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>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
-- ============================================================================== Thomas Delohery | Internet: t-delohery@ski.mskcc.org Manager, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019 1275 York Ave. Box 98 | New York, NY 10021 | ==============================================================================
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