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1. LOGGING Option Value Description Save last value LogOutputPath C Cedex2 CedexLog log Default file name for the log file SERIES DEFAULT FILTER Option Value Description Save last value DataSet CTC Series New Default Filter for DataSet X MeasurementDateFrom CTC Series List Default Filter for Measurement Date From X MeasurementDateTo CTC Series List Default Filter for Measurement Date To X ReactorlD CTC Series List Default Filter for ReactorlD X CTC VIEWS DEFAULT FILTER Option Value Description Save last value CTC Views Default Filter for Measurement Modify Date ModifyDateFrom From X CTC Views Default Filter for Measurement Modify Date ModifyDateTo From X ViewName CTC Views Default Filter for View name X CTC Views Default Filter for Measurement Create Date CreateDateFrom From X CreateDateTo CTC Views Default Filter for Measurement Create Date To X REPORTS Option Value Description Save last value ReportResultPath Report result xml path X ReportTemplatePath The path to report templates files extension Ist REPORT SETTINGS Option Value Description Save last value Operational Data standard Default report template for the report type Report Template Operational Data Measurement Data standard Default report template for the report type Report Template Measurement Data System Configuration standard Default report template for the report type Report Template System
2. 28 Creating a View ES 29 Creating a Cultivation Time Chart 32 4 ADJUSTMENT OF IMAGE ANALYSIS WITH THE LIVE 37 41 Description of the Cell Type List and Live Operator seen 37 The Cell Type List Wind OW ete Pete en ek ten 37 4 2 Working with the Live 40 CedexXSCellOperator and CedexXSObjectOperator parameters 40 Description of the Individual Operator Parameters 41 Cedex XS User Manual 3 12 04 2009 MM DD YY Y Y Cedex XS 5 DATA MANAGEMENT cu 45 5 1 Printing Reports erreur inte Ea 45 5 2 Export to Flle nn ununi etri teri aesti ette tii a eerie ns 45 5 3 Data Exchange entretien eee uei eere 46 E XDOF nietos thee tem ede qe tinte debet eb eme bandes ten ene DA eet 46 ER 47 5 4 Archiving 47 DT EI 47 Restore nena eie le e estie s ys cide e i ee Ee et 48 Deletingrarchived data iecit e a cio elles xke Lo Roi laces 48 5 5 5 enne ese aede Tea raae vanes eheretudewesstvysasuscudeuesued 48 USER PREFERENCES 49 Specifying Default Measurement Parameters sss 49 Specifying oth
3. 6 Sample 10 13 16 Save East uiti con sir 46 Securing 42 45 Selection of Data Series 28 Shortcut area 12 SAUL COW Nis 10 Specific Growth Rate 33 fehl EE 18 System 11 System parameters 42 T Total CONCH te rire t ct Rd 18 Total Object 18 U User 52 V Miability 2 8 18 Viable Cell 18 Viable CONG 18 View 21 62 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Cedex XS User Manual 12 04 2009 MM DD YYYY 63
4. BorderWidth 2 List of the CedexXSObjectOperator parameters After loading a measurement Reactor ID and Sample ID and selecting an image the selected image is displayed in the upper right hand corner of the Live Operator window Each single cell detected by software is marked as dead red circle or living green circle The values New Viable the number of living cells of the current image and New Dead the number of dead cells of the current image are displayed in the Process Data area If an Operator parameter is modified the newest values will appear under New Viable and New Dead after the automatic reprocessing of the selected image is finished The values of the previous processing are displayed under Previous Viable and Previous Dead Thus direct comparison to each previous evaluation is possible In addition New Viability shows the viability of the last processing of the image while Previous Viability shows the viability of the second to last processing of the image Description of the Individual Operator Parameters ClassificationBias Range 20 20 Default 0 Unit absolute value This parameter influences the classification behavior of viable and dead cells Since dying cells of different cell lines absorb Trypan Blue differently dead cells can be variably stained Positive values of this parameter tend to let the operator recognize more living cells and less dead cells Negative values
5. 28 TS 3 onoff 4 onoff 5 on off Figure 21 A view of the Exponential Regression window Only viable cell concentration is shown in this graph The measurement parameters Specific Growth Rate u and Doubling Time T of the culture are computed by means of the differential curve Click on the to close the Exponential Regression window CTC Views window will reappear Printing and Exporting out a Cultivation Time Chart Cultivation Time Charts can be printed out via the Print button in the Cultivation Time Chart and the Exponential Regression window To print out a chart click on the Print button A window will open in which the printer settings can be selected or the chart can be directly printed Click on the Print button to open a window with a preview of the page to be printed It is also possible to export and print out the data from the Cultivation Time Chart see Export to File for more details about export options Cedex XS User Manual 35 12 04 2009 MM DD YYY Y Cedex XS 36 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Adjustment of the Image Analysis 4 4 Adjustment of Image Analysis with the Live Operator The operator selected for a measurement takes care of the image evaluation For various application scenarios Roche Innovatis AG has put in two pre settings that are adapted to a wide variety of cells and objects e CedexXSCellOperator for the analysis of ce
6. Images button to open the Image List window which contains thumbnails of the images from all sample chambers used for the calculation of results Double click on a thumbnail in the Image List window to open a new window called the Image View This window contains an enlarged version of the selected image as well as some further options for viewing the image Double clicking on the image displayed in the Image area of the Measurement window will also open the Image View window 20 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Analyzing the measurement results Viewing a cell image using the Image View window In the Image View window it is possible to see which cells were marked as viable or dead by the analysis software The user can also see how the software analysis evaluated cell shapes and clusters The Cedex XS Software offers a variety of options for adjusting and optimizing the image analysis software in case of unusual cell culture images or for specific internal counting strategies Detailed information can be found in the chapter Adjustment of Image Analysis with the Live Operator The Image View also provides the following information and options e Viable Cells number of viable cells e Dead Cells number of dead cells e Show Circles check box switches on off the marking of cells recognized as living or dead Viable cells are marked with a uniform green circle and dead cells are marked with a red circle The ci
7. e g P001 P002 However the Sample ID can be manually changed if a different Sample ID is preferred 3 Optional Use the Sample Drawing drop down menu to select the date when the sample was drawn 4 Click on the Mix button A window will open in which the amounts of cell sample diluent and trypan blue used to prepare the sample can be entered in uL see Figure 4 After the appropriate volume has been entered for each element the dilution factor is automatically calculated 5 Click on OK to confirm the entries The window will close and the appropriate dilution factor will be automatically entered into the Dilution field in the Measurement window Note NOTE The cell concentration after all dilutions should be no greater than 5 x 10 cells mL Optional Enter remarks in the Comment box in the Sample Parameters area The system name as well as the process date and time of the measurement will be automatically added to the Processing Parameters area Enter volumes DER Sample Volume 50 uL Dye Volume 100 uL Diluent Volume 50 uL resulting dilution 1 4 NUM Figure 4 Window for calculation of dilution factor Defining the measurement and analysis process parameters The measurement and analysis parameters are defined in the Processing Parameters area of the Measurement window In principle all routine measurements can be carried out using the standardized factory set default settings Fo
8. 46 Defaults errat reti dede Lune bd na 46 16 18 Cedex XS User Manual 12 04 2009 MM DD YYYY Diameter 25 26 Diameter Histogram sliders 26 Dilution of cells 14 16 Disclaimer eant nnn tnn nn 5 Disposal Recommendations 56 Doubling 33 5 21 DSOriginal ees 21 Effective 21 Enlarging the diagrams 23 Event window een 10 12 il 42 Exclude images 20 Exponential Regression 33 deo E 42 Export Data Exchange 43 Export to 42 ExportPath 2 2 44 F FCC Notification sees 5 Fit lowest common value button 32 Functions menu sessen 11 G Graphical display 17 23 8 H Hardware Management 11 49 Help menu sse 11 HW Startup 49 HWM 11 12 49 ro 18 Image 19 Image View individual 18 Import Data Exchangge 44 I
9. Configuration Diagnosis Report standard Default report template for the report type Template Diagnosis CTCData Report standard Default report template for the report type Template CTCData 58 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Appendix Cleaning The cleaning can be performed with 80 ethanol Maintenance No maintenance is required Disposal Recommendations Symbol Electrical and electronic equipment marked with this symbol are covered by the European directive WEEE The symbol denotes that the equipment must not be disposed of in the municipal waste system WEEE Directive 2002 96 EC of the European Parliament and the Council of 27 January 2003 on waste electrical and electronic equipment Disposal Recommendations All electrical and electronic products should be disposed of separately from the municipal waste system Proper disposal of your old appliance prevents potential negative consequences for the environment and human health Disposal of the Instrument The instrument must be treated as biologically contaminated hazardous waste Final disposal must be organised in a way that does not endanger waste handlers As a rule such equipment must be sterile before it is passed on for final disposal For more information contact your local Roche Support personnel Cedex XS User Manual 59 12 04 2009 MM DD YYYY Cedex XS Disposal of Control
10. Unit Components Components of your Control Unit such as the computer monitor keyboard etc which are marked with the crossed out wheeled bin symbol are covered by the European Directive 2002 96 EC WEEE These items must be disposed of via designated collection facilities appointed by government or local authorities i For more information about disposal of your old product please contact your city office waste disposal service or your local Roche Support personnel Constraint It is left to the responsible laboratory organization to determine whether 60 control unit components are contaminated or not If contaminated treat in the same way as the instrument Cedex XS User Manual 12 04 2009 MM DD YY Y Y Index A Aggregate Histogram 23 Aggregate Rate Value 18 Aggregate 5 2 24 AggrMaxSize essen 40 40 Analysis parameters 13 18 Analysis results 17 21 23 Analysis software 8 AnalyZer 6 ArchivePath 45 Average Diameter 18 Avg Compactness 18 BorderWidth 40 Cedex Control 8 CedeXSCellOpera
11. by the upper border of the third interval and the maximum value The number of viable cells their percentage of the total viable cell number and the viable cell concentrations x10 cells mL are listed in a table for each of the individual intervals Similarly the number of the objects analyzed their percentage compared to the number of all objects recognized as well as the object concentration x 10 objects mL are listed in a table under the Object Diameter Histogram for each of the individual intervals Compactness Histogram The ratio between the cell circumference and the cell area is based on an ideal sphere 4n which is equivalent to the value 1 on the abscissa of the Cell Compactness Histogram The extent of variation between the cell shape and an ideal sphere is reflected in values that are greater than 1 The shape of a cell is known as the Compactness Click on the Compactness Histogram button to display a histogram that compares viable cell shapes to that of an ideal sphere and shows the distribution of compactness found in a the cell culture The Compactness Histogram can be used to check for changes in the state of the cell culture e g if the stirring speed is too high or nutrient composition is incorrect Histograms Cell Compactness Histogram 8 8 1500 5 5 5 2 2 15 2 0 25 Cell Compactness ym This diagram shows all viable cells with definable co
12. cause the opposite effect with more dead cells and less living cells being recognized This parameter s default setting is optimized for dark looking dead cells and living cells with a bright cell interior An example of a possible need for a change to a more negative value would be a cell line that poorly absorbs Trypan Blue when dead or dying In the default setting with the value 0 these cells would still often be recognized as living If the value is changed to negative value Cedex XS User Manual 41 12 04 2009 MM DD YY YY Cedex XS however more dark objects would be counted as dead cells The viability recorded would therefore be reduced CMinSize Cells minimum size Range 0 200 Default 7 Unit micrometer This parameter determines the minimum cell size diameter values measured in micrometer It serves the purpose of discerning debris from cells LCAppearance Live Cells Appearance Range 20 20 Default 5 Unit absolute value This parameter influences the recognition of living cells When the value of this parameter is set high contrast weak objects are also counted as living cells provided they additionally fulfill the size criterion The lower this value is set the more contrast an object must have for it to be counted as a viable cell If the value is changed to positive shadowy and greyish objects are increasingly counted as living cells Such a change can be sensible if for example the
13. measurement period Geometric optical resolution 0 86 um Pixel Sample chamber height 195 um Operating temperature 10 C to 40 C 50 F to 100 Optimal image quality is achieved between 20 C and 30 C Maximum temperature change 5 C per hour Maximum humidity deviation 596 per hour Operating humidity 20 to 80 relative humidity non condensing Operational altitude Up to 2000 m Energy requirements 100 240 VAC 10 47 63 Hz 0 9 0 34A Hardware Features for the Cedex XS Computer Computer x86 Architecture Intel Pentium IV minimum 2 GHz required Operating system Windows XP Professional Windows Vista Home Premium Windows Vista Business RAM Recommended Windows XP 1 GB Windows Vista 2 GB Hard disk storage USB 2 0 port CD DVD Drive 54 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Appendix List of User Preferences The following list provides a description of all of the user preferences found in the Cedex XS Software via the User Preferences window under the Setup menu of the Cedex Control Center If the column SaveLastValue has been selected for a particular parameter i e there is a checkmark in the column any new value entered in the corresponding location will be automatically saved in the User Preferences windows For example the user has set the default for the Cell Type in the Measurement section of the User Preferences win
14. optical image shows little contrast due to the structure of the medium or of the cells in the medium DCAppearance Dead Cells Appearance Range 20 20 Default 5 Unit absolute measure This parameter influences the recognition of dead cells If the value is set high contrast weak objects are also counted as dead cells as long as they additionally fulfill the size criterion The lower this value is set the sharper the contrast an object must have in order to be counted as a dead cell NOTE In standard situations a change of this parameter can result in the labeling of debris as cells CMaxSize Cells Maximum Size Range 0 200 Default 70 Unit um This parameter serves the purpose of setting the maximum cell size diameter in micrometer used for considering objects for evaluation It distinguishes large dirt particles from cells AggrMinSize um Aggregate Minimum Size Range 8 50 Default 12 Unit um The minimum size of the region that should be analyzed as an aggregate is given here BorderWidth pixel Range 0 20 Default 10 Unit Pixel The border frame around the image not used for evaluation NOTE This parameter should not be changed AggrMaxSize Aggregate Maximum Size Range 10 200 Default 100 Unit um The maximum size of the region that should be analyzed as an aggregate is given here LCAggrApp Live Cells Aggregate Appearance Range 10 10 Default 0
15. sample parameters 14 Defining the measurement and analysis process 14 Preparing the sample and starting the measurement sse 15 End of the Measurement 17 3 ANALYZING THE MEASUREMENT RESULTS 19 3 4 The Measurement Results 19 Result der ek vei Fey 19 Sample Processing Parameters 5 20 p 20 3 2 Using the Measurement 22 Viewing existing measurement results nennen 23 Searching for measurements 23 3 3 Reprocessing Existing Analysis Results Data Sets eese 23 Reprocessing a measurement 24 Effective data Sets i ade e 24 3 4 Graphical Display of the Analysis Results 25 Enlarging the 25 Aggregate Histogram 25 Cell Diameter Histogram 27 Object Diameter Histogram 27 Defining diameter intervals in the Diameter Histograms 28 55 aree deca bid aleae rg a dae de dt eden 28 3 5 The Cultivation Time
16. subject to change without prior notice and should not be construed as a commitment by Roche Innovatis AG Roche Innovatis AG accepts no responsibility or liability for any errors or inaccuracies in this document or for any damage occurring during delivery performance or use of this product Windows XP and Windows Vista are registered trademarks of the Microsoft Corporation All other trademarks are trademarks or registered trademarks of the respective companies Cedex is a registered trademark of Roche Innovatis AG Intended Use The Cedex XS Analyzer determines the cell concentration and viability of cell suspensions The Cedex XS Analyzer is intended for life science research only It is not intended for use in diagnostic procedures It was neither developed nor validated by the manufacturer for any kind of vitro diagnostic application Declaration of Conformity The instrument meets the requirements laid down in Council Directive 2004 108 EC relating to Electromagnetic Compatibility and Council Directive 2006 95 EC relating to Low Voltage Equipment The following standards were applied IEC EN 61326 1 EMC IEC EN 61010 1 Safety The product meets US and Canadian safety requirements for electrical equipment for measurement control and laboratory use Issued by Underwriters laboratories Inc cus UL for Canada and the US UL 61010 1 Electrical Equipment for Measurement Control and Laboratory Use Part LISTED
17. the diagram so that it can be immediately seen where the best position for a series is in the diagram Large time differences can be entered in days d and small ones in hours h by clicking on the appropriate buttons d h The smallest time unit that can be set is 0 10 hours The OK button confirms the offset values for the graph Variable axis calibration The buttons lin or log are used to determine whether the y axis should display in a linear lin or logarithmic log scaling for cell density The default value is lin The x axis units can be displayed as either days d or hours h Click on the Time d below the x axis to switch between days and hours The x axis calibration is automatically recalculated The Exponential Regression Button Use the Exponential Regression button in the View section of the CTC Views window to display cell culture growth patterns with the calculated Specific Growth Rate and Doubling Time T The corresponding values appear in the Data Series list see Figure 21 34 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Analyzing the measurement results atio e a pone al Regre Regression 10 J eee Y Axis C lin y 0 exp u t T log T In 20 amp Viable Cell Density 5 0 10 0 Time h PRINT REPORT Data Series Chart Offset Color Sample ID start Sample ID end 1 on off T P0802 7 602 0024
18. to confirm the modifications for the new Cell Type 8 Click on Cancel to close the Live Operator window without saving changes Editing an existing Cell Type WARNING An existing Cell Type can be modified However it is NOT advisable for data safety reasons to modify an existing Cell Type that has been used for measurements A new Cell Type based on an existing Cell Type should be created instead Follow the instructions below to modify an existing Cell Type not advisable 1 Select the Cell Type to be edited from the list of Cell Types at the top of the Cell Type List window and then click on the Edit button The Cell Type window will open 2 Click on the Live Operator button in order to modify the parameters for the Cell Type within the Live Operator window see Working with the Live Operator 3 Make the desired modifications on a single parameter first then continue to adjust other parameters 4 When an optimized adjustment of the Operator parameters has been found click on Save to confirm the modifications 5 Click Cancel to close the window without saving any changes Viewing an existing Cell Type Select the Cell Type to be viewed from the list of Cell Types at the top of the Cell Type List window and then click on the View button The Cell Type window will open Click on the Live Operator button in order to view the parameter settings for that Cell Type see Description of the Individual Operator Parame
19. 1 General Requirements CAN CSA C22 2 No 61010 1 Second Edition Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements FCC Notification WARNING Changes or modifications to this unit not expressly approved by the party responsible for compliance may void the user s authority to operate the equipment Cedex XS User Manual 5 12 04 2009 MM DD YYYY Cedex XS Note This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications This is a Class A product In a domestic environment this product may cause radio interference in which case the user may be required to take adequate measures Safety Instructions WARNING It is important that the following safety instructions and guidelines be observed in order to ensure safe and reliable operation of Cedex XS Place the device and sturdy surface free of vibrations Prevent the device from getting wet Protect the device from dust and smoke Do not expose the device to direct sunlig
20. 2 The width of the histogram bars CriticalDiskSpace 1 The limit for the free memory space on the non removable disk in gigabyte The user is warned when executing a measurement if the space is below this limit DataBaseDriveMap C The drive on which the database is located this will for Name example be used for checking the free disk space see CriticalDiskSpace Warning Do not change this parameter without consulting with Roche Innovatis AG MEASUREMENT Option Value Description Save last value CellType Std Size Default Cell Type for measurements Measurement 1limage Default precision for the measurement low medium high Precision X ReactorID Default ReactorlD for measurements X SamplelD Default SamplelD for measurements X SamplelnitialDilution 1 2 Default dilution factor for measurements 1 2 1 20 etc X Mask image 1 Mask image required X required Sample volume 50 Default sample volume used for calculating final dilution X default Dye volume default 50 Default dye volume used for calculating final dilution X Diluent volume 0 Default diluent volume used for calculating final dilution X default MEASUREMENT LIST DEFAULT FILTER Option Value Description Save last value CellType Measurement List Default Filter for CellType X ProcessDate From Measurement List Default Filter for Process Date From X ProcessDateTo Measurement List Default Filter for Process Date To X ReactorlD Measurement List Default Filter for ReactorlD X SampleDrawi
21. Cedex XS User Manual INNOVATIS QUALITY THAT COUNTS Cedex XS User Manual Version 1 4 for Cedex XS Software Version 1 0 3 12 04 09 MM DD YY 2 Cedex XS User Manual 12 04 2009 MM DD YYYY Contents cs cc eas Se eae ded eet 5 Be 5 5 Declaration Of Conformity Leere r 5 ae e nce ce 5 Safety IMStructiOns so H 6 1 INTRODUCTION sensu 9 2 WORKING WITH THE CEDEX XSisssscsssisnsdsnstisstssatinaassadinsdssndsuaassadiasdsansdands 11 21 Switching on and Shutting 11 Switching on the analyzer and starting the Cedex XS Control 11 Shutting down the Cedex XS Control 11 2 2 Description of the Cedex XS Control Center eese 11 Ment Bad hieme utu taut tt EE 12 Spice 13 Tracking status of events carried out by the 13 2 3 Carrying out a 5 13 Defining the
22. Cell Type in the list BEE Cell Type 3 a wi IR Operator CedexxSCellOperator CedexXSCellOperator CedexXSObjectOperator _ gt Last Update SET AS DEFAULT SAVE CANCEL Figure 23 Cell Type window To modify the parameters of the new Cell Type do the following 1 Highlight the row of the new Cell Type and click on the Live Operator button The Live Operator window will open see Figure 24 38 Cedex XS User Manual 12 04 2009 MM DD YYYY Adjustment of the Image Analysis 4 2 In the Data Source area select the new Cell Type to be modified from the drop down list 3 Select the Reactor ID Sample ID and Data Set of the measurement for which the image analysis should be improved The parameters for the loaded measurement are preset 4 Select an image of the measurement the Image ID section This image will be loaded and displayed in the window in the upper right hand corner with the living cells marked with a green circle and the dead cells marked with a red circle 5 Make the desired modifications a single parameter first see Description of the Individual Operator Parameters As soon as a parameter has been changed reprocessing of the data is automatic The image display then shows the reprocessed image see Working with the Live Operator 6 Continue to adjust parameters until an optimized adjustment of the Operator parameters has been found 7 Click on Save
23. D8123 169 CHO K1 Test 8000001 Std Size 11 12 2008 10 09 35 V correctdiluion 11 12 20 rH Iv G067D8123 70 CHO Kibatchi 8000002 Size 11 12 2008 10 34 55 7 Original 11 12 2008 Iv Iv G067D8123 CHO Kibatchi 5000003 Std Size 11 12 2008 11 08 07 7 Original 11 12 2008 Iv Iv G067D8123 72 CHO K1 batch 8000004 Std Size 11 12 2008 11 08 38 7 Original 1112 2008 Iv Iv 906708123 4 gt Broo Figure 11 Measurement List window 22 Cedex XS User Manual 12 04 2009 MM DD YYYY Analyzing the measurement results Viewing existing measurement results Existing data sets may be viewed by highlighting the row containing the desired measurement and clicking on the View button in the bottom right hand corner of the Measurement List window A Measurement window will open containing all of the relevant results for the selected measurement see The Measurement Results Window Searching for measurements It is possible to search for specific measurements using a number of different filter criteria including sample drawing date process date Reactor ID Sample ID system name and Cell Type in the Filter By area at the top of the Measurement List window To search for measurements based on various criteria do the following 1 Click on the drop down menu button next to the Sample Drawing Date From or Process Date From sections A calendar will appear in which the desired starting date for filt
24. Unit Absolute value This parameter influences the recognition of living cells in aggregates The higher this value is set the more living cells are recognized in aggregates The lower this value is set the fewer living cells that are recognized in aggregates 42 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Adjustment of the Image Analysis 4 DCAggrApp Dead Cells Aggregate Appearance Range 10 10 Default 0 Unit Absolute value This parameter influences the recognition of dead cells in aggregates The higher this value is set the more dead cells are recognized in aggregates The lower this value is set the fewer dead cells that are recognized in aggregates MinPollutionArea Minimum Pollution Area Range 50 1000 Default 200 Unit um This parameter determines when an image will not be analysed i e the size diameter in micrometer that an object e g dirt or bubbles must have for an image to no longer be analyzed is given here NOTE Note The use of the sliders especially in combination can lead to unexpected results Cedex XS User Manual 43 12 04 2009 MM DD YYYY Cedex XS 44 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Data Management o 5 Data Management 5 1 Printing Reports Print Report buttons can be found in all the appropriate windows e g Measurement List Cultivation Time Chart Measurement window etc In addition a Report menu is also available on the menu ba
25. ambers for that measurement have been analyzed After the final measurement has finished click on OK to confirm the message Measurement is done 1 Please switch to the next chamber of the inserted slide and press OK Press Cancel to end the measurement now Abbrechen Figure 8 Message for switching the sample chamber 16 Cedex XS User Manual 12 04 2009 MM DD YYYY Working with the Cedex XS The Cedex XS can determine cell concentrations in the range of between 1 10 1 107 cells mL Any higher concentration requires a manual dilution Cells with a diameter range of between 5 um to 180 um can be assessed accurately Object recognition performance lies between 2 um and 180 um WARNING Samples should not be left in the sample chamber for more than 15 minutes before measuring If samples are left for longer than 15 minutes evaporation may occur and lead to inaccurate results End of the Measurement The results from the analysis are displayed in the Measurement window after the measurement is finished see Figure 9 The top section of the Result Data area displays the overall results of the measurement If more than one sample chamber was used for the measurement the bottom section displays in succession the results from each sample chamber analyzed When a line is selected in the list of results from single sample chambers the corresponding image will be displayed in the Ima
26. and click on the Reprocess button in the lower right hand corner A Measurement window will open containing all of the result data for that measurement 3 Enter a new name for the data set in the Data Set section of the Sample Parameters area 4 Ensure that there is a check mark in the Effective check box if the new data set should be used as the effective data set see Effective data sets 5 Make the desired changes to the new data set Changes can be made with regard to the dilution factor and Cell Type in the appropriate sections of the Sample Parameters and Processing Parameters areas 6 Click on the Reprocess button in the center of the Measurement window to start reprocessing the measurement based on the new parameters 7 When the reprocessing is finished a window will open asking whether the new data set should be the effective data set Click on Yes if it should be the effective data set A new data set is available once the evaluation has been completed All changes can be retraced by viewing previously created data sets Note NOTE The reprocessing procedure creates additional data sets Original data is not overwritten Effective data sets When measurements are reprocessed it is possible to label one of the resultant data sets as Effective The Effective data set is the version of the measurement original or reprocessed that has the correct parameters Once a data set has been selected as Effective it will be labe
27. below to perform a HW Startup 1 Click on the HWM button in the Cedex XS Control Center or else select Hardware Management under the Functions menu The Hardware Management window will open see Figure 28 2 If the Hardware Management has not been started the HW STARTUP button will be active while the WHITE BALANCE and HW SHUTDOWN buttons will be inactive 3 Click on HW STARTUP The Hardware Management will start and the WHITE BALANCE and HW SHUTDOWN buttons will become active 4 Click on 2 to close the window Follow the instructions below to carry out a HW Shutdown 1 Click on the HWM button in the Shortcuts area of the Control Center to open the Hardware Management window 2 Click on the HW Shutdown button in the lower right hand corner of the window to shut down the Hardware Management 3 Click on 2 to close the window Hardware Management WHITE BALANCE HW SHUTDOWN Figure 28 Hardware management window Performing a White Balance If the brightness in an image is uneven a White Balance should be carried out The White Balance determines the proper adjustment for image brightness To carry out a White Balance do the following 1 Pull the slide carrier completely out of the Cedex XS 2 Click on the HWM button in the Shortcuts area of the Control Center to open the Hardware Management window 3 Click on the White Balance button in the middle The White Bala
28. ble CSV Comma Separated Values generally applicable SML and flat file The use of a Live Operator makes it possible to directly observe the effects on the analysis of a sample when parameters affecting the analysis of the images are adjusted The image acquisition of the samples takes place in the analyzer The 8 sample chambers of the Cedex Smart Slide have been specially developed for the analysis of a defined volume of cell suspension The analysis software is installed on a computer and controls the analyzer see Figure 1 It also controls the analysis and evaluation of the images obtained The Cedex XS Software can be operated on Windows XP Professional or Windows Vista Home Premium and Business versions The analysis software and its graphical user interface GUI known as the Cedex XS Control Center enables the user to work with the Cedex XS The Cedex Software needs to be installed on a computer that is provided either by the user or by Roche Innovatis AG The software analyzes the digital images provided by the analyzer The determination of the number of viable cells cells mL and cell viability of cell suspensions the percentage of viable cells found in the total cell sample is a semi automated procedure based on the manual Trypan Blue Exclusion Method which is based on the principle that viable cells will not take up the dye while dead or dying cells have a compromised membrane that allows for the uptake of the dye Thus d
29. can guarantee optimal functioning of the Cedex XS Analyzer Ensure that the Trypan Blue solution is free of contamination otherwise filter prior to use Secure your measurement data Avoid looking directly into blue LEDs located on the front of the Cedex XS Analyzer as long as they are active Extended and close contact to the LEDs may be harmful to the eyes 6 Cedex XS User Manual 12 04 2009 MM DD YYYY WARNING Installed anti virus software should never be set up to scan the DB directory and especially not the FDB file itself NOTE The software and hardware of your Cedex XS can differ from the information described here but this should not make a difference to the operational functions in general WARNING The manufacturer is not responsible or liable for any damages resulting from improper use or failure to comply with the regulations in the operating instructions gt Cedex XS User Manual 7 12 04 2009 MM DD YYYY Cedex XS 8 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Introduction 1 introduction The Cedex XS determines the cell concentration and viability of cell suspensions semi automatically The analysis is based on the Trypan Blue exclusion method whereby cell suspensions are stained and analyzed with regard to the number of live and dead cells The data obtained allows for the evaluation of the overall state of a cell culture including specific growth rate and doubling time of a cell populat
30. choosing a set of measurements hold the Ctrl button down while selecting the appropriate lines In addition the desired measurements can be searched for using various filters see the section Using the Measurement List 3 Click on Archive to open the Archive window Cedex XS User Manual 47 12 04 2009 MM DD YY YY Cedex XS 4 The path under which the archive file should be stored is entered in the Location area The button on the right of the entry field opens an explorer window in which the path can be selected directly A file name for the data to be archived needs to be entered Click on Save The Archive window will reappear Click on the Archive button to start the archiving process A message window will appear as soon as archiving of information is completed successfully Click on OK T Close the Archive window by clicking on Ba A default entry for the path under which the archive file should be stored can be entered in the User Preferences under the Setup menu Open the Data Exchange tab and look for the ArchivePath parameter In the Value column enter the default path Restore To restore measurement data follow the instructions below 1 Select the Restore function within the Archiving menu to open the Restore window 2 The path of the file that is to be restored is entered in the Location area The button on the right of the entry field opens explorer window which the path the file ca
31. cted view area of the CTC Views window To edit the measurements belonging to a View do the following 1 Highlight the row of the View to be edited in the View area of the CTC Views window 2 Highlight the particular series of the View to be edited in the Series belonging to the selected view area 3 Click on the Edit button This will open the Selection of Data Series window see Figure 18 4 Add measurements from the series list as described above remove measurements by highlighting them and clicking on the Remove button at the bottom of the window 5 Click on the Save button The changes will be saved and the window will automatically close 6 The CTC Views window will become available and other series in the same View can now be edited Measurements can also be excluded from a cultivation time chart without being removed from the series All measurements selected for a series that are marked with in the checkbox of the Use column of the Selection of Data Series window will be displayed in the Cultivation Time Chart Should a particular single measurement not be displayed in the chart the measurement can be unselected by removing the the Use column with a mouse click The measurement will remain in the list but not appear on the chart Viewing list of measurements in a series If measurements have been added to a View a simplified list of the series will appear in the left hand side of the Series bel
32. cts such as cells debris air bubbles and viable and dead cell aggregates Histograms Diameter Histogram for all Objects AGGREGATE HISTOGRAM 8 T CELL DIAMETER HISTOGRAM 8 8 8 HHHH ee pe HHHH COMPACTNESS HISTOGRAM 8 OBJECT DIAMETER HISTOGRAM MEASUREMENT STATISTICS Number of objects E Ep 5 8 10 2 Objects Diameter um Objects as single viable and dead cells debris air bubbles beads aggregates of viable and dead cells medium components contaminants Cell Diameter um 0 lt 10 210 lt 15 Number of cells 225 44 4639 89 9 Concentration 2 01 10 5 41 54 10 5 Figure 15 Object Diameter Histogram Cedex XS User Manual 27 12 04 2009 MM DD YYYY Cedex XS Defining diameter intervals in the Diameter Histograms Both the Cell and Object Diameter Histograms have three sliders that can be used to define separate diameter intervals This allows for dividing a diameter histogram into a maximum of four parts Activate the sliders by clicking on the corresponding boxes and moving the sliders to the desired positions The user can also define the margins of the intervals by entering the numerical values on the right hand side of each slider The borders of the first interval are defined by the minimum value and the first numerical value entered The next two values define the upper borders of the second and third intervals The fourth interval is defined
33. d for a single measurement the bottom section of the Result Data area will display the results for each sample chamber analyzed If only one sample chamber was used for a measurement the results for that sample chamber will appear in the section below Cedex XS User Manual 19 12 04 2009 MM DD YYYY Cedex XS The following parameters are shown in the Result Data area for both the overall results and individual sample chamber results e Viable Cell Conc Concentration of viable cells x10 cells mL e Total Cell Conc Total cell concentration x10 cells mL e Viability 96 The proportion of viable cells 96 to the total number of cells viable and dead cells e Total Cell Count The number of all detected objects counted as cells e Avg Compactness The average variation in shape of viable cells from an ideal sphere with the ideal sphere shape equaling gt 1 lt e Avg Diameter The average diameter of viable cells detected Aggregate Rate Specifies the percentage of all cells Total Cell Count found in aggregates clusters Dead Cell Conc Dead cell concentration x10 cells mL Viable Cell Count The number of viable cells counted Dead Cell Count The number of dead cells counted Total Object Count The number of all detected objects counted Total Object Conc Total object concentration x10 cells mL Avg Object Diameter The average diameter um of objects detected Avg Object Compactn
34. dow to CT1 and has activated the SaveLastValue option If the user then changes the Cell Type parameter to CT2 when running a new measurement CT2 will be automatically saved as the new default in the User Preferences window and will be automatically recommended for the next measurement The SaveLastValue function can be activated for all options in the User Preferences window The parameters for which the last value is saved by default are marked below in grey WARNING Many of the elements in the User Preferences must not be changed without prior arrangement with Roche Innovatis AG Those parameters that should not be changed have been explicitly marked with a warning in the description of the preferences below In all cases Roche Innovatis AG Service and Support must be contacted before any changes are made to those parameters GENERAL Option Value Description Save last value ErrorLevelDisplayTo User Lowest level of error messages to be displayed to the user 1 Fatal 2 Critical 3 Error 4 Warning CellDistMin 3 0 Minimum cell distance um for which cells are ascertained to be single cells with regard to the determination of the aggregate rate Warning Do not change this parameter without consulting with Roche Innovatis AG CellDistRatio Minimum relationship of the cell distance to the sum of the radii used for which cells are ascertained to be single cells with regard to the determ
35. e Add selected measurements button The highlighted measurements will appear in the bottom half of the window under the Measurements belonging to series area 6 Click on Save The Selection of Data Series window will automatically close The CTC Views window will reappear and show the measurements that have been added to the View in the Series belonging to the selected view area 7 To add another series to a View click on the New button again The Selection of Data Series window will open Follow steps 2 6 above to add measurements to the new series 8 Up to 5 separate series can be added to one View Once 5 series have been added to a View the New button will no longer be available for that View Selection of Data Series Reactor ID 1 m Data Set original x Reactor ID Sample ID Data Set Start date time Valid 9 22 2008 1 56 PM __ CHO_K1_batch1 00003 Original 9 23 2008 9 06 AM CHO K1 batch1 00004 Original 9 25 2008 10 34 AM ADD SELECTED MEASUREMENTS Measurements belonging to series Use Reactor ID Sample ID Data Set Ed batchi 500001 Origina REMOVE SAVE CANCEL Figure 18 Selection of Data Series window 30 Cedex XS User Manual 12 04 2009 MM DD YYYY Analyzing the measurement results Editing measurements in a View Measurements can be added or removed from a particular View by using the Edit button in the Series belonging to the sele
36. each other The lowest common value for all the measurement series is automatically determined and the graphs in the diagram are shifted to each other so that the lowest common value falls on the x axis at the same point While doing so the left most point of the graph of all the measurement points is set at value 0 Measurement Offset The graph automatically displays the measurement series so that all series begin at time 0 The user however may prefer to compare different measurement series by setting the offsets so that the beginning of the exponential growth phase log phase starts at the same time for all series The Measurement Offset can be used to shift the curves in the diagram by changing the offset for each series Cedex XS User Manual 33 12 04 2009 MM DD YYYY Cedex XS A button in the Offset column of the Data Series area displays the offset set by the user the button displays 0 h if no offset was set Click on this button to open the Measurement Offset window The measurement offset can be then manually shifted in this window for Measurement Offset Offset 0 00 zi Figure 20 The Measurement Offset window The offset can be separately entered for each of the five measurement series Fix the offset by selecting the desired series number colored boxes and then moving the slider to the desired offset Using the slider will cause an immediate shift of the measurement series in
37. ead cells will appear darkly stained The analysis software recognizes structures and identifies particles thus differentiating cells from cell debris and dirt particles The image processing is integrated into the Cedex XS Control Center in such a way that the user can constantly check on its function in the system Cedex XS User Manual 9 12 04 2009 MM DD YYYY Cedex XS NOTE Trypan Blue has a greater affinity for serum proteins than cellular proteins If the image has a dark background due to high quantities of stained serum proteins centrifuge the cell sample and re suspended it in a protein free medium or physiological saline solution prior to counting Sample chamber position indicator lights Slide carrier for Cedex Smart Slide ON OFF Switch Figure 1 The Cedex XS Analyzer 10 Cedex XS User Manual 12 04 2009 MM DD Y YY Y Working with the Cedex XS ev 2 Working with the Cedex XS 2 1 Switching on and Shutting Down Switching on the analyzer and starting the Cedex XS Control Center Proceed as follows 1 Switch on the Cedex XS Analyzer 2 Switch on the monitor and the PC or laptop 3 Double click on the Cedex XS icon on the desktop to launch the software The Cedex XS Control Center appears after a successful launch of the program and the user has access to all functions WARNING Cedex XS hardware components must be switched on before starting the Cedex XS Software Otherwise the analysis software will no
38. easurements can be run e Cultivation Time Chart Opens a window in which multiple measurement series can be analyzed in graphical form Functions Menu e Operational Data The operating hours and number of measurements that have been carried out are tracked in this window e Live View Opens a window that provides a live view of the sample chamber e Hardware Management Execute start up and shutdown of the hardware in this window e Export to File Measurement results can be exported into various formats including Excel e Archiving Data from the database can be archived or restored e Data Exchange Data can be imported or exported from the database of one system to the database of another system Reports Menu Reports can be generated for the following types of information e Operational data e Measurement data e System configuration parameters Setup Menu e Cell type list For the creation of customized cell types e Diagnosis At the request of Roche Innovatis AG support a diagnosis of the entire system can be run with the options listed in this window e Trace and Logging At the request of Roche Innovatis AG support logs of various functions of the system can be created in this window e User preferences User preferences can be set for default parameters such as measurement parameters measurement lists and the Cultivation Time Chart Help Menu The Help menu provides general information about the analysis sof
39. egate Size Figure 13 The Aggregate Histogram total view sum of living and dead cells here absolute number of cells 26 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Analyzing the measurement results The percentage of cells found in aggregates in relation to the total cell count can also be displayed by using the percent button Cell Diameter Histogram Click on the Cell Diameter Histogram button to display the Cell Diameter Histogram see Figure 14 The Cell Diameter Histogram shows the diameter distribution of living cells only and is based on the individual diameters measured from all living cells detected in the measurement Histograms Cell Diameter Histogram AGGREGATE HISTOGRAM CELL DIAMETER HISTOGRAM MEASUREMENT STATISTICS 3 8 Number of cells 8 2 2 Cell Diameter um This diagram shows all viable cells with definable diameter 10 9 97 15 03 Diameter um 0 lt 9 97 997 lt 15 03 215 03 lt max Number of cells 40 0 9 4068 93 5 244 5 6 Concentration 0 36 10 5 36 43 10 5 2 187 10 5 Figure 14 Cell Diameter Histogram Object Diameter Histogram Click on the Object Diameter Histogram button to open the Object Diameter Histogram see Figure 15 Analogous to the representation of the diameter of the living cells the Object Diameter Histogram shows the diameter distribution of all recognized objects including obje
40. enter offers users the option to modify default values for a variety of parameters via the User Preferences option in the Master Data menu Specifying Default Measurement Parameters Preferred default values for certain measurement parameters cell type process type dilution etc can be set in the User Preferences window Follow the instructions below to change the default values 1 Select User Preferences in the Master Data menu of the Control Center This will open the User Preferences window Select the Measurement tab in the User Preferences window see Figure 26 Manually type in the appropriate default measurement parameters in the Value column The defaults for the following parameters can be determined e MeasurementPrecision e CellType e ReactorlD e SamplelD e SamplelnitialDilution 4 Put a check mark in the check box of the Save Last column if the last value used for a measurement should be used as the default for the next measurement 5 Click on Save to confirm 6 Close the window The new default parameters will apply to new measurements User Preferences General Measurement Measurement List Default Filter Hardware Data Exchange Tracing Loaaina Series Default Filter CTC Views 41 Value 4 The number of images combined to make a bar depends on the number of Combined Image Count Edit field 4 images Default user value MeasurementPrecisionfor a new Measurement Low Medium High Std Si
41. er Default 49 7 ADDITIONAL HARDWARE AND SOFTWARE ELEMENTS 51 Checking the Live View of the sample 51 Operational Data mii 51 Performing HW Startup and HW Shutdown ananas 52 Performing a White Balance 52 8 f iuazppe 53 Contact Addresses ies sess ra alunni EI LLLI 53 ManufactUl er s Ehe ob acetates 53 Belin cS 53 DiStriDUtion im WS A iei cba Aa end eg 53 SUP POUL BER ETE 53 Technical Specifications 54 List of User Preferences 55 2 59 me 59 Disposal Recommendations 59 4 Cedex XS User Manual 12 04 2009 MM DD YYYY Rights Copyright Cedex XS Operating Instructions Copyright 2009 Roche Innovatis AG All rights reserved Operating instructions and manuals are protected by copyright No part of this document may be reproduced edited or translated without prior written consent of Roche Innovatis AG except as permitted under the terms of the copyright Disclaimer The information in this document is
42. ering can be selected 2 When a date has been selected a check will appear in the check box next to the date to indicate that a filter is active based on that date 3 Click on the drop down menu button next to the second check box in order to select the desired ending date 4 Once the dates for filtering the measurements have been selected click on the Find button in the middle of the Measurement List window 5 All of the measurements that match the dates given for sample drawing using the Sample Drawing Date From section or that were run within the dates specified in the Process Date From section will be displayed in the measurement list in the bottom half of the window It is possible to select measurements with regard to both sample drawing date and process date If starting and ending dates for both sample drawing and process dates are selected only those measurements that meet all 4 date criteria will be displayed when the Find button is clicked on Measurements can also be searched for based on Reactor ID and or Sample ID by filling in the information in these sections of the Filter By area In addition it is possible to filter measurements according to data set in the Data Set area and Cell Type using the drop down menu in the Cell Type section NOTE If a measurement has been reprocessed the original and all reprocessed versions of the Note measurement will be listed The data set of a particular measurement that has bee
43. ess The average variation in shape of objects from an ideal sphere with the ideal sphere shape equaling gt 1 lt Sample and Processing Parameters areas The Sample Parameters area of the Measurement window shows the parameters entered by the user before running the measurement If any measurements have been reprocessed the original or reprocessed results can be selected in the drop down menu of the Data Set If a measurement has been reprocessed at some point the Effective checkbox will indicate which data set has been determined to be the effective version see Reprocessing a measurement The Processing Parameters area lists the process parameters of the measurement including Cell Type used to analyze the images see Adjustment of Image Analysis with the Live Operator the precision selected see Defining the measurement and analysis process parameters and the date and time the process was run Image area Viewing cell images The Cedex XS Software enables a close view of an image or an enlarged image display for evaluation Images are viewed in the Image area of the Measurement window When a row with the results of a particular sample chamber is highlighted in this section the image connected with those results is displayed in the Image area To see a different image in this area click on a different row in the Result Data area Only one image is displayed in the Image area of the Measurement window Click on the View
44. ge area see Image area The results of the measurement can be directly printed out via the Print button in the middle of the Measurement window To set up a new measurement click on the New Measurement button If the next measurement belongs to the same Reactor ID as previous measurements the consecutive Sample ID will be automatically entered into the Sample ID area by the analysis software without further entries being necessary If a different Sample ID is desired the new Sample ID can be typed in manually Cedex XS User Manual 17 12 04 2009 MM DD YY YY Cedex XS 18 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Analyzing the measurement results Analyzing the Measurement Results 3 1 The Measurement Results Window Measurement results are displayed in the Measurement window see Figure 9 The analysis results obtained from a measurement are automatically displayed in the same window after the analysis process is completed Measurement results from past measurements can be viewed via the Measurement List window accessed using the List button in the Shortcuts area see Using the Measurement List for more details about working with the Measurement List Measurement Sample Parameters Reactor ID O K1 test 7 Precision 3 images VIEW IMAGES Sample ID Cell Type 1 Size Sample drawing 0 12 2008 13460 Data Set System G067D8123 Dilution 12 E ProcessTi
45. ginal size The brightness and contrast of the image can also be changed for a better analysis Operational Data The Cedex XS System keeps track of some operational data including operating hours and number of measurements Select Operational Data in the Functions menu of the Cedex XS Control Center to open the Operational Data window The values on the left hand side of the window can be set to 0 by clicking on the Reset button This can be useful for tracking the operational information for short periods of time The values on the right hand side track the overall operating hours and number of measurements These values cannot be reset A report of the operational data can be printed out via the Print Report button Cedex XS User Manual 51 12 04 2009 MM DD YYYY Cedex XS Performing a HW Startup and HW Shutdown The HW Startup initializes all of the hardware dependent software modules The HW Startup is automatically carried out when the software is launched However the hardware management can be manually shut down via the HWM button in the Shortcuts area of the Cedex XS Control Center When the Hardware Management is shut down several modules are not yet initialized A HW Startup must be done before a measurement can be performed If a measurement is attempted before the Hardware Management has been started an error message will appear Cannot start a new process Hardware Management is not started Follow the instructions
46. ht Ensure there is sufficient air circulation around the Cedex XS System during operation particularly around the computer and monitor Operate within a constant temperature range of 50 to 100 F 10 to 40 Cedex XS should only be used with the supplied or specified devices Cedex XS Analyzer Cedex XS Software and computer Only trained personnel in a controlled laboratory environment should operate the Cedex XS System All services repairs should be performed by authorized service technicians only Always observe the safety regulations when handling Trypan Blue Wasie contaminated with tissue cells e g Cedex Smart Slides pipette tips etc must be disposed of according to the recommended rules for handling biohazard materials Only use the Cedex Smart Slide from Roche Innovatis AG for the conveyance cell samples to the Cedex XS Other slides can cause irreparable damage to Cedex XS The Cedex Smart Slide is designed for single use only Always wear protective clothing particularly gloves when handling samples Dirt or dust particles should not gain access into the device Ensure that all equipment required for sample preparation e g Cedex Smart Slides pipette tips etc are stored in a dust free environment since coarse dust particles can affect the image quality Check that the Cedex Smart Slide is free of contamination before use Only clean slides
47. ination of the aggregate rate Warning Do not change this parameter without consulting with Roche Innovatis AG CertificateStore My Name of the certificate storage from which the certificate for the check of the license and the signature for the archive is searched Warning Do not change this parameter without consulting with Roche Innovatis AG FlowFactor 1 00 Correction factor regarding movement of liquid Warning Do not change this parameter without consulting with Roche Innovatis AG InnovatisCertificate Innovatis AG Test Specification of the certificate to be used for inspection of the license Warning Do not change this parameter without consulting with Roche Innovatis AG JpegQuality 95 JPEG image encoder quality Warning Do not change this parameter without consulting with Roche Innovatis AG OverallTypeFor Exponential ViableCell Density not adjustable Overall Type for the exponential regression used for the determination of doubling time and Cedex XS User Manual 12 04 2009 MM DD YY YY 55 Cedex XS Option Value Description Save last value Regression specific growth rate MaxCellDensity 5000000 density warning level Maximum total cell concentration in an image without the dilution applied HistogramBarWidth
48. ion as well as documentation of the cultivation process Many parameters including viable cell concentration total cell concentration viability object count average compactness and average diameter are analyzed using result data from the images In addition the aggregate rate which shows the percentage of the total number of cells that appear in aggregates clusters is calculated Graphical representations of the measurement results can be viewed in the Diameter Histogram Compactness Histogram Aggregate Histogram and Cultivation Time Chart The Cell Diameter Histogram shows the diameter distribution of the living cells An additional diameter histogram the Object Diameter Histogram shows the diameter distribution of all objects detected such as cells and debris as well as viable and dead cell aggregates The deviation of the shape of living cells from that of an ideal sphere is represented in the Compactness Histogram The Aggregate Histogram gives an overview of the amount and distribution of aggregation found in the cell culture The Cultivation Time Chart displays the growth curves and cultivation parameters in a time dependent manner The Cultivation Time Chart includes calculations for the doubling time T and specific growth rate u and the results can be separately printed out For further processing of results in other programs it is possible to export the data into formats such as DIF Data Interchange Format Excel compati
49. it lowest common value Viable Cell Density C Dead Cell Density Total Cell Density None FIT ENLTIDITCANM Data Series Regression Chart Offset Color C tog 2 3 onloff mi 4 onoff z 5 ow Figure 19 Cultivation Time Chart window The measurement series including information about which measurements have been used to create the chart are listed in the Data Series area in the bottom left hand corner of the Cultivation Time Chart window A legend at the top of the graph indicates the symbol shape and color used for each parameter The color used for each series is indicated in the Chart area in the lower right hand corner of the window Place the mouse pointer exactly over a measurement point in the diagram to see detailed information about the point including the date and time of the measurement as well as the result for that parameter Also the graphic representation can be enlarged or scaled down again with the use of the mouse see Enlarging the histograms Fitting to the lowest common value The Fit lowest common value button enables automatic shifting of the zero point of single measurement series to each other The following can be selected e Viable Cell Density e Dead Cell Density e Total Cell Density Using this function the zero point of each series will be automatically shifted on the x axis in order to be able to better compare measurement series to
50. led as such in the Measurement List window The data set to be labeled Effective can be later changed by doing the following 1 Select the data set version of the measurement to be labeled Effective in the Measurement List window 24 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Analyzing the measurement results 2 Click on the Reprocess button A Measurement window will open containing the results for that data set 3 Click on the Effective checkbox in the Measurement window A check will appear in the box 4 Close the Measurement and Measurement List windows 5 When the Measurement List window is re opened the newly selected data set will appear as the Effective data set 3 4 Graphical Display of the Analysis Results The Cedex XS Software provides graphic diagrams of the results Histograms can be viewed by clicking on the Histograms button in the Measurement window to open the main Histograms window There are 4 different histograms that can be viewed via this window Aggregate Histogram Cell Diameter Histogram Compact Histogram Object Diameter Histogram Click on the respective button to open the desired histogram All of the results presented in the histograms are based on valid images accepted for evaluation This means that results from individual images that have been manually excluded by the user will not be included in the histograms see Excluding images from the evaluation process Enlarging the hi
51. llow the instructions below to change the settings 1 Select the Cell Type that optimally recognizes your cells or objects from the Cell Type drop down list The following cell types are available by default see Adjustment of 14 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Working with the Cedex XS ev Image Analysis with the Live Operator for more details about cell types e Std Size for the identification of live and dead cells e Objects for the identification of objects If a new Cell Type has been created it can also be selected from the same Cell Type drop down list Detailed information on how to create a new Cell Type can be found in Adjustment of Image Analysis with the Live Operator 2 Select the desired precision in the Precision drop down list The precision level determines the number of sample chambers to be analyzed for a single measurement The results from each chamber analyzed will be combined to give an overall result Results from up to 8 sample chambers can be combined for a single measurement A higher number of samples per measurement requires a somewhat longer measurement time but more cells will be counted which will lead to a more precise calculation Preparing the sample and starting the measurement Once the sample and processing parameters have been defined the sample can be prepared and the measurement started Figure 5 Adding sample to the sample chamber To prepare the sample carry out
52. lls default Cell Type Std Size e CedexXSObjectOperator for the analysis of objects default Cell Type Objects Individual adjustments of the operator can be helpful or necessary for cell cultures that for instance have an unusual optical appearance or for a counting strategy that deviates from the standard counting strategy e g counting aggregates or cell nuclei differently Adjustment of an operator is referred to as creating a new Cell Type A new Cell Type can be created or an already existing Cell Type adjusted This can be done with the help of a number of parameters that influence the way in which an image is evaluated Each of these parameters directly influences the image processing strategy and hence the measurement results WARNING The parameters of the factory installed pre settings are overwritable For data safety reasons the parameters of the factory installed pre settings should not be altered 41 Description of the Cell Type List and Live Operator The Live Operator window in which Cell Types can be created and modified is accessed in two ways e Via the Cell Type List option in the Master Data menu of the Control Center e Via the Measurement window in the View mode The Cell Type List window Select the Cell Type List option in the Master Data menu to open the Cell Type List window see Figure 22 There are several options in this window including New Create new Cell Types Edit Modify existing Cell Type
53. lue Description Save last value ExportCertificate Innovatis AG Certificate that should be used for the designation and Archive Test lexamination of archives for Data Exchange Export Import ExportDelimiter Default value separator for the CSV Excel export measurements ExportPath cA Default for the path that should be used for the Data Exchange export ExportSTring Default for the export file string delimiter Delimiter ArchiveCertificate Innovatis AG Certificate that should be used for the designation and Archive Test examination of archives for Archive Restore Warning Do not change this parameter without consulting with Roche Innovatis AG ArchivePath CA Default path that should be used for Archive Restore ImportPath Default for the path that should be used for the Data Exchange import Measurement Measurement Default file name for CSV Excel export of measurement ExportFileName Export txt__ results AuditTrailExport AuditTrailExp Default file name for the CSV export of Audit Trail entries FileName ort ArchiveFileMask 8 5 6 Archive file name mask parameters 0 9 can be used CTCExportFile CTCExport Default file name for exported CTC files Name TRACING Option Value Description Save last value TraceOutputPath C Cedex2 CedexTrace txt Default file name for the trace file Cedex XS User Manual 57 12 04 2009 MM DD YY YY Cedex XS
54. lusion of images for a particular measurement can be done via the check box in the Excluded column in the Result Data area of the Measurement window A check in the Excluded check box means that the cell image has been excluded from the calculation process The overall results will only be calculated for images that are not excluded 3 2 Using the Measurement List Data from measurements that have been run in the past can be accessed via the Measurement List window Open the Measurement List either by clicking on the List button in the Shortcuts area of the Cedex XS Control Center or by selecting Measurement List in the Measurements menu at the top of the Control Center A number of actions can be carried out in the Measurement List window including searching for measurements using a number of different filters viewing measurements printing reports exporting measurements and reprocessing measurements Filter by Delete selected measurement s mele dste C Archived Measurements Delete onlyimages Delete records Process Date from Alene e Reactor ID Sample ID Data Set Cell Type SJ System Name G067D8123 Y C Original Effective All E ReacriD SamplelD CellType Sample Draw Valid DSNAME DSTME DSORIGINAL DSEFFECTVE CEDEXSYSTEMNAME Commer 69 CHO K1 Test 8000001 Std Size 11 12 2008 10 09 35AM 7 Original 11 12 2008 GB G067
55. me 12 1 2008 1 46 19 PM Comments F Valid Effective w gow LIVE OPERATOR HISTOGRAMS _ 2892609 Result Data ec Viable Cell Conc Total Cell Conc celsimt Viability s Total Cell Count Avg Compactness Ava Diameter um Aggregate Rate Dead Cell Conc gt J 940 850 1 157 13 42 16 7 04671075 4 zw el Dead Cell Conc cells mL Viable Cell Count Dead Cell Count Total Object Count Total Object Conc fobjects mL Avg Object Diameter Avg Object Compactness 05171075 252 19 275 7 44 10 5 12 78 1 245 05171075 252 19 275 744 10 5 1278 1245 0 35 10 5 295 13 302 8 17 10 5 13 03 1 235 Figure 9 The Measurement window with results The Measurement window has 4 areas see Figure 9 Result Data area Sample Parameters area Processing Parameters area Image area Some of the results can be displayed as histograms by clicking on the Histograms button see Graphical Display of the Analysis Results It is also possible to access the Live Operator via the Measurement window using the Live Operator button see Adjustment of Image Analysis with the Live Operator Result Data area The Result Data area in the bottom half of the Measurement window has two separate sections 1 The section just beneath the words Result Data displays the overall results for the measurement 2 If multiple sample chambers were use
56. meters in the User Preferences must not be changed without prior arrangement with Roche Innovatis AG Explicit notes have been attached to the descriptions of those parameters see list of User Preferences in the Appendix In all cases Roche Innovatis AG Service and Support must be contacted before any changes are made to those parameters 50 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Additional Elements B 7 Additional Hardware and Software Elements Checking the Live View of the sample chamber The sample chamber that is in position under the microscope can be viewed via the Live View button in the Shortcuts area of the Cedex XS Control Center see Figure 27 Adjustment x 9 Figure 27 The Live View of a sample chamber Certain areas of the Live View can be enlarged by following the instructions below 1 Place the mouse pointer the top left hand corner of the area to be enlarged 2 Hold the left mouse button pressed down and move it to the lower right hand corner of the area to be enlarged 3 Release the mouse button and the selected area will fill the whole window Return to the original size of the chart by following the instructions below 1 Place the mouse pointer anywhere on the diagram 2 Hold the left mouse button pressed down and form a box from the bottom right corner to the upper left corner 3 Release the mouse button The chart will then return to in its ori
57. mpactness Figure 16 Compactness Histogram 3 5 The Cultivation Time Chart Growth patterns of cell cultures can be graphically evaluated in the Cultivation Time Chart Click on the CTC button in the Shortcuts area of the Cedex XS Control Center or select the 28 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Analyzing the measurement results Cultivation Time Chart option in the Measurements menu to open the CTC Views window In the CTC Views window charts can be created viewed edited exported and deleted A collection of data used for the creation of a particular chart is called a View Each View can have up to 5 separate series of measurements in order to compare data Creating a CTC View The CTC Views window has two main areas see Figure 17 e n View area charts can be viewed exported created and deleted Previously created cultivation time charts can be found via the Filter By section located at the top of the window The user can filter by name of the View or by the date a View was created or modified Enter the desired parameters and click on the Find button When searching by date the check box next to the date must first be checked After checking the box click on the drop down menu to the right of the date to open up a calendar in which the desired date can be selected e Individual series of measurements belonging to the highlighted View can be created edited listed or deleted in the Series belonging to selec
58. mportPath 44 Intended Use sese 5 L LCAggrApp 40 40 Hem 33 Live Operator 8 17 35 38 Live Operator Parameters 38 39 Eom eee 33 M Maintenance 56 Master Data 11 12 61 Cedex XS Measurement data 17 21 27 42 Measurement 11 Measurement parameters 13 18 22 Measurement Series offset 32 Menu Dali cete 11 MinPollutionArea 41 Object Diameter Histogram 25 Object 16 Offset button 32 Operational Data 42 Operators nien eto 38 P Parameters 13 18 22 34 42 Processing parameters 13 18 22 R Reactor h 13 Reports nue eg eg dread 42 Reports 11 Reprocess 22 Result Data 16 17 5 5 Safety instructions
59. n be selected directly 3 Click on Open The Restore window will reappear Click on Restore to start the restoring process A message window will appear as soon as the information has been restored successfully Click on OK 5 Close the Restore window clicking B Deleting archived data Only data that has been archived can be deleted from the Cedex XS Database Archived data can be searched for and displayed in the upper right hand corner of the Measurement List window To delete archived data do the following 1 Click on the Archived measurements s check box in the upper right hand corner of the Measurement List window The list of archived measurements will appear 2 Highlight measurements to be deleted by clicking on the field on the left of the appropriate line When choosing a set of measurements hold the Ctrl button down while selecting the appropriate lines 3 Click the check box beside Delete records if all measurement data is to be deleted from the system Database If the option Delete only images is chosen all data apart from the images will stay in the system Database 4 Click on the Delete button to delete selected measurements 5 5 Securing Data In order to always have enough storage capacity on the Cedex XS computer measurement data must be secured at regular intervals 48 Cedex XS User Manual 12 04 2009 MM DD YY Y Y User Preferences o 6 User Preferences The Cedex XS Control C
60. n selected as the Effective data set will have a check in the check box of the DSEffective column The original data set of a particular measurement will have a check in the check in the check box of the DSOriginal column see Effective data sets 3 3 Reprocessing Existing Analysis Results Data Sets In addition to analysis the Cedex XS Software also allows for modification and reprocessing of existing measurement results or data sets These changes are done on existing software data image and analysis results and therefore do not require any adjustments to the analyzer Cedex XS User Manual 23 12 04 2009 MM DD YY YY Cedex XS Reprocessing a measurement Reprocessing existing measurement results data sets i e viewing a set of image data and re evaluating them with new analysis parameters is done in the Reprocess mode via the Reprocess button in the lower right hand corner of the Measurement List window The reprocess mode offers the following options e Correct the dilution see Defining the measurement and analysis process parameters e Change the Cell Type see Defining the measurement and analysis process parameters for a list of available cell types Follow the instructions below to reprocess existing data sets with changed analysis parameters 1 Click on the List button in the Cedex XS Control Center to open the Measurement List window 2 Highlight the row of the measurement to be reprocessed
61. n the Data Selection area of the Cultivation Time Chart window The following values can be selected for display e Viable Cell Conc Viable cell concentration cells mL e Total Cell Conc Total cell concentration cells mL e Viability Percent viability e Total Cell Count Total number of all cells counted e Avg Compactness Average compactness of living cells without unit e Avg Diameter Average diameter of living cells um e Aggregate Rate Percent of cells found in aggregates e Dead Cell Conc Dead cell concentration cells mL e Viable Cell Count The total number of viable cells counted e Dead Cell Count The total number of dead cells counted e Total Object The total number of objects counted Click on the appropriate checkbox to select the desired values 32 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Analyzing the measurement results Data Selection 1 e ViableCellDensity Viable Cell Conc T T T T 7 cells mL Total Cell Conc cells mL Viability Total Cell Count Avg Compactness Avg Diameter um lev cellsimL Aggregate Rate Pe Cultivation Time Chart 5 ViableCellDensity 1045 5 Dead Cell Conc cells mL Viable Cell Count Dead Cell Count E EE E E Total Object Count 00 0 5 1 0 15 2 0 Time d ma F
62. n the import is successfully completed Click on OK 5 Close the Import from External DB window by clicking on B A default entry for the path under which the import file should be stored can be entered in the System Options under the Setup menu Open the Data Exchange tab and look for the ImportPath parameter In the Value column enter the default path To view imported measurements having a different System ID select the appropriate system name for those measurements in the System Name drop down menu in the Measurement List window 5 4 Archiving Data The objective of the archive function is to secure measurement data by exporting it to external storage media Measurement data must be secured at regular intervals and thereafter deleted from the Measurement List in order to ensure enough storage capacity on the Cedex XS computer All data from a single or a set of measurements or optionally only the images can be deleted once the data has been archived Measurement data can later be restored from the archive All data should also be backed up on a regular basis Archive To archive measurement data proceed as follows 1 Open the Functions menu in the Control Center and select the Archive function found within the Archiving menu The Measurement List window will appear with an Archive button at the bottom of the window 2 Select a single measurement from the list by clicking on the field on the left of the appropriate line When
63. nce will be carried out and the results will be automatically saved 4 When the White Balance is finished the White Balance button will be active again Click X to close the window 52 Cedex XS User Manual 12 04 2009 MM DD YY Y Y 8 Appendix Appendix Contact Addresses Manufacturer Distribution Distribution in USA Support Cedex XS User Manual 12 04 2009 MM DD YYYY Roche Diagnostics Ltd Forrenstrasse CH 6343 Rotkreuz Switzerland Roche Diagnostics GmbH Sandhofer Stra e 116 D 68305 Mannheim Germany Roche Diagnostics 9115 Hague Road PO Box 50457 Indianapolis IN 46250 USA Please contact your local Roche representative for assistance Questions or comments regarding the contents of this manual can be directed to the address below or to your local Roche representative Roche Innovatis AG Meisenstr 96 33607 Bielefeld Germany Phone 49 0 521 2997 298 Fax 49 0 521 2997 285 email support innovatis roche com internet www roche com 53 Cedex XS Technical Specifications Cedex XS Analyzer Operating environment Indoor use only Method of measurement Digital image and pattern recognition Viable dead cell differentiation Trypan Blue exclusion method Detectable cell concentration range 1x 10 1 x 10 cells per mL Detectable cell diameter range 5 um 180 um Detectable object diameter range 2 um 180 um Required sample volume Sample 5 uL Trypan Blue 5 uL Average
64. ng Measurement List Default Filter for Sample Drawing Date DateFrom From X SampleDrawing DateTo Measurement List Default Filter for Sample Drawing Date To X SamplelD Measurement List Default Filter for SamplelD X Measurement List Default Filter for DataSet DataSetType Effective Effective Original All X DataSet Measurement List Default Filter for DataSet X 56 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Appendix HARDWARE Option Value Description Save last value ChamberHeight 0 2 Height of the measurement sample chamber Warning Do not change this parameter without consulting with Roche Innovatis AG PixelsPermmX not adjustable Number of pixels per millimeter in the x 1157 axis of the images taken PixelsPermmY not adjustable Number of pixels per millimeter in the y 1157 axis of the images taken Background 128 Reference gray value used for background correction reference color Warning Do not change this parameter without consulting with Roche Innovatis AG Brightness B Channels to be used for background correction R red channels B blue Warning Do not change this parameter without consulting with Roche Innovatis AG Background 160 Reference target value for use with mask image target value Warning Do not change this parameter without consulting with Roche Innovatis AG DATA EXCHANGE Option Va
65. o allow the cells to settle for 1 3 minutes in the sample chamber before carrying out a measurement 5 Push the Slide carrier into the correct position A soft click can be felt when positioned correctly The chamber position indicator lights will indicate which chamber is in position under the microscope one light indicates that sample chamber position 1 is placed under the microscope and will be measured two lights indicate sample chamber position 2 is in place etc see Figure 7 6 Click on the Start Measurement button to start the measurement Sample chambers A1 A4 to be measured Figure 6 Placing the Cedex Smart Slide into the Slide carrier Figure 7 Chamber position indicator lights The progress of the measurement is tracked in the Processing Progress bar in the Measurement window When the measurement is finished the following message will appear Measurement is done Click on OK to close the window and view the results If multiple samples are to be analyzed for a single measurement i e the Precision level has been set to 2 or more the following message will appear after each sample chamber measurement is finished Please switch to the next chamber of the inserted slide and press OK see Figure 8 Push the Slide carrier in so that the next sample chamber is correctly positioned and click on OK The measurement of the new sample chamber will automatically start Continue with measurements until all sample ch
66. ograms Aggregate Histogram AGGREGATE HISTOGRAM p Detail Selection DETAILED TOTAL Percentage Selection C PERCENT ABSOLUTE Number of Cells 4 Aggregate Size Figure 12 The Aggregate Histogram detailed view divided into living and dead cells here absolute number of cells The Number of Cells identified in aggregates is displayed on the y axis and at the top of the bars The Aggregate Size in terms of the number of cells per aggregate is displayed on the x axis Figure 12 for example shows that 480 living and 132 dead cells of all identified cells were found in aggregates of two cells The percentages can be displayed using the percent button The percentage of cells found in aggregates compared to the total live or dead cell count is displayed on the y axis The aggregate size in terms of the number of cells per aggregate is displayed on the x axis Click on the total button to display Total Cell Count living and dead cells combined per aggregate as shown in Figure 13 Histograms Aggregate Histogram AGGREGATE HISTOGRAM a total 8 COMPACTNESS HISTOGRAM d AU t elias OBJECT DIAMETER HISTOGRAM MEASUREMENT STATISTICS a P Selection DETAILED amp TOTAL Percentage Selection PERCENT ABSOLUTE o E E 5 z 3 Aggr
67. onging to the selected view area of the CTC Views window To see the full list of all measurements from all series created click on the List button This will open the Series List window A search for specific measurements in the list can be done using the Filter by area in the upper right hand corner of the Series List window Searches can be made by Reactor ID or date of measurement Once the filter parameters have been set click on the Find button The measurements that fit the parameters will be displayed in the Measurements area in the lower left hand corner Only series that contain measurements with the correct parameters will be displayed in the Series area in the upper left hand corner of the Series List window Deleting Views and Series Entire Views can be deleted by highlighting the View and clicking on the Delete button in the View area of the CTC Views window If one series of a particular View should be deleted highlight the relevant View in the View area then highlight the series to be deleted in the Series belonging to the selected view area Click on the Delete button in the Series belonging to the selected view area of the CTC Views window Exporting a View or a Series list The series of measurements in a View can be exported via the Export button in the CTC Views window Select the View to be exported in the View area of the CTC Views window and click on Cedex XS User Manual 31 12 04 2009 MM DD YYYY Cedex XS the Expor
68. r of the Cedex XS Control Center This menu serves as direct access to the report functionality for many of the different reports The following information can be printed out via the Report menu e Operational data Allows the user to track the device s operating hours number of measurements and liquid consumption e Measurement data Contains information about measurement parameters and calculated results e g sample identification data name and parameters of the Cell Type used calculated measurement result data etc e System configuration parameters Provides a statement of system parameters as displayed in the User Preferences 5 2 Export to File There are various possibilities for exporting measurement data displayed in the Measurement List window Exporting data is done via the Export to file area of the Measurement List window Four different formats can be chosen including CSV XML Excel DIF and Flat file In addition a sub set of parameters to be included in the file can be selected To export measurement series data to a file follow the instructions below 1 Select the Export to file option in the Functions menu of the Control Center The Measurement List window will appear with the Export to File button located at the bottom of the window 2 Select a single measurement from the list by clicking on the field to the left of the desired line To choose a set of measurements hold down the Ctrl button while selec
69. r this View using the Series belonging to selected view area at the bottom half of the CTC Views window Cedex XS User Manual 29 12 04 2009 MM DD Y Y YY Cedex XS The Series of measurements belonging to selected view area Use this area to create edit view and delete measurements belonging to a particular View In order add to or modify measurement series belonging to a particular View that View must be highlighted in the View area Adding measurements to a View Do the following to find and select measurements for a new or previously created View 1 Highlight the View to be modified in the View area of the CTC Views window and click on the New button on the right hand side of the Series belonging to the selected view area This will open the Selection of Data Series window in which measurements can be selected for the creation of a View see Figure 18 2 Choose the Reactor ID and the preferred Data Set original or effective measurement data see Effective data sets 3 Click on the Find button All of the Sample IDs associated with that particular Reactor ID will appear in the upper section of the Selection of Data Series window 4 Click on the empty column to the left of the measurements to be added to the series in order to highlight that measurement Multiple measurements can be highlighted either by holding down the shift or lt ctrl gt key 5 When all of the desired measurements have been selected click on th
70. rcle diameter corresponds to the cell diameter Objects are marked with a blue circle e Zoom Image enlargement e Brightness and contrast settings e Scroll bars for navigating a window when the image is enlarged scroll bars will appear at the bottom and right edge of the image when the image is enlarged Scrolling through all of the images for a particular measurement using the arrows 3 in the lower right hand corner Figure 10 Viewing a cell image using the Image View window Certain areas of the image can be enlarged by doing the following 1 Place the mouse pointer at the top left hand corner of the area to be enlarged 2 Hold the left mouse button pressed down and move it to the lower right hand corner of the area to be enlarged Cedex XS User Manual 21 12 04 2009 MM DD Y Y Y Y Cedex XS 3 Release the mouse button The selected area will fill the whole window To return to the original size of the image do the following 1 Place the mouse pointer anywhere in the image 2 Hold the left mouse button pressed down and form a box from the bottom right corner to the upper left corner 3 Release the mouse button The image will return to in its original size Click on Close to shut the window or use the x button Excluding images from the evaluation process Images from individual sample chambers used for a single measurement can be excluded from analysis by the user Exclusion and inc
71. rs ClassificationBias 0 REVERT CMinSize LCAppearance pus REVERT 0 REVERT CANCEL Figure 24 Setting parameters for Cell Type Std Size CedexXSCellOperator and CedexXSObjectOperator parameters The parameters available for the CedeXSCellOperator and the CedexXSObjectOperator are displayed on the screen in the Live Operator window when the appropriate Cell Type is selected Parameters that are not immediately visible can be scrolled into view using the scroll bar under the Operator Parameter display The tables below display the positions and settings of the 11 Operator parameters for the CedeXSCellOperator and the 5 parameters for the CedexXSObjectOperator See also Description of the Individual Operator Parameters for a detailed description of each parameter 40 Cedex XS User Manual 12 04 2009 MM DD YYYY Adjustment of the Image Analysis acl ors Min Size Default Default 0 o0 ClassificationBias CMinSze i of 200 7 LCAppearance 20 20 5 12 0 20 70 AggrMinSize 8 50 12 X 12 BorderWidh 0 100 LCAggrApp to 10 O DCAggrApp 40 0f of o0 10 MinPollutionArea um 2000 Table 1 List of the CedexXSCellOperator parameters ERR Default 2 20 OMinSize um 2 OMaxSize um OAppearance MinPollutionArea um
72. rt window The path under which the export file should be stored is entered in the Location area The button on the right of the entry field opens a window in which the path of the database file can be selected directly 6 Click on Export to start the exporting process A Cedex message window will appear as Soon as export has been successfully completed Click on OK 7 Close the Export window by clicking on Bg 46 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Data Management A default entry for the path under which the export file should be stored can be entered in the User Preferences under the Setup menu Open the Data Exchange tab and look for the ExportPath parameter In the Value column enter the default path Import Once measurement data has been exported from one system to external storage the data can then be imported to another system To import measurement data into a Cedex System follow the instructions below 1 Select the Data Exchange option in the Functions menu on the Cedex XS Control Center 2 Click on Import to open the Import from External DB window 3 Select the path of the file that is to be imported in the Location area The button Ed on the right of the entry field opens a window in which the path of the file can be selected directly Click on Open The Import from External DB window will reappear 4 Click on the Import button to start the import process A Cedex message window will appear whe
73. s View View existing Cell Types Delete Delete existing Cell Types Live Operator Open the Live Operator window Cedex XS User Manual 37 12 04 2009 MM DD YY YY Cedex XS o e pe x ID Cell Type Name IR Operator User Name Last Update Std Size CedexxSCellOperato SuperUse 2 Objects CedexXSObjectOperator SuperUser 19 11 2008 00 00 00 3 Min Size CedexxSCellOperator SuperUser 26 01 2009 00 00 00 Filter by v AnA 9 5 ES CANCEL E Figure 22 Cell Type List window Creating a new Cell Type A new Cell Type must be created and saved via the Cell Type window before the Live Operator window can be opened for modification of that Cell Type Do the following to create a new Cell Type 1 Open the Cell Type List window Highlight the existing Cell Type upon which the new Cell Type should be based 2 Click on the New button A message will appear asking if the new Cell Type should be based on the highlighted Cell Type in the list Click on Yes The Cell Type window will open see Figure 23 The Live Operator button will be inactive at this time Fill in the information for the name of the new Cell Type Select the appropriate IR Operator from the drop down menu CedexXSCellOperator for analysis of cells or CedexXSObjectOperator for analysis of objects 5 Click on the Save button The Cell Type window will automatically close The Cell Type List window will be active again and include the just created
74. s to be exported into Excel DER Language English Columns to Export Parameter Delimiter A String Delimiter Cedex ID Cedex system Location Workarea name Reactor identifer Sample identifer Data set name Comment Dilution Sample volume Cell type name Excel DIF C Flatfile Sample draw Time Date finished Username EXPORT SELECTED DATA Avg Diameter All Parameters Cell Data Overall Results Figure 25 The Export window 5 3 Data Exchange The Data Exchange function allows for the exchange of measurement data between different Cedex System databases DBs The user can export selected data from one Cedex System DB and import it into another Export In order to exchange data between systems the data first needs to be exported from the system on which it was created to external storage To export data proceed as follows 1 Select the Data Exchange option in the Functions menu of the Cedex XS Control Center 2 Select Export The Measurement List window will appear with an Export to DB button located at the bottom of the window 3 Select a single measurement from the list by clicking on the field to the left of the appropriate line To choose a set of measurements hold down the Ctrl button while selecting the appropriate lines Alternatively the desired measurements can be searched for using filter criteria Click on Export to DB to open the Expo
75. stograms Certain areas of the histograms can be enlarged by doing the following 1 Place the mouse pointer on the top left hand corner of the area to be enlarged 2 Hold the left mouse button pressed down and move it to the lower right hand corner of the area to be enlarged 3 Release the mouse button The selected area will fill the complete window Return to the original size of the histogram by doing the following Place the mouse pointer anywhere on the diagram and right click with the mouse small window will open with the option Unzoom or Undo all Zoom Pan If the chart was enlarged more than once selecting Unzoom will reduce the image to the point before the last enlargement Select Undo all Zoom Pan to return the histogram to its original size In addition the histograms can be moved around within the window by placing the mouse pointer anywhere in the histogram holding down the middle mouse button and moving the mouse Aggregate Histogram The Cedex XS offers a thorough evaluation of aggregates in cell cultures Click on the Aggregate Histogram button in the Histograms window to open the Aggregate Histogram Cedex XS User Manual 25 12 04 2009 MM DD YY YY Cedex XS The Aggregate Histogram shows the number of cells in the cell culture found in aggregates as well as how many cells each aggregate is composed of Both the actual cell number and the percentage distribution can be displayed Hist
76. t be correctly initialized Shutting down the Cedex XS Control Center Proceed as follows 1 Shut down the software by choosing Exit under the System menu or by clicking on X to close the window The security query Do you really want to exit the Cedex XS Control Center will appear Click on Yes to shut down the program Switch off the individual devices at random 2 2 Description of the Cedex XS Control Center The Cedex XS Control Center is divided into three elements see Figure 2 e Menu bar e Shortcuts area e Event window detailing status of events carried out by the analyzer Cedex XS Control Center Demo System Measurements Functions Reports Setup Help INNOVATIS FRE H Cedex XS 2008 11 12 10 20 24 CedexCore status Operational 2008 11 12 10 20 24 Automatic hardware startup initiated 2008 11 12 10 20 25 Hardware startup completed System status SystemOK Figure 2 The Cedex XS Control Center Cedex XS User Manual 11 12 04 2009 MM DD YYY Y Cedex XS Menu Bar The menu bar at the top of the Control Center has the following menu items e System e Measurements e Functions e Reports e Master Data e Setup e System Menu e Exit exits the Cedex XS Control Center Measurement Menu e Measurement List Provides a list of all measurements that have been carried out Measurements can be sorted or filtered using specific criteria e Measurement Opens a window in which m
77. t button An Export window will open in which the desired settings and parameters can be selected In addition it is possible to export the measurements in an individual Series Select the Series to be exported in the CTC Views window and click on List The Series List window will open To export the data click on the Export button see Export to File for more details about export options Creating a Cultivation Time Chart Once a View has been created with the desired series of measurements the Cultivation Time Chart can be created To create a Cultivation Time Chart do the following 1 Highlight the View in the CTC Views window to be used for the creation of a Cultivation Time Chart 2 Click on the View Chart button this will open the Cultivation Time Chart window see Figure 19 3 Select the parameters to be displayed in the chart by clicking on the desired parameters in the Data Selection area in the upper right hand corner of the window see The Data Selection Area 4 Select the series to be displayed in the Chart area in the lower right hand corner of the window 5 A graph will then be generated showing the selected parameters and series NOTE Only measurements already loaded will be displayed in the Cultivation Time Chart window diagram The Selection of Data Series window must be brought up again to add additional measurements The Data Selection Area The curves to be displayed in the diagram are determined i
78. ted view area at the bottom of the window CIC Views Filter by Name Govs vh 5 9 23 2008 09 oq Y f 9 23 2008 OW Creation date from from Modify date View View Name Modified VIEW CHART EXPONENTIAL REGRESSION EXPORT DELETE Series of measurements belonging to selected view No ReactorID Sample ID from Sample ID to NEW CHO ic S0000 1 batc S00001 LIST DELETE Figure 17 CTC Views window The View Area The View area in the middle of the CTC Views window is used for viewing exporting creating or deleting a View If no filters have been set all Views that have been created will be displayed in the View Name column If filters have been set only those Views that match the filters will appear in this column To open the chart for a particular View highlight the row of the desired View and click on the View Chart button To create a new View click on the New button Highlighted rows of Views can also be exported with the Export button or deleted using the Delete button When the New button is selected a window will open asking for a name for the new View A name must be given before a View can be created Give a name for the new View and click on OK The new View will appear highlighted in the View area It is now possible to select the measurements fo
79. ters Deleting an existing Cell Type Select the Cell Type to be deleted from the list of Cell Types at the top of the Cell Type List window and then click on the Delete button A confirmation box will appear asking the user if Cedex XS User Manual 39 12 04 2009 MM DD YYYY Cedex XS he she wants to delete the cell type If confirmed the Cell Type will be deleted and disappear from the list NOTE All parameters of the measurements are logged Deleted Cell Types have no influence on future measurements but the parameters of deleted Cell Types used for past measurements will still appear in the printed reports of those measurements 4 2 Working with the Live Operator The Live Operator window can be opened via the Cell Type List window the Cell Type window and the Measurement window If the Live Operator button in a Measurement window containing specific results is clicked on the Live Operator will open with an image of that measurement already loaded If the Live Operator is opened via the Cell Type or Cell Type List windows the first measurement in the Measurement List will be loaded Live Operator Data Source System Name Cell Type Size Workarea standard Reactor ID batch Sample ID so0001 Data Set original Image No Process Data Previous viable Previous dead Previous viability 89 1156 New viability Paramete
80. the following steps 1 Prepare the cell sample by staining the sample with the appropriate amount of 0 296 trypan blue typically a 1 1 dilution It may be necessary to first dilute samples with very high cell densities greater than 1 x 10 cells mL with media or phosphate buffered saline PBS prior to staining with trypan blue 2 Mix the sample well then immediately remove 10 pL and add it to the appropriate sample chamber of the Cedex Smart Slide see Figure 5 3 If more than one sample is to be analyzed for a single measurement pipette 10 uL into each sample chamber to be used Mix the cell suspension thoroughly before removing a sample for each chamber 4 Place the Cedex Smart Slide in the Slide carrier in such a way that the sample chambers containing the samples are placed toward the Cedex XS see Figure 6 NOTE Four sample chambers e g chambers A1 A4 can be measured without removing the Smart Slide from the Slide carrier To measure the other four sample chambers e g sample chambers B1 B4 the Smart Slide must be removed from the Slide carrier turned around then placed back into the Slide carrier so that those chambers are positioned towards the Cedex XS The Cedex Smart Slide is designed for single use only Cedex XS User Manual 15 12 04 2009 MM DD YYYY Cedex XS NOTE To achieve optimal results with smaller cells average diameter of approximately 10 or less it may be necessary t
81. ting the appropriate lines Alternatively the desired measurements can be searched for using different filter criteria see Searching for measurements Click on Export to File The Export window will open see Figure 25 4 In the Export window individual Parameter and String delimiters can be defined this only applies to CSV and Flat files 5 In the Location area the target path to be used for storage of the export file is entered The button E on the right of the entry field opens a window in which the path can be selected directly The name of the file will be generated automatically 6 To specify a sub set of parameters to be exported either click the check boxes next to the preset options i e All Parameters Cell Data and Overall Results or select the desired parameters from the Columns to Export area To choose a set of parameters keep the Ctrl button pressed while making selections 7 In the File format area select the file format by clicking on the appropriate check boxes The files will automatically receive dif or csv file name extensions Cedex XS User Manual 45 12 04 2009 MM DD YY Y Y Note Cedex XS 8 Click on the Export Selected Data button 9 A Cedex message window will appear to confirm successful export of information Click on OK to confirm the message The measurement data can then be viewed in the file created NOTE The file format Excel DIF should always be chosen if a file i
82. tor 38 CedexXSObjectOperator 38 Cell 8 18 Cell concentration range 16 Cell Diameter Histogram 25 SIBI 13 35 37 orean ER 36 37 editing 37 ClassificationBias 39 Cleaning 56 CMaxSize 40 CMinSize 40 Compactness 26 Compactness Histogram 26 Control 10 46 Copyright 5 Counting routine 14 17 18 19 35 CSV ete gece 42 View 27 Cultivation Time Chart 27 30 D Data 43 Data 42 Data Selection Area 30 31 Data Serie S isiin ani 28 Data 21 Data Sets 21 DGAggrADpp ett 41 40 Dead Cell 18 Dead Gone 18 Declaration of Conformity 5 Default measurement parameters
83. tware 12 Cedex XS User Manual 12 04 2009 MM DD YY Y Y Working with the Cedex XS ev Shortcut Area Several buttons that act as short cuts to certain functions are available in this area including e LIST Opens the Measurement List window e MEASURE Opens the Measurement window for running a measurement e CTC Opens the CTC Views window for setting up and viewing Cultivation Time Charts e LIVE VIEW Opens a window that shows a live view of the selected sample chamber e HWM Opens the Hardware Management window Tracking status of events carried out by the analyzer The status of events that have been carried out by the analyzer is tracked in the Event window below the Shortcut area If for example the hardware management has started the completion of the routine will be noted in this window Previous events can be viewed by using the scroll bar on the right The window is cleared each time the software is shut down 2 3 Carrying out a Measurement Open the Measurement window by clicking on the Measure button in the Shortcuts area or by selecting Measurement in the Measurements menu see Figure 3 Specify the desired operation and sample parameters in the entry areas of the Sample Parameters and Processing Parameters sections of the Measurement window The parameters should be entered just before measuring in order to ensure a direct relationship between the sample and the data WARNING Before carrying out a measurement ens
84. ure that a HW Startup was automatically performed by the system when the software was launched see Performing HW Startup 7 Measurement ym Sample Parameters Processing Parameters Reactor ID 1 batch Precision 1 image Sample ID 5000002 Cell Type Std Size Sample drawing 12112008 10 23 28 Dats Set rigina z G06708123 Dilution d 11 12 2008 10 23 AM Comments Valid Effective Processing progress Result Data Image No Valid Excluded Viable Cell Conc cells mL Total Cell Conc cells mL Viability 9 Total Cell Count Avg Compactness Avg Diameter um Aggreg Figure 3 The Measurement window Cedex XS User Manual 13 12 04 2009 MM DD Y Y Y Y Cedex XS Defining the sample parameters The sample parameters are defined in the Sample Parameters area of the Measurement window as follows 1 Enter a clearly defined Reactor ID to describe the sample e g the name of the cell culture in the Reactor ID area You may either choose a Reactor ID from the drop down list or enter a new Reactor ID A new entry can be defined by up to 21 alphanumerical symbols 2 Enter a clearly defined Sample ID in the Sample ID area e g P001 The Sample ID can be freely defined If the intended measurement has the same Reactor ID as a previous measurement the analysis software will automatically enter the consecutive Sample ID into the Sample ID area
85. ze Default user value CellType Name for a new Measurement CHO batch Default user value ReactorlD for a new Measurement 800002 Default user value SamplelD for a new Measurement T1 Default user value SamplelnitialDilution ratio for a new Measurement 1 2 1 20 etc 1 Mask image required 4 lt SAVE CANCEL Figure 26 User Preferences window Specifying other Default Parameters The following tabs are also found in the User Preferences window For more detailed information about the parameters see List of User Preferences in the Appendix e General General software and hardware settings e Measurement Create or alter default parameters for settings in the Measurement window Cedex XS User Manual 49 12 04 2009 MM DD YYYY Cedex XS Measurement List Default Filter Create or alter default parameters for filter settings in the Measurement List window Hardware General hardware settings Data Exchange Create or alter default parameters for Export Import and Archive functions Tracing Change the default output path for the tracing Logging Change the default output path for logging Series Default Filter Create or alter default parameters for the filter settings in the Selection of Data Series window CTC Views Default Filter Create or alter default parameters for the filter settings in the CTC Views window Reports Create or alter default paths for reports WARNING Many of the para
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