Hello, I would also be interested in the general practices used to estimate sort purity. The most rigorous method is to re-analyze sorted cells, and use the same gates as used in the sorting to estimate purity. Many people use quadrant analysis, which can make the sort purity seem better than it is, depending on how far the quad. gates are from the sort gates. After checking a number of papers, people very often report sort purity >95% - >99%. I wonder how often this numbers are somewhat inflated by the method of analysis. Perhaps it should be on the list of instructions to authors that this list is contemplating drafting, to include the actual data from the sort re-analysis, showing the gates used. Its seems a somewhat separate issue to discuss the interpretation of sort purity analysis data - there are many possible reasons for cells to fall outside the gates used to define the sort. Because its relevant to something I am working on, I am particularly interested to hear peoples views on how to test for issues other than sorter operation/function that contribute to lower sort purity. We are sorting 2 different populations - one is FITC-neg and PE-high and the other is FITC-high and PE-high. We get 95% or better purity on the FITC-PE-high cells and only ~ 80% purity on the ++ cells. The main source of cells outside the sort gates for the ++ sort are both "PE-lower but not really FITC lower" cells and "FITC-lower but not really PE-lower" cells. I welcome suggestions about how to test what is going on - bleaching ? receptor modulation/internalization ? something else ? Thanks! Rachel ======================================================= Rachel M. Gerstein, Ph.D. Department of Molecular Genetics and Microbiology Graduate Program in Immunology/Virology University of Massachusetts Medical School 55 Lake Avenue North Worcester, MA 01655-0002 (508) 856-1044 (508) 856-5920 (FAX) > ---------- > From: Simon Monard > Sent: Monday, October 29, 2001 2:21 PM > To: Cytometry Mailing List > Subject: Sort purity > > > Hi folks. > Can anyone point me to a discussion of ways to estimate the purity of a > sorted population > of cells. This is clearly easy when you are sorting very bright cells from > negatives > but more problematic when sorting cells from a "shoulder". I remember > seeing a nice > discussion on this subject but cannot remember where. I've been sorting > some very > weakly positive cells from a population, these cells post sort overlap the > negative > cells quite a bit. My customer seems unhappy about that. > Thanks > > Simon Monard > FACS Lab Manager > Trudeau Institute > Saranac Lake > NY12983 > > Ph 518 891 3080 X352 > >
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