Re: Cd34
Wayne Green (Wayne.Green@genetics.utah.edu)
Thu, 26 Oct 1995 14:15:26 -0600
Ed,
The use of CD45 as a gating parameter seems to be increasing in use. BD
has recently released a CD34 enumeration kit (I think its called ProCount) which
uses CD45, a nuclear dye and beads (to get absolute count). There is also a
clinical evaluation currently underway by ISHAGE (International Society for
Hematotherapy and Graft Engineering) which is based on the procedure published
by Sutherland et al. (Expt Hematol 22:1003 1994). Both use CD45 as a gating
parameter in which one includes all CD45 positive cells.
I am currently trying these but have a major concern: They all require a
gate be drawn around CD45 positives which must also include all the CD34's which
express low level CD45. It would be easy to inadvertantly "clip off" some of
the dim CD45's, which would include the CD34's, if someone were unfamiliar with
the technique. I am still running the old standard CD14-FITC/CD34-PE/PI using
logical gating to first exclude dead cells, second exclude monocytes and finally
to gate on CD34 positives versus log side scatter. This is particularly helpful
for apheresis samples of mobilized peripheral blood stem cells in which there
are many monocytes (ie. Fc receptors and autofluorescence). Another problem
with monocytes is that they occupy a region of the light scatter histogram which
coincides with the location of the CD34's. A final benefit is the ability to
count only the "viable" CD34 positives based on PI exclusion.
A final point is how many cells to count. There was a good paper by
Trischmann et al. (J Hematotherapy 2:305 1993) in which they demonstrated the
need to count larger numbers of cells to acheive statistically reliably data.
They reported a minimum of 50,000 cells to be adequate, however the ISHAGE trial
I mentioned above is requiring 75,000 cells.
I look forward to the other responses you receive!
--
Wayne Green
wgreen@genetics.utah.edu
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