Isotype Controls

Robert Pyle pyle at
Sat Feb 22 08:39:03 EST 1997

     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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