From: Alice L. Givan (Alice.L.Givan@Dartmouth.EDU)
Date: Tue Nov 07 1995 - 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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