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PLA 1.2 Analysis of Parallel-Line Assays - User Manual

1. This account is member of the following group Disabled Users Standard Users Inspector System Administrators Administrator can create accounts and modify GLP SOP Settings Figure 5 1 User management of PLA Existing users are displayed in the list in the upper part of the dialog For now only the user Administrator exists Press the Create button to create a new user account This will activate the fields in the center of the dialog Enter the user name in the input field Account Name The password for the new account must be given in the input field Account Password Re type the password in the appropriate entry field to ensure you did not make any typing mistakes The password must have at least five charac ters Tell the new user the initial password for his account After the first time the new user has logged in he can and ought to change his pass word Please refer to chapter 5 2 to learn how to change your password 46 2000 Stegmann Systemberatung www rssystem de PLA Administration Note Please keep in mind that the password is case sensitive The pass word Test is different from test Changing your keyboard settings will change your keyboard layout National characters might become unavail able and the keys for other characters might change e g qwertz and qwerty keyboard layout Account Management Select a user to modify or delete or press the create button to c
2. Di Beispiel Nr 2 s Assay 1 o Assay 2 Standard amp Probe 3 Komtrolle D Assay 3 a D n Biotest Pharma GmbH Figure 6 3 Navigator standards preparations controls All kinds of objects have a different icon as a distinctive feature Project Standard amp Control Assay amp Preparation yObject opened The standard is depicted by an orange test tube the control by a blue green test tube and the preparation has a green reaction vessel icon You can choose the name of all five types of objects freely In our example the objects are called preparation standard and control only for reasons of better understandability Why using the object hierarchy Just to point out only two aspects 1 After some time you will probably have done a multitude of analyses with PLA Of course can delete these measurements but especially in development departments the access to old data is often essential With the help of the hierarchical organization in PLA you can struc ture your work The project object should be regarded as a file for the assays 2000 Stegmann Systemberatung www rs system de 61 Terms and Concepts 2 You can save all your data about your measurements and further information already on the project level If you create a new assay within the project afterwards all data is automatically added to your assay You do not have to enter all common information on the pro j
3. 1 Installation of the software validation module SVM if you have received one with your software 2 Installation of the software 3 First time configuration of PLA Note Ifyou are using Windows NT 4 0 or Windows 2000 the installation have to be done by a system administrator A prerequisite for the installation of PLA 1 2 under Windows 95 is the system component DCOM 95 This component is already installed if you are using the Microsoft Internet Explorer 4 or higher Otherwise you have to install this component which is available on our CD ROM in the subdirectory DCOM95 PLA is a so called node locked software The program can only be in stalled on a single workstation You can either get a SVM with the soft ware which is connected to the parallel port of your computer or receive a license number which is unique for the PC on which you have installed the software If you decided to install a SVM the installation of the mod ule is described in chapter 4 2 2 Otherwise continue with chapter 4 2 3 2000 Stegmann Systemberatung www rs system de 27 System Requirements and Installation 4 2 2 Step 1 Installation of the SVM The SVM is a small device that has to be attached to the parallel port of your computer If you did not receive a SVM with the software simply skip this chapter Please attach the SVM to the parallel port of your PC If you use a printer on your system please remove the
4. Appendices Page 7 of 7 08 10 99 10 07 56 Project Validierungsbeispiele Assay Europ Pharmacop 1993 Additional Annotations Calaulated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs eystem de 2000 Stegmann Systemberatung www rs system de 213 Appendices 11 2 2 PLA Output PLA Complete Statistics Report The PLA Complete Statistics Report of PLA contains all the variance analyses data It is used for the comparison with the EP results 214 2000 Stegmann Systemberatung www rssystem de Appendices Page 1 of 13 03 10 99 10 08 09 Project Validierungsbeispiele Assay Europ Pharmacop 1993 Results of Assay Europ Pharmacop 1993 Project Validierungsbeispiele Current user Dr Matthias Schmitt 1 Complete List of the Object Definitions Standard Sample General settings Code Standard S Preparation U Title Standard S Preparation U Measured substance Batch identification Object status declared closed NO NO Object was created by Dr Matthias Schmitt Dr Matthias Schmitt Creation timestamp 16 06 99 15 14 55 16 06 99 15 14 57 Last modification by Modification timestamp 06 10 99 12 53 12 06 10 99 12 53 12 Definition settings Concentration of undiluted standard 1 7 Concentration units Predilution of standard sample 11 T Number of dilution steps 2 2 Number of data series 10 10 Additional predilution factor 11 11 D
5. Switch between a double logarithmic and a logarithmic analysis By the activation of this function the correct relative potency of a reverse response effect calculation will be calculated This setting is used for e g inhibition assays a value of the Dixon test Refer to chapter 10 1 2 Dixon Test for further details tph Significance of the slope Significance of devia tions from linearity Significance of devia tions from parallelity Statistical confidence of the F test of the sig nificance of the slope Statistical confidence of the F test of the sig nificance of deviation of the measurement points from linearity in the linear range Statistical confidence of the limits for the F test of the deviation from parallelity in the linear range of the standard and the preparation This value will always be taken from the object of which the relative potency is to be calculated 2000 Stegmann Systemberatung www rssystem de 67 Terms and Concepts Property Fiducial limit of the potency estimation Rejection criteria Description Confidence interval for the relative potency of the preparation This value will always be taken from the object of which the relative potency is to be calculated By default the preparation will be rejected when the criteria for the slope linearity or parallelity are not satisfied You can define here which criteria are significant Select
6. ries The number of steps is always three The first step is always a posi tive control the second a negative control and the last step is a blank 2000 Stegmann Systemberatung www rssystem de 151 Reference Selected object Standa Type of object steps _ series Standard E 9 S Apply Press the Apply button to confirm your changes The changed settings will be transferred to the upper list When you edit the preparation B the number of steps and the number of series are already set to the correct values Finally edit the settings for the last object preparation C The last object is not a sample like the standard and the preparation but a control Change the object type accordingly in the Type of Object input field Selected object Control Type of object steps series amp Control fe le El Apply When you choose a control object the field for the number of steps will be grayed out You can only give the number of series which is the number of repetitions for controls Enter eight into the input field Before you confirm the changes you must uncheck the two boxes in the first section of the dialog page because otherwise the number of series will be set to eight for all objects Press the Apply button to confirm the changes You should see the following list of objects now Standard robe You can proceed to the next dialog page of the import wizard now On this page the da
7. 3 0 1250 3 0000 83 84 90 84 75 tep X log Mean S D LAA 1 0 5000 1 0000 601 200 86 817 14 4 2 0 2500 2 0000 326 400 146 933 45 0 3 0 1250 3 0000 83 200 5 357 6 4 50 Response 342 200 Caadatedhy PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany wwwrs system de 2000 Stegmann Systemberatung www rssystem de 199 Appendices Page 3 of 7 03 10 99 100728 Project Validierungsbeispiele Assay Linder 3 Range Selection Linear detection type Fixed range 1 Fixed range 1 Selected Linear Range Step 1 to Step 3 Step 1 to Step 3 4 Test of Linearity Regression equation y a bligo 19x 4 1 Standard Standard Total number of observations 15 _ source dt ____ Sumofsquares ___MeanSquare F Value Model 2 510 603 3333 255 301 6667 26 9098 Error 12 113 847 6000 9 487 3000 Total 14 624 450 9333 Linearity 1 475 240 0000 475 240 0000 50 0922 Quadratic 1 35 363 3333 35 363 3333 3 7274 pacar Estimates tot eg of regression Intercept a 582 4000 43 5598 r 0 817684 Slope b 424 0000 111 0562 Pas 0 787298 Quadratic c 103 0000 53 3497 Root MSE 97 4028 Test passed if Faagate lt Fan P 95 4 7472 Test PASSED 4 2 Sample Probe 1 Total number of observations 15 source et SumofSauares Mean Square F Value Model 2 671 642 1333 335 821 0667 34 5552 Error 12 116 620 8000 9 718 4000 Total 14 788 262 9333 Linearity 1 670 810 0000 670 810 0000 69 0
8. Factor MI PME 2 Testsatz 14 PNF 2 Testsatz 40 PNF DI Dateiname fT est Dateityp PLA Native Format P PME DI Abbrechen In the file dialog switch to the directory in which you want to save the data Enter a file name in the input field and press the Save button to take over the changes to the export wizard Back in the export wizard press the 2000 Stegmann Systemberatung www rssystem de 137 Reference Next button to get to step three of the export process Specify detailed settings PLA Export Wizzard PLA Export Wizzard r Dbiect s to export Choose type of operation Beispiel Nr 1 Select output filename I include child objects Specify detailed settings IV include finished objects Check your selections Export in progress Output options IV include data values IV include assay definition data I include subject information IV include reagent information I include annotations Please choose the elements that will be T include administrative information exported Cancel lt Previuos In the upper part of the dialog you can again choose the object that is to be exported On this page you have two choices Either you keep the object that you have marked in the navigator or you can choose all ob jects which essentially does the same as a complete database backup but does not export any user information In the check boxes below the drop down field you can choose i
9. Reverse response effect correlation NO NO Dixon contamination 0 1 0 1 F Test of the slope 95 95 F Test of linearity 95 95 F Test of parallelity 95 95 Fiducial limit percents 95 95 Slope as rejection criterion NO NO Linearity as rejection criterion NO NO Parallelity as rejection criterion NO NO EC50 calculation NO NO by using Range Selection Settings Linear detection type Fixed range 1 Fixed range 1 Allocation Strategy Minimal Exact of Points k Maximal Allocation Range Allocation Range Step 1 to Step 3 Step 1 to Step 3 Inclusion of 50 Response 50 Response of Cekculaiediby PLA Version 1 2 0 PLAisa product of Siegmann Systemberatung Germany wwnurs system de 198 2000 Stegmann Systemberatung www rs system de Appendices Page20f7 08 10 99 10 07 28 Project Validierungsbeispiele Assay Linder 2 Data Input Data index x technical outlier o outlier found by Dixon test 2 1 Standard Standard Series 2 Series 3 Series 4 Series 1 1 0000 0 0000 487 525 585 600 715 2 0 5000 1 0000 150 350 275 122 410 3 0 2500 2 0000 164 255 64 95 154 tep X loga Mean S LAA 1 1 0000 0 0000 582 400 87 028 14 9 2 0 5000 1 0000 261 400 124 458 47 6 3 0 2500 2 0000 146 400 73 473 50 2 50 Response 364 400 2 2 Sample Probe 1 tep X log Series 1 Series 2 Series 3 Series 4 Series 1 0 5000 1 0000 710 516 620 510 650 2 0 2500 2 0000 560 268 372 247 185
10. Schemes D Els Protected Schemes H User Defined Schemes By clicking on this drop down box you can go to the schemes view I include finished items Project Standard Control assay Preparation yObject opened Figure 9 2 Schemes view in PLA On this page you see two objects 1 Protected schemes can only be created and modified by administra tors The schemes are used to enforce GMP GLP requirements 2 User defined schemes can be created and modified by every user These schemes can be used as templates for the creation of new data objects 2000 Stegmann Systemberatung www rs system de 165 GMP GLP Settings in PLA Administrators can create protected as well as user defined schemes while users can only create the latter Creating a scheme works exactly like generating a data object via the create object dialog Enter all the settings as you would do for a data ob ject Compared to former versions of PLA an important novelty is that the object hierarchy has been transferred to the usage of schemes You can now define schemes for standards preparations and controls that belong to an assay scheme This new feature seemed to be reasonable because the settings for standard preparation and control within an assay do not nec essarily have to be identical When you create a new data object based on an assay scheme PLA checks if the scheme contains subordinate schemes In this case the defini tion
11. You cannot undo this action Are you really sure i Nein Abbrechen Press the Yes button to execute the deletion A second dialog will appear to ask you again If you press the Yes button again the object and all subordinate objects will be deleted 00040 2nd Confirmation E K If you select YES the project Beispiel Nr 1 will be deleted from the database Are you really sure Nein Abbrechen 8 3 3 Showing Finished Items If the checkbox include finished items is deactivated the navigator will only show active objects 100 2000 Stegmann Systemberatung www rs system de Reference 8 4 Creating Objects To create a new object select the parent object from the navigator list first E g if you want to create another sample in the assay Messung 1 in the project Beispiel 1 select the assay and choose New from the task bar Alternatively you can select Create Object from the menu or press CTRL N The following dialog will appear on the screen C New Project ren C New Assay as member of project Beispiel Nr 1 Ne SEE 7 Cancel S New Control as member of assay Messung 1 Input Code and Title Select a creation scheige 2000 Stegmann Systemberatung www rs system de 101 Reference You can enter the essential properties of the new object in this dialog First you can select the object type you want to create Depending on the parent
12. 00000000000000 000a0 000 0e nenen nenenananane 57 6 1 Objects aas aa rare anime 6 2 Object Hierarchy 6 3 UO EE 6 4 Object Propertie Serion enaren ee ee are 6 4 1 General Settings 64 6 4 2 Reagents Settings 65 6 4 3 Definition settings 66 6 4 4 Analys s Settinese unseres erlernen 67 6 4 5 Range Selection Settings ne nena nanas an anananenenoee 69 6 4 6 Annotations z i 6 4 7 GIEP GMP Seite ee dE 71 6 4 8 Dose and Measurement Input 72 My first Parallel Line Assay soooo000000000 0000000000000000 000000000 00000e 73 7 1 Tinitial RE 72 Creating a Project 7 3 Creating an Assay 7 4 Creating the Preparations and the Standard 60eseseseneneenenene 84 7 5 EE EE 87 8 Reference eessesesnesnesssneneonennenesnennenesnennenesnennenesnennenesnennenesnennenennene 93 8 1 General e ee E EE 94 8 2 Deem Age een a NANG gae aa e are ennai kn 8 2 1 File Menu D 8 2 2 Action Mett tege NERE EES 96 8 2 3 Preferences Men s 2ER o Ee 96 8 2 4 Help Menu 97 8 3 The Navigator 98 8 3 1 OpeningObjecisin see aeg a Ae KA KG wa 99 8 3 2 Deleting Objects ice aan adana naa ae aga mad pami ads 100 8 3 3 Showing Finished Items 00000enenenenennanenenanna nenen n nenen 100 8 4 Creating Objects E 8 5 The Object Dialog Seinna messe 104 8 5 1 General SeS et APL ARAL eis nea hicks ads 104 8 5 2 Reagents 8 5 3 Definition Setting
13. 1 Select Automatic Detection common range for standard individual range for prepara tions from the drop down box here This will activate the automatic detection of the linear range Choose Maximum Range from the drop down box to trigger an analysis that will try to find the linear range that will contain the maximum number of points Allo Maximum Range ation Strategy Allocation region Minimal of points 3 IV maximal allocation range Inclusion of 50 response Calkulate the 50 response by using the ignore T 3 Choose the value 3 to find a range with minimal 3 dilution steps After you have entered the range selection settings the definition of the project is finished Close the window and save the settings The newly created project is shown in the navigator You can now continue with the creation of the assays 82 2000 Stegmann Systemberatung www rs system de My first Parallel Line Assay 7 3 Creating an Assay Now you can create the second level of the object definition the assay Assays structure your project Later you will start calculations from this level The procedure to create an assay is easy Select the project you have just created and click New 1 Select New Assay as member of project The code of the project is shown in quotes 2 Enter a code short title for the assay Create object 1 Selec
14. 5 3 0 4 5 6 0 Dose log x Standard PASSED PASSED Probe 1 PASSED PASSED PASSED 0 892 Probe 2 PASSED PASSED PASSED 0 504 Probe 3 PASSED PASSED PASSED 0 633 Probe 4 PASSED PASSED PASSED 2 174 Probe 5 PASSED PASSED PASSED 3 664 Calaulated by PLA Version 1 2 00 PLAisa product ci Stegmann Systemberatung Germany www rs system de 90 2000 Stegmann Systemberatung www rs system de My first Parallel Line Assay PLA 1 2 has successfully analyzed the assay and has found linear and parallel intervals Note PLA offers a plethora of functions and settings that modify the analysis of parallel line assays You can change these settings to a very large extend However many of the settings address explicitly the use of PLA in research and product development In this field many of the pa rameters must be variable to develop standard procedures for the analysis of measurements using parallel line assays Not every parallel line assay that is accepted by PLA is automatically valid The changed settings have to be evaluated and approved as a part of the development process Only those analyses that are performed in the framework of tested parameters are valid in a statistical sense 2000 Stegmann Systemberatung www rssystem de 91 My first Parallel Line Assay 92 2000 Stegmann Systemberatung www rs system de 8 Reference 2000 Stegmann Systemberatung www rs system de 93 Reference 8 1 General I
15. Additionally you have six series of readings one series for the standard and one for each of the five measurements You will need one object for the standard sample and one for each preparation The structure of the project should now look like this Project Assay Standard Preparation 1 Preparation 2 Preparation 3 Preparation 4 Preparation 5 Do you still remember how inheritance works When you enter all set tings that all subordinate objects have in common at the higher level ob ject all settings will be copied to any newly created lower level objects 76 2000 Stegmann Systemberatung www rs system de My first Parallel Line Assay After you have started PLA and logged on the navigator appears Create a new object by selecting New or Create Object You should see the following dialog on the screen now 1 Select New Project to create a new pro ject 2 Enter the code short title here The title will be set automatically Create object Select dell type to create U New Askay as member of project Beispiel Nr 1 Input Code and Title Select a creation scheme A none Hint if there is an active mandatory global or parent scheme this selection will be ignored 3 Press the Create button After you have performed all three actions a new project will be created You should see the following confirmation dialog on the screen 10031 Object created i Th
16. Apply settings Copy to preparations Copy to controls After you have chosen what information is to be copied you have to de fine the destination of the copy command Enter your destination in the input field Select Destination Objects In principle the Apply function does only copy the information to lower level objects 2000 Stegmann Systemberatung www rs system de 134 Reference If you called the apply function from the project level the default setting is to copy the information only to assay level and not beyond Since PLA uses only information from the lowest object level this option prevents the assay from being damaged If you want to pass down the information through the hierarchy tree activate the Standard Preparation or Controls check box For the case you invoked the function on the assay level you can steer the copying process by selecting the target objects in the box Select Destina tion Objects S Apply Settings of Assay Messung 1 to its childs EN You are about to apply the selected settings of the current object to its descendents WARNING You are able to destroy some definitions and set the child objects to an unusable state Apply what apply subject information only T apply reagent information IT apply assay definitions Number of series steps concentration doses etc Tl apply analysis definitions Statistical parameters regression model etc L IV apply linear selection de
17. Back Cancel If you want to change the destination folder choose the Custom option and press the Next button You will see the following screen on which you can change the destination folder 2000 Stegmann Systemberatung www rssystem de 33 System Requirements and Installation Therefore you have to press the Change button and select a different destination directory fe PLA Setup Custom Setup Select the program features you want installed Click on an icon in the list below to change how a Feature is installed rheat re Des PLA Program Files Ion This feature requires 46MB on your hard drive It has 2 of 2 subfeatures selected The subfeatures require 1152KB on your hard drive Install to C Programme PLA Change Installstiield Hep see pa Cancel When you press the Next button the setup wizard has the complete in formation for starting the setup process You will see the following screen 34 2000 Stegmann Systemberatung www rs system de System Requirements and Installation ji PLA Setup Ready to Install the Program The wizard is ready to begin installation Click Install to begin the installation If you want to review or change any of your installation settings click Back Click Cancel to exit the wizard lt Back Cancel If you press the Install button the setup program will start to copy the files to the dest
18. C V 26 1 1 0000 0 0000 248 400 21 996 89 2 0 2500 2 0000 332 000 32 042 9 7 50 Response 290 200 2 2 Sample Preparation U tep X log Series 1 Series 2 Series 3 Series 4 Series 5 1 1 0000 0 0000 230 210 280 261 241 2 0 2500 2 0000 310 290 360 341 321 tep X log Series 6 Series 7 Series 8 Series 9 Series 10 1 1 0000 0 0000 290 223 254 216 235 2 0 2500 2 0000 370 303 334 295 315 tep X log Mean C V 26 1 1 0000 0 0000 244 000 26 808 11 0 2 0 2500 2 0000 323 900 26 926 83 50 Response 283 950 Celoulatecby PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs system de 216 2000 Stegmann Systemberatung www rs system de Appendices Page 3 of 13 03 10 99 100809 Project Validierungsbeispiele Assay Europ Pharmacop 1993 3 Range Selection Linear detection type Fixed range 1 Fixed range 1 Selected Linear Range Step 110 Step 2 Step 1 to Step 2 4 Test of Linearity Regression equation y a bligo Clg 4 1 Standard Standard S LINEARITY IS NOT CALCULATED 4 2 Sample Preparation U LINEARITY IS NOT CALCULATED Calculated by PLA Verson 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs system de 2000 Stegmann Systemberatung www rs system de 217 Appendices Page 4 of 13 Project Validierungsbeispiele 03 10 99 100809 Assay Europ Pharmacop 1993 5 Test of Slope Regression equation y a bligo 5
19. Editor olx Datei Bearbeiten Suchen 672 8 707 0 758 0 672 0 733 0 715 0 704 0 748 8 762 0 744 8 809 8 771 08 796 0 749 0 854 0 708 0 784 0 730 08 846 8 759 8 653 8 648 0 617 0 569 0 594 0 653 0 659 0 615 8 595 8 703 8 494 0 396 0 363 0 456 0 378 0 342 0 387 0 387 8 306 0 394 8 414 0 338 0 301 0 296 0 307 0 288 0 308 0 275 8 204 8 241 8 252 8 241 8 177 0 187 0 208 8 221 0 219 8 182 8 187 8 231 8 128 8 121 8 892 0 113 0 092 0 082 0 095 0 897 8 0861 8 069 8 836 8 811 8 0851 8 0808 6 033 8 010 8 613 08 007 8 028 0 040 8 The measurements are arranged in a 8x12 matrix The values are sepa rated by semicolons The example shows the measurements of a micro titer plate The samples are arranged according to the following scheme NC S1 P1 S1 P1 S1 P1 Sl Pl S1 P1 PC NC S2 P2 S2 P2 S2 P2 S2 P2 S2 P2 PC NC S3 P3 S3 P3 S3 P3 S3 P3 S3 P3 PC NC S4 P4 S4 P4 S4 P4 S4 P4 S4 P4 PC NC S5 P5 S5 P5 S5 P5 S5 P5 S5 P5 PC NC S6 P6 S6 P6 S6 P6 S6 P6 S6 P6 PC NC S7 P7 S7 P7 S7 P7 S7 P7 S7 P7 PC NC S8 P8 S8 P8 S8 P8 S8 P8 S8 P8 PC Where NC negative control PC positive control S1 S8 standard dilution step 1 dilution step 8 P1 P8 preparation dilution step dilution step 8 2000 Stegmann Systemberatung www rs system de 147 Reference A preparation and a standard with eight dilution steps were taken five times Additionally a negative and a posit
20. Fieller s theorem The theorem exceeds the quality that is demanded by the European Pharmacopoeia The calculation can be performed with a level of significance of 90 0 95 0 99 0 and 99 9 The logarithm of the relative potency M is given by the following equa tion ay M b Where Yp and Ys are the intersections with the y axis and b is the com mon slope of both samples According to Fieller one obtains the follow ing confidence interval with a defined error probability tf 1 1 M X X u a GC l g M Xs Xp 2 2 e Crp u DIS according to D J Finney Statistical Method in Biological Assay Lon don 1978 2000 Stegmann Systemberatung www rs system de 183 The Statistics Core of PLA Where Xs averaged log dose of the standard Xp averaged log dose of the preparation s mean square of errors MSE S y sum of square of errors t f p value of the student t distribution for f degrees of freedom and a confidence interval of p n number of measurements of the standard np number of measurements of the preparation The value of the student t distribution is calculated within PLA 184 2000 Stegmann Systemberatung www rs system de The Statistics Core of PLA 10 2 Automatic Detection The crucial novelty in PLA is the possibility to automatically detect the linear and parallel regions of assays This function enables you to find the valid intervals f
21. GLP GMP settings Please refer to this chapter for further information 2000 Stegmann Systemberatung www rssystem de 55 PLA Administration 56 2000 Stegmann Systemberatung www rs system de 6 Tems and Con cept This chapter will introduce you to the terms and concepts of PLA You will learn about the structure and properties of the PLA objects The goal of this chapter is to make working with PLA easier more efficient for you 57 2000 Stegmann Systemberatung www rs system de Terms and Concepts 6 1 Objects PLA knows five different kinds of objects These objects carry out certain functions within the program The five objects are the project the assay the standard the preparation and the control Assay Standard Preparation Control Is equal to the term of an assay in biostatistical litera ture The assay consists of the measurements of a stan dard and at least one preparation The measurements of the standard and the corresponding preparations and controls are analyzed collectively A parallel line assay calculates the relative potency of a preparation in com parison to a standard sample Per definition the relative potency of a standard is always 1 0 100 Refers to the measurements and properties of a stan dard graph of an assay The standard consists of at least two point of measurement collected at least twice Is the term for the measurements and properties of the prepara
22. Import Definition Scheme Control and Summary A r Modify obiect instructions Import in Progress Create the object with this code Objects with same code will be overwritten Please choose or create the destination where the imported data will be saved Rename or select a target where the childs will be appended Note the target will remain unchanged zl import this object Please note the import will not overwrite existing objects Number of objects 5 Number of objects to import 0 lt Previous es Because we have selected New Project in step 3 of the import process the first thing we must do is to create a new project or to assign an existing project to the assay See section 8 12 1 for details 2000 Stegmann Systemberatung www rssystem de 157 Reference For the automated import of assays macros are available Instead of a fixed name you can give a macro value as assay name The following macros are available Return Value GU Will give the user name D Will give the actual date T Will give the actual time F Will give the name and directory of the im ported file You can combine macros In this manner you easily generate unique assay names E g if you enter an assay name F from D the full file name with a date extension will be the assigned name of the assay PLA Import Wizzard D PLA 1 2 1mport 043011 96 PLA Import Wizzard The imp
23. Individual range For the preparations you can choose likewise if the linear range should be optimized freely or if the same dilution steps as for the linear range of the standard should be used The following matrix will give you an over view on the results of combination of the different choices 2000 Stegmann Systemberatung www rssystem de 185 The Statistics Core of PLA Preparation E om kl fo Ss KI Gi Ba Preparation identical individual No optimization The linear region of the preparation will be set to the same interval as the standard The preparation is optimized indi vidually within the linear range of the standard The same region of the standard is used for all prepa rations The linear region of the standard is optimized with respect to all preparations The region of the preparations is set to the same inter val The preparations are optimized with respect to the common linear range of the stan dard Standard Standard Standard fixed common individual The linear region of the standard is optimized for each preparation sepa rately For each stan dard preparation combination the best common range is deter mined Preparations and standards are op timized freely without any re strictions Overall you have nine possibilities to optimize the linear range s with side conditions of varying strength Once you know the valid r
24. Password from the start the following dialog Change your password r Account Password Dy Matthias Schmitt Current Password Retype New Password bei Cancel Enter your old password in the field Current Password Enter the new password in the input field New Password and re type your new password in the field below Press OK to change your password or Cancel to dismiss the changes 50 2000 Stegmann Systemberatung www rs system de PLA Administration 5 3 Program Options Settings The program options contain several settings that are important for the correct output of the analysis reports For this some application paths must be set You have to login as an administrator in order to be able to access these functions 5 3 1 Report Settings Select PreferencesiOptions from the menu The following dialog will appear Options EN Application Settings GLP SOP Settings RTF Preview and Printing Leg fron reaisty IV read ATF file type association from registry Preview DDE Print DE DDE Note use 1 as filename and Application T opic r Default Reports Select one or more reports PLA Condensed Report including graphics PLAO xtf PLA Short Report including values and regression graphics PLA1 xtf PLA Short Report including values and normal graphics PLA2 tf PLA Short Report including values PLA3 xtf PLA Complete
25. Previous Hetz If you click on an arbitrary field on the matrix you will see its properties displayed in the lower sections of the dialog page The left dialog field is actually relevant for the designation of the data objects Additionally you can see the information on the measurement of the corresponding pod Selected datapoint C Control he Dilution step Point Data value Mark column 1 and select Control in the input field Selected Datapoint to assign the measurements Alternatively you can press the key C be cause this letter was given to the control object in the previous dialog You can assign the columns 2 4 6 8 and 10 to the standard key A and the columns 3 5 7 9 and 11 to the preparation key B in the same man ner The columns will be displayed in different colors according to their assigned object U 2000 Stegmann Systemberatung www rssystem de 153 Reference After these steps your matrik should now look like this PLA Import Wizzard D PLA 1 2 Import chi2603c txt PLA Import Wizzard m Assign mode Please the data points to the objects Select Input File Import Definition Scheme Select Destination Scheme Define Objects Sort and Assign Imported Data Choose Destination Save Import Definition Scheme Control and Summary Import in Progress Please assign the displayed data to the objects You may use the keyboard to At least one point was assigned to a
26. SOS differ ence and MS difference are the square sum of the model differences in the parallelity analysis Table 11 1 Comparison between the Linder and PLA results TA Linder Statistische Methoden Basel und Stuttgart 1964 4 Auflage S 162 169 190 2000 Stegmann Systemberatung www rs system de Appendices Standard Sample 2 Linearity SOS quadratic 35 363 35 363 MS quadratic 35 363 35 363 SOS error 113 848 113 848 MS error 9 487 9 487 F value 3 73 3 73 Slope SOS model 475 240 475 240 MS model 475 240 475 240 SOS error 149 211 149 211 MS error 11 478 11 478 F value 41 40 41 41 Preparation Sample 1 Linearity SOS quadratic 832 832 MS quadratic 832 832 SOS error 116 620 116 620 MS error 9 718 9 718 F value 0 09 0 09 Slope SOS model 670 810 670 810 MS model 670 810 670 810 SOS error 117 453 117 453 MS error 9 035 9 035 F value 74 25 74 25 Parallelity of Sample 1 and Sample 2 SOS difference 8 405 8 405 MS difference 8 405 8 405 SOS error 266 664 266 664 MS error 10 256 10 256 F value 0 82 0 82 Relative Potency of Sample 1 in Comparison to Sample 2 RP 2 04 2 04 RP upper limit 1 63 1 63 RP lower limit 2 56 2 57 2000 Stegmann Systemberatung www rs system de 191 Appendices 11 1 1 PLA Output PLA Short Report The following output is generated as a so called PLA Short Report It contains the shown values 192 2000 Stegmann Systemberatung www rs system de Appendices Pag
27. Select output filename nu ZE ak os Exporting the selected objects Check your selections Exporting object 15 of 783 Probe 1 Export in progress Please wait The blue progress bar shows you how far the export process has suc ceeded After the export has been successfully finished press the Close button to leave the export wizard 2000 Stegmann Systemberatung www rssystem de 139 Reference 8 12 Data Import The import interface is a new function in PLA 1 2 with which you can migrate external data from various sources into the program On the one hand PLA can import PNF files that have been exported from PLA Users can exchange data from different PLA installations in this way On the other hand PLA can read virtually data from any measurement system that is able to write files You will need import modules which you can purchase from the Stegmann Systemberatung for the import of third party data formats The standard installation contains an import module for 96 well plates in CSV comma separated values format Depending on the type of the object import as PNF file or by an external import module that is imported into PLA the steps in the PLA Import Wizard are different We will introduce you to the procedures in the next chapters 140 2000 Stegmann Systemberatung www rs system de Reference 8 12 1 Importing PNF Files In this section the import of an existing PNF file is described Firs
28. Standard S nU g Selected linear Range Test of Linearity Fayadraie Fass p e Test of Slope Fee Fariica P r Test of Parallelism Fetttctmadets Faitca P e Step 1 to Step 2 Step 1 to Step 2 Linearity is not calculated Linearity is not calculated PASSED PASSED 46 2695 44 2211 6 5146 6 5146 98 0 98 0 0 7199 0 7107 PASSED 0 046 5 927 98 00 0 9922 Relative Potency 95 Fiducial Limits Relative fiducial limits Preparation U vs Standard S 1 112 0 830 1 497 74 6 134 7 60 0 4 Calculation of EC50 Method of EC50 calculation 50 Response of EC50 concentration Standard S Preparation U Calculated by PLA Version 1 2 00 PLAis a product of Stegmann Systemberatung Germany wew rs system de 208 2000 Stegmann Systemberatung www rs system de Appendices Page 3 of 7 08 10 99 10 07 56 Project Validierungsbeispiele Assay Europ Pharmacop 1993 5 Figure 3907 A a A 4 Standard S a 3307 EI a E Q 8 d g i 27 m Preparation U 210 d d d d 0 0 0 5 1 0 15 2 0 Dose log x 6 Annotations Calaulated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs eystem de 2000 Stegmann Systemberatung www rssystem de 209 Appendices Page 40f7 08 10 99 10 07 56 Project Validierungsbeispiele Assay Europ Pharmacop 1993 Results of Assay Europ Pharmacop 1993 Proj
29. Systemberatung www rssystem de 59 Terms and Concepts 6 2 Object Hierarchy Projects assays and standards preparations controls are organized in a hierarchical way The topmost level is the project level You can see a part of the PLA navigator in Figure 6 1 You see several entries EX Navigator E EE Factor Vill sz Dy Novartis Pharma e DEN Systemvalidierung e DEn Test e DEN Validierungsbeispiele Figure 6 1 Navigator Projects Open the project Beispiel Nr 2 by clicking on the symbol left of the project name In this way you get access to assays the second level ob jects In Figure 6 2 you can see that project Beispiel Nr 2 contains three assays which are called Assay 1 Assay 2 and Assay 3 A Navigator Data Objects eispiel Nr 2 Assay 1 Assay 3 Biotest Pharma GmbH Factor Vill Novartis Pharma Systemyalidierung Figure 6 2 Navigator Assays Each of the assays contains another level of objects the standards the preparations and the controls Those objects are on the third and lowest 60 2000 Stegmann Systemberatung www rs system de Terms and Concepts level of the object hierarchy You can access the object from the assay level in the same way as you can access the assays from the project level In Figure 6 3 you can see that the assay 2 contains one standard one preparation and one control EI Navigator
30. criterion Linearity is rejection criterion Parallelity is rejection crite rion 114 2000 Stegmann Systemberatung www rs system de Reference The general section allows you to set all the analysis properties according to the European Pharmacopoeia 1997 If you check this option the whole dialog page will be will be deactivated The option facilitates the control of objects The second option in the general analysis box is the activation of the Dixon test which performs a check for statistical outliers Please refer to chapter 10 1 2 for further information on the Dixon test PLA can do the analysis of parallel line assays with two different regres sion models You can choose between a simple logarithmic lin log and a double logarithmic analysis log log The simple logarithmic option in which only the dose inputs are treated logarithmically is the default In the double logarithmic version the dose inputs as well as the responses are transformed The logarithm is always calculated to a base of 2 Note Never use different regression models within the same assay Another feature of the general analysis settings is the ability of PLA to calculate reverse response effect correlation Check this feature if an inverse dose response relation exists in your assay This option is neces sary for the measurement of e g inhibition assays where the increase of the dose leads to a decrease of the reading The relative potency of the
31. de office rs system de 2000 Stegmann Systemberatung www rs system de 21 Introduction 3 3 Whatis new in this Version In comparison to previous versions PLA 1 2 offers a multitude of new important and productivity increasing features The following list gives you but a quick overview 1 The graphical user interface has been redesigned A task bar has been added to allow you even quicker access to the functions that are most important FrameBar Lei 2 D Er Wii x 7 a g A Navigator New Definitions Edit data Close So Delete Import Export Calculate Check Inf The function for the automatic detection of linear regions has been extended considerably You can now limit the region in which the program searches for linearity and parallelity and the number of points in the linear region Furthermore you can enforce the inclusion of the 50 response value in the linear range The calculation of the 50 response value as well as the calculation of the 50 concentration of the standard ECs are new features In addition the new outputs contain mean values standard deviations and coefficients of variation of the measured values In PLA 1 2 controls are now available as part of an assay You can enter positive and negative controls as well as blanks The input of individual dilution steps direct dose input has been improved You can now enter dilution steps already at project or as say level These values wi
32. is their suitability for the definition of standard operating procedures Schemes are used as templates for the creation of new objects projects assays standards preparations and controls In comparison to data ob jects templates are defined by a set of object settings but they do not contain any measurement data When you start the create object dialog you have the opportunity to select an object definition scheme see topic 3 in Figure 9 1 By the use of a scheme you bypass the normal way of inheritance The created object is not based on the definitions of the parent object It is based on the settings of the selected scheme Select object type to create 1 Bien Prec y as member of project Test Cancel Input Code and Title Select a creation scheme 4 none Hint if there is an active mandatory global or parent scheme this selection will be ignored Figure 9 1 The create object dialog 164 2000 Stegmann Systemberatung www rs system de GMP GLP Settings in PLA For the usage of PLA you should always define a scheme for every assay In this scheme all information on the number of dilution steps the number of data series the statistical parameters as well as the analysis method is saved Create a new scheme by clicking on the drop down box in the upper part of the navigator Select the schemes view from the list see Figure 9 2 A Navigator IDI
33. now either pick a substance name from the list or add a new substance to the database You cannot enter the substance name directly S Select a substance for meassurement EN none AD 3 Cancel Input the substance code short name Input field for the substance title Add a substance to the database Edit a substance Delete a substance from the database Cancel the dialog Apply changes and close dialog 106 2000 Stegmann Systemberatung www rs system de Reference In analogy to preparations and standards PLA uses a substance code and a substance name For the analysis you can use the code to corresponding to the labels e g identification numbers you normally use in your labora tory while using the correct name for the printed output Choose a code from the list and press the Select button to pick a substance from the list You will also get back to the object settings Click on the New button to add a new substance to the database Enter the substance code and name in the appropriate input fields Press the Save button to apply the changes to the database The substance is now dis played in the pick list If you do not supply a code and a name of the substance a warning mes sage will appear E0190 Missing Information CH Please input the code and the name for the new entry Choose the code from the list and press the Edit button to change a sub stance name 2000 Stegma
34. parallel line assay according to the European Pharmacopoeia with fixed values for the linear and parallel regions of your measured values If you have chosen an automatic detec tion method the program will first do a selection of the measured values as described in section 10 2 As a result all preparations and standards will be assigned a linear and parallel region which will be analyzed statisti cally according to the following section The methods for the automatic detection partially utilize the functionality depicted in section 10 1 but the technical implementation is completely separated from the statistical cal culation Therefore the analysis is independent from detection method and in full accordance with the European Pharmacopoeia 176 2000 Stegmann Systemberatung www rs system de The Statistics Core of PLA 10 1 Statistical Calculations In figures Figure 10 1 and Figure 10 2 the flow chart of a statistical analy sis in PLA is shown To some extend the analysis settings have effect on the course of the calculation The analysis corresponds to the EP proce dure although the sequence of steps is slightly altered for technical rea ES With Al A Statistical Evaluation of EFSER the Single Objecis Dixon Test activated Yes Execute Dixon Test No Yy Linear Regression wl Slope is a KEE Slope significant siaction criteria Yes v No Linear Regressio
35. select direct dose input With this setting you can enter the dose values directly in the data editor ATTENTION If you change the dilution scale from direct dose input to in 2 series you loose the already entered dose values 2000 Stegmann Systemberatung www rssystem de 113 Reference 85 4 Analysis properties The settings on the fourth page of the object dialogs refer exclusively to the analysis definitions These settings are divided into three sections EP 1997 accordance Slope hypothesis Linearity hypothesis A Regression model Parallelity hypothesis IN Inverse response effect Confidence interval Y A relation AN Bi Assay Europ Pharnacop 1993 Bisi E V General Reagent Defrtieinz ara Range Selection Annotations GLP Gener Perform Don test for data outlier AtA A5 series Regressio Model lin log e Reverse Ak e k ffect Correlation m Probabilities Dixon test contamination alpha Significance of the slope ST rejection enterion By che Significance of deviations from parallelity SI y Lf Fiducial limit of potency estimation 25 95 0 d Significance of deviations from linearity ST T nten TEJESHGTTENTENGN Values marked are used only by objects whose pot n y will be calculated EC50 Calculation Method Do not calculate late 50 Response by s object Slope is rejection
36. start you can call the Run function from the Windows Start menu and type D SETUP where D is the drive letter of your CD drive 30 2000 Stegmann Systemberatung www rs system de System Requirements and Installation 4 2 4 Step 3 Welcome to the Setup Program After the setup program has been started you should see the following welcome screen fi PLA Setup EN Welcome to the PLA Setup The Setup program will install PLA on your computer To continue click Next Cancel You will always find the following option to navigate through the setup wizard on the following screens Next Next step Cancel Abort the setup program Back Go to the previous step The welcome screen introduces you shortly to the setup program Press the Next button 2000 Stegmann Systemberatung www rssystem de 31 System Requirements and Installation 4 2 5 Step 4 License Agreement Please read the License Agreement carefully You have to agree to the licensing contract in order to install PLA The setup program shows you the license agreement on the screen You can use the scroll bar to move up and down the text If you agree to the licens ing conditions select the I accept the terms in the license agreement button and go to the next screen Otherwise the setup program will termi nate fe PLA Setup License Agreement Please read the Following license agreement carefully License
37. steps identical for all objects Import Definition Scheme number of series identical for all objects Select Destination Scheme Apply general settings Define Objects List of raw data objects Sort and Assign Imported Data steps series Choose Destination Save Import Definition Scheme Control and Summary Import in Progress Please define the type count and general settings number of steps and series of the r Selected object objects you want to create eg Type of object H steps series G i L E Hint You have to create the objects that will contain the raw data lt Previous Hez Let us discuss this dialog page with the data from our example In the first section of the page you have to give the number of object that are con tained in the import data In our example we have three objects a prepara 150 2000 Stegmann Systemberatung www rssystem de Reference tion a standard and a control Because all controls positive and negative controls are treated by PLA as one object each assay can only have one control Enter 3 in the input field Number of Objects With the check boxes left of the spin control you can define that the number of dilution steps and the number of repetitions are the same for all objects This true for the preparation and the standard but not for the controls in our exam ple Nevertheless let us check the two boxes Two activate the settings pr
38. take a moment PLA now performs the automatic detection of the linear interval and analyses the assay afterwards When the calculation is finished the print save and preview buttons will be acti vated Select one or more reports from the upper box in the dialog and press the preview button to view the report 88 2000 Stegmann Systemberatung www rs system de My first Parallel Line Assay Calculation Control Select your reports Ihn PLA Condensed R eport including graphic SW In PLA Short Report including values and regression graphics In PLA Short Report including values and normal graphics In PLA Short Report including values hg PLA Complete Statistics Report og PLA Validation Report Progress The calculation has been finished Time needed 23 secs Options print immediatly when calculation finished Save Print Preview If you choose the PLA Condensed Report you should see the following results 2000 Stegmann Systemberatung www rs system de 89 My first Parallel Line Assay Page 1 of 1 09 09 99 09 30 50 Beispiel Nr 1 Project Beispiel Nr 1 Current user Dr Matthias el dE Date Signature ZZ T i est of Test of Test of Relative Assay Standen Sample inearity Slope Parallelity Potency Messung 1 7714 A Standard ma m Probe 1 Q 2 H e Probe2 N a 453 v Probe3 S Probe 4 D a 294 4 Freee 0 0 1
39. the field Create the object with this code If you confirm the change by pressing the Rename button the name of the project will be changed in the object tree Furthermore you can exclude objects from the import by selecting the object and unchecking the box Import this object If you exclude an assay or project from the import all the lower level objects will also be ex cluded 144 2000 Stegmann Systemberatung www rs system de Reference If your object is an assay you will first see the following dialog PLA Import Wizzard D PLA 1 2 Import Ec99061_pnf rte The import tree contains Select Input File ER a DI EC99061 Choose Destination X Standard 200 ng ml Control and Summary X amp 50 potency 100 ng ml Import in Progress X amp 150 potency 300 ng ml x Control D Modify obiect instructions Create the object with this code Objects with same code will be overwritten Please choose or create the destination where the imported data will be saved Rename or select a target where the childs will be appended Note the target will remain unchanged zl IV import this object Please note the import will not overwrite existing objects Number of objects 6 Number of objects to import 0 lt Previous des The object is displayed in the object tree but it is marked with a red cross Before you can import the assay you must select the project to which the assay wil
40. the largest number of points for which the hypotheses are fulfilled Exact Range Finds the best linear interval with exactly the number of points given By giving the minimum number of points you can specify the minimum interval of the linear range Note The bigger the given value the faster is the automatic method For normal analyses you should leave the value on the default of three If you have selected the exact range allocation strat egy this input defines the exact number of point for the linear range With the settings for the allocation region you can limit the search for a linear range to a particular interval Set the allocation region by deactivat ing the field maximum allocation region and giving the interval manually Another option for automatic methods is the possibility to include the 50 response in the linear region You can again choose from three op tions ignore The 50 response is ignored for the selec tion of the linear range 120 2000 Stegmann Systemberatung www rs system de Reference include if possible inclusion is mandatory The program tries to include the 50 re sponse if possible However if the range containing the 50 response is not valid the 50 is ignored The 50 response must be included in the linear range even if no valid linear range is found and the assay is rejected The same four options as for the calculation of the EC50 are available for the calculation of t
41. the object with this code Objects with same code will be overwritten EEN Rename Please choose or create the destination or select a target where the childs will be appended Note the target will remain where the imported data will be saved unchanged IV import this object Please note the import will not overwrite existing objects Number of objects 40 Number of objects to import 40 lt Previous Next gt In the upper part of the window you will see the structure of the import object displayed You should be familiar with this representation from the navigator window Besides the usual icons for the object types assays standards preparations and controls several other symbols might be shown These symbols have the following meaning Symbol Cross The object already exists in the database You will have to rename the object if you want to continue the import process Bend arrow The object already exists but you can import the lower level objects This symbol is used for assays and projects only 2000 Stegmann Systemberatung www rssystem de 143 Reference Symbol Rectangle The object does not exist in the database It can be imported without limitations If all objects are marked with a rectangle you can continue the import process without limitations You can change the name of the project if you want to Select the project you want to rename and enter a new name in
42. yy hh mm ss with dd for day mm for the month and yy for the year in the date input and with hh for the hour mm for the minute and ss for the second in the time input E g Date of Meassurement 03 12 96 20 05 59 will set the time stamp to December 3 five minutes past 8 a m 104 2000 Stegmann Systemberatung www rs system de Reference Preparations and standards on the lowest object hierarchy can be con verted into each other By changing the setting in the switch objeci type group box you can change a standard into a preparation and vice versa In this way it is possible to compare different standards against each other Batch number from which the sub stance was taken Bi Assay Europ Pharmacop 1993 General Title Batch identification of the substance Status Date of Meassurement Ty finished o display in navigator 16 06 99 15 14 55 History 2000 Stegmann Systemberatung www rssystem de 105 Reference 8 5 1 1 The Substance Database Substances often have long and complicated names PLA offers a sub stance database to facilitate the substance input for you In this database the program keeps a list of all substances that have been measured al ready You only need to enter a substance name only once After that you can always pick the substance from database list Pressing the Select Substance button will open the substance dialog You can
43. 1 Standard Standard S Total number of observations 20 Source di Sumofsquares _MeanSqure ___FVdue Model 1 34 944 8000 34 944 8000 46 2695 Error 18 13 594 4000 755 2444 Total 19 48 539 2000 RRE CR Intercept a 248 4000 8 6905 e 0 719929 Slope b 41 8000 6 1451 Pa 0 704370 Test passed if Fmosa gt Fain P 98 6 5146 Test PASSED 5 2 Sample Preparation U Total number of observations 20 __ Source di ____ SumofSquars ___Mean Square F Value Model 1 31 920 0500 31 920 0500 44 2211 Error 18 12 992 9000 721 8278 Total 19 44 912 9500 prom Estimates Error Quality of regression Intercept a 244 0000 8 4960 d 0 710709 Slope b 39 9500 6 0076 Pas 0 694638 Test passed if Fmosa gt Fan P 98 6 5146 Test PASSED Calculated by PLA Verson 1 2 00 PLA is a product of Stegmann Systemberatung Germany wee steen de 218 2000 Stegmann Systemberatung www rssystem de Appendices Page 5 of 13 Project Validierungsbeispiele 03 10 99 100809 Assay Europ Pharmacop 1993 6 Test of Parallelity 6 1 Estimation of Common Slope Total number of observations 40 Source a ____ __SumofSquars _MeanSquwe _F Vaue__ Model 3 3 363 703 4750 Error 37 26 621 5250 Total 40 3 390 325 0000 1 121 234 4917 719 5007 ___________ _Param Estimates mer Quality of regression Intercept a1 243 0750 7 3459 Intercept a2 249 3250 7 3459 Slope b 40 8750 4 2412 ig 0 9921
44. 1 for a more detailed description of schemes 83 1 Opening Objects To open the object definition dialog select the object in the navigator by either clicking on it or highlighting it with the cursor keys EN Navigator DI Data Objects D 2 05 Beispiel Nr 1 mM Messung 1 D Standard amp amp Probe 2 amp Probe 3 amp Probe 4 amp Probe 5 DI Messung 2 DI Messung 3 E Messung 4 U Messung 5 E Messung 6 E Messung 7 E Messung 8 E Messung 9 Di Beispiel Nr 2 n Biotest Pharma GmbH Di Factor vill a Di Novartis Pharma T7 include finished items F H E E Project Standard Control Assay Preparation yObject opened You can open the object in four different ways Choose FilelOpenlObject Definitions from the menu Select Definitions from the task bar Press CTRL O Double click on the object DD 2000 Stegmann Systemberatung www rssystem de 99 Reference 8 3 2 Deleting Objects Click once on the object you want to delete ATTENTION All lower level objects will be deleted as well The deletion cannot be undone Once the object is selected either press the DEL key or choose FilelDelete Object Tree from the menu You will be asked to affirm the deletion 90038 Please confirm the deletion request x K You are about to DELETE the following project and all of its descendants from the database Beispiel Nr 1
45. 247 Quadratic 1 832 1333 832 1333 0 0856 rom Ssimstes imr E ofregresdon Intercept a 907 6000 192 1716 E 0 852053 Slope b 322 2000 218 2204 Pa 0 827396 Quadratic c 15 8000 53 9956 Root MSE 98 5819 Test passed if Feuaraic lt Fan P 95 4 7472 Test PASSED Calculated by PLA Verson 1 2 00 PLA is a product of Stegmann Systemberatung Germany wee steen de 200 2000 Stegmann Systemberatung www rssystem de Appendices 08 10 99 10 0728 Page 4 of 7 Project Validierungsbeispiele Assay Linder 5 Test of Slope Regression equation y a b lg x 5 1 Standard Standard Total number of observations 15 Source dt Sumofsquares Mean Square __Fvaue Model 1 475 240 0000 475 240 0000 41 4053 Error 13 149 210 9333 11 477 7641 Total 14 624 450 9333 Parem Estimates Enor Gualityofregression Intercept a 548 0667 48 7374 e 0 761053 Slope b 218 0000 33 8788 Pad 0 742672 Test passed if Fmosa gt Fai P 95 4 6672 Test PASSED 5 2 Sample Probe 1 Total number of observations 15 Sowce __df_ __SumofSquares_ Mean Square Fade Model 1 670 810 0000 670 810 0000 74 2470 Error 13 117 452 9333 9 034 8410 Total 14 788 262 9333 Paa Estimates Error ____Gnelityofregressin Intercept a 854 9333 64 9327 r 0 850998 Slope b 259 0000 30 0580 Fa 0 839536 Test PASSED Test passed if Frode gt Fain P 95 4 6672 Calculated by PLA Version 1 2 00 PLA isa product of Stegmann Systembera
46. 3 10 99 10 08 09 Project Validierungsbeispiele Assay Europ Pharmacop 1993 Results of Assay Europ Pharmacop 1993 Project Validierungsbeispiele Current user Dr Matthias Schmitt 1 Complete List of the Object Definitions Standard Sample General settings Code Standard S Preparation Z Title Standard S Preparation Z Measured substance Batch identification Object status declared closed NO NO Object was created by Dr Matthias Schmitt Dr Matthias Schmitt Creation timestamp 16 06 99 15 14 55 16 06 99 15 14 53 Last modification by Modification timestamp 06 10 99 12 53 12 06 10 99 12 53 12 Definition settings Concentration of undiluted standard 1 7 Concentration units Predilution of standard sample 11 T Number of dilution steps 2 2 Number of data series 10 10 Additional predilution factor 11 11 Dilution scale direct input direct input Analysis settings Analysis according to EP YES YES Dixon test enabled YES YES Regression model linNog linlog Reverse response effect correlation NO NO Dixon contamination 0 02 0 02 F Test of the slope 98 98 F Test of linearity 98 98 F Test of parallelity 98 98 95 Fiducial limit percents 95 Slope as rejection criterion YES YES Linearity as rejection criterion YES YES Parallelity as rejection criterion YES YES EC50 calculation NO NO by using Range Selection Settings Linear detection type Fixed range 1 Fixed range 1 Allocatio
47. 48 Root MSE 26 8235 6 2 Estimation of error due to non parallelity Total number of observations 40 P Source dt Sum of Squares Mean Square F Vaue Model 3 3 363 703 4750 1 121 234 4917 Difference of Models 1 34 2250 34 2250 0 0463 Model Total 4 3 363 737 7000 840 934 4250 Error 36 26 587 3000 738 5361 Total 40 3 390 325 0000 Intercept a1 244 0000 8 5938 r 0 992158 Intercept a2 248 4000 8 5938 Root MSE 27 1760 Slope b1 39 9500 6 0767 g 0 0221 Slope b2 41 8000 6 0767 Test passed if Fat todas lt Fan P 98 5 9268 Test PASSED Calculated by PLA Version 1 2 00 PLA isa product of Stegmann Systemberatung Germany wee steen de 2000 Stegmann Systemberatung www rssystem de 219 Appendices Page 6 of 13 03 10 99 10 08 09 Project Validierungsbeispiele Assay Europ Pharmacop 1993 7 Estimation of the Relative Potency Relative potency of sample Preparation U in comparison to standard Standard S Preparation U vs Standard S Estimated relative potency 1 11 95 Fiducial Limits 0 83 1 50 Relative fiducial limits 74 6 134 7 60 0 8 Calculation of EC50 Standard S Preparation U Method of EC50 calculation We nc 50 Response of EC50 concentration 9 Annotations Gacdatedby PLA Version 1 2 00 PLAisa product of Siegnann Systemberatung Germany waw rs system de 220 2000 Stegmann Systemberatung www rs system de Appendices Page 7 of 13 0
48. 9 645 693 696 4 1 8 593 587 628 647 5 1 16 522 521 569 572 6 1 32 463 459 502 517 7 1 64 417 416 456 466 Table 7 1 Measurements of the example 74 2000 Stegmann Systemberatung www rssystem de My first Parallel Line Assay Your task is to determine the relative potency of the preparation samples in comparison to the standard The preparations cannot be analyzed over the full range of dilution steps because of saturation effects You do not know which interval can be analyzed 2000 Stegmann Systemberatung www rssystem de 75 My first Parallel Line Assay 7 2 Creating a Project First you should create a new project This is not really necessary for only one assay but it will help you if decide to measure further assay later These assays can then be added to the project Do you remember chapter 6 2 Object Hierarchy and 6 3 Inheritance There you will find all essential information about the creation of objects and how they relate to each other Inheritance has the advantage that all settings of the higher order objects are passed on to a lower level object You can immediately take advantage of this feature in our example Take a moment to think about which objects you will need First all assays that you create will be regarded as part of a project Therefore you will need a project Second all samples are part of the same assay You will need one assay for the preparations and the standard
49. Agreement All products sold by Stegmann Systemberatung or companies associated therewith including demonstration sets hardware media and manuals hereinafter referred to collectively as Product as well as all future orders shall be subject to the Following provisions IF you do not accept these provisions please return the product to us within seven 7 days of having purchased or received the Product We will refund the purchase price minus fees for shipping and processing 1 License Stegmann Systemberatung holds all rights to the Product and herewith assigns to you a non transferable nonexclusive right of use to exploit utilize the Product according to the Following sl I accept the terms in the license agreement Cancel 32 2000 Stegmann Systemberatung www rssystem de System Requirements and Installation 4 2 6 Step 5 Choosing the Setup Type On the following screen you will be asked for the type of installation If you choose the Complete option all PLA program files are being in stalled in the default destination directory which is C Program Files PLA N PLA Setup Setup Type Choose the setup type that best suits your needs Please select a setup type All program Features will be installed Requires the most disk space C Custom Choose which program features you want installed and where they ke will be installed Recommended for advanced users lt
50. Bs In our example the intersection with the y axis b of the preparation is 5 5 and for the standard 6 0 The common slope is 2 0 By solving the above equation we obtain a relative potency of 0 84 for the preparation 16 2000 Stegmann Systemberatung www rs system de Introduction In biological and pharmaceutical research and guality control the analysis is often not as trivial as in the given example Many possible sources of error require an analysis of the measurements that is statistically valid Legislative bodies and regulatory authorities often require a proof of va lidity The most important questions arising from this requirement are proof for the linearity of the measurements proof for the parallelity of the measurements and the confidence interval of the relative potency These questions must be answered by the analysis A Standard Response m Probe 1 5 0 0 0 5 1 0 1 5 2 0 Dose log x Figure 3 1 Schematic Analysis of a Parallel Line Assay The European Pharmacopoeia describes in its appendices the necessary preconditions for a valid analysis of the relative potency using parallel line assays These requirements cover the construction of the experimental design as well as the mathematical analysis 2000 Stegmann Systemberatung www rs system de 17 Introduction The implemented mathematical methods in PLA 1 2 perform an analysis of parallel line assays according
51. Close button to return to the main window Note If you have selected an automatic method for the detection of the linear range the results can be different from those of the calculate func tion The check function always tests one preparation against one standard 126 2000 Stegmann Systemberatung www rs system de Reference while the calculate function might test several preparations against a stan dard For the latter situation you might obtain a different linear range that is optimized with regard to all preparations You should only use the check function to test if you input data is reasonable You should always use the calculate function for the complete analysis 2000 Stegmann Systemberatung www rs system de 127 Reference 8 8 The Calculate Function The calculate dialog s purpose is the control of the analysis Select an assay from the navigator window and choose Calculate from the task bar The following dialog will appear on the screen Selection of output reports Print results immediately after calculation Status of the calculation S Calculation Control lin PLA Validation Report Progress No calcylation has been started yet Preview of the reports Print reports Start calculation If you have selected an automatic method for the detection of linear ranges it is significant from which object you start the calculation In the case you have chosen the setting Automat
52. DAPLAT 2 Repot a Standard Import Path JDAPLAT 2 mpot 00 Fi Standard Export Path JD PLAT 2 Expot ei PTDADS Path DNPLA1 2 Converter E Report XTF Path D PLA1 2 ReportTemplates Ki You can change the following paths 1 Report Output Path This is the default path to which your analysis reports will be saved The default setting is the Report subdirectory of you PLA program directory 2 Standard Import Path The default path from which you want to import data files The default setting is the Import subdirectory of you PLA installation 3 Standard Export Path The default path to which you want to write your exported data files The default setting is the Export subdirec tory of your PLA program directory 54 2000 Stegmann Systemberatung www rs system de PLA Administration 4 PTD IDS Path The path where your input converters and your im port definition schemes reside The default setting is the Converter path of you PLA installation See chapter 8 12 2 for further details 5 Report XTF Path The path where the program looks for the report template files The default setting is the Report Templates subdirec tory of your PLA program directory Confirm the changes by pressing the Save button Otherwise press the Cancel button The last page of the options dialog contains the GLP GMP settings of PLA These are not directly relevant for the installation of PLA The whole chapter 9 is dedicated to the
53. Figure 10 2 Statistical Analysis Part B 10 1 1 Transformation of the Measurement Values PLA supports two different kinds of regression schemes for the analysis of parallel line assays The default method is based on a simple logarithmic transformation of the values The logarithm of the dose values is calcu lated while the response values are not transformed As an alternative scheme PLA can use a double logarithmic method by calculation the dose as well as the response on a logarithmic scale PLA works corresponding 2000 Stegmann Systemberatung www rs system de 179 The Statistics Core of PLA to the EP and common biostatistical literature with the logarithm to the base 2 10 1 2 Dixon Test With samples that have been measured more than twice a so called Dixon test can be performed to rule out statistical errors The test is applied to every value In PLA the two sided version of the test is implemented PLA supports contamination levels a of 0 3 0 2 0 1 0 05 0 02 0 01 and 0 005 These levels can be set for each object separately in the analysis definitions The smaller you choose the estimated contamination level the stricter every value is examined PLA can test up to 25 repetitions of measurement with the Dixon test To perform the Dixon test the readings are sorted by value In a second step the value with the largest deviation from the average is determined either the largest or the smallest value Referr
54. LA Import Wizzard D PLA 1 2 Import chi2603c txt PLA Import Wizzard Select Input File Import Definition Scheme Select Destination Scheme Define Objects Sort and Assign Imported Data Choose Destination Save Import Definition Scheme Control and Summary Import in Progress Please check the settings you made on the previous pages You can start the import by selecting Finish r Summary of operation Please check carefully Import file D PLA 1 2 Import chi2603c tst The import file contains raw data Creation scheme none The following objects will be imported 1 Project Beispiel Nr 2 0 Assay DAPLA 1 2 lmport chi2603c txt vom 07 10 99 0 Standard Standard Preparation Preparation Control Control Cancel lt Previous If you are content with your settings start the import procedure by pressing the Finish button When the import process has finished successfully press the Close button to leave the import wizard PLA Import Wizzard D PLA 1 2 Import chi2603c txt PLA Import Wizzard Select Input File Import Definition Scheme Select Destination Scheme Define Objects Sort and Assign Imported Data Choose Destination Save Import Definition Scheme Control and Summary Import finished The import process has been finished You may now close this window r Import process finished Finished Errors 0 160 2000 Stegmann Systemberatung www rs system de Re
55. PLA 1 2 Analysis of Parallel Line Assays User Manual Version dated 08 04 2000 2000 Stegmann Systemberatung Auestra e 31 D 63110 Rodgau Phone 49 6106 82619 0 Fax 49 61 06 82 619 2 www rs system de Software Lizenzvertrag der Stegmann Systemberatung Auestra e 31 D 63110 Rodgau Alle Produkte der Stegmann Systemberatung oder mit ihr verbundener Unternehmen einschlie lich Demonstrations Sets Hardware Medien und Handb chern nachfolgend zusammen als Produkt bezeichnet sowie alle zuk nftigen Bestellungen unterliegen den unten angef hrten Bestimmungen Falls Sie diese Bestimmungen nicht akzeptieren senden Sie bitte das Produkt mit Handbuch innerhalb von sieben 7 Tagen nach Erhalt an uns zur ck Sie erhalten dann von uns den von Ihnen bezahlten Kaufpreis abz glich Transport kosten und Bearbeitungsgeb hr 1 Lizenz Die Stegmann Systemberatung ist Inhaberin aller Rechte am Produkt und bertr gt Ihnen hiermit ein nicht bertragbares nicht ausschlie liches Nutzungsrecht das Produkt entspre chend der nachfolgenden Bedingungen zu verwenden Sie sind nicht berechtigt das Produkt oder Teile davon Dritten zu bertragen oder in anderer Weise zug nglich zu machen oder die Software oder andere Teile des Produkts zu ver ndern zu zerlegen zu dekompilieren reverse engineering durchzuf hren zu berarbeiten zu verbessern oder au er unter den Voraussetzungen von 69e UrhG den Quellcode der Sof
56. Statistics Report PLA4 fl PLA Validation Report PLAS xtf print immediatly after calculation On the first tab of the options dialog you have access to several options that are important for viewing and printing reports 2000 Stegmann Systemberatung www rs system de 51 PLA Administration PLA creates reports as RTF files rich text format that can be viewed with various text processor applications You have to define with which program you want to view and print your report files The default setting is to read RTF file association from registry PLA will find the program that is linked to RTF files You do not need to change any of these set tings If you deactivate the check box you can enter DDE commands for the preview and printing of RTF files Only experienced users should manipu late these settings If you press the Load from Registry button the actual values from the registry are loaded into the input fields If you want to use the program WordView for the preview of your reports for example you must give the exact path to the program Alternatively you can press the button right of the input field to open up a standard file dialog You must give the parameter 1 to hand over the name of the RTF file to the program ATF Preview and Printing read ATF file type association from registry Load from registry Preview D Program Files WordView W ordview exe 1 nae DDE Pri
57. Strategy Minimal No of Points Maximal Allocation Range S Allocation region Step 1 to Step 2 Step 1 to Step 2 Inclushion of 50 response 50 Response of 3 Results Standard S nz rat Selected linear Range Test of Linearity Fquactatic Faitca p r Test of Slope Fee e P r Test of Parallelism Festtoimodeis Faitca P KH PASSED 46 2695 6 5146 98 0 0 7199 Step 1 to Step 2 Linearity is not calculated Step 1 to Step 2 Linearity is not calculated FAILED 6 3249 6 5146 98 0 0 2600 Parallelity is not calculated Relative Potency 95 Fiducial Limits Relative fiducial limits Preparation Z vs Standard S Assay Rejected Relative Potency is not calculated 4 Calculation of EC50 Standard S Preparation Z Method of EC50 calculation 50 Response of EC50 concentration ne Goler by PLA Version 1 2 00 PLAisa product of Stegmann Systemberatung Germany wwnurs system de 2000 Stegmann Systemberatung www rs system de 211 Appendices Page 60f7 08 10 99 10 07 56 Project Validierungsbeispiele Assay Europ Pharmacop 1993 5 Figure 3907 A a Standard S 6 Response m Preparation Z L 0 0 0 5 10 1 5 2 0 Dose 4og x Annotations Calaulated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs system de 212 2000 Stegmann Systemberatung www rs system de
58. System Requirements and Installation After you have received the unlock code green code press the Next button in the registration wizard Enter the unlock code in the field below the registration code green backround If the code is accepted the PLA registration wizard will not show up again PLA Registration Wizard PLA Registration version 1 2 1999 Stegmann Systemberatung Thank you for trying out our product Please follow these instructions for extending this trial 1 Communicate the value in the red box to us 2 Enter our response in the green box 3 Press the Ok button If you have any questions please do not hesitate to contact us Registration Number Unlock Code 42 2000 Stegmann Systemberatung www rs system de 5 PLA Administration This chapter describes the administrative functions of PLA You will learn how to use the account management and where the program preferences are modified 43 2000 Stegmann Systemberatung www rs system de PLA Administration 5 1 Account Management One of the most important functions in PLA 1 2 is the completely revised account management You have to identify yourself to the program with a user name and a password to access PLA 1 2 and the data stored in the program The user management serves two purposes e Different access privileges depending on the status of the user must be assigned in order to es
59. a format was recognized successfully Click the Next button to proceed to the following dialog page Here you can choose a so called import definition scheme This scheme is a template in which all the information on the arrangement of the data can be saved If you import a file for the first time this field will be empty 148 2000 Stegmann Systemberatung www rs system de Reference Import definition schemes will be generated at the end of the import pro cedure after all other settings of the import have been selected PLA Import Wizzard D PLA 1 2 1mport 043011 96 PLA Import Wizzard r Import Definition Scheme IDS Select Input File The settings you will have to make on the following pages are somewhat complex at all When you finished to make these settings you are asked to save them in a Import Definition Scheme special format which is called Import Definition Scheme or in short IDS ae You may now select a previously saved import definition scheme IDS Define Objects Sort and Assign Imported Data Choose Destination Save Import Definition Scheme Control and Summary Import in Progress You may select an Import Definition Scheme IDS if you saved one in a previously run of this wizzard Cancel lt Previous Because no import definition schemes exist you can directly go on to the next dialog page Here you can give the information into which PLA object the data is import
60. actors of the standards and preparations are the same the correct predilution factor will have no influence on the result of the calculation 2000 Stegmann Systemberatung www rssystem de 79 My first Parallel Line Assay Click on the Analysis Settings page now Before you fill out this dialog consider which settings for the statistic parameters of the analysis you will need For the evaluation of the tests for the slope linearity and parallelity al ways choose to work with a confidence level of 98 0 Set the confidence interval for the relative potency to 95 0 If one of the tests is not passed the relative potency should not be calculated and the assay should be rejected To achieve this your analysis settings should look like the fol lowing figure 1 In the following 3 fields set the 2 Set the fiducial value to 98 0 and check the re limit to 95 0 jection criterion box Project Beispiel Nr 1 lolx General Reagents Definitions Analysis Range Belektion Annotations GLP General I Analysis according to Europ Pharmacd r Regression Model lin log D I Reverse Reshonse Hffekt Correlation m Probabilities Dixon test contamination alpha 0 2 Significance of the slope ST 98 0 IV rejection criterion Significance of deviations from linearity 2 198 0 zl rejection criterion Significance of deviations from parallelity 25 98 0 zl rejection criterion Fiduci
61. al limit of potency estimation DT 950 Values marked are used only by objects whose potency will be calculated r EC50 Calculation Method Do not calculate Calculate 50 Response by 3 Click on the Range Selec tion tab to go to the range selection dialog 80 2000 Stegmann Systemberatung www rs system de My first Parallel Line Assay Leave all other settings on the default values On the tab Range Selec tion you can decide which method is used for the selection of the linear and parallel interval For our example choose the following settings The linear and parallel range of our preparations and standards are to be determined automatically in a way that 1 the linear range for each preparation can be found in different inter vals 2 the linear range for the standard must always be in the same interval Each preparation is compared with the standard individually With this restriction you prevent that different linear regions are chosen for the standard in each comparison 3 in each preparation there must be at least three dilution steps in the linear range 4 the range selected should be as large as possible meaning as many measurements as possible should be within the region To achieve this you have to change the settings in the range selection dialog to the following 2000 Stegmann Systemberatung www rssystem de 81 My first Parallel Line Assay
62. al settings of the project on the page you can see on the screen 78 2000 Stegmann Systemberatung www rs system de My first Parallel Line Assay Keep in mind that the settings on the project level are not used immedi ately for the calculations Instead they serve as a template for the object you create in the next steps L Set the number of dilution steps to 7 Project Beispiel Nr 1 Bisi x General Reagents Analysis Rangd Selection Annotations GLP m Concentrations and Predilutions Concentration of the undiluted standard 1 0000 in units Predilution factor of the standard 1 11 0000 Predilution factor of the preparatio 1 11 0000 m Dilution series _ Number of dilution steps in a data series d SH pa Number of data series S Additional predilution factor for the Det step 1 1 si Dilution scale 1 in 2 series DI 2 Set the number of data 3 Go to the analysis settings by series to 2 clicking on the Analysis tab All measurements in this example have the following settings in common the number of dilution steps seven in our case and all data series have been taken twice When you enter the dilution values the numbers will be copied to the assays standards and preparations Furthermore the predilu tion factors in the preparations and standards are the same In this case the respective input fields can be left unchanged Note If all the predilu tion f
63. ard Users System Inspectors System Administrators Administrator can create accounts and modify GLP SOP Settings Save Cancel You can now change the name the password and the user group of this account You will probably use this function most often because a user has forgotten his password Press the Save button to activate the changes To dismiss the changes press the Cancel button 48 2000 Stegmann Systemberatung www rs system de PLA Administration 5 1 3 Deleting an Account To delete a user account from the account management you have to login to PLA as an administrator Perform the following three steps to delete the account 1 Start the account management 2 Select the user that you want to delete 3 Press the Delete button 00068 Delete Request E S You have selected the account Dr Thomas Wagener to be deleted Do You want to delete this account Nein Abbrechen To delete the user account finally press Yes in the pop up dialog other wise press No In both cases you will get back to the account manage ment dialog 2000 Stegmann Systemberatung www rssystem de 49 PLA Administration 5 2 Changing Your Password PLA offers two options to change a password The first option is available to administrators only and can be used if a user has forgotten his password see 5 1 2 The second possibility is available for any user Select Prefer encesIChange your
64. asurement values devi ate significantly from linearity The model for the analysis is y atbxtcx y corresponds to the transformed values x is the logarithmic concentra tion a b and c are the regression coefficients The deviation from linear ity is checked by comparing the F value of the quadratic regression coef ficient to the critical F value If the calculated F value is less than the critical F value one can assume that the measurements do have a signifi cant deviation from linearity and that the validation criterion is fulfilled 10 1 3 3 Significant Deviation from Parallelity The proof for the parallelity is based on two variance analyses over the measurement of all objects The first analysis uses the model y a bx 182 2000 Stegmann Systemberatung www rs system de The Statistics Core of PLA n 1 for the standard n 2 for the preparation With this model the slope of both regression lines is calculated The second analysis is done with the model y a 6 x With this variance analysis both regression lines are estimated together The deviation from parallelity is determined by the F value of the differ ence between the square sums of both models If the F value is less than the critical F value both regions are assumed to be parallel 10 1 4 Calculation of the Relative Potency according to Pet ler The calculation of the relative potency and of the confidence intervals is done according to
65. ates the 50 response from all ob jects 2000 Stegmann Systemberatung www rs system de 117 Reference 8 5 5 Linear Selection The fifth page of the object definition dialog contains the settings for the selection of the linear and parallel range of the measurements End of linear range Linear range start Selection method Bi Assay Europ Pharmacop 1993 General Reagents Definitions Analys Al et de Selection Annotations GLP m Linear Range Automatic Detection individual range K m Manual Selection Wptans Fixed Range m Automatic Selection Options used by automatic detection methods Allocation Strategy Allocation region Best Range DI ANG 3 S Minimal of points B z maximal allocation range Inclusion of 50 response Calculate the 50 response Py using the ignore this of Sa First point of Tr ES Calculate 50 response range of the following object Use maximum number of Minimum number of points points for allocation range Allocation strategy Last point of allocation range Include 50 response 118 2000 Stegmann Systemberatung www rs system de Reference On the top of the page you can pick the selection method you would like to use from the list You can choose from four different methods three automatic approaches and one manual scheme By selecting an automatic mechanism all inp
66. be overwritten Sort and Assign Imported Data ee P Choose Destination Define Objects Save Import Definition Scheme Control and Summary Import in Progress If you need to use the settings you have made in a later run of this wizzard you can save the settings in an import definition scheme IDS Cancel lt Previous The purpose of import definition schemes is to faciliate the import proc ess The settings you have to manipulate during the import process are quite complex In most laboratories the assays are measured in a standard arrangement e g of the 96 well plates However the import settings are normally the same for all assays With the import definition schemes you can save all your import settings so you can use them for the import In this way import definition schemes will save you a lot of time because you load all your settings from a file Furthermore this also will avoid errors because you only have to check you import settings once Choose Yes on the dialog page and enter a name for you scheme in the input field below The next time you call the import function the scheme will be available On the next dialog page you will see a summary of your input data Check if your settings are correct If you find an error on this page you can always go back using the Previous button and do changes on the according dialog page 2000 Stegmann Systemberatung www rs system de 159 Reference P
67. c positive controls are assigned to dilution step one negative controls are dilution step 2 Blanks would have been as signed to dilution step 3 if they had been taken in the assay In our exam ple you have mark column 1 and assign the value 2 for the dilution step Process column 12 in the same way but assign the column to dilution step 1 The data assignment is now finished and you can go to the next page The Next button will be available now 2000 Stegmann Systemberatung www rs system de 155 Reference PLA Import Wizzard D PLA 1 2 1mport 043011 96 PLA Import Wizzard Select Input File Import Definition Scheme Select Destination Scheme Define Objects Sort and Assign Imported Data Choose Destination Save Import Definition Scheme Control and Summary Import in Progress Please assign the displayed data to the objects You may use the keyboard to assign selected data points to an object select the abbriviation characters ab etc and numbers to assign the step Assign mode Please the data points to the objects op a oo ro rPrrrrrrr EEGEN r Selected datapoint Grid display options fe Control C z display by row gt Edit ciel Dilution step Point Data value display point H of columns P gf gm GE 2 a VW color the grid lt Previous Next gt The dialog box Grid Display Option has been explained yet Let us leave the example for a moment to disc
68. ction of the linear and parallel range The following choices are available 1 Best Range 2 Maximum Range 3 Exact Range Allocation strategy Inactive if manual detection is activated Minimal of steps Depending on the selected allocation strategy Exact of steps either the minimum or the exact number of points is given Inactive if manual detection is activated 2000 Stegmann Systemberatung www rs system de 69 Terms and Concepts Property Allocation Region Maximum Allocation Range Inclusion of 50 response Calculate the 50 response by using 6 4 6 Annotations Property Annotations Description You can restrict the interval in which the auto matic detection tries to allocate the linear and parallel range You must select Maximum Allo cation Range in the Allocation Strategy field for this feature Inactive if manual detection is activated If you activate this option the whole concentra tion range will be used for the automatic detec tion Inactive if manual detection is activated With this feature you can select if the 50 response of an object is to be taken into ac count for the determination of the linear and parallel range The following choices are possi ble I Ignore 2 Inclusion if possible 3 Inclusion is mandatory Inactive if manual detection is activated Choose the object of which the 50 response for the selection of the linear and
69. d you must register PLA PLA Registration Wizard PLA Registration version 1 2 1999 Stegmann Systemberatung welcome to a 14 day triall Press the Ok button to start reducing allocated usage Press the Cancel button to postpone starting until later does not start reducing allocated 40 2000 Stegmann Systemberatung www rs system de System Requirements and Installation If you decide to use PLA the program will display the following screen until you register your program Press the OK button PLA Registration Wizard PLA Registration version 1 2 1999 Stegmann Systemberatung Your trial has 14 days left Please contact us for instruction on how to acquire this product Thank you for trying out our product Please press the Next button to view options Registration Number The shown number red backround is your registration code red code Please copy this number to the supplied registration form and send it to the Stegmann Systemberatung We will send you an unlock code which you enter in the registration wizard to lift the time limitation Note The registration code is unique for the computer It will not work on any other machine Before you send us the red code please be sure you have installed PLA on the computer you want to use for production with PLA 2000 Stegmann Systemberatung www rs system de 41
70. damages to you or a third party for any reason whether for claims arising from this agreement or tortious liability including ordi nary negligence the amount of the damages to be paid is limited to the sum invoiced for the Product that caused the damage or for the Product on which a claim for compensation from Stegmann Systemberatung is based However this limitation shall not apply if Stegmann Systemberatung is responsible for the damage due to malice aforethought or gross negli gence Stegmann Systemberatung shall in no way be liable for damages caused by violation of duty on your part consequential damages economic losses damages due to loss of data or for claims from third parties put forth against you 6 Termination of the Agreement Should you fail to comply with the provisions of this agreement your license and this agreement shall be terminated The provisions in paragraphs 3 4 and 5 shall remain in full force and effect even after termination of this agreement Version dated 02 09 1999 vi 2000 Stegmann Systemberatung www rs system de 2000 Stegmann Systemberatung www rs system de vii viii 2000 Stegmann Systemberatung www rs system de 1 Contents 1 C E ix 2 How to read this manual sesesuesesnesuesesnennesesnennesesnennesesnenussennen 13 3 Introduction ussessesnesesnennenesnennenesnennenesnennenennennesennennesennennssesnenusnen 15 3 1 Parallel Line Assaysa 2 22 22 Ze
71. dialog you see that we will create a protected scheme You must be logged in as an administrator user to be able to perform this task If you belong to the standard user group you can only create user 2000 Stegmann Systemberatung www rs system de 167 GMP GLP Settings in PLA defined schemes However user defined schemes are not compliant with GLP GMP regulations Under topic 2 you can enter a name for the scheme that you want to cre ate Press the Create button to open the object dialog where you can enter the desired definitions Change to the definitions tab Enter the number of data series 5 and dilution steps 8 These are all the settings we will need for our example Create the scheme for the controls now Select the scheme you have just created in the navigator and choose New from the task bar r Select object type to create 1 e New Protected Scheme Special Standard Scheme as Part of Protected Scheme amp Special Preparation Scheme as Part of Protected Scheme ea Special Control Scheme as Part of Protected Scheme Cancel r Input Code and Title 2 a Kontrole Kontrolle r Select a creation scheme 3 E none DI Hint if there is an active mandatory global or parent scheme this selection will be ignored Select Special Control Scheme as Part of Protected Scheme under topic 1 because we want to create a scheme especially for controls After you have
72. e 10f4 08 10 99 10 06 47 Project Validierungsbeispiele Assay Linder Results of Assay Linder Project Validierungsbeispiele Current user Dr Matthias Schmitt 1 Data Input Data index x technical outlier o outlier found by Dixon test 11 Standard Standard tep X log Series 1 Series 2 Series 3 Series 4 Series 1 1 0000 0 0000 487 525 585 600 715 2 0 5000 1 0000 150 350 275 122 410 3 0 2500 2 0000 164 255 64 95 154 tep X log Mean S D LAA 1 1 0000 0 0000 582 400 87 028 14 9 2 0 5000 1 0000 261 400 124 458 47 6 3 0 2500 2 0000 146 400 73 473 50 2 50 Response 364 400 1 2 Sample Probe 1 Series 2 Series 3 Series 4 1 0 5000 1 0000 710 516 620 510 650 2 0 2500 2 0000 560 268 372 247 185 3 0 1250 3 0000 83 84 90 84 75 tep X log Mean S D LAA 1 0 5000 1 0000 601 200 86 817 14 4 2 0 2500 2 0000 326 400 146 933 45 0 3 0 1250 3 0000 83 200 5 357 6 4 50 Response 342 200 Caadatedhy PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany wwwrs system de 2000 Stegmann Systemberatung www rssystem de 193 Appendices Page 2 of 4 Project Validierungsbeispiele 03 10 99 10 06 47 Assay Linder 2 Definitions Standard Probe 1 Perform Dixon Test YES YES Regression Model linlog linNog Reverse Response Effect Correl NO NO Selected Method Fixed range Fixed range Allocation Strategy Minimal No of Points Maximal Allocation Range Allocati
73. e 16 3 2 PLA 132 Sasa aa cates tes we 19 3 3 What is new in this Version 22 4 System Requirements and Installation 00006060000000000000000000 25 4 1 System Requirements ucasessesnenesessensennennennennenennennennennnnnenenononnennn 4 2 Installation akas bang et are E etlech 4 2 1 Preparing the Installapon ce eceeseeseeeeeeseeeeeeeeeeeeeeneeeaees 4 2 2 Step 1 Installation of the SVM fe 4 2 3 Step 2 Start the Installation Program 29 4 2 4 Step 3 Welcome to the Setup Program 31 4 2 5 Step 4 License Agreement 4 2 6 Step 5 Choosing the Setup Type 4 3 First Time Configuration of PLA oz 4 3 1 Starting PLA for the first Time 4 3 2 Administrator Login 0e0eseseseeoenenenesenennanenenenenannananananene 4 3 3 The PLA Registration Wizard sesen0e0enenenenaenenanannnene 5 PLA Administration rsorsssssssosssnssnsssssussnssonsnnsnnsnnssnsnnsnnsnnsnnnnnne 43 5 1 Account Management 44 5 1 1 Creating a New Account 45 5 1 2 Changing an Existing User Account 1 48 5 1 3 Deleting an Account 1 49 5 2 Changing Your Password 50 5 3 Program Options Bet nges ona nanena wanan enasanonoa anane 51 5 3 1 Report Settings 2 Ta AAN a NANANG WANA SENA A A A A 51 5 3 2 Setting the Application Paths eseesessessessensennenneneeneennnn 54 2000 Stegmann Systemberatung www rs system de ix Contents 6 Terms and Concepts 00000000000000
74. e Produkt inklusive aller Komponenten den Medien und Drucksachen allen Upgrades und diesen Lizenzbe stimmungen bertragen und der Dritte diesen Lizenzbestimmungen zustimmt Falls dieses Produkt ein Upgrade ist muss die bertragung alle vorherigen Versionen des Produkts einschlie en 3 Garantie 2000 Stegmann Systemberatung www rs system de iii Die Stegmann Systemberatung garantiert Ihnen f r zw lf 12 Monate ab Auslieferungsda tum folgendes a dass die Leistung der Software in allen wesentlichen Merkmalen der im Handbuch gegeben Beschreibung entspricht vorausgesetzt sie wird auf einer geeigneten Hardware eingesetzt und b dass das Medium auf der die Software gespeichert wird im wesentlichen frei von Material und Herstellungsfehlern ist 4 Haftungseinschr nkungen Im Falle der Verletzung dieser Garantie ist die Stegmann Systemberatung ausschlie lich verpflichtet nach eigener Wahl das Produkt oder Teile davon kostenlos zu reparieren oder zu ersetzen Im Falle dass die Stegmann Systemberatung nicht in der Lage sein sollte dieser Verpflichtung nachzukommen sind Sie berechtigt vom Kaufvertrag zur ckzutreten Garan tieanspr che m ssen innerhalb der Garantiezeit und sp testens sieben 7 Tage nach Auftre ten des Defekts schriftlich an die Stegmann Systemberatung gemeldet und belegt werden Die defekten Produkte sollen an den Distributor zur ckgesandt werden bei dem sie bezogen wurden falls sie nicht direkt b
75. e Substance Database Reagent information refer to chapter 8 5 2 Reagents Definitions information refer to chapter 8 5 3 Definition Settings Please be very careful with this option Analysis information refer to chapter 8 5 4 Analysis properties e Linear selection information refer to chapter 8 5 5 Linear Selection Most common application of this function GMP GLP definitions refer to chapter 1 S 2000 Stegmann Systemberatung www rssystem de 133 GMP GLP Settings in PLA Copy the substance information Copy the reagent information Copy the assay definitions Copy the analysis definitions Copy the linear selection definitions Copy the GLP GMP definitions S Apply Settings cf Project Beispiel Nr 1 to its childs E You are aboyf tg apply She sefected settings of the current object to its descendents WARNING You ate able to destroy some definitions and set the child 6 yabfe state Apply Aupjezt information only apil deiert information Apal Assay definitions Number of series steps concentration doses etc Apply analysis definitions Statistical parameters regression model etc 1 Apply linear selection definitions Detection type strategy linear ranges etc apply GLP SOP settings Select Destination Objects Standard Preparations Controls Copy to assays a Copy to standards
76. e deviation from linearity must not be significant in any object e The deviation from parallelity of a preparation in comparison to the standard must not be significant The checks for the above criteria are done with the help of several F tests PLA supports statistical reliabilities 1 0 of 90 95 98 99 99 5 99 8 99 9 99 95 99 98 and 99 99 in every F test These values correspond to a significance level of 0 1 0 05 0 02 0 01 2000 Stegmann Systemberatung www rssystem de 181 The Statistics Core of PLA 0 005 0 002 0 001 0 0005 0 0002 and 0 0001 The critical F values quantiles are calculated within PLA 10 1 3 1 Significance of the Slope PLA does a variance analysis ANOVA to prove the significance of the slope The parameters of a linear regression over y atbx are determined y is the transformed measured value x the logarithmic concentration a the intersection with the abscissa and b the slope of the regression line PLA always uses the logarithm to the base 2 in logarith mic calculations A F test is performed in which the F value of the model y a bx must be greater than the critical F value If the F value is greater the linear regression has a significant slope and the validation criterion is fulfilled 10 1 3 2 Significant Deviation from Linearity PLA performs a variance analysis ANOVA for each object that contains more than two dilution steps to determine if the me
77. e input field You can change the status of the box by pressing the Toggle status indicator button Hint You can use the TAB and RETURN key to move between the input fields Close the input dialog or return to the object dialog to save your data 2000 Stegmann Systemberatung www rssystem de 125 Reference 8 7 The Check Function You can start the check function from the PLA task bar or from the menu It can only be applied to standards and preparations The purpose of the function is to give you a quick overview on a single object The check function performs a test of the respective object against itself S Preliminary check of Probe 2 vs Standard EN A Standard o o c o o Ki E m Probe 2 1 5 3 0 Dose log69 Hypothesis Testing Potency Estimation Preparation Standard Test of Linearity PASSED PASSED Estim Potency 0 504 Test of Slope PASSED PASSED Fiducial Limits 043 T 052 Test of Parallelity PASSED Hint These results are preliminary Use Calculate for final results and printing Finished When you call the check function the calculation starts immediately After it is finished you should see the above overview screen of the results The results shown are always with regard to the chosen confidence interval for the relative potency the slope linearity and parallelity hypothesis The graph of the mean values is shown as a solid line Press the
78. e object Beispiel Nr 1 was created successfully The creation was not based on anything 2000 Stegmann Systemberatung www rs system de 77 My first Parallel Line Assay Additionally you will get further information from which object the defini tions have been copied parent object or scheme After you have pressed the OK button the object dialog appears You should see the page with the general settings now Most of the settings in this tab have no influence on the calculation In the following steps we will limit ourselves to the settings that are important for the analysis These are the definition of the dilution steps the data series and the analy sis settings Project Beispiel Nr 1 OF Xx Reagents Dei tions Analysis Range Selection Annotations GLP Code Beispiel Nr 1 Title Beispiel Nr 1 m Measured Substance none Select substance Charge identification of the substance Status r Date of Meassurement 7 Switch Object Type IT finished REECH AF Eieren no display in navigator 03 12 96 20 05 17 nja akan m History Last modified by Dr Matthias Schmitt at 16 06 99 15 14 12 Created measured by Stegmann Systemberatung at 03 12 96 20 05 17 Parent Object 1 Click on the Definitions tab to enter the general definitions When you click on the index tab Definitions the appearance of the dia log changes You can change the gener
79. e relative potency according to Fieller s Theorem You choose from 90 95 99 and 99 9 here The last section on this page covers the calculation of the EC50 The EC50 is the effective concentration of a test substance at 50 response The calculation of this value is done with the equation for a regression line y mx b Two variables must be known for the calculation of the EC50 the slope and the 50 response You can choose one of the follow ing options Do not calculate The ECs will not be calculated Linear regression of the The ECs is calculated from the linear re standard gression of the standard Common regression of The ECs value will be calculated from the the assay linear regression of the whole assay For the calculation the common slope of the stan dard and the preparations will be used This slope is also used for the calculation of the relative potency 116 2000 Stegmann Systemberatung www rs system de Reference In the input field calculate 50 response b you can specify from which object the 50 response will be calculated Essentially the 50 response is the difference between the maximum and the minimum reading divided by 2 The following objects can be chosen This object The 50 response will be calculated from the respective object Standard Calculates the 50 response of the standard Control Calculates the 50 response from a positive and negative control All objects Calcul
80. ect Validierungsbeispiele Current user Dr Matthias Sch saseg aana a adha bak naga aa a ag aa AE EE anaa aa Date Signature 1 Data Input Data index x technical outlier o outlier found by Dixon test 11 Standard Standard S tep X log Series 1 Series 2 Series 3 Series 4 Series 1 1 0000 0 0000 289 221 267 236 250 2 0 2500 2 0000 300 310 330 290 364 1 1 0000 0 0000 231 229 269 233 259 2 0 2500 2 0000 328 390 360 342 306 tep X loga Mean S D LAA 1 1 0000 0 0000 248 400 21 996 89 2 0 2500 2 0000 332 000 32 042 9 7 50 Response 290 200 1 2 Sample Preparation Z tep X log Series 1 Series 2 Series 3 Series 4 Series 1 1 0000 0 0000 236 213 283 269 251 2 0 2500 2 0000 250 268 273 240 307 tep X log Series 6 Series 7 Series 8 Series 9 Series 10 1 1 0000 0 0000 294 223 250 216 265 2 0 2500 2 0000 270 317 312 320 265 tep X log Mean S D LAA 1 1 0000 0 0000 250 000 28 012 11 2 2 0 2500 2 0000 282 200 29 234 10 4 50 Response 266 100 Calaulated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs system de 210 2000 Stegmann Systemberatung www rs system de Appendices Page 5 of 7 Project Validierungsbeispiele 08 10 99 10 07 56 Assay Europ Pharmacop 1993 2 Definitions Standard S Preparation Z Perform Dixon Test YES YES Regression Model linAog linNog Reverse Response Effect Correl NO NO Selected Method Fixed range Fixed range Allocation
81. ect again Be careful If you change your project data in a project where you have already created assays the changes are not automati cally copied to the existing assays The project data is only copied on the creation of an assay 62 2000 Stegmann Systemberatung www rs system de Terms and Concepts 6 3 Inhentance A central element in the usage of PLA is the concept of inheritance To illustrate this concept one might use the analogy to genetic inheritance An ancestor passes its genetic information on to its descendants Within PLA inheritance works in the following way The first generation the topmost hierarchy level is always a project The second generation second level is always an assay The third level is a standard a preparation or a control If you create an assay it will inherit the information of the project it belongs to Analogously all the standards preparations and controls will inherit the information of the parent assay on creation In this way you need to enter the common information for the assays or the standard preparations controls only once on the next higher level of the object hierarchy If you later create a new object all the information from the next higher level in this case the assay level will be copied to the new object automatically It is important to understand that the analogy to genetic inheritance is valid only on the creation of new objects If you modify a higher l
82. ed The object will be imported to a new project by default You can change this setting by giving an existing project in the input field Destination Project The second input field allows you to attach object definitions to the im ported data from an object definition scheme This feature is important if you want to add GLP GMP information to the project For further infor mation on the GLP GMP settings refer to chapter 9 2000 Stegmann Systemberatung www rssystem de 149 Reference PLA Import Wizzard D PLA 1 2 1mport 043011 96 PLA Import Wizzard Select the Destination Project Select Input File Import Definition Scheme Select Destination Scheme Define Objects Default Scheme for Creation of Objects Sort and Assign Imported Data EISE Choose Destination Save Import Definition Scheme Control and Summary Import in Progress Objects in PLA can be created by using schemes You use these schemes in every creation process Please select the one you want to use here Please also select the destination project of your imported data Cancel lt Previous Switch to the next dialog page on which you have to supply general in formation on the number and type of the objects that you want to import PLA Import Wizzard D PLA 1 2 1mport 043011 96 PLA Import Wizzard Raw data objects general settings Select Input File Number of objects D Tal number of
83. egion of the standard you should define the fixed range in the definitions of the standard Depending on whether you want to allow a free choice of the linear region for the preparations you should set the allocation strategy to Automatic detection Individual range or to Automatic detection Identical range if you want to keep the linear range of the preparation within the bounds of the standard Besides you have the possibility to influence the results of the analysis by changing the evaluation criteria The allocation strategy allows you to 186 2000 Stegmann Systemberatung www rs system de The Statistics Core of PLA choose between the linear range with the best agreement Best Range the range with the maximum number of points Maximum Range and a range with a given number of points Additionally you can enter a lower limit for the number of points in the linear region Minimum of points You can narrow the interval in which PLA tries to detect the linear region by giving an allocation region Furthermore you can include the 50 re sponse within the linear region You can either force the program to do so inclusion is mandatory or propose the program to include the value include if possible With the latter option the program will include the 50 response only if the assay is still valid 2000 Stegmann Systemberatung www rssystem de 187 The Statistics Core of PLA 188 2000 Stegmann Sys
84. egmann Systemberatung www rssystem de 3 Introduction 2000 Stegmann Systemberatung www rs system de 15 Introduction 3 1 Parallel Line Assays Parallel line assays are used in biological and pharmaceutical research A parallel line assay determines the effectiveness of a prepared sample in comparison to a standard The standard sample has a defined effectiveness of 1 0 The result of such an analysis is the so called relative potency of the prepared sample The relative potency is a dimensionless factor so it can be given in percent For example a relative potency of 2 0 correlates to an effectiveness that is twice as big as the standard The calculation of the relative potency is based on measurable properties observables that correlate to the logarithm of the concentration If the observable depends linearly on this function a relative concentration can be calculated by a parallel line assay An example is shown in Figure 3 1 The relative potency can be calculated as the distance of the regression lines at a fixed value of the observable We can use the following two equations for the calculation of the potency Both lines are supposed to be parallel their slope m is equal Thus for every point y on the regression line y mx bp mx by P prepared sample S standard sample where x is the logarithm of the concentration The relative potency RP is then given as log RP Ax x Xp Pp
85. ei der Stegmann Systemberatung erworben wurden Die Kosten f r Versand und Transportversicherung sind von Ihnen zu bernehmen AUSSER DER OBEN ANGEF HRTEN GARANTIE WERDEN VON DER STEGMANN SYSTEMBERATUNG WEDER AUSDR CKLICH NOCH STILLSCHWEIGEND KEINE WEITEREN ZUSICHERUNGEN GEGEBEN BEZ GLICH DES PRODUKTS INSBESONDERE NICHT BEZ GLICH DESSEN WIRTSCHAFTLICHEN VERWERTBARKEIT UND VERWENDBARKEIT F R BESTIMMTE ZWECKE 5 Schadensersatz Sollte die Stegmann Systemberatung gegen ber Ihnen oder Dritten aus welchen Gr nden auch immer sei es aus Vertrag oder deliktischen Anspr chen einschlie lich leichter Fahr l ssigkeit schadenersatzpflichtig sein so wird die H he des zu leistenden Schadensersatzes auf die Summe beschr nkt die f r das Produkt das den Schaden verursacht hat oder auf grund dessen die Schadenersatzpflicht der Stegmann Systemberatung entstanden ist in Rechnung gestellt wurde Diese Beschr nkung gilt jedoch nicht falls die Stegmann Sys temberatung den Schaden wegen Vorsatz oder grober Fahrl ssigkeit zu vertreten hat Auf keinen Fall haftet die Stegmann Systemberatung f r Sch den die auf von Ihnen zu vertre tenden Pflichtverst en beruhen f r Folgesch den Verm genssch den Sch den aufgrund Datenverlusts oder f r Anspr che die von Dritten gegen Sie geltend gemacht werden 6 Vertragende Sollten Sie Bestimmungen dieses Vertrages nicht einhalten f hrt dies zur Beendigung Ihrer Lizenz und dieses Vertrage
86. els If you have selected a 1 2 dilution series J in 2 series as the dilution scale the dose values will already be filled in when you open the data editor Dose of series Data input field Status of the measurement ed ee Ol x Concentration Series N 1 Series N 2 14 67 0o00 M 67200000 12 623 00000 4 617 00000 4 1 4 572 00000 M 562 00000 M 1 8 513 00000 M 50600000 M 1 16 454 00000 M 447 000000 1 32 390 oooooW 383 000004 1 64 339 oooooW 345 00000 M L D Toggle status indi gor Me status indicator Un check the indicator at the right side of the value for exclusion Graphical explanation Change the status of the focussed measurement 124 2000 Stegmann Systemberatung www rs system de Reference You can open the data input window by pressing CTRLAD by selection Edit Data from the PLA task bar or by choosing FilelOpenlEdit Object Data from the menu The dimension of the input for the number of dilu tion steps and the number of data series is given in the object dialog The data is entered in the input field of the respective dilution step When you leave the input field the check box for the measurement status will be activated By checking or unchecking this option you control if the step is used for the analysis E g if you discovered a technical outlier in your measurement data you can exclude this value from the analysis by un checking the box right of th
87. en by the head of the laboratory or department The PLA administrator has the highest position in the user hierarchy Additionally to all the rights of a standard user an administrator has the possibility to manage accounts This does include the creation of new account changing passwords and deactivating users Furthermore the PLA administrator can use all the functions for the GLP GMP management With these functions he can define standard operating procedures for the standard user For a description of the GMP GLP settings refer to chapter 1 The laboratory staff members that perform and analyze the biological tests are the standard users of PLA Standard users can use all the functions of PLA except for the account management and the GMP GLP settings Administrators can limit the access to some functions however In this way administrators can enforce standard operating procedures on the standard users The PLA inspector is the lowest level of the PLA user hierarchy Inspec tors can examine the settings and data within PLA but they are not al lowed to change them 2000 Stegmann Systemberatung www rssystem de 45 PLA Administration To create new user accounts choose PreferenceslAccount Management from the menu Account Management Select a user to modify or delete or press the create button to create a new onel Create adify r etmas or aecaunt val Account Name Account Password Retype password
88. entered a name press the Create button to open the object dialog 168 2000 Stegmann Systemberatung www rs system de GMP GLP Settings in PLA Scheme Control Kontrolle Iof x General Reagents Definition amp Analysis Annotations GLP Number of control values Maximum number of control repititions ja Dixon test for data outliers perform Dixon test for data outliers Dixon test contamination alpha 0 1 bu On the tab Definition amp Analysis enter the number of repetition in the appropriate input field This is all you need for the control scheme You should see the following hierarchy in the navigator window EX Navigator Bisi E Schemes D TH Beispiel Schema Kontrolle Di User Defined Schemes If you create a new object using your scheme the default settings will be taken from your scheme settings However if you create a new control object the settings will be taken from the subordinate control scheme In this way you do not have to create a scheme for every object type but only for those that differ from the default settings 2000 Stegmann Systemberatung www rssystem de 169 GMP GLP Settings in PLA 9 2 GLP GMP Settings of the Ob ject Definition The access protection is attached directly to the objects Change to the last tab of the object definitions see Figure 9 3 It does not matter if you define a data object or a scheme because both input masks are t
89. entry Delete a reagent Add a reagent You cannot edit the list directly Instead you have to use the three buttons under the list field Press the Add button to add a reagent to the list Press the Edit button to edit an entry Both functions will open the reagent database With the Delete button you can delete entries from the list 2000 Stegmann Systemberatung www rssystem de 109 Reference Reagent selection and definition Select a reagent Entry field for code Pick list for reagents Batch number Concentration Concentration Edit reagent entry Delete reagent Cancel dialog Apply changes and close dialog You can add delete and edit reagents in this dialog In principle the name batch number and concentration characterize every reagent The concentration input accepts only numerical values The dimension of the concentration should be given with the description of the reagent 110 2000 Stegmann Systemberatung www rs system de Reference Description for the selected reagent Please enter a code Wasserstoffperosid Please enter a description umtited Note the description is displayed in reports instead of the short one shown in the user interface of the program After you have supplied all the necessary information the reagent defini tion is saved in the reagent database The reagent will be available the next time the database is opened I
90. ents substance information protected reagent information protected object definitions protected IV analysis settings protected range selection settings protected direct dose values protected IV data values not editable 2000 Stegmann Systemberatung www rs system de 171 GMP GLP Settings in PLA 2 To activate the protection level you have to press the enable GLP SOP protection button first If you select one of the protected pages you will see that you cannot change the settings on that page Assay Messung 1 Bisi x General Reagents Definition ange Selection Annotations GLP General JE Analysis according to Europ Pharmacopoeia 97 Jv Eeron Divan test for date outliers pita 25 series Regression Model finos z E Reverse Response Elect Corelation Gbsblpes Dixon test contamination alpha 2 d Significance of the slope IT 38 0 A J ejection citenan 98 YA rejection criterion Significance of deviations from parallelity CT bon z J ejection enterion Fiducial limit of potency estimation SO F Values marked are used only by objects whose potency will be calculated m EC50 Calculation Method Calculate 50 Response by This object z Significance of deviations from linearity Do not calculate The kind of GMP GLP protection you can activate is dependent on whether you a
91. ess the button Apply General Settings PLA Import Wizzard D PLA 1 2 1mport 043011 96 PLA Import Wizzard r Raw data objects general settings Select Input File Number of objects 3 lal IV number of steps identical for all objects Import Definition Scheme IV number of series identical for all objects Select Destination Scheme Define Objects List of raw data objects Sort and Assign Imported Data Choose Destination Standard A Save Import Definition Scheme B Preparation B 2 1 C Preparation C 2 1 Control and Summary Import in Progress Please define the type count and general settings number of steps and series of the objects you want to create steps series p E 0 EI Cancel lt Previous Next gt As you can see all three objects are created automatically standard A preparation B and C The number of steps and series is identical for all objects Since the numbers are not correct you have to edit the entries You can edit the settings of the related object in the lower fields of the dialog page Change name as you like e g Standard and change the number of steps and the number of series according to our example eight steps five series Controls are special compared to preparations and standards because three objects positive negative control and blank are combined within one object The number of repetitions is thus given as the number of se
92. essible for PLA administrators 2000 Stegmann Systemberatung www rssystem de 173 GMP GLP Settings in PLA The global GMP GLP settings are shown in Figure 9 4 Access to these options is limited to users with administrator privileges You can set the following options 1 GLP SOP Master protection active By activating this function the field Enable GLP SOP protection mandatory on the GLP tab will be activated in all PLA objects within the PLA database All fields checked under the protection level topic will be locked If there are no fields marked the protection will have no further effect The stan dard users cannot unlock these settings Projects may be created by administrators only This option forbids the standard users to create new projects The administrator has the opportunity to enforce GMP GLP settings in every project that cannot be changed by the user Every assay in the project will automatically inherit the protected information Default scheme for the creation of projects With the help of this option you can choose the basis on which scheme the user can create new projects and assays With the GLP settings of the selected scheme the protected object properties are already defined Note The protection of the object created with this scheme is not activated automatically Either you will have to initiate the protection manually or you must set the option GLP SOP master protection active to a
93. evel object later the changes will not occur in the existing descendants Only newly created objects inherit the modified data However you can influence the inheritance of a new object by giving the name of the object from which the new object will get its data explicitly You can also use the apply function to transfer data e g changed analysis settings from a higher level object to an existing object 2000 Stegmann Systemberatung www rssystem de 63 Terms and Concepts 6 4 Object Properties All objects in PLA project assay standard and preparations contain the same possibilities for the definition of properties The program dialog for the input of the settings is the same for all object types Controls are the only exception because they are described by a reduced set of information On the following pages we will introduce you to all the settings They are divided into seven categories that reflect the structure of the program Settings that are marked with a star are optional They are not neces sary for a successful calculation 6 4 1 General Settings Setting Description Code Abbreviation of the object s the full name This name is mainly used for the screen display e g in the navigator Title Full name of the object This name is used for the printout of the calculation results Measured Substance Name of the examined substance Batch identification Batch identification number of the exa
94. f you want to edit an existing reagent select the entry from the list and press the Edit Description button The reagent description dialog appears and you can change the code as well as the name of the reagent To delete a reagent from the list select the reagent code and press the Delete button The following dialog appears and you will be asked to confirm the deletion 90170 Please Confirm Deletion x k You have selected to delete Reagent Anti lgG 1 from the database The deletion will be rejected if the reagent is used by any object Are you sure Nein Abbrechen If no other object uses the reagent it will be deleted from the list Other wise the deletion process will be terminated 2000 Stegmann Systemberatung www rs system de 111 Reference 8 5 3 Definition Settings The third page of the object definitions contains the fundamental settings for the data The dilution steps and the number of readings must be given here Concentration standard Concentration units redilution standard redilution preparation Number of measurements Number of data series Predilution factor Dose input Bi Assay Europ Pharmacop 1993 x General Reagents 10 W E weni Annotations GLP Concentrations and Predilutions d NO Concentration of the undiluted standard A D in units Predilution factor of the standard Predilution factor of the preparation Dilu
95. f you want to export the lower hierarchy levels as well and if you want to include finished objects On the lower part of the page you can define which data of the object shall be exported The meaning of the check boxes is the same as with those of the apply function E g if you only want to export the measure ments of the object uncheck all other fields Again press the Next button to get to the next page of the export wizard There you will see a summary of all the settings you have selected so you can check them before you export your data 138 2000 Stegmann Systemberatung www rs system de Reference PLA Export Wizzard PLA Export Wizzard Summary of operation Choose type of operation Export Type Standard Export Select output filename Focus Beispiel Nr 1 incl child amp amp finished objects Specify detailed settings Check your selections Options Export in progress Definitions Data Values Subject Information Reagent Information Annotations Export format PLA Native Format PNF Filename Test PNF Directory D PLA 1 2 Export Please check your settings to ensure the correct process definition Cancel lt Previuos If all the settings are correct start the data export by pressing the Finish button PLA Export Wizzard PLA Export Wizzard d Export in progress please stay patient Choose type of operation
96. ference 8 13 About PLA Parallel Line Assay Version 1 2 00 C Copyright 1996 1999 by Stegmann Systemberatung This product is licensed to Stegmann Systemberatung Dr Matthias Schmitt Auestrasse 31 D 63110 Rodgau 101 License Code 101 3041 3595 0230 7415 6637 Current user Dr Matthias Schmitt Stegmann Systemberatung Auestrasse 31 D 63110 Rodgau Germany Phone 4961 06 82 61 90 Fax 49 61 06 82 61 92 E Mail office rs system de Internet www rs system de 2000 Stegmann Systemberatung www rssystem de 161 Reference 162 2000 Stegmann Systemberatung www rssystem de 9 GMP GLP Settings in PLA During the development of this version of PLA special emphasis was laid on the implementation of GMP GLP compliant standards as regulatory authorities demand them For this reason an account management has been closely revised All PLA users now belong to a user group c f chap ter 5 1 Account Management In the following chapter we will intro duce you to additional functionalities of PLA that will allow you to work under GLP GMP conditions 2000 Stegmann Systemberatung www rssystem de 163 GMP GLP Settings in PLA 9 1 Schemes Another tool for the implementation of GMP GLP requirements is the usage of schemes Originally schemes in PLA were developed to facili tate working procedures that are repeated regularly A new aspect within the GMP GLP framework
97. finitions Detection type strategy linear ranges etc apply GLP SOP settings Select Destination Objects E Essay IV Standard IV Preparations Apply Close V Controls As a default all three object types are selected To start the copying proc ess presses the Apply button To cancel the process presses the Close button 2000 Stegmann Systemberatung www rssystem de 135 Reference 8 11 Data Export In this version PLA offers you two new very helpful functions the im port and export of data With these functions you can easily exchange data between PLA and other applications The export function is useful for backup purposes because it allows you to export the complete PLA data base as well as single objects Select Export from the task bar to activate the export wizard PLA Export Wizzard PLA Export Wizzard r Choose the type of operation Choose type of operation Select output filename Export the selected object including definitions amp values Specify detailed settings Note GLP Informations will be lost by exporting the object Check your selections Complete Database Backup Export in progress Please select wether to export a single object tree or the complete database The PLA export wizard will guide you through the data export step by step The blue square on the left always shows you the actual step of the process The first thing that you have t
98. he 50 response namely this object standard control and all objects This object Standard Control All objects The 50 response will be calculated from the respective object Calculate the 50 response of the standard Calculate the 50 response from a positive and negative control Calculate the 50 response from all objects 2000 Stegmann Systemberatung www rs system de 121 Reference 85 6 Annotations The sixth page of the object settings allows you to add text to the com ment or evaluate the object Bi Assay Europ Pharmacop 1993 iof x General Reagents Definitions Analysis Range Selection Annotations cur 122 2000 Stegmann Systemberatung www rs system de Reference 8 5 7 GLP GMP Settings The last page of the object settings allows you to define the protection level of the respective object Since the GLP GMP settings are described later please refer to chapter 9 2 for further information fi Assay Messung 1 gt Enable GLP SOP protection Mandatory a Jl LU a N a x 2000 Stegmann Systemberatung www rssystem de 123 Reference 8 6 The Data Editor The PLA data editor is used for the data input You can enter measure ments as well as dose values here if you have selected direct dose input While the input of the measurements is only possible at the lowest level of the object hierarchy you can enter dose values on all hierarchy lev
99. he same Assay Messung 1 Bisi x Object uses the following scheme it will be used on creation of child objects none r GLP SOP Protection Level Settings E Enable GLP SOP protection Mandatory Enable GLP SOP protection User Level Protection Level Please choose the protected elements substance information protected T reagent information protected object definitions protected analysis settings protected range selection settings protected direct dose values protected data values not editable Figure 9 3 GLP settings within the object definitions 170 2000 Stegmann Systemberatung www rs system de GMP GLP Settings in PLA The protection of an object is a two step process 1 First you have to select the protection level you want to apply to the project For example if you would like to prevent access to the analy sis settings you should chose the respective field If you want to pro tect the access to the measurements you should check the data val ues not editable field Note that the protection of the object has not been activated yet Assay Messung 1 Ol x General Reagents Definitions Analysis Range Selection Annotations GLP Object uses the following scheme it will be used on creation of child objects none m M GLP SOP Protection Level Settings r r m Protection Level Please choose the protected elem
100. ial settings are based on object Messung 1 Press the OK button to open the object setting dialog If you press the Cancel button instead of the Create button the dialog will be closed immediately without generating a new object 2000 Stegmann Systemberatung www rssystem de 103 Reference 8 5 The Object Dialog Every object in PLA except controls has a full set of properties These have already been introduced in chapter 6 4 You can edit the settings in the object dialog The dialog contains seven different tabs structuring the information on the object If you change any of these settings the program will ask you to save the changes upon closing the dialog In the following sections every object setting is described in detail To bring a particular dialog page to the front simply click on the respective tab with the mouse 85 1 General Settings The first dialog page contains the general settings for the object You can change the object s code short title and title here Furthermore objects can be declared finished Checking this box has the effect that the object and all its descendants will not be displayed in the navigator To display finished objects in the navigator you have to check the box include fin ished items there The date of measurement will always be set to the creation date of the object in PLA but you can change the date on this dialog page The format of the time stamp input is dd mm
101. ialog appears As you have seen in the previous chapter all settings you have entered at project level have copied to the assay object These settings are now cop ied to the standard Take a look around the settings To enter the data for the standard you have to start the data editor by choosing Edit Data from the menu The object dialog will now close and you should see the following screen 84 2000 Stegmann Systemberatung www rs system de My first Parallel Line Assay Standard Standard Of x Concentration Series N 1 Series N 2 11 mE 00 12 pawar 00 1 4 ECK non 18 pono noon 1 16 TE CR CC 1 32 ooh 00 1 64 pooh 000 anang Il D Toggle status indicator k lt status indicator Un check the indicator at the right side of the value for exclusion This is the edit mask for the measurement values Now enter your read ings The check box on the right side of the measured values is activated automatically as soon as a value is entered into the edit field The check box is called the status indicator It shows you if the respective value is used for the analysis For further information on the status indicator please refer to chapter 8 You can go to the next field by pressing the tab key After you have entered the data for the standard your screen should look like this 2000 Stegmann Systemberatung www rssystem de 85 My first Parallel Line Assay S
102. ic detection Common range for 128 2000 Stegmann Systemberatung www rs system de Reference the standard and you start the calculation from a preparation object in an assay that contains several preparations the linear range might differ from the one that the program would have found if you had started the calcula tion from the assay level Nevertheless the results are nevertheless correct and valid They may not be the best possible however In the above case PLA will show the following warning w0007 Informational Warning H The results of the calculation may be different from the true results if you start the calculation Gi on the preparation level and have choosen automatic detection common range for the standard To obtain the true results start the calculation on the assay level The following table correlates the type of calculation performed to the object from which the calculation is started Start calculation from Calculation of Project all assays in the project Assay the whole assay Standard the whole assay Preparation the selected preparation in comparison to the standard Calculate is a two step function First the calculation is performed The time that is required depends on the selected method the performance of your computer and the complexity of your assay Before the calculation has finished the Save Print and Preview buttons are deactivated When the calculation is finished the
103. ier o outlier found by Dixon test 1 1 Standard Standard S tep X log Series 1 Series 2 Series 3 Series 4 Series 1 1 0000 0 0000 289 221 267 236 250 2 0 2500 2 0000 300 310 330 290 364 Series 6 1 1 0000 0 0000 231 229 269 233 259 2 0 2500 2 0000 328 390 360 342 306 tep X loga Mean S D LAA 1 1 0000 0 0000 248 400 21 996 89 2 0 2500 2 0000 332 000 32 042 9 7 50 Response 290 200 1 2 Sample Preparation U tep X log Series 1 Series 2 Series 3 Series 4 Series 1 1 0000 0 0000 230 210 280 261 241 2 0 2500 2 0000 310 290 360 341 321 tep X log Series 6 Series 7 Series 8 Series 9 Series 10 1 1 0000 0 0000 290 223 254 216 235 2 0 2500 2 0000 370 303 334 295 315 tep X log Mean S D LAA 1 1 0000 0 0000 244 000 26 808 11 0 2 0 2500 2 0000 323 900 26 926 83 50 Response 283 950 Caadatedhy PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany wwwrs system de 2000 Stegmann Systemberatung www rssystem de 207 Appendices Page 2 of 7 Project Validierungsbeispiele 03 10 99 10 07 56 Assay Europ Pharmacop 1993 2 Definitions Standard S Preparation U Perform Dixon Test Regression Model Reverse Response Effect Correl Selected Method Allocation Strategy Minimal No of Points Maximal Allocation Range Allocation region Inclushion of 50 response 50 Response of YES YES linlog linlog NO NO Fixed range Fixed range Step 1 to Step 2 Step 110 Step 2 3 Results
104. ilution scale direct input direct input Analysis settings Analysis according to EP YES YES Dixon test enabled YES YES Regression model linNog linlog Reverse response effect correlation NO NO Dixon contamination 0 02 0 02 F Test of the slope 98 98 F Test of linearity 98 98 F Test of parallelity 98 98 95 Fiducial limit percents 95 Slope as rejection criterion YES YES Linearity as rejection criterion YES YES Parallelity as rejection criterion YES YES EC50 calculation NO NO by using Range Selection Settings Linear detection type Fixed range 1 Fixed range 1 Allocation Strategy r Minimal Exact of Points e A Maximal Allocation Range Allocation Range Step 1 to Step 2 Step 1 to Step 2 Inclusion of 50 Response 50 Response of Calculetedby PLA Version 1 2 00 PLAis a product of Siegmann Systemberatung Germany wwnurs eystem de 2000 Stegmann Systemberatung www rs system de 215 Appendices Page 2 of 13 08 10 99 10 08 09 Project Validierungsbeispiele Assay Europ Pharmacop 1993 2 Data Input Data index x technical outlier o outlier found by Dixon test 2 1 Standard Standard S tep X loga Series 1 Series 2 Series 3 Series 4 Series 1 1 0000 0 0000 289 221 267 236 250 2 0 2500 2 0000 300 310 330 290 364 tep X log Series 6 Series 7 Series 8 Series 9 Series 10 1 1 0000 0 0000 231 229 269 233 259 2 0 2500 2 0000 328 390 360 342 306 tep X log Mean S D
105. ination folder If you install the program from diskettes you will be asked to change the installation medium for several times The program will tell you when the installation is finished You will see the following screen ie PLA Setup InstallShield Wizard Completed The InstallShield Wizard has successfully installed PLA Click Finish to exit the wizard PLA is ready for use now Read the following chapter to learn about the necessary steps to configure PLA 2000 Stegmann Systemberatung www rs system de 35 System Requirements and Installation 4 3 First Time Configuration of PLA After the successful installation of PLA you have to configure the pro gram before you will to be able to work with it These configuration steps must be completed when you start PLA for the first time You have to perform the following steps for the successful configuration of PLA H Start PLA for the first time The next step is only necessary if you did not receive a SV module with PLA Node lock PLA with the PLA Registration wizard 36 2000 Stegmann Systemberatung www rssystem de System Requirements and Installation 4 3 1 Starting PLA for the first Time You can now start PLA for the first time Choose PLA from the Start Programs menu The program might ask you for the conversion of existing data at start up This conversion is not destructive and can be readily done After the successful conversion PLA
106. ing to this extreme value the r quotient is calculated and then compared to a critical r value from the literature If the r quotient is greater than the critical r value the measurement value will be marked as a statistical outlier These values will not be considered in further calculations This procedure is repeated until there are only two values left or the extreme value is accepted Depending on whether the largest or the smallest value is chosen the r quotient is calculated as follows Iw Dixon Processing Data For Outliers Biometrics 1953 9 74 89 180 2000 Stegmann Systemberatung www rs system de The Statistics Core of PLA Number of Extreme is the largest Extreme is the smallest measurements value N 3 7 Ay Xy A TA Per or rer An TA An TA 8 10 An Xy A TA e a KN Ta An TM 11 13 Xn An Az A r r Xy 8 ANA TM 14 25 An TANI Az A K r Xn E Xy 2 TAI x are the measurements sorted by value Thus xy is the largest and x is the smallest value 10 1 3 Linear Regression In order to be able to perform a valid calculation of the relative potency several criteria have to be fulfilled These criteria are determined by a variance analysis with each object standards and preparations and by a variance analysis with each preparation and the standard together The three criteria that must be fulfilled are e The regression line of each object must have a significant slope e Th
107. ive control were taken with each dilution step With the shown example we will now demonstrate how the import wizard works with an external import module The import wizard starts with the same dialog as for the import of a PNF file You first have to select which data format you want to import PLA Import Wizzard D PLA 1 2 1mport 043011 96 PLA Import Wizzard 1 What type of file do you want to import Select Input File g Plate 96 PLA Native Format PNF PNF Softmax Pro 2 4 1 txt Select Destination Scheme Victor2 txt Define Objects Import Definition Scheme Sort and Assign Imported Data EE Choose Destination Select File D PLA 1 2Mmport043011 96 Save Import Definition Scheme Analysation results Selected File D PLA 1 2 Import 043011 96 Selected Format 96 Well Plate CSV Control and Summary Import in Progress Analyzing the input file Please wait complete Please select the file you want to import Press Select File to choose the import file Input file contains D valid objects There is raw data present Select the data type 96 Well Plate and choose the file and the directory by pressing the Select File button Please note that the files must have the extension 96 for PLA to recognize the data format In the field Analysis Results a summary of the selected object is shown Most important the program will report if the dat
108. l belong You have two options to continue the import process either you create a new object or you append the assay to an existing pro ject Select a project from the drop down list Select a target where the childs will be appended to join the assay to an existing project All projects in the database are displayed in this list The program will check if the same assay already exists in the project If this is not the case you can continue with the import process When you have finished the entries on this page press the Next button to see a summary of the settings you have chosen If you want to change settings use the Previous button to get back to the import wizard 2000 Stegmann Systemberatung www rs system de 145 Reference PLA Import Wizzard D PLA 1 2 Import Ec99061 pnf If all the settings are correct press the Finish button to finally impot the data into the PLA database 146 2000 Stegmann Systemberatung www rs system de Reference 8 12 2 Importing Extemal Data The import module of PLA enables you to migrate your measurements with other systems to the PLA database Because the data format is differ ent for every system you must install an import module for your format In the standard installation PLA comes with an import module for meas urements of 96 well plates in CSV comma separated values format The following example shows you a file with the correct data format amp 043011 96
109. ll be passed to the samples Probably the most fundamental improvements are the import and export feature This new feature allows you to transfer your data automatically into PLA The import interface is designed to easily migrate any data format You can purchase individual import and export converters separately If you do not find your system in the list of available converters we can easily create a new import converter for any measurement instru ment The only requirement for the development of a new converter is 22 2000 Stegmann Systemberatung www rs system de Introduction that you can supply us with the data format Do not hesitate to contact the Stegmann Systemberatung 7 GLP GMP support PLA now contains vastly improved functions for the work according to GLP GMP regulations PLA users are assigned access rights as they are demanded by GLP GMP 2000 Stegmann Systemberatung www rssystem de 23 Introduction 24 2000 Stegmann Systemberatung www rssystem de d System Requirements and Installation ratung www rs system de System Requirements and Installation 4 1 System Requirements PLA needs some RAM for the database management as well as for the generation of graphics and reports Besides the statistics core of PLA needs a certain amount of core memory for the efficient evaluation of the assays This leads to the following minimum requirements for a PC run ning PLA In
110. mined of the Substance substance Status finished If this field is marked the object will be con sidered closed The object is no longer dis played in the navigator but will not be deleted from the database Date of measurement The creation date of the object You cannot edit this field 64 2000 Stegmann Systemberatung www rs system de Terms and Concepts Setting Switch Object Type History Description This option is only active for standards and preparations A preparation object will be switched to a standard and vice versa This field contains information about the crea tion the last modification and the ancestors of the object 6 4 2 Reagents Settings The next index of the object definition gives you the opportunity to enter a list of the reagents in the sample You can document the way the prepara tion was processed by filling out these fields These settings are optional They are not necessary for the analysis Property Reagents Batch Concentration Description Name of the reagent Batch id of the reagent You can only enter the concentration of the reagent as a numerical value Thus you should enter the dimension of the concentration in the name field 2000 Stegmann Systemberatung www rssystem de 65 Terms and Concepts 6 4 3 Definition settings Property Description Concentration ofthe The original concentration of the standard For undiluted standard the analy
111. n Significant Non Linearity on Lineratiy isa Yes SE SE ejection criterion Further Objects i Object rejected ml Figure 10 1 Statistical Analysis Part A 2000 Stegmann Systemberatung www rs system de 177 The Statistics Core of PLA sons First PLA inspects all objects that are necessary for the calculation For every object a Dixon test is performed if this option has been acti vated Subsequently all measurement values within the linear region are tested for significant slope and non significant deviation from linearity If one criterion is not fulfilled the object and the assay as a whole is re jected Depending on the analysis settings PLA may nevertheless proceed with the calculation see rejection criterion in the analysis settings However the assay is not valid in a statistical sense because one of the starting hypotheses is not fulfilled 178 2000 Stegmann Systemberatung www rs system de The Statistics Core of PLA With Every lt Combination Do tandard o Preparation rejected No D B Common Calculation of all Preparation Standard Kombinations Common Linear Regression ignifican Deviation from arallelity No D Estimation of the Relative Potency Further combinations No Assay rejected u
112. n Reagent Database Close Window Save Object Delete Object Tree F2 CTRL N CTRL O CTRL D CTRL F4 CTRL S Open a navigator window Open a create dialog Open the object definitions dialog of the selected object Open the data editor Open the substance database Open the reagent database Close the active window Save the settings for the active object without closing the dialog Delete the selected object including all lower level objects 2000 Stegmann Systemberatung www rs system de 95 Reference Short Cut Apply CTRL A Open the apply function Import Start the import module Export Start the export module Logout Log out from PLA Exit ALT F4 Terminate the program 82 2 Action Menu Short Cut Calculate amp CTRL C Open the calculate dialog Report Check CTRL K Quick analysis Display CTRL I Open a graphical representation of the Graphical Info assay for visual control 8 2 3 Preferences Menu Short Cut Some Menu for the customization of PLA Toolbars Options Open a dialog for application paths and the GLP GMP settings of PLA Bee Open the account management dialog agement Change your Password Open a dialog for changing the password 96 2000 Stegmann Systemberatung www rssystem de Reference Short Cut Check Data f Perform a check of the database consis base Consis tency tency 8 2 4 Help Menu Short Cut Ab
113. n Strategy r Minimal Exact of Points e A Maximal Allocation Range Allocation Range Step 1 to Step 2 Step 1 to Step 2 Inclusion of 50 Response 50 Response of Calculetedby PLA Version 1 2 00 PLAisa product of Siegmann Systemberatung Germany wenurs eystam de 2000 Stegmann Systemberatung www rs system de 221 Appendices Page 8 of 13 08 10 99 10 08 09 Project Validierungsbeispiele Assay Europ Pharmacop 1993 2 Data Input Data index x technical outlier o outlier found by Dixon test 2 1 Standard Standard S tep K logos Series 1 Series 2 Series 3 Series 4 Series 5 1 1 0000 0 0000 289 221 267 236 250 2 0 2500 2 0000 300 310 330 290 364 tep X log Series 6 Series 7 Series 8 Series 9 Series 10 1 1 0000 0 0000 231 229 269 233 259 2 0 2500 2 0000 328 390 360 342 306 tep X log Mean S D C V 26 1 1 0000 0 0000 248 400 21 996 89 2 0 2500 2 0000 332 000 32 042 9 7 50 Response 290 200 2 2 Sample Preparation Z tep X log Series 1 Series 2 Series 3 Series 4 Series 5 1 1 0000 0 0000 236 213 283 269 251 2 0 2500 2 0000 250 268 273 240 307 tep X log Series 6 Series 7 Series 8 Series 9 Series 10 1 1 0000 0 0000 294 223 250 216 265 2 0 2500 2 0000 270 317 312 320 265 tep X log Mean S D C V 26 1 1 0000 0 0000 250 000 28 012 11 2 2 0 2500 2 0000 282 200 29 234 10 4 50 Response 266 100 Caadatedhy PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www r
114. n Systemberatung www rssystem de 131 Reference keep the CTRL key pressed during the selection If you would like to see all objects that are displayed activate the include all field Hint for the graphical representation Linear regions of the mean value s graph are displayed as solid lines Non linear regions are shown as dashed lines 132 2000 Stegmann Systemberatung www rs system de Reference 8 10 The Apply Function Eventually you will be in a situation where you want to copy information from higher level to lower level objects For this purpose PLA offers you the apply function You can call this function from the menu by choosing FilelApply or by pressing CTRL A You should be very careful with the apply function because the settings will be copied without any check of the information s validity E g if you copied the assay definitions the apply function would copy among other things the number of dilution steps and the number of data series A very common way of using this function is the actualization of changed analy sis and linear selection method settings The apply function needs two inputs 1 Which information is to be copied 2 Where should the information be copied Enter the selection of the information which is to be copied in the input field Apply what You can select six different groups of information that can be copied H Substance information refer to chapter 8 5 1 1 Th
115. n this case because the test does only contain two concentration values In the EP the slope and parallelity are calculated with a slightly different scheme of the variance analysis For that reason a direct comparison of the numbers is not possible for all values Annotations on the table SOS is the sum of squares MS is the mean sum of squares The SOS and MS for the respective models are shown SOS error and MS error are the residual errors SOS difference and MS difference are the sums of squares of the differences between the models in the parallelity analysis EP 1997 Parallelity of Preparation U and Standard S SOS difference 34 2 34 2 MS difference 34 2 34 2 SOS error 26587 3 26587 3 MS error 738 5 738 5 F value 0 05 0 05 Relative Potency of Preparation U in Comparison Standard S RP 1 11 1 11 RP upper limit 0 82 0 83 RP lower limit 1 51 1 50 2000 Stegmann Systemberatung www rs system de 205 Appendices 11 2 1 PLA Output PLA Short Report The following output is generated as a so called PLA Short Report It contains the shown values 206 2000 Stegmann Systemberatung www rs system de Appendices Page 10f7 08 10 99 10 07 56 Project Validierungsbeispiele Assay Europ Pharmacop 1993 Results of Assay Europ Pharmacop 1993 Project Validierungsbeispiele Current user Dr Matthias Serni ap a janane nga gp saa Sa a Date Signature 1 Data Input Data index x technical outl
116. nd quality assurance depart ments Furthermore the need for modern program systems with user friendly interfaces arose from the laboratory staff Another aspect that was not taken care of by statistical programs was the electronic archiving of meas urements Especially in research departments the access to older meas urements and analyses is essential for the development of new ideas or standard operating procedures On the one hand PLA is flexible enough to allow the user to vary the analysis side conditions quickly for develop ment of new analysis procedures On the other hand PLA offers all possi bilities for the work under GLP GMP conditions in quality assurance Older systems normally did not offer this flexibility The following list gives you an overview on the features of PLA E Statistical analysis according to the European Pharmacopoeia H A user friendly and modern graphical user interface Anyone who has ever worked with Windows applications will find his way around PLA quickly E Database approach for all data You can access your assay data fast and efficiently E Efficient organization of data You can structure your assays in pro jects and access them with the Navigator that allows you to find your data easily 2000 Stegmann Systemberatung www rs system de 19 Introduction Multi document interface You can work simultaneously on different measurements Extended analysis functionality The program fi
117. nds linear and parallel regions in assays automatically You can fine tune this feature with many available program options Management of additional assay information You can manage all your measurement information with PLA RTF reports are generated including graphics You can print out your reports on any printer that is supported by Microsoft Windows You can load the reports in any standard text processors that support the RTF format e g Microsoft Word Report templates can be easily adapted for special requirements Check function You can immediately see a graphical representation of your data on screen with a preliminary analysis Speed The statistics core of PLA is highly optimized Depending on the number of measurements the analysis normally takes only a few seconds to complete For example the analysis of the Linder standard example preparation and standard with three measurements each taken three times overall 18 measurement values takes less than one second on a standard PC Pentium 133 MHz 32 MB RAM Windows NT 4 0 20 2000 Stegmann Systemberatung www rs system de Introduction We do our best to improve our software If you have any suggestions for new features or improving existing features please let us know You can reach us under the following address Stegmann Systemberatung Auestra e 31 D 63110 Rodgau Phone 49 0 61 06 82 61 90 Fax 49 0 61 06 82 61 92 www rs system
118. nfomation PLA 1 2 uses the common Windows user interface If you have already used common Windows applications you will be able to find your way around PLA quickly All PLA functionalities can be accessed from the menu Alternatively you can use the task bar for the most common tasks In this chapter you will find a description of every element of PLA The only exception is the user management that has already been introduced in chapter 4 System Requirements and Installation Generally you can operate PLA either with the mouse or with the keyboard Use the tab key to move within the dialogs Nearly every window offers the following options SC Parallel Line Assay Minimize Window Maximize window Close window program If you have changed anything within the window you will be asked if the changes should be saved when you close the window 94 2000 Stegmann Systemberatung www rs system de Reference 8 2 The Menu You can access all functions of PLA from the menu bar Many of these functions can also be accessed by short cut key combinations or via the task bar icons Yet some of the functions are only accessible from the menu All functions available from the menu are listed in the following sections A more detailed description is given in the following sections 82 1 File Menu Short Cut Navigator Create Object Open Object Definitions Edit Object Data Open Sub stance Data base Ope
119. nn Systemberatung www rssystem de 107 Reference S Select a substance for meassurement E Code kr 1 Echt Kolnisch Wasser You can change the input fields code and name now To apply the changes to the database press the Save button To delete an entry from the list select the substance code with the mouse and press the Delete button If the substance is used in any object you will see the following error message and the procedure will terminate without any changes to the database E0192 Request Denied amp Sorry the entry is in use You cannot delete entries while they are in use by other objects Generally you can only delete substances that are not in use by any ob ject However this only applies to objects that have already been saved 108 2000 Stegmann Systemberatung www rs system de Reference 85 2 Reagents The second page of the object dialog serves only informative purposes You can specify the reagents that you have used for the preparation of the samples standards and controls on this page The information on the re agents you can specify is the name the batch number and the concentra tion Again you can give acode and a name for the reagent You can add as many reagents as you like Assay Europ Pharmacop 1993 Bisi EN General Reagents Definitions Analysis Range Selection Annotations GLP Reagent Concentration Double Click or Enter to Edit Edit a reagent
120. nnssnsnnsnnssnsnnsnnnnnn 189 11 1 Check A Linder nk es nen Ban as essen 190 11 1 1 PLA Output PLA Short Report 192 11 1 2 PLA Output PLA Complete Statistics Report 197 11 2 Check B European Pharmacopoeia s sesesseseeseseeesrserrrsrerereeseee 205 11 2 1 PLA Output PLA Short Report 206 11 2 2 PLA Output PLA Complete Statistics Report 214 2000 Stegmann Systemberatung www rs system de xi Contents xii 2000 Stegmann Systemberatung www rs system de 2 How read this manual For a general introduction to the program read e chapter 3 Introduction and e chapter6 Terms and Concepts If you also need some introduction to the practical work with PLA refer to e chapter 7 My first Parallel Line Assay If you want to install and configure PLA read e chapter 4 System Requirements and Installation and e chapter 5 PLA Administration If you are working with PLA for the first time read e chapter 6 Terms and Concepts and e chapter 7 My first Parallel Line Assay For more in depth information read e chapter 8 Reference If you want to work under GLP GMP conditions read e chapter 9 GMP GLP Settings in PLA If you are interested in the mathematical background of PLA read e chapter 10 The Statistics Core of PLA 2000 Stegmann Systemberatung www rssystem de 13 How to read this manual 14 2000 St
121. nt O m DDE Note use 1 as filename and Application Topic The DDE commands must be given in the syntax Applica tion Topic Command 1 Command 2 e g Win Word System FileOpen 1 52 2000 Stegmann Systemberatung www rs system de PLA Administration You can change the following settings in the lower part of the dialog 1 Default reports You can select one or more reports from the list These reports will already be activated when you start a calculation Together with the option Print immediately after calculation you can minimize the effort for an analysis To choose more than one re port keep the CTRL key pressed while selecting the report types from the list Note The list of available reports may differ between instal lations 2 Print immediately after Calculation This will activate the setting Print immediately in the calculation dialog as a default Press the Save button to confirm the new settings Press the Cancel button otherwise 2000 Stegmann Systemberatung www rssystem de 53 PLA Administration 5 3 2 Setting the Application Paths You can change the PLA application paths on the next page of the options dialog If you press the ellipsis buttons right of the input fields you can open a standard file dialog to select the directories SE Options E Reporting Application Settings GLPASOP Settings Application Path Settings Report Output Path
122. o decide is if you want to export the whole database Complete Database Backup or a single object from the data base Export the Selected Object To export a single object you have to select that object in the navigator before you call the export function 136 2000 Stegmann Systemberatung www rs system de Reference After you have decided which type of export you want to do press the Next button to get to the next page of the wizard In the second step you select the output format the file name and the directory to which the data will be saved PLA Export Wizzard PLA Export Wizzard Select your output format PLA Native Format PNF Choose type of operation Select output filename Specify detailed settings Check your selections Export in progress r Select output file directory Directory Select Filename Please input or select the output file name and format Cancel lt Previuos Select the output format from the list Normally the only option available is the PNF format Press Select to choose file name and directory Speichern in 3 Export D cl el 043010 pnf gt Sicherung 14 7 PNF Testsatz 15 PNF gt Beispiel Nr 1 PNF gt Sicherung 21 6 PNF Testsatz 17 PNF gt ec99061 PNF gt Sicherung 30 9 PNF 2 Testsatz 18 PNF gt ef PNF sl System Validation PNF Testsatz 38 PNF gt ef390 PNF gt Systemvalidierung PNF 8 Testsatz 39 PNF S
123. of the object some options might not be available If you have not chosen any object from the navigator hierarchy only projects can be gen erated If you have chosen a project as parent only assays can be created If the selected parent is an assay new preparations standards and control can be generated New Object Mandatory Parent Selection Project Any Assay Project Preparation Standard Control Assay Second you can enter a short title code for your new object This name will be copied to the full name title but you can change this name after wards The input for the code and the title of the new object is mandatory Third you can give the name of a template that is used for the creation of the object creation scheme Defining a scheme is a comfortable and efficient way to define default settings for the properties of you newly generated object If you do not select a scheme the settings of the parent will be copied to the new object For the case that you have already cre ated schemes you can pick one of them from the drop down box Please refer to chapter 9 1 if you want to know more about schemes 102 2000 Stegmann Systemberatung www rs system de Reference When you are finished with your input press the Create button to gener ate the object If the operation is successful you will see the following affirmative dialog 10033 Object created E The object Probe 6 was created successfully The init
124. on of the Relative Potency Relative potency of sample Probe 1 in comparison to standard Standard Probe 1 vs Stand Estimated relative potency 2 04 95 Fiducial Limits 1 64 2 57 Relative fiducial limits 80 1 125 8 45 6 8 Calculation of EC50 Standard Probe 1 Method of EC50 calculation We ne 50 Response of EC50 concentration 9 Annotations Calculetedby PLA Version 1 2 00 PLAis a product of Siegnann Systemberatung Germany weww rs system de 2000 Stegmann Systemberatung www rs system de 203 Appendices Page 7 of 7 08 10 99 10 07 28 Project Validierungsbeispiele Assay Linder Additional Annotations Calaulated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs system de 204 2000 Stegmann Systemberatung www rs system de Appendices 11 2 Check B European Phar macopoeia For the second check of the PLA results the examples of the European Pharmacopoeia 1997 section 5 3 Statistical Analysis example 3 2 8 1 are recalculated with PLA Because the EP does not include dilution se ries but only measurements 1 1 1 0 unit per 100g of body mass and 1 4 0 25 units per 100g of body mass a test for the standard S and the prepa ration U will be defined with three dilution steps All values for the dilu tion step 2 the missing 1 2 step will be excluded from the analysis how ever A test for linearity is not possible i
125. on region Step 110 Step 3 Step 110 Step 3 Inclushion of 50 response 50 Response of H 3 Resulis Standai ob TD Na AA Selected linear Range Test of Linearity Fayadraie Fass p e Test of Slope Fee Fariica P r Test of Parallelism Fetttctmadets Faitca P 2 Step 110 Step 3 PASSED 3 7274 4 7472 95 0 0 8177 PASSED 41 4053 4 6672 95 0 0 7611 Step 110 Step 3 PASSED 0 0856 4 7472 95 0 0 8521 PASSED 74 2470 4 6672 30 0 8510 PASSED 0 819 4 225 95 00 0 9439 Relative Potency 95 Fiducial Limits Relative fiducial limits Probe 1 vs Standard 2 040 1 635 2 567 80 1 125 8 45 6 4 Calculation of EC50 Standard Probe 1 Method of EC50 calculation nc nc 50 Response of EC50 concentration Galculatedby PLA Version 1 2 00 PLAisa product of Siegnann Systemberatung Germany www rs system de 194 2000 Stegmann Systemberatung www rs system de Appendices Page 3 of 4 08 10 99 10 06 47 Project Validierungsbeispiele Assay Linder 5 Figure a Standard Response m Probe 1 L 0 0 08 15 2 3 3 0 Dose log x 6 Annotations Calaulated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs eystem de 2000 Stegmann Systemberatung www rssystem de 195 Appendices Page 4 of 4 08 10 99 10 06 47 Project Validierungsbeispiele Assay Linder Additional Anno
126. or the parallel line analysis quickly and efficiently Assays of biological systems often show saturation effects in the region of high and low concentration These regions are unsuitable for a parallel line assay The automatic detection method has been developed to facilitate the analysis of such measurements The purpose of this chapter is to help you to utilize this method efficiently Overall there are four methods for the determinations of the linear and parallel region one manual fixed range and graphical selection and three automatic approaches These methods can be combined arbitrarily You can for example detect the linear region of the standard with a man ual method and the linear region of the preparations with an automatic detection scheme to optimize with respect to the manually selected range There are two groups of automatic methods for each the standard and the preparations Take into account that the analysis with PLA always takes place at the assay level The program always focuses on the standard and all preparations at the same time This leads to the problem that the auto matic optimization of the linear range of the standard can be done either with respect to all preparations or for each probe individually It possible to select if the linear range of the standard should be optimized for all preparations together Automatic detection Common range for standards or for each preparation individually Automatic detection
127. ormance of the software corresponds in all essential characteris tics to the description in the manual provided the software is used with the appropriate hardware and b that the medium on which the software is to be stored is free of material and manufacturing errors 2000 Stegmann Systemberatung www rs system de V 4 Limitation of Liability In the event of a breach of warranty Stegmann Systemberatung shall be exclusively obli gated at its own discretion to repair or replace the Product or parts thereof In the event that Stegmann Systemberatung should not be in a position to fulfill this obligation you are entitled to rescind the purchase agreement Warranty claims must be submitted to Stegmann Systemberatung in writing within the warranty period and at the latest within seven 7 days of the defect s appearance and the defect must be documented Defective Products should be returned to the distributor from whom the Product was originally purchased provided that the Product was not purchased directly from Stegmann Systemberatung You shall bear all costs for shipping and shipping insurance WITH THE EXCEPTION OF THE WARRANTY STATED ABOVE STEGMANN SYS TEMBERATUNG GRANTS NO OTHER WARRANTIES NEITHER EXPLICIT NOR IMPLIED WITH REGARD TO THE PRODUCT AND EXPRESSLY EXCLUDES WARRANTIES ON THE PRODUCT S ECONOMIC EXPLOITABILITY AND USE FOR SPECIFIC PURPOSES 5 Compensation for Damage Should Stegmann Systemberatung be liable for
128. ort tree contains 2 2V a Beispiel Nr 2 target no overwrite Select e 5 0 El vom D Import Definition Scheme D Standard A Select Destination Scheme D amp Preparation B Define Objects D 45 Control C Sort and Assign Imported Data Choose Destination Save Import Definition Scheme Control and Summary A r Modify object instructions Import in Progress Create the object with this code Objects with same code will be overwritten eege or select a target where the childs will be appended Note the target will remain unchanged Beispiel Nr 2 hed IT append to the given target Please choose or create the destination where the imported data will be saved Number of objects 5 Number of objects to import 5 Cancel lt Previous Next gt 158 2000 Stegmann Systemberatung www rs system de Reference Macros only make sense if you want to use import definition schemes These templates are used for the automated import of external data Change to the next page where you can save your settings in an import definition scheme PLA Import Wizzard D PLA 1 2 Import chi2603c txt PLA Import Wizzard Save Import Definition Scheme IDS Select Input File Do you want to save the import settings for further use Import Definition Scheme C yes no Select Destination Scheme a Input the name of the scheme If you select an existing one this one will
129. ou have created the account you can log on to PLA 38 2000 Stegmann Systemberatung www rssystem de System Requirements and Installation 4 3 2 Administrator Login You have to identify yourself to the program in order to gain access to PLA Choose one of the symbols on the left side PLA Administrator for the first login and enter the password in the lower input field Please remember the password is case sensitive Press ENTER or the Logon button to log in Please identify yourself Select yourself PLA Administrator License Information Unregistered Version DON T USE FOR PRODUCTION 7042 9755 8920 0937 3853 Product Information Enter your password Eat If your password is correct you will gain access to the program 2000 Stegmann Systemberatung www rssystem de 39 System Requirements and Installation 4 3 3 The PLA Registration Wizard If you have received a SV module with the software you can skip this section As an alternative to installing a SVM PLA supports the usage of a license number logic This licensing technique is performed by the PLA Registra tion Wizard After a successful login to PLA the PLA Registration Wizard will start up You can choose either OK or Cancel to proceed If you press the Cancel button the program it will terminate immediately If you press the OK button you will get a 14 day trial access to the program Within this perio
130. out Open the about dialog 2000 Stegmann Systemberatung www rs system de 97 Reference 8 3 The Navigator The central element in PLA is the navigator This window controls the access to all objects It includes a hierarchical list of the different object types To learn more about the hierarchical organization of PLA refer to chapter 6 2 EX Navigator OF x Data Objects ha e Di Beispiel Nr 2 a D n Biotest Pharma GmbH Drop down list that Di Factor VIII changes between the data Ei Novartis Pharma and the schemes view Di Report Testing Di System Validation Check this box to include finished project in the data display Explanation of the object include finished items symb ols Project Standard Control Assay Preparation yObject opened The list displays all objects within the database The first level contains the project Click on the symbol on the left side of the project to access the assay level below the projects You can access the samples and con trols from the assay level in the same way With the help of the drop down box in the upper part of the navigator you can switch between the view of the data objects and a view of the defined schemes Schemes are templates for the generation of new data objects 98 2000 Stegmann Systemberatung www rs system de Reference Furthermore they are important for working under GMP GLP conditions Please refer to chapter 9
131. p Pharmacop 1993 Additional Annotations Caadatedhy PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs eystem de 2000 Stegmann Systemberatung www rs system de 227 Appendices 228 2000 Stegmann Systemberatung www rs system de
132. parallel range is used You can choose from this object stan dard control and all objects Inactive if manual detection is activated Description Annotations and further information on the object This information is not passed on by inheritance 70 2000 Stegmann Systemberatung www rs system de Terms and Concepts 6 4 7 GLP GMP Settings On this page of the object definitions you can select the settings for the protection of the object These settings are used for the work under GLP GMP conditions where well defined operation procedures have to be followed A more detailed description of the GLP GMP functions is given in chapter 9 Property Object uses the fol lowing scheme Enable GLP SOP Protection Manda tory Enable GLP SOP Protection User Level Substance informa tion protected Reagent information protected Object definitions protected Description Give the scheme for the object definitions here If you activate this button you can lock the selected protection levels for the user Only PLA administrators can activate or deactivate this setting This button activates the protection levels for the user In contrast to the above option PLA administrator as well as normal users can ma nipulate this function By activating this option you can protect the access to the substance information and prevent changes by the user Activate this option to protect the access
133. plug of the printer cable first and mount the SVM on the printer port with the side that is marked with PARALLEL Attach the printer cable to the other side of the SVM that is marked with SERIAL The SVM will not interfere with the printer and you can use the device normally Printer Cable Printer Computer DI e S lt D 28 2000 Stegmann Systemberatung www rs system de System Requirements and Installation 4 2 3 Step 2 Start the Installation Program 4 2 3 1 Installation from Diskette On the first diskette you will find the program setup exe This pro gram will install PLA on your PC Insert the diskette with the label PLA 1 2 Disk 1 into drive a From the Windows Start menu choose the Run function Dei Programme 4 zl Favoriten gt CH Dokumente 4 Eh Einstellungen gt A Suchen 4 amp Hilfe Ausfuhren Stegmann abmelden CT Beenden In the appearing dialog enter Windows NT Workstation A SETUP 2000 Stegmann Systemberatung www rssystem de 29 System Requirements and Installation Ausf hren 21x Dokuments an das bzw der ge ffnet werden soll ffnen SC v A Gretrennter Speichergereich al Abbrechen Durchsuchen 2 Geben Sie den Namen des Programms Ordners oder 4 2 3 2 Installation from CD ROM Insert the CD ROM into your CD drive The setup program should start automatically If the program does not
134. r2 D bal Import in Progress Choose Destination 2 Select the import file parta 1 2 Import Ec99061 pr f Analysation results Selected File D PLA 1 2 lmport Ec99061 prif Selected Format PLA Native Format PNF Please select the file you want to import Analyzing the input file Please wait complete Press Select File to choose the import file ser el Press the Next button to change to the next dialog page of the import wizard Choose the location to which the object is imported on this page Of course the location is dependent on the type of object you want to import 142 2000 Stegmann Systemberatung www rs system de Reference If you are trying to import a project the program checks if a project of the same name already exists in the PLA navigator If this is the case all subordinate objects assays preparations standards and controls are inspected in turn If all subordinate objects are identical PLA terminates the import process with an error message Otherwise you will get to the next page of the import wizard PLA Import Wizzard D PLA 1 2 Export Beispiel Nr 1 PNF re ett The import tree contains Select Input File 1 Choose Destination ies x Ee Control and Summary D amp Probe 1 Import in Progress D amp Probe 2 D amp Probe 3 DN amp Probe 4 Q amp Probe 5 o D Messung 2 H A d Chandar Modify object instructions Create
135. re logged on as user or administrator As a standard user you can only activate the object access protection on a user level Any other user can lift this protection If you activate the access protection on the administrator level man datory standard users will not be able to change any of the GLP set tings Thus for the realization of GLP GMP requirements especially the administrator protection is important 172 2000 Stegmann Systemberatung www rs system de GMP GLP Settings in PLA 9 3 Global GLP GMP Options In the previous sections you have learned how to protect objects or how to create new objects with the help of schemes But how can you lead your coworkers to work with PLA according to standard operation procedures SOP In this regard PLA offers several possibilities that you can achieve with the help of the GMP GLP settings Select PreferencesiOptions from the menu From the dialog choose the GLP SOP settings tab S Options x Reporting Application Settings M GLP SOP Settings may be changed by administrator only I GLP SOP Master protection active all objects are protected The GLP SOP master protection protects all the objects in the database regardless of the settings in the object definition Projects may be created by administrators only Default scheme for the creation of projects none e Figure 9 4 GMP settings in the options dialog These options are only acc
136. reate a new onel Administrator Heger Dess m Please enter the setting of the new account Enter account name Dr Matthias Schmitt Enter account password Retype password fH This account is member of the following group Disabled Users System Inspectors System Administrators Administrator can create accounts and modify GLP SOP Settings Create Cancel Ease Select the user group of the new account from the pick list in the lower part of the dialog Normally you will choose Standard User System Ad ministrator or System Inspector Press the Create button to generate the new account Press Cancel to abort the account generation The next time the login screen of PLA is displayed the new user will be available 2000 Stegmann Systemberatung www rs system de 47 PLA Administration 5 1 2 Changing an Existing User Account If you want to change an existing user account you can do this with the account management dialog Pick the user name from the list in the upper part of the dialog and press the Modify button Account Management Select a user to modify or delete or press the create button to create a new onel a Administrato a Dr Matthias 5 hmitt r Settings for account Dr Thomas Wagener Change account name Dr Thomas Wagener Change account password Retype password This account is member of the following group Disabled Users Stand
137. s 8 5 4 Analysis properties ike 8 5 9 Ismear Selection date ih ag aaa E aa a dagan paka cet 8 5 6 VAIOLA TOI a KINE aa Anaa a a a KANE AGA Web aa NGGER 8 5 7 GLP GMP Settings ada 8 6 Whe Data Editor os a a sages ahaaa iannis EES 8 7 TDhecheckbuuegen eebe geet aaa Et baang eet 8 8 The Calculate Function u aaa naanin anana khan K 2000 Stegmann Systemberatung www rssystem de Contents 8 9 The Ihfo Fun6tiOn sasasi sasa icr KANG SANGKA NAHEN ENG NE KASANGA Ee 131 8 10 TheApply Function sakahanan an A a e GANG Ea ak bia anah 133 SIL Datt EE EE 136 GEERT 140 8 12 1 Importing PNP File Smisen e a EARE E 141 8 12 2 Importing External Data 147 E E EE 161 9 GMP GLP Settings in PLA 00000000000000 0000000000000000 00000000000000 163 9 1 Schemes a En ee edel 164 9 1 1 An Example on Using Schemes 167 9 2 GLP GMP Settings of the Object Defien 170 9 3 Global GLP GMP Options unensessenesessessersensennennennennenennanan 173 10 The Statistics Core of PLA 000000000000000000000000 0000 anana a0ananan 10 1 Statistical Calculations eses eeeeee eee wanen anna waana n anana nana anan 10 1 1 Transformation of the Measurement Values 10 1 2 Dixon KEE 10 1 3 Linear Regression as sama See eegener 10 1 4 Calculation of the Relative Potency according to Fieller 183 10 2 Automatic Detection een E 185 11 Appendices cusousssssnssnssossnnsonsnnsnnsnnennssossnnsons
138. s Die Bestimmungen in den Paragraphen 3 4 und 5 bleiben auch nach Beendigung des Vertrages wirksam Fassung vom 02 09 1999 iv 2000 Stegmann Systemberatung www rs system de Licensing Agreement for Stegmann Systemberatung Auestra e 31 D 63110 Rodgau Germany All products sold by Stegmann Systemberatung or companies associated therewith includ ing demonstration sets hardware media and manuals hereinafter referred to collectively as Product as well as all future orders shall be subject to the following provisions If you do not accept these provisions please return the product to us within seven 7 days of having purchased or received the Product We will refund the purchase price minus fees for ship ping and processing 1 License Stegmann Systemberatung holds all rights to the Product and herewith assigns to you a nontransferable nonexclusive right of use to exploit utilize the Product according to the following You are not authorized to transfer the Product or parts thereof to third parties or to make them accessible in any way or to modify disassemble decompile reverse engi neer process or improve the software or other parts of the Product or to attempt to dis cover the source code of the software with the exception of the conditions as stated in Section 69e of the German copyright law You have the right to install and use the Product on one 1 computer If you want to install and use the Prod
139. s of lower level objects will be taken into account Otherwise all defi nitions will be taken from the assay scheme 166 2000 Stegmann Systemberatung www rs system de GMP GLP Settings in PLA 9 1 1 An Example on Using Schemes Let us elucidate the usage of schemes with the example given in chapter 8 12 2 The data is from a standard 96 well plate containing a standard a preparation and a control with a positive and negative control The preparation and standard are identical regarding their object definition Both consist of eight dilution steps taken five times The control differs in this regard because positive and negative controls were taken eight times Because the number of repetitions of the standard preparation and the control are different is reasonable to define two schemes For this proceed in the following manner 1 Create a scheme that contains the definitions for the standard and the preparation 2 Assign a specific control scheme as a sub object to the created scheme that accounts for the special settings of the controls different number of data series To create the higher level object select Create Object Select object type to create 1 G il New Protected Scheme Cancel Input Code and Title m Select a creation scheme 3 none X Hint if there is an active mandatory global or parent scheme this selection will be ignored From topic 1 of the
140. s system de 222 2000 Stegmann Systemberatung www rssystem de Appendices Page 9 of 13 03 10 99 100809 Project Validierungsbeispiele Assay Europ Pharmacop 1993 3 Range Selection Linear detection type Fixed range 1 Fixed range 1 Selected Linear Range Step 110 Step 2 Step 1 to Step 2 4 Test of Linearity Regression equation y a bligo Clg 4 1 Standard Standard S LINEARITY IS NOT CALCULATED 4 2 Sample Preparation Z LINEARITY IS NOT CALCULATED Calculated by PLA Verson 1 2 00 PLA is a product of Stegmann Systemberatung Germany waw rs system de 2000 Stegmann Systemberatung www rssystem de 223 Appendices Page 10 of 13 Project Validierungsbeispiele 03 10 99 100809 Assay Europ Pharmacop 1993 5 Test of Slope Regression equation y a bligo 5 1 Standard Standard S Total number of observations 20 Source di Sumofsquares _MeanSqure ___FVdue Model 1 34 944 8000 34 944 8000 46 2695 Error 18 13 594 4000 755 2444 Total 19 48 539 2000 RRE CR Intercept a 248 4000 8 6905 e 0 719929 Slope b 41 8000 6 1451 Pa 0 704370 Test passed if Fmosa gt Fain P 98 6 5146 Test PASSED 5 2 Sample Preparation Z Total number of observations 20 __ Source di ____ SumofSquars ___Mean Square F Value Model 1 5 184 2000 5 184 2000 6 3249 Error 18 14 753 6000 819 6444 Total 19 19 937 8000 prom Estimates Error Quality of regre
141. se assays would be the inverse of the potency for normal assays PLA will give the right value for this kind of assays if you check this option The second section of this dialog page relates to the statistical analysis of the assays You can set the necessary statistical parameters and the rejec tion criteria for the assay here In the input field Dixon test contamination alpha the confidence level of the Dixon test can be given You can enter values from 0 005 to 0 3 here The input has no effect if you have deactivated the function Perform Dixon test for data outliers 2000 Stegmann Systemberatung www rssystem de 115 Reference The following three fields significance of the slope significance of devia tions from linearity and significance of deviations from parallelity refer to give the probabilities for the F value for achieving the respective hy pothesis for the slope linearity and parallelity You can enter values from 90 to 99 99 here If you want to reject the calculation of assays that do not fulfill the hy pothesis mark the rejection criterion check box In the case the option is activated the calculation of the assay will immediately stop after failing the test If you checked the field Analysis according to Europ Pharmaco poeia 1997 all three hypotheses would be rejection criteria The value for the Fiducial limit of potency estimation determines the confidence interval for the calculation of th
142. se functions will become available while the Calculate button will be grayed out 2000 Stegmann Systemberatung www rssystem de 129 Reference If you have activated the print immediately when calculation finished function your report will be printed automatically after the completion of the analysis Otherwise you can check the report on screen by pressing the Preview button and send the output to your standard printer with the Print button afterwards Furthermore you can archive your reports on hard disk by pressing the Save button and use the typical file dialog shown to save your results to a rich text format RTF file Speichern nja Report D cl pla1 rtf W Validate ATF pla2 rtf pla3 rtf pla6 rtf Complete Validation Report ATF Dat gt Dateityp Rich Text Format D RIP zi Abbrechen 130 2000 Stegmann Systemberatung www rssystem de Reference 8 9 The Info Function This dialog shows you a graphical representation of your assay You can use the info function to gain an overview on the measurements and control them visually Standard m Probe 1 Probe 2 Response Probe 3 Probe 4 Probe 5 Doe log69 Include the following objects E W include all From the pick list Include the following objects you can select one or more objects to be displayed If you want to select more than one object 2000 Stegman
143. sis this value is taken from the object definitions of the standard in units Units dimension of the concentration Predilution factor of For the analysis this value is taken from the the standard object definitions of the standard Predilution factor of For the analysis this value is taken from the the Preparation object definitions of the preparation Number of dilution Number of measurements in a dilution series e steps in a data series g points on a titration curve Number of data series Number of data sets taken e g two titration curves Additional predilution You can enter a predilution factor e g from the factor for the first step introduction of a prediluted medium to the object here Values from 1 1 to 1 10 are possi ble Dilution scale It is possible to switch between 1 2 dilution series and direct dose input here The input of the dose can already be given at project level 66 2000 Stegmann Systemberatung www rs system de Terms and Concepts 644 Analysis Settings Property Analysis according to Europ Pharmaco poeia 97 Dixon test for data outliers Up to 25 series Regression model Reverse response effect relation Dixon test contamination Description All analysis settings will be set according to the values given by the European Pharmacopoeia 1997 Activation of the Dixon test for statistical out liers This test is limited to a range from 3 to 25 data sets
144. ssion Intercept a 250 0000 9 0534 d 0 260019 Slope b 16 1000 6 4017 Pas 0 218909 Test passed if Fmosa gt Fan P 98 6 5146 Test FAILED Calculated by PLA Verson 1 2 00 PLA is a product of Stegmann Systemberatung Germany wee steen de 224 2000 Stegmann Systemberatung www rssystem de Appendices Page 11 of 13 08 10 99 10 08 09 Project Validierungsbeispiele Assay Europ Pharmacop 1993 6 Test of Parallelity PARALLELITY IS NOT CALCULATED Calaulated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs eystem de 2000 Stegmann Systemberatung www rssystem de 225 Appendices Page 12 of 13 03 10 99 10 08 09 Project Validierungsbeispiele Assay Europ Pharmacop 1993 7 Estimation of the Relative Potency Relative potency of sample Preparation Z in comparison to standard Standard S Preparation Z vs Standard S Estimated relative potency ASSAY REJECTED 95 Fiducial Limits RELATIVE POTENCY IS NOT CALCULATED Relative fiducial limits 8 Calculation of EC50 Standard S Preparation Z Method of EC50 calculation nc nc 50 Response of EC50 concentration 9 Annotations Calculated by PLA Version 1 2 00 PLAisa product of Stegmann Systemberatung Germany wenurs eystam de 226 2000 Stegmann Systemberatung www rs system de Appendices Page 13 of 13 08 10 99 10 08 09 Project Validierungsbeispiele Assay Euro
145. step or object only and not to both assign selected data points to an object G t A select the abbriviation characters al bletc and numbers to assign the step Selected datapoint Grid display options fe Control z display by row gt Ett Grill Dilution step Point Data value L display paint H 8 of columns p 4 a pa display data eg 2 I color the grid The data input is not finished yet as you can see from the red error mes sage that is displayed and the grey Next button Since the measurements are defined by their object type and the dilution step you have to assign a dilution step to each data point in the second step of the input Mark all the fields that belong to the same dilution step e g the first row in our example Either press the key 1 or enter 1 in the Dilution Step input field to assign the dilution step with the highest concentration to the data points Assign the dilution steps 2 to 8 in the same way 154 2000 Stegmann Systemberatung www rs system de Reference Your data matrix should look like this afterwards 1 2 3 4 5 6 i 8 At least one point was assigned to a step or object only and not to both As a last step you have to assign the controls in the first and last column The logic of control objects is somewhat different from standards and preparations Column 1 contains a negative control column 12 a positive control Ac cording to the PLA logi
146. t select PLA Native Format in the upper part of the first page of the dialog PLA Import Wizzard PLA Import Wizzard 1 What type of file do you want to import Select Input File Plate 6 PLA Native Format PNF PNF Softmax Pro 2 4 1 txt Victor D bal 2 Select the import file Select File Analysation results Please select the file you want to import Press Select File to choose the import file Second choose the directory and the name of the file you want to import into PLA By pressing the Select File button a standard file dialog is opened where you can choose the file to import 2000 Stegmann Systemberatung www rs system de 141 Reference Select Input File PLA Native Format P PNEU 21x Suchen in CH Import D cl el 3 Ec99061 pnt gt ec99065 PNF Ec99071 pnt Dateiname Ec99061 pnf Dateityp Pa Native Format P PME x Abbrechen Select the input file that contains the data you would like to import into PLA and press the Open button to get back to the PLA Import wizard The program will check the file structure and data in the file A summary of the analysis results will be displayed in the import wizard PLA Import Wizzard D PLA 1 2 Import Ec99061 pnf PLA Import Wizzard 1 What type of file do you want to import Select Input File lak 596 PLA Native Format PNF PNF Softmax Pro 2 4 1 txt Control and Summary Victo
147. t t type to create s member of project Beispiel Nr 1 Input Code and Title Messung 1 Messung 1 Select a creation scheme 3 E none D Hint if there is an active mandatory global or parent scheme this selection will be ignored 3 Press Create to create the new assay After you clicked Create the object dialog for the assay appears on the screen In this example no changes are necessary for this object Look at the properties of the assay You will see that the project settings have been copied to the assay 2000 Stegmann Systemberatung www rs system de 83 My first Parallel Line Assay 7 4 Creating the Preparations and the Standard The next thing you will have to do is to create the preparations and the standard Start with the standard If you have not closed the creation dia log for the assay yet please do so In analogy to the creation of the assay select the newly created assay in the object navigator window and click New or Create Object from the menu Now you have the choice to create either a preparation or a standard Create the standard first Do always check that you have selected the cor rect type of object For each of the five preparations select New Prepara tion as member of assay If you want to create a standard choose New Standard as member of assay After you have executed the Create command for the standard the object d
148. ta values of the import file which correspond to the pods on the micro titer plate are assigned to the data objects The upper part of the dialog page is arranged like a 96 well plate to give you a better over If the number of repetitions is not the same for all types of controls you must give the maximum number of repetitions 152 2000 Stegmann Systemberatung www rs system de Reference view on the data With the help of the matrix you can assign every pod of the plate to the corresponding dilution step You can work with the matrix as you would use a Microsoft Excel data sheet To select a complete row or column click on the corresponding number PLA Import Wizzard D PLA 1 2 Import chi2603c txt PLA Import Wizzard Assign mode Please the data points to the objects Select Input File Import Definition Scheme Select Destination Scheme Define Objects Sort and Assign Imported Data Choose Destination 4 2 3 4 5 6 7 8 Save Import Definition Scheme Control and Summary Import in Progress Please assign the displayed data to the objects You may use the keyboard to assign selected data points to an object select the abbriviation characters S SCH a a b etc and numbers to assign the step Selected datapoint gt Grid display options display by row gt Edi il Dilution step Point Data value T display point H 8 of columns Par gpm Beer CR lt
149. tablish GLP GMP environments The user management is a prerequisite for the operation of the program under GLP GMP conditions e All changes of the data should be logged PLA uses the account man agement to save the user name with the changes in the data With this security feature it not only possible to determine when the data had been changed but also who has done the changes However the login mechanism does not prevent the manipulation of data intended or un intended but you are able to recognize modifications more easily Note The identification mechanism itself does not guarantee the security of your data You should use the security mechanisms of the operating system e g Windows NT to protect the access to the workstation with your data If you fear that your data might be manipulated you should end PLA and lock the workstation every time you leave it unattended The following sections will show you the usage of the PLA account man agement Only PLA administrators have access to the user management 44 2000 Stegmann Systemberatung www rs system de PLA Administration 5 1 1 Creating a New Account The user management of PLA tries to depict the organization of an ana lytical laboratory As you can see from Figure 5 1 PLA distinguishes between four groups of users 1 Standard users 2 System administrators 3 System inspectors 4 Deactivated users The position of the PLA administrator is normally tak
150. tandard Standard Concentration Series N 1 Series N 2 1 1 70400000 Mf 7900000 12 541 00000M f 645 00000 1 4 57500000M 576 00000 1 8 51200000M 51400000 1 16 M 45000000 1 32 34 0000 m SEEEEKER 1 64 338 00000 4 339 000 Lal gt Toggle status indicator bk lt status indicator Un check the indicator at the right side of the value for exclusion Close the data editor and answer the question if the values should be saved with Yes This will lead you back to the navigator Create the five preparation objects in the same manner Keep in mind that you have to select preparations in the create dialog of the object Enter the measurement values for all preparations When you have finished the creation of all preparation samples your navigator window should look like this Data Objects E Ge Beispiel Nr 1 gt Standard amp Probe 1 amp Probe 2 amp Probe 3 amp Probe 4 amp Probe 5 The identifiers you see in the navigator correspond to the names that you gave to the objects Since you have finished the data input successfully you can now analyze the assay 86 2000 Stegmann Systemberatung www rs system de My first Parallel Line Assay 7 5 Analysis In this chapter you will do your first PLA analysis The analysis settings are identical in all objects that you have created You can start your analy sis immediately Before you start the analysis you sho
151. tations Calaulated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany www rs system de 196 2000 Stegmann Systemberatung www rs system de Appendices 11 1 2 PLA Output PLA Complete Statistics Report The PLA Complete Statistics Report of PLA contains all the variance analyses data It is used for the comparison with the Linder results 2000 Stegmann Systemberatung www rssystem de 197 Appendices Page 1 of 7 Project Validierungsbeispiele 08 10 99 10 07 28 Assay Linder Results of Assay Linder Project Validierungsbeispiele Current user Dr Matthias Schmitt 1 Complete List of the Object Definitions Standard Sample eee u TB Z _ m EE EE General settings Code Standard Probe 1 Title Standard Probe 1 Measured substance Batch identification Object status declared closed NO NO Object was created by Dr Matthias Schmitt Dr Matthias Schmitt Creation timestamp 16 06 99 15 14 50 16 06 99 15 14 52 Last modification by Modification timestamp 06 10 99 12 53 13 06 10 99 12 53 13 Definition settings Concentration of undiluted standard Concentration units Predilution of standard sample 11 1a Number of dilution steps 3 3 Number of data series 5 5 Additional predilution factor qA 12 Dilution scale 12 12 Analysis settings Analysis according to EP NO NO Dixon test enabled YES YES Regression model linNog linlog
152. tel 486 processor with 66 MHz 32 MB of RAM 30 MB free hard disk space Screen resolution of 800 x 600 pixels Microsoft Windows 95 Windows 98 Windows NT 4 0 SP4 or higher The application performance and speed of analysis is greatly enhanced with the following configuration Intel Pentium processor with 133 MHz 64 MB RAM Ca 30 MB of free hard disk space Microsoft Windows NT 4 0 Workstation TM SP4 Technical Note Additionally the program installs the following necessary components runtime module for Powersoft Powerbuilder 6 5 applications runtime module for Heiler Graphics Server 5 0 OCX control Sybase SQL Anywhere 5 5 desktop server The modules are protected products by their respective manufacturers Powersoft Sybase Heiler Software and Microsoft These components are only sold and may only be used in conjunction with PLA 1 2 26 2000 Stegmann Systemberatung www rs system de System Requirements and Installation 4 2 Installation PLA comes on a CD ROM or upon request on diskettes The setup pro gram on the CD or the first diskette installs PLA on your system If you do not give any other drive letter PLA is installed on drive C of your hard disk Note Please note that PLA must be configured after it has been installed Refer to chapter 4 3 First Time Configuration of PLA 4 2 1 Preparing the Installation The installation consists of three steps
153. temberatung www rs system de 11 Appendices 2000 Stegmann Systemberatung www rs system de 189 Appendices 11 1 Check A Linder The textbook on statistical methods in science by A Linder contains a complete analysis of a parallel line assay The test data set has been recal culated with PLA to check the program results In Table 11 1 the results of Linder and PLA are both shown The values of PLA have been rounded for this comparison You see that for preparation 2 the F value of the slope and the upper limit of the confidence interval of the relative potency differ slightly Both differences are due to rounding errors in the Linder calculation While Linder rounds all the sums of squares and the mean sums of squares to integer numbers PLA uses double precision numbers 8 byte double preci sion number format compliant to IEEE Linder himself mentions the possibility of deviations from computer results because of rounding errors The table shows that PLA reproduces the literature values Annotations on the table Linder s sample 2 is used as the standard in PLA Linder s sample 1 is used as the preparation SOS is the sum of squares MS is the mean square For each sample the SOS and MS for the applied model are shown SOS error and MS error are the respective residual errors SOS quadratic and MS quadratic are the quadratic regression coefficients SOS model and MS model are the square sums of the regression
154. the method for the calculation of the nen effective concentration of the 50 response There are three options available 1 No calculation of the EC50 2 Calculation of the EC50 by linear regres sion of the standard 3 Calculation of the EC50 by linear regres sion of the assay Calculate EC50 by Selection from which object s the EC50 will using be calculated You can choose from this ob ject standard control and all objects 68 2000 Stegmann Systemberatung www rs system de Terms and Concepts 6 4 5 Range Selection Settings You can choose the method for the selection of the linear and parallel range on this tab of the object definition dialog The range can be allo cated either manually or automatically Both methods are mutually exclu sive By selecting one method the input fields for the other method are grayed out Property Description Selection of the linear range There are four methods available for the selection of the linear and parallel range 1 Fixed Range 2 Automatic Detection individual range 3 Automatic Detection common range for standard individual for preparations 4 Automatic Detection common range for standard identical for preparations Refer to chapter 10 2 Automatic Detection for further details Linear Range Fixed Range Manual input of the linear and parallel range Inactive if automatic detection is activated Evaluation criterion for the automatic sele
155. tically and cannot be changed by the user Input of the measurement reading Manual exclusion of a reading from the analy sis technical outlier 72 2000 Stegmann Systemberatung www rs system de 7 My first Parallel Line Assay This chapter will show you how to run a full parallel line analysis of an assay You should be able to do all the steps by yourself with your PLA installation After reading this chapter you should be able to run parallel line assays with PLA 1 2 The chapter does not introduce you to all functions of PLA however It just shows you the steps that are normally necessary to run the analysis For a description of all functions please refer to chapter 8 2000 Stegmann Systemberatung www rssystem de 73 My first Parallel Line Assay 7 1 Initial Stuation You have done a measurement with five preparations and one standard sample The measured concentration range extends from 1 1 to 1 64 All dilution steps have been taken twice The measurements are in Table 7 1 No Dilution Series1 Series2 Series 1 Series 2_ 1 1 704 719 701 704 2 1 2 641 645 637 632 3 1 4 575 578 566 565 4 1 8 512 514 502 505 5 1 16 450 450 439 438 6 1 32 394 384 383 380 7 1 64 338 339 334 333 1 1 652 674 675 677 2 1 2 582 585 603 602 3 1 4 512 514 530 529 4 1 8 447 454 470 474 5 1 16 394 396 412 412 6 1 32 336 333 350 354 7 1 64 294 294 307 310 1 Ll 710 707 768 771 2 1 2 668 659 732 739 3 1 4 64
156. tion series Number of dilution steps in a data series Number of data series 1 1 in 2 series Additional predilution factor for the first step Dilution scale 112 2000 Stegmann Systemberatung www rs system de Reference The first three input fields are related to the settings for the standard The input of the concentration of the undiluted standard is optional The di mension of the standard concentration should be given in the units field The input of the predilution of the standard is mandatory The predilution is given as a ratio 1 X If your predilution is 1 100 enter 100 in this field The predilution of the preparation is given in the same way The input for this field is compulsory too In the following input fields the number of dilution steps and the number of data series must be given E g if your measurement has four dilution steps and has been repeated three times enter 4 as the number of dilution steps and 3 as the number of data series In addition to the given predilution you can enter an additional predilu tion factor for the first step because many measurement procedures con tain an extra dilution step by bringing the preparation into a medium for the measurement You can only enter integer ratios from 1 1 to 1 10 here The last entry concern with the input of the dose values The default set ting is J in 2 series If you want to enter other dose values you have to
157. tions of an assay The preparation consists of at least two points of measurement from repeated read ings The task of the program is to determine the relative potency of a preparation referring to the corresponding standard of the assay Is the term for the measurements and properties of an assay control There are three different kinds of con trols positive controls negative controls and blanks In contrast to the preparation the properties of all controls are managed in one object Controls are optional for the calculation of relative potencies They serve as a check for the examined test system The concept of controls is fundamentally dif ferent from that of standards or preparations Controls 58 2000 Stegmann Systemberatung www rs system de Terms and Concepts Project are only measured as repetitions and not as dilution series The project is an object for the management of a group of assays The organization of assays in projects allows you to keep track of a multitude of assays Examples Project storing experiments contains all assays that were calculated during a storing experiment Project measurements April 1 contains all assays taken on April 1 PLA encourages you to manage all assays in projects This allows you to organize your work better if you keep the hierarchy of the objects in mind The object hierarchy is described in the following paragraph 2000 Stegmann
158. to the reagent information and prevent changes by the user By activating this option you can protect the access to the general object settings and prevent changes by the user 2000 Stegmann Systemberatung www rs system de 71 Terms and Concepts Property Analysis settings pro tected Range selection set tings protected Direct dose values protected Data values not edit able Description Activation of this option protects the access to the analysis settings and prevents changes by the user By activating this option you can protect the access to the linear selection settings and pre vent changes by the user By activating this option you can protect the access to the given dose values and prevent changes by the user By activating this option you can protect the access to the measurement points and prevent changes by the user 6 4 8 Dose and Measurement Input For the input of the dose values and measurements the same input mask is used in PLA While the dose values can be set on a higher object level the input of the measurement values is only possible on the lowest level of the object hierarchy standards preparations controls The following proper ties are available for each measurement Property Concentration Series No Status indicator Description Concentration of the respective dilution step If you have preselected 1 2 dilution series the value will be given automa
159. to the European Pharmaco poeia The program can work with either the measurements of dilution series or with freely defined doses It can be used for arbitrary measurement meth ods e g ELISA measurements or flow cytometry Furthermore PLA contains many additional functions for the efficient administration of measurements and the development of statistically valid parallel line assays Typically measurements of biological systems show saturation effects at very high and very low concentrations These regions cannot be analyzed with the parallel line method PLA offers methods for the automatic detection of linear regions to make analysis of measurement curves more efficient in product development 18 2000 Stegmann Systemberatung www rs system de Introduction 3 2 PLA 1 2 PLA is the first commercially available program solution for the analysis of parallel line assays The actual version is the third significantly im proved release of the program Until the release of PLA biological assays had to be analyzed either with the help of extensive statistical program packages or with software that had been developed for the special system in question The statistical programs had the disadvantage that they were not optimized for the analy sis of biostatistical assays They had to be adapted to the task with great effort Normally the results were not flexible enough and too slow to be used in day to day routine of development a
160. tung Germany wee steen de 201 2000 Stegmann Systemberatung www rs system de Appendices 08 10 99 10 0728 Page50f7 Project Validierungsbeispiele Assay Linder 6 Test of Parallelity 6 1 Estimation of Common Slope Total number of observations 30 Sowce dt ____ SumofSquares ___Mean Square F Value Model 3 4 474 666 1333 1 491 555 3778 Error 27 275 068 8667 10 187 7358 Total 30 4 749 735 0000 PeramEsimtes Emr Quality of regression Intercept a1 813 9333 52 1223 e 0 942088 Intercepi a2 568 5667 34 4756 Root MSE 100 9343 Slope b 238 5000 22 5696 6 2 Estimation of error due to non parallelity Total number of observations 20 Source TTT TTT Tag of Squares Mean Square F Vaue Model 3 4 474 666 1333 1 491 555 3778 Difference of Models 1 8 405 0000 8 405 0000 0 8195 Model Total 4 4 483 071 1333 1 120 767 7833 Error 26 266 663 8667 10 256 3026 Total 30 4 749 735 0000 TTT TT Quality ofregressin Intercept a1 854 9333 69 1829 r 0 943857 Intercept a2 548 0667 41 3447 Root MSE 101 2734 Slope bi 259 0000 32 0255 g 0 0079 Slope b2 218 0000 32 0255 Test PASSED Test passed if Fest otmocas lt Fan P 95 4 2252 Calculated by PLA Version 1 2 00 PLA is a product of Stegmann Systemberatung Germany wee steen de 202 2000 Stegmann Systemberatung www rs system de Appendices Page 6 of 7 08 10 99 10 07 28 Project Validierungsbeispiele Assay Linder 7 Estimati
161. tware herauszufinden Sie haben das Recht die Software auf einem 1 Computersystem zu installieren und auf diesem System zu nutzen Wenn Sie das Produkt auf anderen Computern installieren oder nutzen wollen m ssen Sie f r jeden Computer eine Lizenz erwerben und sie diesem Computer zuordnen Sie haben das Recht eine 1 Kopie der Software anzufertigen um diese zu archivieren 2 Weitere Rechte und Bestimmungen Falls das Produkt als Upgrade bezeichnet ist m ssen Sie ber eine g ltige Lizenz ber ein Produkt verf gen das von der Stegmann Systemberatung als f r das Upgrade berechtigend bezeichnet wurde um das Produkt nutzen zu d rfen Ein Produkt das als Upgrade bezeich net wird ersetzt und oder erg nzt das Produkt das die Basis f r die Upgrade Berechtigung bildet Sie d rfen das resultierende aktualisierte Produkt nur in berstimmung mit diesen Lizenzbestimmungen verwenden Falls das Produkt eine Aktualisierung einer Komponenten eines Pakets von Softwareprogrammen ist die Sie als ein einziges Produkt lizenziert haben d rfen Sie das Produkt nur als Teil dieses Pakets von Softwareprogrammen nutzen oder bertragen und es nicht separieren um es auf mehr als einem Computer zu nutzen Sie d rfen das Produkt nicht mieten leasen oder vermieten Sie d rfen alle Ihre Rechte die sich aus diesen Lizenzbestimmungen ergeben vollst ndig auf einen Dritten bertragen sofern Sie keine Kopien des Produkts aufbewahren Sie das vollst ndig
162. uct on other computers you must acquire and dedicate a license for each separate computer You have the right to make one 1 copy of the software in order to archive it 2 Other Rights and Limitations If the Product is labeled as an upgrade you must be properly licensed to use a product identified by Stegmann Systemberatung as being eligible for the upgrade in order to use the Product A Product labeled as an upgrade replaces and or supplements the product that formed the basis for your eligibility for the upgrade You may use the resulting upgraded product only in accordance with the terms of this license agreement If the Product is an upgrade of a component of a package of software programs that you licensed as a single product the Product may be used and transferred only as part of that single product package and may not be separated for use on more than one computer You may not rent lease or lend the Product You may permanently transfer all of your rights under this license agreement provided you retain no copies you transfer all of the Product including all component parts the media and printed materials any upgrades and this license agreement and the recipient agrees to the terms of this license agreement If the Product is an upgrade any transfer must include all prior versions of the Product 3 Warranty For a period of twelve 12 months after the delivery date Stegmann Systemberatung shall guarantee a that the perf
163. uld check if your data is correct Select the assay you have created in the navigator To get a plot of your data use the Info command from the menu When you select this function you should see the measurement values of the preparation and the stan dard on a graph For our example you should see the following graph on your screen Standard Probe 1 Probe 2 Response Probe 3 Probe 4 Probe 5 1 5 3 0 Dose log6d Include the following objects d E VW include all Close 2000 Stegmann Systemberatung www rssystem de 87 My first Parallel Line Assay The purpose of this function is to give you a quick overview over the quality of your data For the moment the visual control is sufficient Please close the window Back in the navigator window you select the function Calculate from the menu to start the analysis You should see the following dialog on the screen now S Calculation Control xl Select un reports IPL ndensed Repo at ine sluding graph gt Ihn PLA Short Report including values and regression graphics Ihn PLA Short Report including values and normal graphics In PLA Short Report including values In PLA Complete Statistics Report Ihn PLA Validation Report Progress No calculation has been started yet Options print immediatly when calculation finished Calculate Press the Calculate button to start the analysis The calculation might
164. uss the available options With the op tions in this dialog field you change the display of the data matrix in the upper part of the dialog page The best way to learn the effect of these settings is to play with them and observe the changes in the data display r Grid display options display by row D gt Eat era I display point of columns display data 12 W color the grid The following table gives you a short description of the available options display by Select one of the three possible data display arrangements display point Show the counter of every data field display data Show the measurement of every data field 156 2000 Stegmann Systemberatung www rs system de Reference Effect color the grid The coloring of the table can be switched on or off with this option of columns Limit the number of columns that are dis played to the given value Probably you will rarely use these functions You will possibly recognize the next dialog page form the previous chapter You have to give the des tination and the name of the object that will be imported PLA Import Wizzard D PLA 1 2 1mport 043011 96 ESI Ne The import tree contains Select Input File ax 5 t Definition Sch TK DI New Assay Import Definition Scheme X Standard A Select Destination Scheme X amp Preparation B Define Objects X amp Control C Sort and Assign Imported Data Choose Destination Save
165. ut fields for the manual method will be grayed out and vice versa Fixed range Automatic detection individual range Automatic detection common range for stan dard individual for preparations Automatic detection common range for stan dard identical for preparations Manual method You must give a linear interval from which the relative potency will be calculated Automatic method The linear range of preparations and stan dards will be determined individually for every combination Automatic method The linear range of the standard will be de termined once while the linear interval of the preparations will be calculated individu ally Automatic method The linear range of the standard will be de termined once The same interval is used for the preparations Please consult chapter 10 2 for further information on the selection meth ods If you choose the manual selection method you gain access to the manual selection options where you can set the linear range 2000 Stegmann Systemberatung www rssystem de 119 Reference If you choose the automatic method instead you can modify the automatic selection options You can set several criteria for finding a linear interval here First you should select an allocation strategy from one of the available options Best Range Does a best fit of the linear and parallel range where all hypotheses a fulfilled Maximum Range Looks for
166. utomatically activate the protection As you can see administrators have several options at hand to selectively limit the access privileges of the standard users With the flexible interpretation of the GMP GLP restrictions PLA is suit able for the use in product development on the one hand as well as for the GMP GLP conform use in quality assurance on the other 174 2000 Stegmann Systemberatung www rs system de 10 The Statistics Core of PLA The topic of this chapter is the statistical methods for the calculations of PLA are In the first part we will give you an overview on the execution of the calculations The operating procedures are oriented towards those Automatic Detection see chapter 10 2 Automatic Detectio Method Selected Statistical Evaluation see chapter 10 1 Abb 10 1 Flow diagram 2000 Stegmann Systemberatung www rs system de 175 The Statistics Core of PLA proposed by the European Pharmacopoeia 1997 Please note that some of the requirements of the European Pharmacopoeia refer to the design of the experiment If you are in doubt of the correctness of your experimental setup please check those issues separately If you meet the requirements e g the normal distribution of the measurements you will get a correct and valid analysis of your assay provided that you validation parameters are correct Section 10 1 describes the analysis of a
167. will display a message for your information concerning the improved user management of this version Only PLA administrators can now establish new user accounts 10080 Account Management Information PLA has an enforced user account management User accounts can be created by al Print members of the group System Administrators only Since there is no member of this group in the database of this license a default administrator called PLA Administrator will be created now You are prompted to give this administrator a password WARNING WARNING WARNING If you forget this password you are locked out of PLA and cannot modify your user accounts WARNING WARNING WARNING z At the time of the first start up a user account PLA Administrator is auto matically created You will be asked to enter and confirm a new password for this account The password is case sensitive 1 5 S In previous versions any user was able to create new accounts 2000 Stegmann Systemberatung www rs system de 37 System Requirements and Installation Enter password for default administrator r Account Password LA Administrator Current Password New Password at least five characters long Retype New Password Pate o ooo Cancel ATTENTION Remember your password well You will not be able to create new accounts or to manage existing accounts without the PLA Administrator password After y

PLA 1.2 Analysis of Parallel-Line Assays - User Manual

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