Re: selective loss of CD8+ cells during ficol

Be careful--did they use a FITC CD8 reagent?  (Or biotin, or 
unconjugated)?	I'll bet a nickel that if this were done with PE CD8, 
it wouldn't happen.

This may be an "artefact" of the disappearing CD8 cells--an issue I 
brought up about 2 years ago on this list (and generated quite a bit 
of interest, but we never found the time to track down the reason for 
this behavior).  We found that when you using antibodies to CD8 
conjugated to small molecules (FITC, Biotin, Alexa dyes), after 
staining AND centrifugation, many CD8's "jumped" into the monocyte 
gate.  This happened to varying extents according to the individual 
from whom the sample was isolated, etc. etc.  If you stained but did 
not centrifuge (wash), this did not happen.

This process did not occur at all when cells were stained with CD8 
conjugated to phycobiliproteins (e.g., PE, Cy5PE, APC, etc).

What seems to be happening is that the CD8's aggregate with a myeloid 
cell after centrifugation when labeled with a small-molecule 
conjugate of anti-CD8--an aggregation that seems to be of a fairly 
high strength.	When staining in whole blood, this probably doesn't 
happen.

We are trying very hard to avoid using FITC (or Alexa-dye or 
biotin)-conjugated anti-CD8's in our phenotyping experiments so as to 
avoid this problem, and relying instead as much as possible on 
phycobiliprotein-conjugated CD8.  If you must use FITC-anti CD8, then 
be sure not to use a lymphocyte gate!

mr

>In a 1987 article "Analysis of T cell subsets in normal adults. 
>comparison of whole blood lysis technique to Ficoll-Hypaque 
>separation by flow cytometry by  P Renzi & L Ginns in Journal of 
>Immunological Methods, it was found that, in normal individuals, 
>ficoll separation of lymphocytes selectively decreases the CD8 
>subset compared to whole blood analysis on a buffy coat in all 32 
>individuals tested.  The average CD4/CD8 ratio was 1.8 in the buffy 
>coat and 2.8 in the ficolled specimen. We have seen results here 
>that are not too different.
>
>Does anyone have any more recent information, experiences, or fixes 
>they would like to share on this issue?
>
>Thanks.
>
>Marty
>--
>Marty Bigos
>Director, Flow Cytometry Core Laboratory
>Gladstone Institute of Virology and Immunology