compensation calculation

Flow Forum (Inactive)
compensation calculation arein  2007-10-19 13:59
Status: Closed
 
Could you help me understand a little bit more about how compensation is done in CPAS? Is the formula pretty much the same as what Flow Jo uses for compensation? I assume so, because compensation in CPAS looks basically identical to compensation in Flow Jo, but I was just wondering how that is calculated. Along these lines, when we edit our compensation matrix and set the positive and negative peaks based on single color controls, one must assign a value for the positive gate and another line for the negative gate. Does CPAS look at the positive line and consider everything to the right of it positive and then look at the negative line and consider everything to the left of it negative? I guess I was just wondering why those peaks have to be set twice, given that you could just cut the histogram in half (like in Flow Jo) and it will know that cells on the left are negative and cells on the right are positive. If we should be setting two separate peaks for positive and negative populations, then what happens to the cells that fall in the middle - are they just excluded? Thanks for your help!
 
 
nicksh responded:  2007-10-19 14:57
I believe we use the same formula as FlowJo. The results we get are nearly identical, and the formula is very simple.
The spill value for FL-1 into FL-2, for instance, is the difference, for the FL-1 compensation control, between the median FL-2 fluorescence values of the positive and negative populations, divided by the difference between the median FL-1 fluorescence values of the positive and negative populations.

When you are using LabKey to calculate the compensation matrix for multiple runs, we recommend that you use gates which are smaller than you would use if you were drawing the gates by hand for each run. This is because there will be variation between the runs, and you want to make sure that the indeterminate cells in the middle are not used in the calculation.

In practice, it does not make much difference whether these cells in the middle are included in the positive or negative populations. The only values that matter in the calculation are the median fluorescence values, and including these small numbers of cells does not change the median by very much.
 
arein responded:  2007-10-22 12:49
Thank you! That definitely helps.