VenturiOne – Manual Compensation in Flow Cytometry

 

VenturiOne is our rapid data analysis platform to process, interpret, and report on your FCS data files – using features such as manual compensation to interpret your flow cytometry data. In our VenturiOne tutorial series, you can find out how to process multiple files, use our compensation wizard, autogating and more on VenturiOne.

 

 

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 the guides here.

Manual Compensation in Flow Cytometry

By default VenturiOne will read the compensation that is saved with the FCS file for your flow cytometry data. It does allow you to change the settings or to calculate the compensation settings and then apply them to a series of files.

Firstly, we will look at how we can change the compensation settings on a single FCS file. On this particular sample, there are a number of plots where the populations are quite hard up against the axis and they look like they may be over compensated.

What we can do here is use some of the tools in VenturiOne to help us recompensate a sample. So firstly, we can look at setting everything to a hyper log axis.

Setting the fluorescence to a hyper logger axis allows you to see both very high and very low off scale overcompensated data. Very clearly the first thing is to change all of the fluorescence plots to show a high pull of display.

So, we highlight all of the plots and deselect the forward scatter side scatter plot and now change everything to a hyper log scale. We need to adjust the zero position and the number of negative decades present to create something that is a little easier to see.

Click on a plot which is in hyper log and two sliders will appear on each axis. The purple slider allows you to set the position of the zero point and the red slider allows you to adjust the linear to log percentage.

So, now we’ll go to separate plots and straighten up some of the data there so we can actually see exactly what is overcompensated and what isn’t. Having adjusted several parameters it becomes obvious that there are several populations that are overcompensated.

We can change the compensation on a plot by highlighting a plot and going to the compensation tab of the ribbon and selecting plot compensation. In this case we know that this population which is heading towards the axis should be level with the population on the left.

We have a couple of tools which can be used to manually alter the compensation. The first being to simply click on that population and drag that population upwards.

When you release the data is recalculated and all the plots are updated. Again click on a plot and drag the population upwards and one last time and click and drag and release.

If you wish to return to the settings that were initially saved with the FCS file you can go to the ribbon and select load compensation from the headers and that will return the compensation settings back to their own initial values.

Another method for manual compensation for your flow cytometry data is putting a quadrant on each plot this can be done separately or if you do wish to do many at once and you highlight all the plots you need the quadrant on.

Select the quadrant region and then press control and click the mouse button over a highlighted plot. When you select a plot you can select plot compensation on the ribbon and then the current compensation settings will be displayed here together with the statistics.

For quadrants within that plot so we can look at the x statistics or the y statistics. When we look at the x statistics we look at quadrants one and three and when we look at the Y statistics we look at quadrants three and four.

For a correctly compensated population the exit is 6 for 1 and 3 should approximately be the same value as should be Y values for 3 and 4. So, looking at the plot on the right first looking at the Y statistics for quadrants three and four we can see that the Y value for quadrant three is 267 and the Y value for quadrant 4 is minus 483 which is significantly overcompensated.

So, you can either drag this population backup or use the controls in the ribbon. The single arrow up will move the population up slowly or the double arrow up will move the population more quickly.

So, as this is quite overcompensated we will use the double arrow until the values are approximately the same. So using the single arrows as a more fine adjustment for your manual compensation until they’re around the same values, so that’s the Y statistics if we check the X statistics look okay but let’s check them anyway.

We can see that the numbers are already correct. Let’s have a look at the second plot.

In this case we are looking at the X statistics and in quadrant one the population is slightly over compensated, so we can reduce that value and then we can have a look at quadrants three and four. Again it’s overcompensated so we can manually adjust that compensation, so we can bring that population into the correct position again using the fine adjustments.

We can repeat this process for any population and plots that need recompensating. You can save the manual compensation settings for future use.

You can go to file save and then save compensation as and then enter a file name and then save. That file name then appears in the playlist compensation column.

As another alternative manual compensation can be performed using the preview plots if you click on a region this will gate the previews on that region then on the compensation tab of the ribbon.

Choose manual compensation and then click on the preview plot to drag a population where you want it to go. You can also use the preview plots to look at the data in hyper log by going to the preview tab of the ribbon and selecting the hyper log option, then using that in conjunction with manual compensation option in the compensation ribbon tab.

You can get a quick overview of what the compensation looks like and make any quick adjustments if you need to. To view the compensation matrix go to the compensation tab of the ribbon and select the show matrix option.

The window that comes up will show you the current compensation settings these can be printed if needed. This is the end of our manual compensation tutorial.

If you would like to see how to calculate flow cytometry compensation using our wizard, please see the compensation wizard tutorial.