M.G.Kelly at queens-belfast.ac.uk
Tue Sep 26 09:45:42 EST 2000
we have worked with BAL and performed flow cytometry - both in
normal subjects and those with COPD. The COPD patients
occasionally have slightly gloopy BAL, but on the whole, I have
found it to be not too bad. We do not filter or use DTT, so as
to interfere with the sample as little as possible. Some people
will filter like you have done, and you may have to do that on
occasion, tho' we have avoided it. We did have one BAL which was
very gloopy, and even after centrifuging, we didn't get good
separation. However, what we did was simply to lift out the
gloopy, mucusy bit. Yes, we probably lost some cells, but we
could not think of any other option.
I would suggest caution with DTT, as it may interfere with
some cell surface markers, and there is some literature on this.
A recent study in the ERJ (below) though done on blood, would
hold this up, and there are other papers:
D. Loppow, M. Böttcher, G. Gercken, H. Magnussen, R.A. Jörres
Flow cytometric analysis of the effect of dithiothreitol on
leucocyte surface markers Eur Respir J 2000; 16: 324-329
I have seen the reply by Bruce Greig in Nashville, and that
would seem eminently sensible.
Best of luck with your work.
Department of Clinical Biochemistry,
Institute of Clinical Science,
BELFAST. BT12 6BJ
Tel No: (028) 90263267
On Fri, 22 Sep 2000 16:51:26 +1000 Rees Beth
<beth.rees at dchs.tas.gov.au> wrote:
> Hi Flowers
> Here's one for the clinical flow cytometrists - how do you separate the
> cells from a slimy snotty goopy bronchial lavage ( have I totally put off
> the non-clinical cytometrists yet?). I have tried diluting it and flushing
> it through a 100um filter, but as soon as I centrifuge it the gunk just
> condenses again.
> The microbiologists use something called mucolyse (dithiothreitol) which
> reduces mucoprotein disulphide bonds, but I'm a bit scared to use this in
> case it interferes with the cell surface in any way.
> Any suggestions much appreciated.
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