Re[2]: that pesky CCC article

Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
31 Oct 1996 08:05:35 Z

I actually wonder how you can manage not to separate the
beads. Perhaps I should read thast article.
There is no magic behind the optics or electronics
and unless either screw up the compensation completely or
you pour coffee over you dichroics you can hardly go wrong.
Even the fact that the Coulter system deflects the light in
a plane perpendicular to the laser polarization can not
avoid that signal discrimination. I remember the
disappointed faces of the Becton Dickinson people when they
visited us after having rejected their offer and having
bought one of the first EPICS Elite's in the UK. After
having run their test beads they had to admit that that
machine was measuring as good (or bad) as theirs. They could
only complain about my attitude to put forward scatter on
the Y axis and the side scatter on the x-axis. I always do
that as I look at 'size' going up and 'obesity' going
sideways (I think I have to dig out that cartoon of Haegar
the Horrible from Dick Brown as a nice way to illustrate
that perhaps for Paul's next CD). Even worth, I look at
scatter distributions in a log display which really goes too
far for most users. Apart from giving far better cluster
definition for your monocytes (and significant effects on
cluster analysis like automated gating algorithms) it's
perhaps because it makes the 'fat' granulos look slimmer and
the slim lymphos look fatter (my part of wishful
thinking).
As the fluorescence collection in our old Elite system is
not corrected for colour aberration you can either
compromise or optimize on green or red. As we do a lot of
'green' work, we run it at a sensitivity of approximately
150 FITC equivalents which lowers the PE sensitivity but not
the separation of the peaks.

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

I forgot, there was a bug in the very early electronics,
only apparent when running the beads with a low flow rate
(not like when the beads were poured in by the people who do
not have to order them themselves) . It made the separation
look worth in parameter2 compared to parameter3 in the
parameter list, i.e. when selecting PMT2/FITC/log as
parameter 3 and PMT3/PE/log as parameter 4, the separation
of the FITC beads looked better in the uncompensated PE
channel than in the FITC channel, but that was (should have
been) corrected long time ago.

______________________________ Reply Separator _________________________________
Subject: re: that pesky CCC article
Author: tshanke@bsd.medctr.luc.edu at INTERNET
Date: 30/10/96 04:18

On Thu, 24 Oct 1996, Tom Mc Closkey wrote:
>
> I have run those beads on my Elite and was able to resolve all populations.
>
> The apparent discrepancy may actually be stated in the discussion, "The
> reason for the diminished resolution ... could have been due to ...operator
> error."
>
> -------------------------------------
> Name: Tom Mc Closkey

Since its getting cold here in Chicago, and to add a bit more fuel to
that fire... We have run those same pesky beads on our Epics (I call it a
minus V, since it's a 541) and on an XL numerous times, and in each
instance we get separation of the six different bead populations that is
at least comperable to that shown in Fig. 4 (A,B, or D) of that now
famous CCC article (Chance, et al) in both the FITC and PE channels. From
my experience, most flow cytometers that are properly maintained
(including filters that actually filter, clean optics, stable fluidics)
and operated give good separation of these types of beads.
This thread brings up the uncomfortable issue of - how are we EVER going
to have a serious impact on clinical applications if we can't even
overcome the basics?
Vince Shankey
Loyola University Med Cntr.

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