Re: Gating revisited.
/G=Gerhard/S=Nebe-von-Caron/OU=1890CHPE/O=TMGB.URC/@LANGATE.gb.sprint.com
Mon, 6 Nov 1995 05:30:00 -0500
Hello Wal
I thought that by now lymphocyte scatter gates would have
been abolished altogether. There is indeed a relationship
between scatter and cell type (and instrument manufacturer).
Most of us will probably have seen it for cd8 and cd4
positive t-cells. The subject has been studied in detail by
Leon W.M.M.Terstappen "Flow cytometric characterization of
white blood cells of healthy donors and patients with
lymhocytic diseases". His thesis has been published under
ISBN 90-9002197-3 and some articles out of it have been
published in the 80th.
In principle you can probably run normal samples with
scatter gates, but as soon as you look at abnormals the
trouble starts. There is the problem of escape-lymphocytes
who do not lyse properly (cell type specific) and thus don't
come in you light scatter gate and as I showed in my poster
on the last ISAC, there are even sometimes problems with
specific binding of lymphocytes to monocytes or macrophages
via adhesion molecules which can not be seen by the cd45/14
measurement used to check the lymphocyte gate. When you then
set a lifegate you won't see any of these effects.
If you analyse your pictures in side scatter versus
fluorescence you can sometimes correct your results but you
better avoid scatter gate on its own altogether and use
antibody gates instead.
Gerhard Nebe-v.Caron
Unilever Research, Colworth Laboratory Sharnbrook,
Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
E.mail: gerhard.nebe-von-caron@urcgb.sprint.com
______________________________ Reply Separator _________________________________
Subject: Gating revisited.
Author: 102675.320@compuserve.com at INTERNET
Date: 05/11/95 22:11
Does anyone else out there feel that the current widely held concept of
tightening Lymphocyte gates/bitmaps until Monos are excluded (CD14/45) is a bit
like a catch 22 situation ?
The premise is that lymphocyte subsets are evenly distributed throughout the
lymphocyte population as isolated by FS and SS - my experience is often the
opposite.
High degree of pleomorphism of CD 8's compared to CD4's in reactive pictures,
especially AIDS and I.M.
Conversely, my experience of normal bloods is that the CD3+/CD4+ population is
slightly more variable in size (i.e.FS) than normal CD3+/CD8+ and tightening of
the gate, or allowing the instrument to do it for you, exclude some of the
target population with consequent inaccuracy.
My opinion on tightening the lower end via CD45 can wait until later.
The reason I put this to the mailing list is that I am putting together a
"devil's advocate" lecture and would appreciate other views.
On the other hand I may just like a good argument !
Get back to me via the Mailing list or on <102675.320@compuserve.com>
Wal Sharp.
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
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If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
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EMAIL robinson@flowcyt.cyto.purdue.edu
