Re: Bckgrnd subt.
David_Burleson_at_USAISR1__FSHTX@ftdetrck-ccmail.army.mil
Fri, 10 Nov 95 14:29:25 EST
Fellow Cytometrists,
Please pause to consider that your assumption about the isotypic control is that
it binds to the cell and produces fluorescence in the same way as your test
antibody. This assumes that only Fc binding is involved, that Fc binding is
identical between conjugated antibodies of the same isotype and the
fluorochrome/protein ratio between the antibodies is exactly the same. It is
possible that the Fc binding and other nonspecific binding (usually hydrophobic
in nature) are not the same as the isotypic control. This is rarely exactly
true, so the isotypic control is really just an estimate of this 'background'
binding. If you want an absolute control you would have to develop a control for
each antibody that you use, which is clearly impractical. For immunophenotyping
it is usually justified to move the cut-off to compensate for a change in a
uniform shift in a negative population. Obviously you must be careful that you
are compensating only for the 'nonspecific' antibody binding and not for
biological differences. It may be important to run the antibody on several
controls to verify this. Trying to report a corrected fluorescence only
complicates matters. To get an accurate correction factor you need to know the
effective molecules of fluorochrome for your negative peak, the positive peak
and that they are log-normal in distribution. Even if you get an accurate
correction factor how does it help you interpret immunocytometry data? At best
you will have a measure of the difference in fluorescence between your isotypic
control and your antibody. Perhaps that could be useful quality control
information but it says nothing about your sample. Quantifying surface or
internal antigens is different, there you must calibrate each antibody
specifically.
David Burleson
US Army Institute of Surgical Research
Fort Sam Houston, Texas
______________________________ Reply Separator _________________________________
Subject: Bckgrnd subt.
Author: t-delohery@ski.mskcc.org (Thomas Delohery) at Internet-Mail
Date: 11/9/95 5:53 PM
Eric Martz wrote:
>Changing subject a bit, what bothers me is the occasional use of the cut
>line set on the control (e.g. 99% of control below the line) to estimate %
>positive when the entire distribution shifts slightly to the right (left
>edge as much as right edge) without changing shape. Although 20% may now
>be above the cut line, seems to me the result is that approximately 100% of
>the cells are (weakly) positive. The 20% is meaningless since the cells
>which were dimmest before staining have become just as much brighter as
>have the cells which were brightest before staining. Thus, seems to me the
>cut line should be used to estimate % positive as <100% only when the shape
>of the distribution changes, consistent with bimodality (at least a bump or
>tail on the right).
I completely agree with Eric. When the entire population shifts but is not
resolved from the negative control I recommend reporting mean fluorescence
corrected for background; ie, net fluorescence. Using cutoffs for positive
and negative inadequately describe the data. What may be 30-50% positive by
using cutoffs may be 3-5 fold increase in fluorescence above the control.
There was an article in Cytometry some years ago on measuring pgp (MDR pump)
in normal, primary tissue that dealt with this problem. I can only remember
one of the authors names: Mark Mueller. If anyone is interested I'll look
for it.
td
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