Re: Lysis of Heparinised blood, fixation and PE fluorescence

From: PLM Enterprises and Designs (plem@erols.com)
Date: Fri Jun 20 1997 - 15:58:17 EST


What I do for lysis of heparin or EDTA, post antibody staining, is use
"Whole Blood Lyse and Fix" by Gentrak....e-mail me for the address.
First, antibody is added to 100 ul whole blood, then the lysing solution
is added followed by the fixing solution, washed then I add 300-500 ul
2% paraformaldehyde. I read 1-2 days later.


> 
>           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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