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From: Walter Sharp, 102675,320
TO: INTERNET:KWatkins@aol.co, INTERNET:KWatkins@aol.com
DATE: 06/11/95 19:05
RE: Copy of: Re: Gating revisited 2.
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From: INTERNET:KWatkins@aol.com, INTERNET:KWatkins@aol.com
TO: Walter Sharp, 102675,320
DATE: 06/11/95 07:19
RE: Re: Gating revisited.
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Date: Mon, 6 Nov 1995 07:09:47 -0500
Message-ID: <951106070946_99041821@mail06.mail.aol.com>
To: cyto-inbox
Subject: Re: Gating revisited.
Wal --
I'm a little confused by your query. You mentioned tightening up the gate
based on 14/45. I agree that a tight lymph gate based on just FS/SSC is
dangerous. But if you set or analyze your gate based on 14/15, you know if
you're excluding lymphocytes from your scatter gate.
--Kirk Watkins
Hi Kirk,
Thanks for the reply, hopefully I'll get a few more.
I am assuming that you are using 14/45 in the standard manner - i.e. Isolating
the 14-/45+ and using these to build a second FS/SS scatter display within which
you can see Lymphs irrespective of their scatter properties.
Following that, I again assume that you then use this gate for subsequent
analyses such as 3/4 or 3/8.
If I am wrong and there is a more advanced way of utilising 14/45 I would like
to hear it - & no, I'm not being facetious, simply curious.
It's simply that we're out here in the Middle East and getting hold of new (as
opposed to currently accepted) views and/or methodologies is difficult.
Hence my initiation into the Cytometry mailing list - we've only just got a
server !
Back to my point :-
It's a great improvement over using simple scatter gates, I'm sure we all agree.
However - said scatter gate is then applied to tubes which do not have a 14/45
discriminator and any non-lymphs can now stand up and be counted as negative.
Under normal circumstances this will make very little difference clinically but
when one is processing AIDS (as oppsosed to early HIV positive) samples, for
example, the scatter properties of lymphs and monos tend to merge dramatically
so one is still left with the dilemna of either tightening the scatter gate and
losing high scatter lymphs or leaving it where it is and including negative
monos thus also biasing the result.
Our studies indicate that these very large lymphs are cytotoxic CD8's.
The only two ways out of this I can see at the moment are:-
1) A true 4 colour, one laser, method using 3/4/14/45 or 3/8/14/45 for example.
(Any of you out there with more than one laser need not reply !)
2) Using triple colour 45/3/19, 45/3/4 and 45/3/8 on the total WBC population
and calculating accordingly.
Assuming non-specific binding to Granulocytes can be overcome, of course !
Wal.
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