Re: Gating revisited.
David_Burleson_at_USAISR1__FSHTX@ftdetrck-ccmail.army.mil
Tue, 07 Nov 95 07:44:27 EST
Walter,
Nothing comes without cost or compromise.
My concept of the CD45/14 use is to allow wider gates, not narrower gates and
use the CD45/14 analysis to correct for contaminating non-lymphoid cells. The
compromise here as you have already alluded to in your second message is that
the contaminating cells may not be passive bystanders but may pick up antibody
nonspecifically. You can use antibody gating, as Gerhard suggests but with added
antibody expense, loss of some of the power of multi-parameter analysis and you
still have to worry about non-specific staining. In the end I think each user
has to evaluate the method to use based on their needs and requirements, fully
realizing that their is no perfect solution and that each solution has
compromises. We have chosen to use wide gates with CD45/14 correction and an
attempt to evaluate and correct for the level of nonspecific staining that
occurs. It's not perfect but it seems to work best for us.
Dave Burleson
US Army Institute of Surgical Research
Fort Sam Houston, Texas
______________________________ Reply Separator _________________________________
Subject: Gating revisited.
Author: Walter Sharp <102675.320@compuserve.com> at Internet-Mail
Date: 11/5/95 8:26 PM
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
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
