Dear Christopher, Good questions which nicely address a valid concern. In our experience, it is more often useful to consider which populations the blasts should be considered as part of a whole. That is, intact, viable nucleated cells = the denominator. Toward that end I would those events using a combination of gates with CD45/SSC as the primary and FSC/SSC as the secondary. If you wanted to be real specific, you could also include viability as a third criteria by adding 7AAD to your CD45 tube. Nucleated rbc's should not be excluded because they are part of typical marrow elements. Light scatter gating should allow you to visualize the location of most non-viable cells, debris, etc. which should be excluded. By logic with this combination, the intact, viable cells should be considered the denominator taking an argument from the CBC on whole blood which performs its differential based on intact, nucleated cells. If you wish to express blasts in terms of only the WBC's then you may wish to include Glycophorin A in your profile to exclude any rbc, nrbc elements. The phenotype of the previously described blasts would serve as the numerator. Hope this helps, Bruce Greig Technical Director, Flow Cytometry Vanderbilt Univ. Medical Center Nashville, TN. (615)322-2682
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