Try
http://cmgm.stanford.edu/~roederer/compensation/index.html
for an excellent course on compensation and you know the
limit of quadrants.
If you really want to be convinced that it is not a good
idea to move regions or think about alternative ways of
analysis you should get in touch with Jim Watson or read the
chapter on Immunofluorescent Data in :Flow Cytometry Data
Analysis, basic concepts and statistics, Cambridge
University Press (1992) ISBN 0-521-41545-4
Gerhard Nebe-v.Caron
Unilever Research, Colworth,
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
gerhard.nebe-von-caron@unilever.com
______________________________ Reply Separator _________________________________
Subject: Isotype Controls
Author: pyle@smtpgw.kfshrc.edu.sa at INTERNET
Date: 23/02/97 03:23
We have been recently taking a retrospective look at some CD45 values
in acute leukemias that were originally interpreted as negative. The
issue arose as a query into what is believed to be an unexpected high
number of negatives. This has led me to question what the current use
of isotype controls are among other flowers. Here are my current
thoughts: the age old use of isotypes has been as a bench mark for
analyzing other markers of the same isotype class. This allowed the
user to determine the amount of non-specific binding that might take
place with that particular isotype class and determine that the
correct amount of compensation has been taken. The quadrant cursors
are set on the isotype and that setting is used to analyze all the
tubes containing the same isotype(s). I have never been the kind to
take this quadrant placement as cast in stone and have always used
experience and intuition to reposition these cursors when indicated.
I remember being at the Bench Top Flow Cytometry course in New Castle
a couple of years ago and hearing one lecturer say that he would
consider the data of anyone who never moved their cursors as suspect.
Anyway, we are now having a dialog about the value of isotypes in a
panel and whether they actually have any value at all. One major
laboratory in the States has actually dropped their isotypes from
their panels save perhaps a single one. I know about the use of CD45
in every tube as a third color and how well it works in peripheral
blood but question its use in bone marrow analysis where the CD45 is
either very dim or negative in the blast population of interest. In
many cases, the advice has been to simply disregard isotypes and go
strictly with intuition and perhaps an unhealthy amount of what is
expected, to analyze specific histograms.
So, my question is: How are isotype controls being used by other
laboratories? How much importance do you place on the isotype control
in placement of your quadrants and later in analysis of other tubes?
Do you still use isotype controls in your panels at all?
Thanks in advance for your invaluable advice.
Haywood Pyle
pyle@kfshrc.edu.sa
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