>
> 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
>