background subtraction

Alice L. Givan (Alice.L.Givan@Dartmouth.EDU)
07 Nov 95 10:13:05 EST

Here's an old problem: What do people do about subtraction of isotype control
values from flow determination of the so-called "% positive" population? In
most lab with which I am acquainted, if the isotype control marker is set at
3%, then 3% is subtracted from every subsequent sample. Although I have never
heard it discussed in public, I have known for a long time that this approach
is obviously incorrect -- as can be seen if one imagines a sample with clearly
ALL the cells brightly stained. By subtracting the control, it would then
appear that only 97% of the cells were stained. The problem with this
"classical" approach is that we should be subtracting 3% of the true negative
cells only because those are the ones that are mistakenly contributing to the
determination of the positive population.

When I follow this through algebraically, I come up with the following
formula:

y=(x-c)/(1-c/100)

where y is the actual % positive
x is the measured % positive
c is the % positive set for the isotype control

Am I wrong about this (or is it so obvious that everyone else does it already)?
Does someone have the patience to check my derivation?
Thanks.

Alice L. Givan
Englert Cell Analysis Laboratory
Dartmouth Medical School
Lebanon, NH 03756
tel 603-650-7661 or 7907
fax 603-650-6130

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