MERGE FILES TUTORIAL. Version 1.4

Transcription

1 MERGE FILES TUTORIAL Version 1.4

2 1. INTRODUCTION The merge process consists on adding two or more sample data files from the same sample or from different samples. The file resulting from the merging will contain all the events and markers of all the files selected in the merging process. For more detailed description of all the available options, please consult User s Manual (Chapter 6). As you can see in the following figures, after the merging process the user will be able to study the expression of all markers of the panel in the same data file. Tube FITC PE PerCP-Cy5.5 APC 1 FSC-H SSC-H CD103 CD25 CD19 CD11c 2 FSC-H SSC-H CD22 CD23 CD19 CD20 3 FSC-H SSC-H FMC7 CD24 CD19 CD34 4 FSC-H SSC-H LAMBDA KAPPA CD19 CD5 Merge process Figure 1. File Merge matrix. CY-XX Rev.:001 Issue date: May 31,

3 Common parameters, will allow us to analyze the populations of interest at the same time in all merged files. Notice that common parameters should have the same appearance in all tubes. Therefore, please, before designing and acquiring a panel of tubes to merge, have a look to the following advices in order to correctly use the File Merge option: -Acquisition settings and sample preparation protocol have to be the same for each tube to be merged. -The appropriate marker panel design is essential in order to perform global analysis of the merged sample. The design of the necessary antibodies panel must be done taking into account which are the most appropriate common markers: The more common parameters we use, the more precise the identification of the population of interest will be, e.g. to identify the population of B Lymphocytes from a peripheral blood sample, FSC,SSC and CD19, at least, could be used as common parameters. The common markers used in the panel must be marked with the same fluorochrome. In fluorescences not occupied by common parameters, the rest of antibodies to be used in our study will be included. Infinicyt TM includes an internal control system to verify that the previously mentioned conditions are applied (See chapter 6.5. User s Manual). In this tutorial, we will review in detail the File Merge option. Figure 2. Merge process CY-XX Rev.:001 Issue date: May 31,

4 2. OBTAIN SAMPLE DATA FILES This tutorial introduces you some basic features of Infinicyt TM software. You will perform a typical process of merge data files. Please, go to to download the files needed for this tutorial. These folders correspond to sample files which can be used as example. Go to Documentation/Tutorials to select the files. Figure 3. Download samples directory. Download Merge Tutorial PDF and the second Sample Folder which is named demo_ merge _files.zip. Download this folder to your hard disk. In this folder you will find 4 files from a B-cell Chronic Lymphocytic Leukemia (B-CLL) sample with different markers studied on B-cells (see the box below). In order to analyze the merged file with a single gating we have designed a specific panel. The common parameters are: FSC, SSC and CD19-PerCP-Cy5.5. Tube FITC PE PerCP-Cy5.5 APC 1 FSC-H SSC-H CD103 CD25 CD19 CD11c 2 FSC-H SSC-H CD22 CD23 CD19 CD20 3 FSC-H SSC-H FMC7 CD24 CD19 CD34 4 FSC-H SSC-H LAMBDA KAPPA CD19 CD5 Figure 4. Parameter chart of the data files to merge. CY-XX Rev.:001 Issue date: May 31,

5 3. MERGE MODE 3.1. Open Infinicyt TM. Launch Infinicyt TM by double-clicking on the icon that appears in the desktop and open the option File Merge Select files to Merge. Figure 5. Infinicyt TM main menu The following File Merge dialog box appears. Click on Select Files button and a new window will appear to select the files to be merged. Figure 6. Select Files Window. CY-XX Rev.:001 Issue date: May 31,

6 Go to Sample Data Files folder you have already downloaded and select files in the folder demo_merge_files. Figure 7. Folder selector window Click the Ctrl key to select the files you want to merge. Once the selection is done click Select. Figure 8. Files to be merged. CY-XX Rev.:001 Issue date: May 31,

7 3.3. Merge Matrix. Now the files will appear in the File Merge Window in the same order you have selected them. You will see also a matrix with all the merged files and the information about which parameters were measured in each tube. Once the files are selected, using the icons you can select three different views: 1. Both file list and the parameter matrix (Default). 2. File list only. 3. Parameter matrix only. 1. Both file list and the parameter matrix (Default). List of files to merge Change the order of the files Change view Change the order of the parameters in the matrix Figure 9. File Merge window matrix. Parameter matrix CY-XX Rev.:001 Issue date: May 31,

8 2. File list. Figure. 10. File List View. 3. The Parameter matrix. Figure 11. Parameter Matrix. To register a parameter as a common one for the different tubes, the software must recognize exactly the same name in the parameter description in the FCS file of each different original tube. In case there are differences, e.g. regarding capital letters, dots, hyphens, commas, script, etc., the software will not identify the corresponding parameters as common. Therefore, it is essential to check the spelling of the parameters that are going to be common. CY-XX Rev.:001 Issue date: May 31,

9 3.4. How to match the legends of the parameters. The following four files of the chart belong to the same patient. FSC and SSC and CD19 should be common parameters, but they are not. On each file the labels are written in a different way. File 1 FSC- A SSC- A CD19 PerCP-Cy5-5- A:PerCP-Cy5-5-A File 2 FSC-A SSC-A CD19 PerCP-Cy5-5-A:PerCP-Cy5-5-A File 3 FSC SSC CD19 PerCPCy5-5-A:PerCP-Cy5-5-A File 4 FSC-Height SSC-Height CD19 PerCP-Cy5.5:PerCP-Cy5-5-A Figure 12. Different way of writing for the common parameters. To solve these label discrepancies, just click on Group. Figure 13. Solve Label discrepancies. 1. Match FSC It will appear a dialog box in which you may click on the labels that should be grouped. Let s see an example of how to group the parameter FSC. As we have seen in fig. 12 we have four different labels to indicate the parameter FSC: FSC- A FSC-A FSC FSC-Height Click on. A column with all parameters will appear. Fig 14. Group. CY-XX Rev.:001 Issue date: May 31,

10 Select the box of the first parameter, FSC- A. Some parameters will disappear from the list (the ones that belong to the same file). A new group will be created called FSC- A. That name will appear in the field. Figure 15. Make groups of labels for FSC. Select the following parameters we want to group with FSC-A from the list. The next one is FSC-A, then FSC-Height and FCS. Figure 16. FSC: Group of labels. Click. Check in the merge matrix that FSC now appears as a common parameter. Figure 17. FSC grouped in all files. We must do the same procedure with parameters that should be common: SSC and CD19. CY-XX Rev.:001 Issue date: May 31,

11 2. Match SSC As we have seen in fig. 12 we have four different labels to indicate the parameter SSC: SSC- A SSC-A SSC SSC-Height Click again on. A column with all parameters will appear at the right side. Note: There is a folder named FSC-A which correspond to the group we have previously created. Figure 18. Previous group of parameters: FSC Repeat the same operation as before but now select the parameters to group as SSC. Figure 19. SSC: Group of labels. CY-XX Rev.:001 Issue date: May 31,

12 Click. Check if in the merge matrix SSC now appears as a common parameter. Figure 20. SSC grouped in all files. As we have seen in fig. 12 we still have four different labels to indicate the parameter CD19: CD19 PerCP-Cy5-5- A:PerCP-Cy5-5-A CD19 PerCP-Cy5-5-A:PerCP-Cy5-5-A CD19 PerCPCy5-5-A:PerCP-Cy5-5-A CD19 PerCP-Cy5.5:PerCP-Cy5-5-A 3. Match CD19 Click once again on. A column with all parameters appear on the right side of the window. (Notice that there is a folder named FCS- A and a folder named SSC- A. Those are the groups previously created). Figure 21. Previous groups of parameters: FSC and SSC. CY-XX Rev.:001 Issue date: May 31,

13 Follow the same procedure as before but selecting parameters to group CD19 as a common parameter. Figure 22. CD19: Group of labels. Click. Check in the merge matrix that CD19 now appears as a common parameter. Figure 23. CD19 grouped in all files. At the same time we have created groups of labels, the software has created a dictionary. The second time we load these files to make a merge, the software will make the groups automatically. If you click on Manual Analysis or in Save as.. the changes on the dictionary will apply. CY-XX Rev.:001 Issue date: May 31,

14 3.5. Check the dictionary The software has saved the groups previously done. Follow the instructions to use the previous groups. Repeat steps 3.2 and 3.3. Click on but this time, Just Click on to use the dictionary. The software will automatically group the labels. Figure 24. Option: Use previous groups. Click on Previous groups to check the dictionary. Figure 25. Groups into the dictionary Change the order of the parameters. It is possible to change the order of the parameters in the merge matrix. 1. Select the parameters to move. 2. By clicking on you can change the order of the parameters in the merge matrix. Figure 26. Parameter order selector. CY-XX Rev.:001 Issue date: May 31,

15 3.7. Save merged files If you agree with the merge, select to save the merged file or select to see the result of the merge process without saving it. You will always have the option for saving the merge file later during the analysis. The software will make the merge of the files. We have generated a new file with the following parameters: FSC/SSC/CD103/ CD25/CD19 /CD11c /CD22/CD23/CD20/ FMC7/CD24/CD34/LAMBDA/KAPPA/CD5 If you click Save as give a name to the new data file and the directory where you want to save it, then select Save Finish the merge process Click on the Main Menu box to finish the process. The new data file generated with this process is a FCS (Flow Cytometry Standard) file, and it is ready to analyze whenever you want clicking in. BEFORE STARTING THE ANALYSIS OF THIS SAMPLE, PLEASE, READ IN DETAIL THE TUTORIAL OF MANUAL ANALYSIS. CY-XX Rev.:001 Issue date: May 31,

16 4. ANALYSIS OF A MERGED FILE Select the merge file to analyze Select Manual Analysis if you want to analyze this file immediately. If it is the first time you open the software, several default plots will automatically appear. You will also see the Manual Analysis Window menu bar and a Population Tree with different labels for different populations If you have saved the merged file, and you want to do later the analysis, go to the main screen and select Manual Analysis. On the Manual Analysis Window menu bar Select File/Open from the menu and select the recently merged file, named as Analysis. The dot plots you need for the analysis are at least: FSC vs. SSC and FL3 vs. SSC to identify B-cells. However you can open new diagrams for each marker in order to show the phenotype of those B-cells (see chapter of the User s Manual to create new diagrams) Change the parameters displayed on the graphs. By right clicking on a label of a diagram; you will see the list of parameters contained in the file. Common parameters are identified with the symbol. See figure below: LABEL OF A DIAGRAM Figure 27. Common parameters. CY-XX Rev.:001 Issue date: May 31,

17 4.4. Population Tree. Regarding the population tree, you will need a label for B-cells to assign the gate on those cells. If you do not have it created in your population tree, visit chapter of the User s Manual. Figure 28. Population Tree with the population B-Cells created Gate on Common Parameters. Draw a region on FSC vs. SSC (DotPlot A) including the events contained in the lymphoid area. (Click on the upper-side bar for full screen mode). Then, draw a region in the CD19PerCP-Cy5-5 vs. SSC (DotPlot B) including CD19 positive events. (Click on the upper-side bar for full screen mode). Some of the CD19 positive events which are not in the lymphoid area will be eliminated from the selection. Now, only the intersection of both regions is selected in black. A B Figure 29. Intersection of two regions in order to get a better identification of our cells of interest (B-cells). Click Ctrl+Z for Undo or Ctrl+R to Reset the analysis and start from the beginning. CY-XX Rev.:001 Issue date: May 31,

18 4.6. Assign events to the population of interest. Go to the analysis window and click on the pink label of B-Lymphocytes to assign the pink color to the selected population. Click on this label Figure 30. Manual Analysis window with the population B-cells characterize Options of visibility. Unselect the VIS box and select only the pink label of B-cells, so we will visualize on the Dot Plots only events classified as B-cells. In this type of merging process, the file generated will content each marker on a different parameter within the same data file, so you can obtain new diagrams with the parameters you want. Figure 31. Phenotype of B-cells with the selected surface markers. CY-XX Rev.:001 Issue date: May 31,

19 4.8. Display different combinations of markers. Remember that you can choose different combinations of markers in a dot plot only if they were measured in the same tube. A B Figure 32. Parameters measured in the same tube. Figure 33. Parameters measured in different tubes Display all markers in a unique diagram. Once we have selected the population of interest, we can ask for diagrams that will allow us to study all parameters at the same time. Those graphs are Parameter Band Dot plot and Parameter Band Histogram. Go to Diagram/New diagram in the Manual Analysis Window menu bar and select a Parameter Band Histogram with 15 dimensions (see chapter User s Manual). Figure 34. How to select a Parameter Band Histogram graph. CY-XX Rev.:001 Issue date: May 31,

20 Click on OK and study the Parameter Band-DotPlot. Figure 35. Parameter Band Histogram graph performed with 15 parameters Add comments to the population of interest. Go to the Population Tree. Double click on the pink label of B-Cells in the column named Comments. Fill the gaps of each parameter with comments about them. After that, comments will appear in the report. Figure 36. B-Cells comments. CY-XX Rev.:001 Issue date: May 31,

21 4.11. Report of a merged file. Here is shown an example of an edited Report. On the Manual Analysis Window go to File/Report. You will find a default Report. (See chapter 5 in the User s Manual for more detailed information) Figure 37. Report CY-XX Rev.:001 Issue date: May 31,

22 5. OTHER INFORMATION RELATED TO MERGE FILES o STATISTICS In order to obtain statistics in this merged file, click on the drop-down menu at the upper right corner (see picture below). You will see different options to show your statistics: All files: Statistics related to all events in the new file. Population Files: Statistics of a given population only related to the files in which this population is present. File n : Statistics related to each independent file. Figure 38. Manage statistics of merged files. Note: You will find the value NA when the statistic is not applicable. o SELECT FILES Go to Edit/Select File/File number. Select a file and you will have a selection (gate) done in all the events included on that original file. Figure 39. How to analyze merged files one by one. CY-XX Rev.:001 Issue date: May 31,

23 o MERGE CONTROLS Infinicyt includes a process of Coherence control to determine if the user is performing the merge process with the optimal files, the similarity of expression in the common parameters. The control contains different threshold values, so the user can modify them in the corresponding option. Nevertheless, the users are strongly advised not to change the threshold values predefined in the program, when working with real data, unless they understand perfectly these processes. Failure to comply with this warning may produce unreliable results. There are two major types of coherence controls: -Global coherence control (Merge Control): the software, regarding the common parameters analyzes the similarity of the total events from each file between all files merged. -Populations coherence control (Population Control): whenever the user analyzes a merged file, and tries to define a group of events from different tubes as a unique population, the software analyzes the similarity between these populations from each file. In this sense, the Merge Control allows to compare the main populations represented in the file, to detect, for example, that a common MoAb has not been added, while the Populations control allows comparing populations less represented which can go unnoticed in the global coherence control. In both controls, there are some threshold values to define the strictness of the software controls to show a warning of no coherence between files or populations. Configuration of the merge controls Click on Configure on the File Merge dialog box (also available for in the Manual Analysis Window menu bar on Profile/Configure/Controls ). Figure 40. Merge Controls. CY-XX Rev.:001 Issue date: May 31,

24 In this box, you can select your threshold parameters for coherence controls. For this tutorial we have selected the following threshold for warning, (see picture bellow). It means that whenever the difference on the common parameters is more than 10%, we will obtain a warning before saving the new file. Figure 41. Control Threshold box (Infinicyt V1.3 Advanced). Merge Control. Once we have done the merge, we have two indicators to identify if we have any global difference among our files in common parameters. Those indicators are: -Global coherence control (Merge Control): -Populations coherence control (Population Control): As we saw before, we had differences between some files in common parameters. Due to those differences, in the Manual Analysis Window we will find a red sign. Figure 43. Coherence control. CY-XX Rev.:001 Issue date: May 31,

25 Population Control. Click on the red sign and you will get information about the parameters and files in which we have differences bigger than 10%. Population differences: If the software detects any differences in a population inside the merged file we will see a warning sign as well. Figure 44. Coherence control. Click on Population differences label and you will get the following window. This window tells us in which population we have differences once we have analyzed the data. In this case we have differences in the population we named as B-Cells. The differences are higher than 2%. Figure 45. Population difference box. CY-XX Rev.:001 Issue date: May 31,

26 o REVIEW and SAVE GATE STRATEGIES REVIEW ANALYSIS On Infinicyt TM is possible to review the strategy of analysis in merged files. With this option, you will be able to check your global analysis. Remember that the selection has been made based on gating on common markers. It is important to check if the selection in all tubes has been made properly and in a comparable way. In our case we have identified the population B-Cells in all the files. We have classified the population B-Cells gating on FSC/SSC and SSC/CD Ask for the gating strategy. By right-click on the label of B-Cells select Load Gates Review. In this moment a new column appears with this symbol. CY-XX Rev.:001 Issue date: May 31,

27 2. Check gate strategies. Click on the symbol. The software will show all steps of gating you have made to classify the population. The information of the gate strategy will appear in three places: 1º. The software will show you the gates in the diagrams and the visibility at the moment you classified the population. You can see in the gates made in the same color as the population. If you want to modify your strategies of gating just click on the gate and modify it. All statistical values will be modified automatically CY-XX Rev.:001 Issue date: May 31,

28 2º. When you have activated the option gates review, on the Manual Analysis Window additional information about the gates appears. We will find information about which steps were made in order to select a population. The software shows us the population to review, in which diagrams we made the gates and which parameters were used to identify each population. Pressed button: Review Strategy activated Population to review Parameters used for gating on each diagram Diagrams used for gating 3º. Go to Profile/Analysis Info in Manual Analysis Window menu bar. In the Analysis Info Window you will find information about the whole strategy of gating made during the analysis. CY-XX Rev.:001 Issue date: May 31,

29 3. Save and apply a gating strategy Save gating strategies. On version 1.4 is possible to save strategies of gates and then apply it over others files. To save strategies of gates follow the steps: A. Click on Profile/Save Analysis Strategy in the Manual Analysis Window menu bar. B. Name the new strategy. The new gate strategy will be saved in the profile. CY-XX Rev.:001 Issue date: May 31,

30 3.2. Apply gating strategies. Once we have saved a strategy of gating we can apply it over other files. To apply strategies of gates follow the steps: A. Click on Profile/Load Analysis Strategy in the Manual Analysis Window. B. Select the more appropriate strategy of gating to apply. As we saw in this tutorial (Section 2 Check Strategies of Gating), it is possible to modify the gates applied. Just click on the gate and move it. CY-XX Rev.:001 Issue date: May 31,