Cedex XS System Customer Training Guide

Transcription

1 Cedex XS System Customer Training Guide For life science research only. Not for use in diagnostic procedures.

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3 Table of Contents Prologue 5 I Revision History... 5 II Trademarks... 5 III Intended Use... 5 IV Conventions Used in this Guide... 6 A Installation 7 Unpacking... 7 Setting Up Environmental Requirements System Installation... 8 B Start-up 9 Starting the Cedex XS Analyzer... 9 Starting the Cedex XS Control Unit Performing a White Balance... 0 C Main Application: Counting Cells and Determining Viability Preparing the Samples... Loading the Samples... 3 Setting up a Measurement in the Software Open the Measurement Dialog Box Define the Sample Parameters Define the Measurement and Analysis Parameters Starting the Measurement Viewing the Results Viewing the Results in the Measurement Dialog Box Viewing the Results in the Histograms Viewing the Images Viewing Images in the Image View Optimization: Performing a White Balance... 8 Measurement List... 9 Shutting Down the Cedex XS Analyzer... D Further Applications 3 Monitoring Growth of Cells over Time Carrying out Measurements for Growth Curve Analysis Result Analysis Create a CTC View Add One or More Series of Measurements to the CTC View Create a CTC Chart Associated with the View to Analyze Pattern of Growth Over Time Work with the Exponential Regression Function to Determine the Specific Growth Rate and Doubling Time of the Cell Culture Based on the Log Phase of Growth Checking Regression Curve Fitting Editing Measurements in a View

4 Table of Contents Analyzing Unusual Cells Overview Example of Cells not Recognized by the Cell Type Std. Size Create a New Cell Type Modify the Parameters for the New Cell Type via the Live Operator Window Reprocess the Measurement Using the New Cell Type as the Basis for the Image Analysis E Appendix 39 Index Cedex XS Analyzer

5 Revision History Prologue I Revision History Version Revision Date.0 March 00 Copyright 00, Roche Diagnostics GmbH. All rights reserved. Information in this document is subject to change without notice. No part of this document may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the express written permission of Roche Diagnostics GmbH. Questions or comments regarding the contents of this Training Guide can be directed to the address below or to your Roche Diagnostics representative. Roche Diagnostics GmbH Roche Applied Science Customer Support Nonnenwald 8377 Penzberg, Germany Every effort has been made to ensure that all the information contained in the Cedex XS Analyzer Customer Training Guide is correct at the time of printing. However, Roche Diagnostics GmbH reserves the right to make any changes necessary without notice as part of ongoing product development. II Trademarks CEDEX and INNOVATIS are trademarks of Roche. Other brands or product names are trademarks of their respective holders. III Intended Use The Cedex XS Analyzer determines the cell concentration and viability of cell suspensions. It is intended for life science research and must be used exclusively by laboratory professionals who are trained in laboratory techniques and have studied the instructions for use of this system. The Cedex XS Analyzer is for life science research only. Not for use in diagnostic procedures. It was neither developed nor validated by the manufacturer for any kind of in vitro diagnostic application. Prologue 5

6 Conventions Used in this Guide IV Conventions Used in this Guide Text Conventions To present information consistently and memorably, the following text conventions are used in this Operator's Guide: Numbered list Italic type Bold type Blue italic type Steps in a procedure that must be performed in the order listed. Used for operating instructions for the Cedex Software. In addition, important notes and information notes are shown in italic type. Laptop keys. In addition, important phrases within the text. Refers to a different section in this Training Guide, which should be consulted. Symbols In this Training Guide the following symbols are used as visual signals: Symbol Heading Description IMPORTANT NOTE INFORMATION NOTE Information critical to the success of the procedure or use of the product. Additional information about the current topic or procedure. Procedure continued on next page. End of procedure. The following symbols appear on the analyzer: Symbol Description The instrument meets the requirements laid down in Council Directive 004/08/EC relating to Electromagnetic Compatibility and Council Directive 006/95/EC relating to Low Voltage Equipment. The following standards were applied: IEC/EN 636- (EMC) and IEC/EN 600- (Safety). The product meets US and Canadian safety requirements for electrical equipment for measurement, control and laboratory use. Issued by Underwriters laboratories, Inc. (UL) for Canada and the US. UL 600- Electrical Equipment for Measurement, Control and Laboratory Use; Part : General Requirements. CAN/CSA-C. No (Second Edition) Safety Requirements for Electrical Equipment for Measurement, Control and Laboratory Use, Part : General Requirements. Electrical and electronic equipment marked with this symbol is covered by the European Directive WEEE. The symbol denotes that the equipment must not be disposed of in the municipal waste system. 6 Cedex XS Analyzer

7 Unpacking 0 Installation Unpacking A The Cedex XS System comes in a box with: The Cedex XS Analyzer EBS Power Supply Operator s Guide and Customer Training Guide Install CD A Cedex XS Accessory Box, containing Cedex Smart Slides for Cedex XS Analyzer HR USB cable A/B male Power cord Fuse 5 x A T 50V ULR/IEC The Cedex XS Mobile Control Unit with Laptop computer Power supply for laptop Laptop accessory Open the box containing the Cedex XS Analyzer. You should see A cardboard box containing the Cedex XS Analyzer incl. accessories. A cardboard box containing the Cedex XS Mobile Control Unit. Open the box containing the Cedex XS Mobile Control Unit; remove the stuffing and the laptop computer. Open the cardboard box and remove the cutting protection cardboard. You should see: The Cedex XS Operator s Guide and the Cedex XS Customer Training Guide Install CD Certificate of Quality Remove the styrofoam cover. Take out the Cedex XS Accessory Box and the Cedex XS Analyzer. 6 Remove the plastic bag wrapped around the Cedex XS Analyzer. Installation 7

8 Setting Up Environmental Requirements A Setting Up. Environmental Requirements Cedex XS Analyzer is a precision measuring instrument. To ensure error-free measuring, you must choose the location carefully. Read the handling requirements and general precautions in the section Warnings and Precautions in the Cedex XS Operator s Guide.. System Installation Plug the power cord into the Cedex XS Analyzer. Bolt the screw to avoid accidental removal of the plug. Plug the USB cable into the Cedex XS Analyzer. Plug the power cord into the socket. Plug the Cedex XS Analyzer into an interference-free measuring instrument socket. Do not operate the Cedex XS Analyzer from the same socket as laboratory equipment that works with high switching loads. Ensure that all laboratory equipment situated in the vicinity is working without interference or faults. Connect the USB cable to the laptop computer. 8 Cedex XS Analyzer

9 Starting the Cedex XS Analyzer 0 Start-up Starting the Cedex XS Analyzer Press the ON/OFF switch on the Cedex XS Analyzer. The switch should light up with a blue light indicating that the Cedex XS Analyzer is on. B ON/OFF switch Starting the Cedex XS Control Unit Switch on the laptop. The Windows operating system starts. Double-click on the Cedex XS Icon on the desktop to launch the software. The Cedex XS Control Center opens. The first time a Cedex XS System is set up or when the Cedex XS System is moved to a new location, a White Balance must be carried out. See section Performing a White Balance below. Start-up 9

10 Performing a White Balance 3 Performing a White Balance A White Balance must be carried out the first time the Cedex XS Analyzer is set up and any time the system is moved. The White Balance determines the proper adjustment for image brightness. To carry out a White Balance: B Pull the slide carrier completely out of the Cedex XS Analyzer. Click on the HWM button in the Shortcuts area of the Cedex XS Control Center to open the Hardware Management dialog box. 3 4 Click on the White Balance button. The White Balance will be carried out, and the results will be automatically saved. When the process is finished, the White Balance button will be active again. Click on the icon to close the dialog box. 0 Cedex XS Analyzer

11 Preparing the Samples 0 Main Application: Counting Cells and Determining Viability Preparing the Samples Dilute samples with very high cell concentrations (higher than x 0 7 cells/ml) with media or phosphate buffered saline (PBS). The cell concentration after dilution should not be higher than 5 x 0 6 cells/ml. 3 4 Stain the sample with the appropriate amount of 0.% Trypan Blue (typically part cell sample and part Trypan Blue). 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-suspend it in a protein-free medium or physiological saline solution prior to counting. Mix the cell suspension thoroughly. Pipette 0 µl of the cell suspension into each sample chamber of the Cedex Smart Slide to be measured. You can measure up to eight samples within one overall measurement or run individual measurements on each chamber filled with cell samples. C If you are working with smaller cells (average diameter of approximately 0 µm or less), let the cell suspension settle in the sample chamber for about three minutes before starting a measurement. Main Application: Counting Cells and Determining Viability

12 Loading the Samples Loading the Samples The Cedex XS Smart Slide has eight chambers for testing samples. Four sample chambers (e.g., chambers A-A4) can be measured without turning the Cedex XS Smart Slide. Pull out the slide carrier. Place the Cedex XS Smart Slide in the slide carrier with the chambers containing the samples going in first. C 3 Push the slide carrier with the Cedex XS Smart Slide back into the Cedex XS Analyzer until you feel a soft click. The chamber position indicator lights indicate which chamber of the Cedex XS Smart Slide is under the microscope: For the first chamber, only the left-most lamp lights up. For the second chamber the two left-most lamps light up, and so on. Chamber position indicator lights (here the second chamber is in position under the lens, so lamps light up) Cedex XS Analyzer

13 Setting up a Measurement in the Software Open the Measurement Dialog Box 3 Setting up a Measurement in the Software To set up a measurement in the Cedex XS Control Center, you have to: Open the Measurement Dialog Box Define the Sample Parameters in the Sample Parameters group Define the Measurement and Analysis Parameters in the Processing Parameters group 3. Open the Measurement Dialog Box In the Cedex XS Control Center shortcuts area, click on the MEASURE button. The Measurement dialog box opens. C 3. Define the Sample Parameters 3 In the Sample Parameters group of the Measurement dialog box, enter a Reactor ID. The Reactor ID describes the sample (e.g., the name of the cell culture). Select a name from the drop-down list or enter a new Reactor ID. A Reactor ID consists of up to alphanumerical characters. Enter a Sample ID (e.g., P00). Select a name from the drop-down list or enter a new Sample ID. A Sample ID consists of up to alphanumerical characters. If the intended measurement has the same Reactor ID as a previous measurement, the analysis software will automatically enter the consecutive Sample ID (e.g., P00, P00,...). However, you can change the Sample ID. If you wish, select a date from the Sample Drawing list. The date indicates when the sample was drawn. An additional date and time will be added automatically by the software (the process time), reflecting the actual date and time the sample was measured. Main Application: Counting Cells and Determining Viability 3

14 Setting up a Measurement in the Software Define the Measurement and Analysis Parameters 4 Click on the Mix button. The Enter volumes dialog box opens. C Enter the amounts in µl of the cell sample (Sample Volume) Trypan Blue used to prepare the sample (Dye Volume) diluent used to dilute the sample, if applicable (Diluent Volume) used for the mix. When you have entered the appropriate volumes for each liquid, the resulting dilution is automatically calculated. The cell concentration after all dilutions should not be higher than 5 x 0 6 cells/ml. Click on OK to confirm the entries. The Enter volumes dialog box closes and the appropriate dilution factor is taken over automatically into the Dilution field in the Measurement dialog box. If you wish, you can enter comments on the sample in the Comments text field. 3.3 Define the Measurement and Analysis Parameters 3 In the Processing Parameters group of the Measurement dialog box, select the desired precision level from the Precision list. The precision level determines the number of sample chambers to be analyzed for a single measurement. Results from up to 8 sample chambers can be combined for a single measurement. The results from the analyzed chambers are combined to give an overall result. 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. Select the cell type that optimally recognizes your cells or objects from the Cell Type list. The following cell types are available by default: Std. Size for normal-sized cells with a diameter of approximately - 4 µm Objects for identification of objects Min. Size for small cells with a diameter of approximately 8 µm The Cedex XS Control Center automatically adds the System Name Process Time 4 Cedex XS Analyzer

15 Starting the Measurement 4 Starting the Measurement In the Measurement dialog box, click on the START MEASUREMENT button. The progress of the measurement is indicated in the Processing progress bar. If multiple samples are to be analyzed for a single measurement (i.e., the Precision is set to or more), the following message is displayed after a sample chamber is measured: Push the slide carrier further into the Cedex XS Analyzer so that the next sample chamber is positioned under the microscope. Click on OK to close the message box. The measurement of the next sample chamber starts automatically. Repeat steps and 3 for the other sample chambers to be measured. If you have more that four samples to be measured, the Cedex Smart Slide has to be turned around in order to measure the other chambers: Pull the slide carrier out of the Cedex XS Analyzer. Extract the Cedex Smart Slide and turn it around. Insert the Cedex Smart Slide into the slide carrier. Push the slide carrier with the Cedex Smart Slide back until you feel a soft click and the first chamber position indicator lamp lights up. To achieve optimal results with smaller cells (average diameter of approximately 0 µm or less), you may need to allow the cells to settle for -3 minutes in the sample chamber before carrying out a measurement. When the final sample chamber has been analyzed, the following information box opens: C 7 Click on OK to close the information box and complete the measurement. Main Application: Counting Cells and Determining Viability 5

16 Viewing the Results Viewing the Results in the Measurement Dialog Box 5 Viewing the Results 5. Viewing the Results in the Measurement Dialog Box After the measurement process is completed, the measurement results are displayed automatically in the Result Data area of the Measurement dialog box. The Result Data area consists of two tables: The first table displays the overall results for the measurement, while the second table displays the individual results for each sample chamber analyzed for the single measurement. C The following parameters are listed in both tables: Viable Cell Conc.: Concentration of viable cells [x 0 5 cells/ml] Total Cell Conc.: Total cell concentration [x 0 5 cells/ml] Viability (%): The proportion of viable cells (%) to the total number of cells (viable and dead cells) Total Cell Count: The number of all detected objects counted as cells Avg Compactness: The average variation, in shape, of viable cells from an ideal sphere, with the ideal sphere shape equaling >< Avg Diameter: The average diameter [µm] 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 [x 0 5 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 [x 0 5 cells/ml] Avg Object Diameter: The average diameter [µm] of objects detected Avg Object Compactness: The average variation, in shape, of objects from an ideal sphere, with the ideal sphere shape equaling >< In the individual result data table, the following three colomns precede the above: Image No.: Number of the image Valid: Indicates whether the software considered the image quality to be acceptable for analysis Excluded: Checkbox to exclude/include the measurement from the calculation process 6 Cedex XS Analyzer

17 Viewing the Results Viewing the Results in the Measurement Dialog Box To exclude a single measurement from evaluation Excluding a single measurement means that the image and the result data are excluded from the calculation process. The overall results will only be calculated for data that is not excluded. C In the Result Data area of the Measurement dialog box, select the checkbox in the Excluded column of the relevant result line. The overall results are automatically recalculated. Data Output Measurement results can be printed out. They can also be exported in various formats for further analysis. For more information on these functions, see the Cedex XS Operator s Guide. Main Application: Counting Cells and Determining Viability 7

18 Viewing the Results Viewing the Results in the Histograms 5. Viewing the Results in the Histograms All the results presented in the histograms are based on valid images accepted for evaluation. This means that results from individual images that you have excluded from evaluation are not included in the histograms (see To exclude a single measurement from evaluation). The following histogram types are available: Aggregate Histogram Cell Diameter Histogram Compactness Histogram Object Diameter Histogram C In the Measurement dialog box, click on the HISTOGRAMS button. The Histograms dialog box opens. Select one of the following histogram types for result analysis: Cell Diameter Histogram Compactness Histogram Object Diameter Histogram Aggregate Histogram For a more detailed description of general histogram functions, see the Cedex XS Operator s Guide. 8 Cedex XS Analyzer

19 Viewing the Images Viewing Images in the Image View 6 Viewing the Images The image area on the right of the Measurement dialog box shows the image corresponding to a selected row in the individual sample chamber result data table. To view an image In the Result Data area, select a row in the individual sample chamber result data table. The corresponding image is displayed in the image area. To view all images In the image area, click on the VIEW IMAGES button. The Image List dialog box opens containing a thumbnail of each image of the measurement. C 6. Viewing Images in the Image View The Image View dialog box provides an enlarged view of the selected image. The number of viable and dead cells is analyzed and the cells are marked with green and red circles respectively. The Image View dialog box provides the following further options for viewing the image: Adjust contrast, brightness and zoom in the Contrast/Brightness/Zoom group. Show or hide the cell marker circles in the Marker group. View the following or previous image using the arrows in the Images group. Right-click on the image to save it or copy it to the clipboard. Main Application: Counting Cells and Determining Viability 9

20 Viewing the Images Viewing Images in the Image View C To open the Image View dialog box from the image area Double-click on the image displayed in the image area. The Image View dialog box opens. To open the Image View dialog box from the Image List Double-click on a thumbnail in the Image List. The Image View dialog box opens. To enlarge a certain area of an image in the Image View In the Image View dialog box, select a rectangle by left-clicking and dragging the mouse from top left to bottom right. Release the left mouse button; the selected rectangle fills the whole display area of the dialog box. Resize the enlarged image In the Image View dialog box, select a rectangle by left-clicking and dragging the mouse from bottom right to top left. Release the left mouse button; the image is displayed in its original size. 0 Cedex XS Analyzer

21 Optimization: Performing a White Balance 7 Optimization: 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: Pull the slide carrier completely out of the Cedex XS Analyzer. Click on the HWM button in the Shortcuts area of the Cedex XS Control Center to open the Hardware Management dialog box. 3 4 Click on the White Balance button. The White Balance will be carried out, and the results will be automatically saved. When the process is finished, the White Balance button will be active again. Click on the icon to close the dialog box. 8 Measurement List C Once the Measurement dialog box containing measurement results and images has been closed, it is possible to re-open it with the results loaded via the Measurement List dialog box. To open the Measurement List and access previous measurements, proceed as follows: Click on the LIST button in the shortcuts area of the Cedex XS Control Center, or select Measurement List in the Measurements menu of the Cedex XS Control Center. Main Application: Counting Cells and Determining Viability

22 Shutting Down the Cedex XS Analyzer The following actions can be carried out in the Measurement List dialog box: Searching for measurements using a number of different filters Viewing general measurement data Deleting measurements Printing reports Exporting to file Starting the Measurement via the NEW button Viewing measurements Reprocessing measurements Searching for measurements Search for measurements using the following criteria in the Filter by group: C Sample drawing date Process date Reactor ID Sample ID System Name Archived Measurements / All Measurements Data Set Cell Type Select the appropriate filter criteria. Click on the FIND button. The measurement list is adjusted according to the filter criteria set. For more information on functions available via the Measurement List dialog box, see the Cedex XS Operator s Guide. 9 Shutting Down the Cedex XS Analyzer Close the Cedex XS Control Center by selecting Exit in the System menu or clicking on the icon on the Cedex XS Control Center. 3 Shut down the Cedex XS Control Unit. Switch off the Cedex XS Analyzer. Cedex XS Analyzer

23 Monitoring Growth of Cells over Time Carrying out Measurements for Growth Curve Analysis 0 Further Applications Monitoring Growth of Cells over Time. Carrying out Measurements for Growth Curve Analysis Perform the following steps: 3 Immediately after sub-culturing the cell culture of interest, remove a sample and carry out a full analysis with regard to cell concentration and viability using the Cedex XS Analyzer as described in the chapter Main Application: Counting Cells and Determining Viability. Measure the cell culture with regard to cell concentration and viability at regular intervals for 7 to 0 days. Intervals between measurements should typically range between 8 and 8 hours. Approximately or 3 days after the cell culture has stopped increasing in viable cell concentration, the culture can be discarded and a growth curve analysis prepared as described in the following section. Many cell cultures reach their growth limits within 7 to 0 days after being sub-cultured, particularly if there is no exchange of media or addition of nutrients during that time. However, the time needed for a cell culture to reach maximum growth and either decline in cell concentration or reach senescence (the plateau phase of a growth curve) can vary depending on the cell type and conditions in which the culture is cultivated.. Result Analysis After all measurements have been carried out as described in the previous section, the Cultivation Time Chart (CTC) for the analysis of measurement results and cell culture growth pattern is prepared as follows:. Create a CTC View. Add One or More Series of Measurements to the CTC View 3. Create a CTC Chart Associated with the View to Analyze Pattern of Growth Over Time 4. Work with the Exponential Regression Function to Determine the Specific Growth Rate and Doubling Time of the Cell Culture Based on the Log Phase of Growth D.. Create a CTC View 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. Further Applications 3

24 Monitoring Growth of Cells over Time Result Analysis To create a View Click on the CTC button in the Shortcuts area of the Cedex XS Control Center or select the Cultivation Time Chart option in the Measurements menu to open the CTC Views dialog box. The CTC Views dialog box has two main areas: The View area, which is the top half of the window. This is where charts are viewed, exported, created and deleted. The Series of measurements belonging to selected view area at the bottom of the window. This is where individual series of measurements belonging to the highlighted View are created, edited, listed or deleted. D 3 Click on the NEW button in the View area. You are prompted for a name for the new View. A name must be given before a View can be created. Enter a name for the new View and click on OK. The new View is displayed highlighted in the View area. It is now possible to select the measurements for this View using the Series of measurements belonging to selected view area in the bottom half of the CTC Views dialog box as described in the next section. 4 Cedex XS Analyzer

25 Monitoring Growth of Cells over Time Result Analysis.. Add One or More Series of Measurements to the CTC View To find and select measurements for the new View Select the View to be modified in the View area of the CTC Views dialog box and click on the NEW button on the right-hand side of the Series of measurements belonging to the selected view area. The Selection of Data Series dialog box is displayed, in which you can select measurements for the creation of a View. 3 4 Select the appropriate Reactor ID used for the growth curve measurements from the dropdown menu. Select the preferred measurement Data Set to be used from the drop-down menu (original or effective measurement data, see section Effective Data Sets). Click on the FIND button. All Sample IDs associated with the selected Reactor ID will be dispayed in the upper table of the Selection of Data Series dialog box. D 5 Click on the empty column to the left of the measurements to select a measurement. Multiple measurements can be selected by holding down the Shift or Ctrl key on the keyboard. Further Applications 5

26 Monitoring Growth of Cells over Time Result Analysis 6 When you have selected all desired measurements, click on the ADD SELECTED MEASUREMENTS button. The selected measurements will be displayed in the Measurements belonging to series table in the lower bottom of the dialog box. 7 Click on the SAVE button. The Selection of Data Series dialog box will close. The CTC Views dialog box displays the measurements that have been added to the View in the Series of measurements belonging to selected view area. D 8 To add another series of measurements to a View, click on the NEW button again. The Selection of Data Series dialog box is displayed. Perform steps through 7 above to add further measurements to the new series. You can add up to 5 separate series to one View. Once 5 series have been added to a View, the NEW button will no longer be available for that View. 6 Cedex XS Analyzer

27 Monitoring Growth of Cells over Time Result Analysis..3 Create a CTC Chart Associated with the View to Analyze Pattern of Growth Over Time Once you have created a View and added the desired series of measurements for that View, you can create the Cultivation Time Chart. To create a Cultivation Time Chart In the CTC Views dialog box, select the View to be used to create a Cultivation Time Chart. Click on the VIEW CHART button. The Cultivation Time Chart dialog box opens with the selected measurements automatically displayed. By default the results for the Viable Cell Conc. are displayed. The figure below shows a typical growth pattern of a cell culture from the initial seeding to the limit in growth: Lag phase Log phase Plateau D To display curves for other types of measurement results, choose the desired parameters in the Data Selection area. The following values can be selected for display: Viable Cell Conc.: Viable cell concentration [cells/ml] Total Cell Conc.: Total cell concentration [cells/ml] Viability: Percent viability [%] Total Cell Count: Total number of all cells counted Avg. Compactness: Average compactness of living cells [without unit] Avg. Diameter: Average diameter of living cells [µm] Aggregate Rate: Percent of cells found in aggregates [%] Dead Cell Conc.: Dead cell concentration [cells/ml] Viable Cell Count: Total number of viable cells counted Dead Cell Count: Total number of dead cells counted Total Object: Total number of objects counted A graph will be generated showing the measurement results for the selected parameters. If desired, click on the log option button to display the y-axis in a logarithmic scale. Click on the lin option button to display the y-axis in a linear scale. The x-axis units can be displayed as either days [d] or hours [h]. Click on Time [d] below the x-axis to switch between days and hours. The x-axis calibration is automatically recalculated. Further Applications 7

28 Monitoring Growth of Cells over Time Result Analysis 6 7 To view detailed information on a measurement point, position the mouse pointer exactly over a point in the diagram. The information is displayed, including the date and time of the measurement as well as the result for the displayed parameter. If you wish, you can enlarge the graphic representation or scale it down again using the mouse. For more information on general histogram functions, see the Cedex XS Operator s Guide...4 Work with the Exponential Regression Function to Determine the Specific Growth Rate and Doubling Time of the Cell Culture Based on the Log Phase of Growth Specific growth rate and doubling time for a cell culture are generally calculated based on the change in viable cell concentration during the log phase of growth only. This is the part of the growth curve during which the cell culture grows at its quickest (see the Cultivation Time Chart in section To create a Cultivation Time Chart, above). Since the calculations for specific growth rate and doubling time should only be carried out using measurement results from the log phase, the measurement results from the lag and plateau phases need to be removed from the chart before the calculations are performed. To determine the specific growth rate and doubling time of a culture based on the log phase of the growth curve D 3 In the CTC Views dialog box, select the View to be used for the analysis of specific growth rate and doubling. Click on the VIEW CHART button. The Cultivation Time Chart dialog box is displayed showing the change over time in the viable cell concentration of the cell culture. Study the curve to determine which points on the curve are part of the log phase of growth. In the figure below, for example, the first three points are most likely part of the lag phase, while the last two points are most likely part of the plateau phase. These points will need to be deactivated before the specific growth rate and doubling time can be calculated. Log phase 8 Cedex XS Analyzer

29 Monitoring Growth of Cells over Time Result Analysis 4 5 In the CTC Views dialog box, select the appropriate View in the top part of the window and then highlight the relevant series of measurements in the bottom part. Click on the EDIT button in the bottom part of the dialog box to edit the list of measurements included in the chart. The Selection of Data Series dialog box is displayed showing all measurements used for the chart in the bottom of the window. 6 7 Measurements can be temporarily eliminated from the Cultivation Time Chart without being permanently removed from the list. Deselect the checkbox in the Use column for the measurements to be temporarily removed from the CTC. If there is no check mark in the Use box, the results for that measurement will no longer be shown in the CTC. To add the measurement back to the chart again, select the Use box again. Temporarily remove all measurements from the lag and plateau phases via the Use box, then click on SAVE to save the changes. The window closes and the CTC Views window is displayed. Click on VIEW CHART to confirm that only results from the measurements relating to the log phase are shown: D 8 Confirm that only the log phase is shown, and close the dialog box. Further Applications 9

30 Monitoring Growth of Cells over Time Result Analysis 9 In the CTC Views dialog box, click on the EXPONENTIAL REGRESSION button. The Cultivation Time Chart - Exponential Regression dialog box is displayed: D The Cedex XS Software carries out an automatic calculation of the specific growth rate and doubling time based on the viable cell concentration results from the measurements actively included in the chart. The graph only shows the viable cell concentration. The measurement parameters specific growth rate µ (/d) and doubling time Time [d] of the culture are computed by means of the differential curve and shown in the Data Series area at the bottom of the Exponential Regression chart...5 Checking Regression Curve Fitting If the regression curve does not fit well to the measurement results for viable cell concentration, the specific growth rate and doubling time will not be as expected for the culture. See, for example, the figure below. 30 Cedex XS Analyzer

31 Monitoring Growth of Cells over Time Result Analysis The measurement results for viable cell concentration should fit relatively closely to the regression curve determined for the measurements in the Exponential Regression chart. In the case shown above, measurement results from samples of the cell culture when it was in the lag and plateau phase were included in the Cultivation Time Chart and Cultivation Time Chart - Exponential Regression chart. Therefore, the fit and calculated doubling time are not as expected for the culture. Perform the following steps: Check the curve for viable cell concentration in the Cultivation Time Chart. Temporarily remove measurement points that are not part of the log phase of growth, using the Use checkbox in the Selection of Data Series dialog box (see section Work with the Exponential Regression Function to Determine the Specific Growth Rate and Doubling Time of the Cell Culture Based on the Log Phase of Growth)...6 Editing Measurements in a View Measurements can be added or removed from a particular View at any time by using the EDIT button in the Series of measurements belonging to the selected view area of the CTC Views window. To edit the measurements belonging to a View: Select the row of the View to be edited in the View area of the CTC Views window. Select the particular series of the View to be edited in the Series of measurements belonging to the selected view area. Click on the EDIT button. The Selection of Data Series dialog box is displayed. Add measurements from the series list as described above, or remove measurements by selecting them and clicking on the REMOVE button at the bottom of the dialog box. Click on the SAVE button. The changes will be saved and the dialog box will automatically close. The CTC Views dialog box will be displayed and other series in the same View can be edited. D Measurement results from a Cultivation Time Chart can be printed and exported to various formats. You can also export a View or Series list and delete Views and Series. For more information on the CTC functions available, see the Cedex XS Operator s Guide. Further Applications 3

32 Analyzing Unusual Cells Overview Analyzing Unusual Cells. Overview The Image Analysis Operator takes care of the software-based image evaluation. Two operators, adapted to a wide variety of cells and objects, have been pre-installed for various application scenarios: CedexXSCellOperator for the analysis of cells, default Cell Types: Std. Size for normal sized cells with a diameter of approximately - 4 µm Min. Size for small cells with a diameter of approximately 8 µm 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 existing Cell Type can be adjusted. This is done using 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. D Once a new Cell Type has been created and modified, measurements can be carried out with the new Cell Type and existing measurements can be reprocessed with the new Cell Type. 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. 3 Cedex XS Analyzer

33 Analyzing Unusual Cells Example of Cells not Recognized by the Cell Type Std. Size. Example of Cells not Recognized by the Cell Type Std. Size In the image from a measurement shown below, some live and dead cells were not identified using the standard Cell Type Std. Size. This particular culture, therefore, requires a new Cell Type that includes changes to certain parameters in the image analysis software (Operator). The following steps must be taken to create a new Cell Type, adjust the parameters for the Cell Type, and re-analyze the images using the new Cell Type:. Create a New Cell Type. Modify the Parameters for the New Cell Type via the Live Operator Window 3. Reprocess the Measurement Using the New Cell Type as the Basis for the Image Analysis D.. Create a New Cell Type To create and save a new Cell Type: Open the Cell Type List dialog box by selecting the Select the Cell Type List option in the Master Data menu. There are several options in this dialog box, including: NEW: Create new Cell Types. EDIT: Modify existing Cell Types VIEW: View existing Cell Types DELETE: Delete existing Cell Types Further Applications 33

34 Analyzing Unusual Cells Example of Cells not Recognized by the Cell Type Std. Size 3 Select the existing Cell Type on which the new Cell Type should be based, and click on the NEW button. A message will be displayed asking if the new Cell Type should be based on the highlighted Cell Type in the list. Click on YES. The Cell Type dialog box will open with the LIVE OPERATOR button inactive: 4 Enter the name of the new Cell Type. D 5 6 Select the appropriate IR Operator from the drop-down menu: CedexXSCellOperator for analysis of cells or CedexXSObjectOperator for analysis of objects Click on the SAVE button. The Cell Type dialog box will automatically close. The Cell Type List dialog box will be active again and include the newly created Cell Type in the list... Modify the Parameters for the New Cell Type via the Live Operator Window Click on the LIVE OPERATOR button in the Cell Type List dialog box. The Live Operator dialog box will open: 34 Cedex XS Analyzer

35 Analyzing Unusual Cells Example of Cells not Recognized by the Cell Type Std. Size In the Data Source area, select the new Cell Type to be modified from the drop-down list, in this example called NewCellType. Select the Reactor ID, Sample ID and Data Set of the measurement for which the image analysis is to be improved. The parameters for the loaded measurement are pre-set. Select an image of the measurement in the Image No. field. This image will be loaded and displayed in the dialog box with the living cells marked with a green circle and the dead cells marked with a red circle. Make the desired modifications to a single parameter first. In the example shown in the figure above, some live and dead cells were not recognized. This is most likely due to the fact that some of the cells fell below the cut-off for minimum size and also had an unusually weak contrast against the background. For this particular cell culture, the following changes were made to the Cell Type NewCellType (for more detailed information on available parameters, see the Cedex XS Operator s Guide): ClassificationBias set to: - CMinSize set to: 3 LCAppearance set to: 0 DCAppearance set to: 0 See the figures below: Image analysis with Std. Size before parameter adjustments Image analysis with NewCellType after parameter adjustments D 6 As soon as each parameter is changed, the data is automatically reprocessed. The image display then shows the reprocessed image, as well as changes in the number of live and dead cells identified by the new Cell Type: 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. 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. Thus it is possible to directly compare the evaluation with each previous evaluation. Further Applications 35

36 Analyzing Unusual Cells Example of Cells not Recognized by the Cell Type Std. Size 7 Continue to adjust parameters until you find an optimized adjustment of the Operator parameters (see step 5). Use of the sliders, especially in combination, can lead to unexpected results. 8 Click on the SAVE button to confirm the modifications for the new Cell Type. Click on the CANCEL button to close the Live Operator window without saving changes. 0 parameters are available for adjusting the Cell Type. For details of the parameters and the effects of changing them, see the Cedex XS Operator s Guide. Existing Cell Types can be edited and deleted. For more information on these actions, see the Cedex XS Operator s Guide...3 Reprocess the Measurement Using the New Cell Type as the Basis for the Image Analysis To reprocess existing data sets using the new Cell Type (e.g., NewCellType): D Click on the List button in the Cedex XS Control Center to open the Measurement List dialog box. Select the row of the measurement to be reprocessed and click on the REPROCESS button in the bottom right-hand corner. The Measurement dialog box opens containing all the result data for that measurement. Enter a new name for the data set in the Data Set field of the Sample Parameters area (e.g., reprocess, as shown in the figure below). Select the new Cell Type to be used for the image analysis (e.g., NewCellType) in the Processing Parameters area. You can also change the dilution factor in the appropriate field of the Sample Parameters area. Click on the REPROCESS button in the center of the Measurement dialog box to start reprocessing the measurement based on the newly selected parameters. 6 When the reprocessing is finished, a dialog box will open asking whether you want the new data set to be the effective data set. Click on YES if this is the case (see section Effective Data Sets). 36 Cedex XS Analyzer

37 Analyzing Unusual Cells Example of Cells not Recognized by the Cell Type Std. Size The new data set is available in the Measurement List dialog box once the evaluation is complete. All changes can be retraced by viewing previously created data sets via the Measurement List dialog box. 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 resulting 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 labeled as such in the Measurement List dialog box. To change the data set to be labeled Effective later: 3 4 Select the data set version of the measurement to be labeled Effective in the Measurement List dialog box. Click on the REPROCESS button. A Measurement dialog box will open containing the results for that data set. Check the Effective option in the Measurement dialog box. Close the Measurement and Measurement List dialog boxes. When the Measurement List dialog box is re-opened, the newly selected data set will appear as the Effective Data Set. D Further Applications 37

38 Analyzing Unusual Cells D 38 Cedex XS Analyzer

39 Appendix Index A Analysis parameter...4 C Cedex Smart Slide... 7, Cedex XS Accessory Box...7 Cedex XS Control Center...3 Cell counting... Cell Type...4 Create new...33 Live Operator...34 Chamber position indicator lights... Counting cells... CTC (Cultivation Time Chart)...7 Exponential regression function...8 CTC View...3 Cultivation Time Chart (CTC)...7 D Determining viability... Diluent Volume...4 Doubling time...8 Dye Volume...4 E Environmental requirements...8 Evaluation parameter...6 Exponential regression function...8 F Fuse...7 G Growth curve analysis...3 H Histogram...8 I Image Viewing...9 Image View dialog box...9 Installation... 7, 8 System installation...8 Unpacking...7 Intended use...5 L Live Operator...34 Appendix M Measurement Dialog box... 3 Exclude... 7 Growth curve analysis... 3 Parameter... 4 Results... 6 Searching... Setting up... 3 Starting... 5 Measurement list... Monitoring growth of cells over time... 3 N New Cell Type O Optimization White Balance... P Process Time... 4 Processing parameter... 4 Cell Type... 4 Process time... 4 System name... 4 R Reactor ID... 3 Result CTC (Cultivation Time Chart)... 3 Viewing... 6 Result data table... 6 Resulting dilution... 4 S Sample Loading... Preparing... Sample Drawing... 3 Sample ID... 3 Sample Parameter Define... 3 Diluent volume... 4 Dye volume... 4 Reactor ID... 3 Resulting dilution... 4 Sample Drawing... 3 Sample ID... 3 Sample volume... 4 E 39

40 Sample Volume...4 Setting up...8 Shutting down... Slide carrier... Specific growth rate...8 Starting Cedex XS Analyzer...9 Cedex XS Control Unit...9 Measurement...5 System installation...8 System Name...4 T Trypan Blue... U Unpacking...7 Unusual cells...3 V Viability determination... View CTC (Cultivation Time Chart)...3 Viewing Histogram...8 Images...9 Result...6 W White Balance...0, E 40 For life science research only. Not for use in diagnostic procedures. Cedex XS Analyzer

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42 Published by: Roche Diagnostics GmbH Roche Applied Science Werk Penzberg 837 Penzberg Germany Roche Diagnostics All rights reserved. For life science research only. Not for use in diagnostic procedures.