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>
> 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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web