalculate the response to the dose. The response of unknown values can be entered into the corresponding field and the software will calculate the corresponding dose values. If the fit has been performed the software immediately calculates the dose value if a new unknown response value is entered. Is the value lower than the value in the curve it will indicate < and the minimal dose. If it it higher than the curve, it will indicate > and the maximum dose value. With the default setting the program does not extrapolate. The limit values however can be changed in the backfit limits fields. When the fit is performed, the program calculates the fitted curve at the data points and it will calculate the % bias. This is the bias between the fitted curve and the real data points. Also the fit parameters are calculated. In this case, the formula uses four parameters, A, B, C_ and D that are used by the fit function. The program calculates the optimal values for A, B, C_ and D and these can 14 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 be found in the table below the graph. 15 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual Weight settings In the fit above the weight settings of all the calibrators were set to 1. This means that the calculation of the bias al the calibrators equally contributed. You can adjust the weight of a calibrator, making this calibrator more important for correct fitting. In this example we have set the weight of the fifth. After performing the fit, the fitted curve fits this point better than before. The weights can also be set with a formula for example 1/Y^2 is commonly used. 16 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Calibrator order and empty values The fit is independent on the order of the calibrators in the table, it also can accept empty values. This is very easy if you would like to see what effect dropping a point in the curve would have on the fit. In this example both the order of the calibrators and empty values are introduced, giving exactly the same fit as above. Please note that both the dose and response value have to be removed. 17 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Fit parameters and start values The fit parameters are in the table below the graph. When the program starts fitting it will use the start values, enter these into the fit function and from there will try and find the most optimal values for the parameters. A linear regression function y= Ax + B will only use the two top parameter, while a 5PL function will use all five parameters. The start values for a linear regression and for a 4PL or 5PL are different. When entering a custom function the correct start values will need to be applied, because otherwise the start values may be so far off, the software cannot find the optimal values. The R2 column represents the goodness of fit. The number in the left column is the total sum of squares. This adds up all the differences of the values from the actual values, though the least squares method. The program will try and make this value as small as possible. In the right column the actual correlation coefficient is displayed. 18 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Solver parameters The program uses the excel solver to optimize the parameters. Some settings of the solver can be adjusted in the AssayFit program. The options are in the Solver parameters table. The solver of excel 2007 and 2010/2013 is different and the 2010 solver is not using all of the parameters indicated. Runs is the number of runs that the solver performs. Additional settings of the solver (if needed) can be set by selecting “solver” on the data tab. Please make sure you use an unprotected sheet if the solver is activated as it will not work with a protected sheet. 19 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Entering a new custom fit function By selecting the menu item “Enter new custom functions” a form appears. In this form new fit functions can be added. You can enter new functions in this form. X as unknown needs to be present in the functions as well as some of the parameters A, B, C_, D, E. Please write the C parameter as C_ , otherwise this will result in errors. Some functions like Ln need to be written as application.worksheetfunction.ln. After pressing the button “Save functions” the functions will be saved and available under the fit function 20 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 select menu. Copying of AssayFit sheets You can easily copy a AssayFit tab by right clicking onto the tab and then selecting the “move or copy option”. You can then either copy the sheet to a new file or to the same file. 21 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 A second sheet is created. If you modify the calibrators, run the fit again to ensure proper fit. 22 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 Creating an overview of all the AssayFit sheets in a file If you have multiple sheets and want to create an overview of all the calibration curves select the option Curve fit overview. You have two options: Overview large, with all the calibrators and overview small, with a smaller number of calibrators. The program will create a sheet called “Graphoverview” with a copy of the calibration curve and values. The name of the sheet will be added as a title. 23 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual Settings Under the settings and layout menu, color and default settings can be saved and restored. You can remove the purple color from the sheet by selecting the “Decolorize” option, the Colorize option brings the color back again. You can save all settings by selecting “Save current sheet as custom sheet”. All settings, like fit function, calibrator values, color and formatting settings, solver parameters, start values and limits are stored by using this option. If you then add a new sheet it will have these settings. You can also apply settings on an existing sheet. This is done by the option “restore saved settings with/no cal values.” “Reset settings to default” restores the default settings. Fit functions structure in Excel When you select a cel of the fitted curve, the fit function will show. It will have references to the input value (K8) , the fit model (B5) and the five parameters. 24 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 When you would like to add an AssayFit function to a cell, click the fx sign it will show the available functions. You can select the IVDtools option to see all IVDtools functions 25 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual The four possible functions are shown with an explanation of the parameters. The use of the point to point and the spline function does not work from a different sheet. The other functions will also work from a different sheet. To use the point to point backfit function use: =PtoPb(calibrators, unknown) . The spline backfit cannot be used from a different sheet. 26 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 You can then add or select the reference to the different cells. 27 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual Automation of AssayFit with visual basic scripts If you wish to automate the fit function using VBA, then “runAssayFit” and “runAssayFitvalues” can be called to trigger curve fitting. Below is a very simple script. Sub automate() runAssayFit End sub More complicated scripts can be used to copy data into the AssayFit sheet, perform curve fitting and copy the output to a new sheet. Trial version and full version A trial version is available that has all the functionality of the full version but only with the Linear regression and 4PL function. This trial can be used to test the suitability of the software. It can be downloaded for free from the IVDtools website (www.IVDtools.com). The full version can be obtained from the IVDtools webshop (www.IVDtools.com) and downloaded directly. It will perform the full set of curve fitting including custom formula entry Troubleshooting If you have trouble curve fitting you can use the following suggestions 1. Make sure the Excel solver is installed and active. 2. Start with the example sheet which is installed on the desktop and see if AssayFit will run from this sheet 3. Make sure the location you install AssayFit to is writable 4. Make sure your Excel security settings will allow VBA macro’s to be run 5. Old excel sheets may have too less rows and columns, copy the data to a new xlsx sheet 6. If you are still having problems contact IVDtools at [email protected] or use the website’s contact box 28 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual Version Info Version 1.6 Version changes April 24 2014 Added 96 well fitting support to facilitate fitting of ELISA plates Version 1.5 Version changes March 15 2014 Changed name to AssayFit. Changed SST to sum of squares total, without devision. And clarified this in the header Version 1.4 Version changes March 3 2014 Minor changes Version 1.3 Version changes February 24 2014 Name change back to IVDtools and AssayFit RUO research use only. Addition of the Excel Ribbon user interface. Update of the manual. Version 1.2 Version changes January 12 2014 Name change to IVRtools and IVRfit throughout all the functions and sheets Version 1.1.5 Version changes December 12 2013 Minor bug fix, changes in the license and manual Version 1.1.4 Version changes. November 14 2013 Fixed a small bug related to sheet locking Version 1.1.3 Version changes. November 7 2013 Fixed a bug with run-time error 5, which prevented running AssayFit in certain languages Version 1.1.2 version changes. April 2 2013 Added a warning when using xls and csv sheets because the number of rows and columns is too small to add a new AssayFit sheet. Version 1.1.1 version changes. December 12 2012 Installer is compatible with office 2013 Changed color settings of some items Changed menu item order Light version 1.1.1 introduced January 20 2012 29 all rights reserved copyright © 2014 by IVDtools 2014 AssayFit 1.6 user manual Version 1.1 version changes: September 24 2012 Added correlation coefficient calculation Plugins are independent on the location of the solver Plugins are now office version independent Software is compatible with Mac OS office 2011, but the solver on Mac runs slow Warning added for custom functions with formulas Start parameter D for 4PL and 5PL set to 0 Version 1.0 original version April 10 2012 Original version 30 all rights reserved copyright © 2014 by IVDtools AssayFit 1.6 user manual 2014 License Before downloading you have to agree to the software license agreement. It is also in the software under the license menu. Software License Agreement The AssayFit software add-ins (AssayFit.xlam, AssayFitcust.xlam) together with installer and accompanying documentation (referred to as "Software" in this Agreement) is the property of IVDtools.com ("IVDtools"). By downloading, installing or using this Software or parts of this software, you (or you on behalf of your organization) are agreeing to the terms and conditions of this Agreement. This Software is protected under International Copyright Laws. 1. License Grants One purchased single user copy from the IVDtools website (http://www.IVDtools.com) allows the use of the Software by one user on one computer in your organization. 2. License Restrictions You may not sell, copy, emulate, clone, rent, lease, adapt, translate, sublicense, decompile, disassemble, otherwise reverse engineer Software except as specified in this agreement. Any unauthorized use shall result in immediate and automatic termination of this license and may result in prosecution. 3. Transfer You may make a one time permanent transfer of all your rights of use of the Software to another person or legal entity provided you also transfer this Agreement and you retain no copies of the Software. The trial or light version of the software may be transferred to other persons for the trial period, provided you also transfer this Agreement. 4. Termination This Agreement takes effect upon your use of the Software and remains effective until terminated. It will be terminated automatically without notice from IVDtools if you fail to comply with any provision contained here. You agree on termination of this Agreement to destroy all copies of the Software in your possession. 5. Limitation of liability In no event will IVDtools be liable to you for any damages, including any lost profits, lost savings, or other incidental or consequential damages arising from the use or the inability to use the software, even if an authorized dealer or distributor has been advised of the possibility of these damages. 6. Warranty Disclaimer IVDtools disclaims all warranties, whether expressed, implied, or otherwise, including the warranties of merchantability or fitness for a particular purpose. IVDtools does not warrant that the software is error-free or will operate without interruption. 7. Intended Use The software is for research use only. All rights reserved © 2014 by IVDtools 31 all rights reserved copyright © 2014 by IVDtools
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