As I mentioned recently, I will also ask fairly basic
questions, as short version for people that are in a hurry,
the full story for interested people further down:
- Has anyone experience with the lysis of heparinised blood
and scatter gating. How can I get rid of the thrombo
aggregates, what lysing solution is recommended?
- Has anyone looked into the effect of formalin fixation on
the fluorescence of PE, or compared fluorescence intensities
of FITC and PE in different lysing solutions, perhaps NH4Cl
of distilled water.
And here the long winded story:
Admittingly I am not an expert in clinical FCM, but want to
undertake a clinical study. I have consulted a number of
people and got the panel of appropriate antibodies. I
already asked some while ago about the use of transport
media for blood samples, but eventually we got transport
organized to get the samples 5 hour fresh. I would have
loved to get separate blood samples with heparin and EDTA,
but as 50ml heparinized blood is taken from the volunteers
for cell activity assays I eventually agreed that 3ml
heparinised blood would be fine to do both phagotest and
antibody staining (I remembered that heparin was a commonly
used anticoagulant in the early days), and this is were the
trouble starts.
I want to look at granulocytes (no gradients). I also would
like to avoid to spend the money on CD45 in every tube. As I
look at healthy volunteers, lymphocytes should not be rare
events, but a nice fat cluster. Fat it is indeed, covered in
a comet tail of activated thrombocytes. Initially I
compared a number of commercial lysing systems. They all
work fine - on EDTA blood, but can not handle Heparin. In
desperation and absence NH4CL lysing solution I used a
distilled water lyse neutralized with 10X PBS. This method
allows to distinguish the lymphos from the thrombo
aggregates already in a non-wash system but the lifetime of
the sample is limited (~2 hours). In addition my
fluorescence intensities are much higher, in FITC and PE
when compared to the commercial non-wash lysing solutions
(apart from the Dako Uti-Lyse). For CD14 this is a problem
as it goes out of scale and thus screws up the compensation.
Washing of the A.D.-lyse makes the gating easier as the
thrombos disappear. Whilst that is not the case with the
commercial reagents, at least the FITC fluorescence of those
samples recovers. Further investigation revealed that only
the Uti-lyse has a final pH that does not quench the FITC
fluorescence, but this does not explain the difference in
the PE fluorescence.
In the hope to keep my samples stable for longer I spun the
lysed samples and resuspendet them in PBS containing 0.4%
praformaldehyde (diluted to 289mOsmol, pH 7.4). Within two
hours my PE fluorescence comes down as well, but I have not
yet determined whether there is a final level or if it goes
down continuously. Perhaps the PFA only interferes with the
outer parts of the molecule.
If my observation is correct, it makes me wonder how to
compare signal intensities, or can't you do that with
fixative lysing solutions? Also the PE equivalents derived
from the Sperotech beads will thus be misleading as will be
the FCSC beads unless they have been lysed and fixed the
same way.
I have searched the Howard 2nd edition (as someone else has
the 3rd), medline and cytometry, but without success.
Perhaps someone can give me a related reference or any
useful tips regarding the stabilization post lysis or any
minimum fixation time to wait for.
Probably having opened a can of nematodes I am looking
forward to lots of comments
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
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