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Hi my name is Jo Crofts, welcome to the Applied Cytometry flow cytometry software VenturiOne. We’ve recorded some tutorials to help you get started using the software.
To get the most out of the software the tutorials are designed to be used alongside the getting started guide and the operation manual. You will find these at the website below.
Custom Fields Flow Cytometry Tutorial
We have already seen how we can add plots and statistic boxes onto a report. In addition to base, VenturiOne allows us to create custom fields which can be added onto a report as well.
The custom field can either be individual results or a calculation based on more than one result or they can be rules which is based on an outcome of either a field or a calculation. In this example I have a very simple two color experiment loaded consisting of four tubes which is a nicer type control
A CD34 a CD 38 and CD3+ CD19 custom fields allows us to do things like create a cd4 cd8 ratio and put that on the report and create a calculation that is the average CD3. We can also do a limb foursome which enables us to add various components together and check the validity of our results.
We can then add an interpretation to say whether the calculation or the result is within range or out of range. To flag on the reports if an abnormal result or if a problem has occurred custom fields are created from regions and FCS information embedded within the file.
Within each sample I have created a simple workspace, I have a region A picking up the lymphocytes. I have fluorescence plots gated on those lymphocytes with region B picking up the FITC labeled events region C picking up the PE labeled events and the quadrant region picking up the dual labelled events and those regions are repeated for the other samples in the playlist as I’m showing.
To create custom fields go to the reports tab of the workspace and select the custom fields in the ribbon options. The custom fields dialog opens up and here are three tabs.
The sample fields calculations and the rules sample fields are pieces of information that you are extracting from each sample. This could be a statistic or information from the FCS header.
In this case we are going to add sample fields to pick up the CD3 results from each of these tubes and then we will pick up the cd4 and cd8 results in order to calculate the CD48 ratio.
So, first I’m going to add three fields to pick up each of the individual CD three results. You can give the fields a custom name which I will do here.
Next we will need to identify which sample and region the result we need comes from. In this case I’m going to pick playlist index sample number two and then the source can be either the FCS keyword, the compensation setting or the region name that you want the results to come from.
In this case I want the CD3 which is region B. We will now need to select the result type that you want displayed and in this case I want the percentage gated.
The system then goes and extracts the value from the region and displays it in the field value column. Next I need to do the same for sample 3, so I will select playlist index 3 region B again because we want the CD3 result and percentage gated as a result type.
The result is extracted from sample 3 and displayed. Then I will repeat the process for sample 4.
I can now use those three sample fields to create calculation for example to display the average cd3 result. To create a calculation I go to the calculations tab, add a calculation and give the calculation a name.
Then we enter the equation for that calculation. To enter the equation we can use the drop down menu to pick the sample fields, any operators and other items that we need to construct the equation.
In this case I’m going to add my three sample fields together. I’m going to add parentheses and then sum my three sample fields together, then I will divide the results by three to get the average cd3 result, this is then extracted and calculated and shown in the resort column.
If I also want to create a CD48 ratio I need to add fields to pick up the relevant results for me to create the calculation. In this case the CD4 will be coming from sample 2 and region C and I want the result type to be count and then I want the CD8 to come from Sample 3 and region C and then the result type being count again.
Next we will create a calculation, so give the calculation a name. In this case the calculation is fairly straightforward, it’s simply the CD4 result divided by the CD8 results.
To create a rule, go to the rule tab and press the add rule button. I want to add a rule for the CV 4:8 ratio, so I will give it the appropriate name and in this case I’m going to ask for the cd4 cd8 ratio to be between one and four and to flag that as being normal.
So I can say the CD 4 CD 8 ratio is greater than or equal to one, then pick the and operator and select the CD4 CD 8 ratio calculation, then add less than or equal to four. Then these comments are what will be displayed.
It shows here that the output is true which means that the result for the cd4 cd8 ratio is between 1 and 4. If there are any errors in the calculation this will be stated underneath the comment column.
The output if true column, is the comment you wish to show if like in this example the output is true, so we will change this to normal ratio.
The output if false is the comment you wish to be shown if the result is outside of the stated range so for this I’m going to put abnormal ratio.
The outputs if error column shows the text that you want to be displayed in the event of calculation error. Having created that rule I can now send that rule to the report and it now appears on the report page.
We can configure exactly what appears on the report using the setup button and then checking or unchecking the boxes for these options as you need. Now if we zoom in on the report page, it shows here the default options of rule name and the output displayed.
I can manually choose a sample field a calculation or a rule to be displayed on the report using the field option on the ribbon. I can also configure exactly what is displayed using the custom fields button on the format area of the ribbon.
So, I can have for example just the rule output then I can insert a text field and enter the heading that will go with it. So, here I will use CD 4 CD 8 ratio and then I can choose the calculation to be displayed so I want to choose the CD 4/8 ratio calculation and from that data I only want the result
So, I’ve created the text the result and the flag to indicate if this is normal or not. Using this technique we can build up some quite complicated rapport tables as I’ll show you now.
So, for example in this playlist we have 18 samples set up in group mode. In group mode you can create one report and then that report is repeated for each group so here the group size is set to one so the report will repeat for each sample.
In this report we have the date and time. The date and time are inserted via the fields drop-down using the keywords for date and time.
We have fields where we can enter patient information and lot information by typing in text we have a barcode number which is a field from an FCS keyword again. We have plots displayed that are taken from the workspace and underneath that are the individual results generated using custom fields both as a percentage and absolute counts.
We have various calculations generated from the custom fields, working out the T-sum percentage, the lymphoma and the CD 4/8 ratio. Next to these are the rules associated with these calculations which tells us if the result is normal or abnormal.
As this is in group mode, as you click between the different samples everything on the right updates to show the information for that sample, the plots, the results for calculations, and rules all update according to the information within the file selected so VenturiOne can prove to be a very powerful reporting tool.
That’s it for this tutorial, thank you very much for watching, bye!