Re: CD34 cell enumerations
Margaret Cooley (m.cooley@arnie.cfi.unsw.EDU.AU)
Wed, 17 May 1995 09:02:25 +1100
>
>To:G R BARCLAY <100066.300@compuserve.com>
>From:m.cooley@cfi.unsw.edu.au (Margaret Cooley)
>Subject:Re: CD34 cell enumerations
>
>We routinely do CD34+ estimations on growth-factor mobilised peripheral blood
>leukapheresis specimens. The set up we use is CD45-PerCP from B-D, then
>CD34-FITC or CD34-PE (both HPCA-2 from B-D, but some labs use a cocktail of
>HPCA-2 and Qbend anti-CD34 from Immnotech, as these see different epitopes and
>the combination may give better results). This gives you a third colour to
>use if you want to look at subsets of CD34+ cells, e.g. CD33+ or CD38+. We
>just stain for 10 min room temp and treat the specimen on Coulter Q-prep, wash
>once afterwards.
>
> The machine set up is:
>
>Hist 1: CD45-PerCP as single parameter histogram, then set gate on all CD45POS
>cells (n.b., stem cells are relatively dim CD45 so be generous on the lower
>limit): this just excludes debris and any unlysed red cells.
>
>Hist 2 (gated on Hist 1): CD34 versus side scatter. This gives you a very
>clear population of low side scatter CD34-bright cells which represent the
>true stem cells, and means the dim CD34+ cells which are more mature and have
>limited repopulating potential are excluded.
>
>This set up enables you to calculate the CD34+ as a percent of total nucleated
>cells without trying to estimate % mononuclear from sometimes indeterminate
>scatter pictures.
>
>N.b. count a minimum of 100,000 nucleated cells, unless your percent CD34+ is
>over 1%.
>
>We use a Coulter EPICS XL but I see no reason why this strategy could not be
>used on any analyser or sorter.
>
>Bst of luck,
>
>
Margaret A. Cooley, PhD
Centre for Immunology
St Vincent's Hospital
Darlinghurst NSW 2010 AUSTRALIA
Ph: 61-2-3617700; Fax: 61-2-3612391.
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