Isotype Controls

Anna Porwit-MacDonald anpo at
Mon Feb 24 03:47:18 EST 1997

After introducing double- and three colour stainings in all routine panels 
the value of isotype controls have diminished, since there is almost always 
a negative population which one can evaluate the "negativity" on!
We still have one negative control in the panel (FITC/PE/PercP) but we look 
much more on negative cells in the actual triple staining than on the controls.
Using CD45 in all triples is not applicable in the ALL diagnostics since there
quite most cases are dim (just a little above negative) or simply negative! 
While looking for residual cells in the BM of ALL patients after treatment 
we use
gating on CD19/SSC and look for two other markes (like CD10/TdT) in this 
With best wishes
Anna Porwit-MacDonald MD, PHD
Haematopathology Lab.,
Karolinska Hospital, Stockholm

>     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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