isotype controls anad PFC

Wayne Harris waharri at
Tue Jan 16 18:26:39 EST 2007

Hello everyone,

I just wanted to throw out a question to see what people's opinions  
are. i know that several times before the subject of the utility of  
isotype controls in analysis of multicolor cytometry has come up. The  
basic perspectives that seem to repeat are there are those that use  
isotypes because they always do, or it's better to use isotypes than  
not to. And still others favor the use of FMO controls. But somehow  
it all seems incompletely accepted what is best.

My question is really if you are using a six or seven color  
experiment (perhaps more) with tandem conjugates on a digital system  
can you really use isotype controls to aid in your analysis at all  
since they will be even more different from the actual reagents used  
in comparison to the case for our "normal" reagents (FITC,PE, PerCP,  
APC)  where the argument against the utility of isotypes focussed on  
differences in Ig concentrations etc. And furthermore, if the tandems  
can vary so significantly from lot to lot,  to what degree might we  
see shifts in the staining patterns as the levels of background or  
just simply staining characteristics of the reagents vary for the  
different antibodies in our panels....

ie. even though we create antibody specific compensations in the  
matrix, do the differences in the panels because of the different  
antibodies cause significant shifts in background etc that your  
threshold for signal determination may itself shift from panel to  
panel thus making isotypes virtually useless as a control other than  
to say whether or not there is significant background.

Simply stated, does polychromatic flow cytometry further  
significantly increase the problems in analyzing data using isotype  
controls because you may have greater problems with backgrounds etc   
for different panels even for samples acquired on the same machine  
with the same settings... Are FMO controls the only option then for  
each reagent in each panel you use?

just wanted to get an idea how people are dealing with this question.


Wayne A. C. Harris
Bone Marrow Transplant Laboratory
Immunology and Flow Cytometry Assays Laboratory
Winship Cancer Institute, Emory University
1701 Uppergate Drive
WCI, building C, Rm#4032
Atlanta GA  30322

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