Re: More on the mysterious case of the biexponential transformation...

Diana,

It is hard to give a definitive  answer to your question ". do I even need 
to compensate PE v. APC?  Obviously I can't do the biexponential 
transformation if I don't, but what are the rules about compensating 
channels with little or no spectral overlap? "	Most likely the answer is 
Yes.

There appear to be some confusion here.  First, biexponential plots are 
not just to be used on compensated data.  They are useful for all data. 
The reason people connect them with compensation is that biexponential 
plots easily reveal poorly compensated data (as it does in your lower 
right hand plot).  They won't necessarily tell you why the compensation is 
off.

As to when to compensate, the best and safest answer is "always and for 
all channels".	Differences of as little as 0.5% can significantly affect 
the data for dyes with 1-2% spillovers.  You don't mention the spillover 
values for your expt so it's not easy to judge what going on in your 
situation.

Given your graphs I suspect that one problem you have had in setting your 
comps is in your gating of the "lymphocyte" population.  A key rule for 
accurately setting compensation (even more important for low spillovers) 
is that the background/autofluorescence of the negative cells MUST be 
equal to the background/autofluorescence of the positive cells.  Your 
positive cells are a clean population of CD8+ lymphocytes.  I'm not so 
sure your "negatives" are only lymphocytes.  It looks as if your 
"lymphocyte" population also include some monocytes.  These will be 
included in the negative population and will adversely affect your 
compensation.  That is why many people use Ig-capture beads to perform 
compensations.	 This problem is avoided. (Disclaimer: BDB sells such 
Ig-capture beads, but that is not my reason for the recommendation). If 
you still want to use stained samples, put a much tighter gate around the 
lymphocytes.  It really doesn't matter if 50% of the lymphocytes are gated 
out.  Its more important to exclude all other types of cells.

A second potential problem which you note is that the comp control was a 
week old fixed sample.	A second key rule for compensation is that the 
fluorescence spectrum of the Comp control fluorophore must match the 
fluorescence spectrum of the reagent being comped.  Normally I would not 
expect the spectrum of FITC to change over time but the same may not be 
true for APC.  APC is a multimeric protein which can breakdown over time 
affecting the fluorescence emission spectrum.  And you are correct, small 
differences in spectral emission (not intensity) can have a big effect on 
the perceived spillover especially on low spillovers.

With the right controls compensation really is fairly straight-forward. On 
digital instruments it's a simple mathematical calculation.

Good luck,

Alan Stall
BD Biosciences
==============================================




Diana Martin <zigra@uga.edu>
10/07/05 07:56 AM
	To:	Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
	cc:	(bcc: Alan Stall/SDCA/BDX)
	Subject:	More on the mysterious case of the biexponential 
transformation...


Flowers,

I've had time to go back and look at this data again.  I've 
included my comp controls and what to (my) naked eye looks 
like a bright enough PE comp control is not, in fact, true. 
The MFI (median) of the PE+ comp control is 1059, for the 
sample it is 1267.  I used CD8 PE for both samples, but the 
comp control was about a week old (fixed mouse SC).  We have 
been doing this pretty regularly the past year, but 
apparently a small loss of fluorescence over time, or 
differences in day to day staining, can make a big 
difference.  I've included a graph with:

Top = lymphocyte gate
Middle = PE single stain (left), APC single stain (right)
Bottom = CD8 PE v. IFNg APC  left = uncompensated, right = 
compensated

So my next question... do I even need to compensate PE v. 
APC?  Obviously I can't do the biexponential transformation 
if I don't, but what are the rules about compensating 
channels with little or no spectral overlap?  Thanks for 
everyone's comments.

Diana
Diana L. Martin
Post-Doctoral Fellow
Center for Tropical and Emerging Global Diseases
University of Georgia
Athens, GA  30602
(706)542-3396
This attachment - 'IC stain.jpg' -  357.04 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/29147e1f6be05cf6678981feeb4580106a11a184.jpg 






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