Dear Cheryl / Peter / Readers
One other thing to cause more unfixed or 'bigger' cells
(higher in Forward Angle Light Scatter (FALS)) with the FACS
lysing solution seems to be antibody incubation on ice prior
to cell lysis. Those cells are still negative to PI uptake,
thus not permeabilised. Since we do antibody incubation in
out 15oC incubator I haven't seen it again.
With regards to different CD3 intensities I currently look
at some elderly patients where I found a number of high
expressing CD3 subpopulations, mostly positive to CD16/56.
Keep it flowing
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
______________________________ Reply Separator _________________________________
Subject: Re: Lymphocyte Populations
Author: zug@asklepios.umfiasi.ro at INTERNET
Date: 24/08/97 06:36
Cheryl M. A. Palmer wrote:
>
> I use a FACScan along with Simulset software to enumerate T- lymphocyte
> subsets on HIV positive patients. On a few occasions I have seen two
> distinct lymphocyte populations. On gating these populations, whether
> individually or together, the T+B+NK cells are much lesser than the
> ideal 100%.
> Can someone give me some ideas as to why this is happening and how I can
> account for the 'missing' cells.
Dear Cheryl,
Double images of the leukocytes on FS - rarely on SS, also - may occur
(not only in the HIV+ patients) when lysis of the erythrocytes fails
and, sometimes, when cells are damaged (difficult blood
drawing/homogenization with the anticoagulant, long delay - with
apoptosis, perhaps - before labeling and analyze, evantually some drug
treatments).
Repeating the tube, after labeling and vortexing during incubation,
eventually by manually delivering the lysing reagents may help to obtain
a better image of the lymphs. I use a Coulter system and I prefer to
lyse manually the red cells in order to optimize the timing of the steps
according to (I believe) the hematocrit. The length of the first step
(formic acid,0.12%) appeared to be critical. In the worst cases, in HIV+
specimens, I repeat the labeling with a cocktail having CD45 on one
fluorescence and CD3, CD2, CD20 one another(s). The last 3 markers
should produce a reasonable estimation on the lymphs. By the way, some
of the "silent lymphs-like populations" may be CD86+.
I wish to add to Peter's reply (although I wonder if this point was
also addressed in your question) that distinct levels in CD3 and CD2
expression might occur even in normal (and more evident in HIV+)
individuals. "Activated" / "memory" lymphs - especially TCD8+ might have
~2 fold more CD2 expression and ~1.5fold less CD3expression when
computing the mean fluorescence channel ratios. Resting TCD4+ and
resting TCD8+ lymphs express also slight differences in these two
markers (it might be seen in nwe borns with CD3,CD4 versus CD2). The CV
of these markers, in simple labeling, might be higher in HIV+ specimens.
Relevant data concerning surface expression of many markers might be
found in Int Immunol 8 (1): 1-11 (1996), Changes in antigen densities on
leukocyte subsets correlate with progression of HIV disease, Roederer M,
Herzenberg LA, Herzenberg LA, and related.
florin ze
Laboratorul de Imunologie Tumorala Iasi, Romania.
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