purity and isotype issues in 3 and 4 color staining

From: Maciej Simm (simmmmer@yahoo.com)
Date: Thu May 10 2001 - 15:50:11 EST


Dear group,

I have a question regarding the following statements, in context of
immunophenotyping CD4/8/19/3/16+56 in HIV+ patients:

1. If you run 3 or 4 color flow, isotype control is not required
2. If you run 3 or 4 color flow, and define lymphs on SSCvs.CD45,
purity is assumed 100%

my problem with 1 is that every once in a while I'll see a trail of
cells in the isotype tube (usually IgG1Fitc/IgG2aPE, BD) which does
not go away with 3 colors (as in .. fsc/ssc gate vs. ssc/CD45 gate)

my problem with 2 is if I run CD45Fitc/CD14PE/CD45PE-Cy5, gate on
ssc/CD45PE-Cy5, and look at the scatter plot of CD45Fitc/CD14PE, the
CD45Fitc+/CD14PE- cells will be almost always less than 100% (usually
greater than 97%)

These statements come from our set of guidelines for a inter-lab
quality assessment.

What does everyone else think about this?

=====
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