> 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
>
>
>