Isotype Controls

Gerhard Nebe-von-Caron Gerhard.Nebe-von-Caron at
Mon Feb 24 04:48:04 EST 1997


          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 at     

______________________________ Reply Separator _________________________________
Subject: Isotype Controls
Author:  pyle at 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 at

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