Maciej Simm <simmmmer@yahoo.com>
cc:
Subject: Re: RBC lysis problem in lymphocyte studies (Document link not
converted)
Hi there,
I had a customer recently with the same problem, although not in a patient
with microcytic red cells.
One option (perhaps the simplest) is to spin the sample down on Ficoll,
wash and then stain.
Additionally, if using a CD45/SSC plot to define an initial lymphocyte
gate, you may be able to get rid of more red cell debris by increasing the
CD45 Flx (in many 3 colour systems, this will be Fl3) threshold to channel
300 or greater. But take care not to move the threshold so far as to
encroach on the lymphocytes...
However, this customer had already taken the Fl3 threshold as high as they
dared, had no Ficoll and was in a rush...
So, in their lyse-wash (LW) assay, I suggested adding a few drops of
distilled water for 5 seconds before adding the regular (FACSLyse) lysing
solution, and then washing with PBS (FACSFlow, or similar). If need be,
this can be repeated until you're happy that all red cells have been lysed.
In this particular case, 2 cycles were sufficient and they then got good
results. (As you probably know, microcytic red cells have relatively more
membrane than normocytic ones, which I think is at least part of the reason
that they can be harder to lyse).
If you're worried about losing lymphocytes because of the distilled water,
they are surprisingly resistant.... This can be easily tested/validated by
taking an EDTA sample, running it through a haematology analyser and
noting the differential WBC. Then spin it down and carefully remove an
exact volume of plasma, say 1-2 mLs, using a Gilson pipette, or similar.
Replace with an exactly equal volume of distilled water. Mix, spin, count,
and repeat. After 3-4 cycles you will have effectively replaced all the
isotonic plasma with water, but the lymphocyte count remains constant for
quite a while. Other WBCs are not as tough, however. I did this using a
Coulter MAXM analyser-the plots look bizarre (it wasn't designed for this
application!)...however it may not work on all routine haematology
platforms. You can probably do the same using beads (eg TruCount) and a
flow-cytometer.
I would anticipate that the same manoeuvre would work in a lyse-no wash
(LNW), but don't have any direct experience.
I hope this helps.
Regards and best wishes,
Nigel LLEWELLYN-SMITH
Applications Specialist
Becton Dickinson UK Ltd
Oxford
UK
OX4 3LY
Maciej Simm <simmmmer@yahoo.com> on 18/06/99 22:29:26
To: Cytometry Mailing List
cc: (bcc: Nigel LLEWELLYN SMITH/Europe)
Subject: RBC lysis problem in lymphocyte studies
Hello,
We're experiencing consistnat difficulties w/lysing of RBC in
lymphocyte immunoaphenotyping. Our diff. is in the "noise" around
the lymphocyte population, so that we can't do significant numerical
analysis. The problem is usually conicident with young patients who
are receiving a lot blood transfusions (such as THAL patients). We
think that their RBC's are too strong for our lysis protocol. But at
the same time we need to preserve the lymphocyte cell membranes b/c
we're staining for a variety of CD markers. If anyone has any insight
in this are please don't hesitate to share ;)
Maciej Simm
Research Technician
Weill Medical College of Cornell University
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