Hi Carl-Magnus, If I am right in interpreting what you are saying, you are sorting the lower part of a positive peak and the re-running it to check purity, is that right? The general way to assess purity would be to re-run the sample on the machine you sorted on, leaving all parameters unchanged. When you sort a defined population, say, double positive, you would want most (>90%) of the cells to fall within the original sort gate, but I would accept that there may be some drift (generally toward the negative); as long as there isnt significant contamination with other populations though I would be happy. However, I have often done sorts on, for example, integrin-stained keratinocytes where the user wnats to sort the top and bottom 10% to see if they have different growth characteristics. In this case what they want to see is a precipitous cut off at one end of the re-run sample. However this is unlikely ever to happen and you will see what you are seeing. This stems from the fact that if a cell goes through the machine twice it will not necessarily be in the smae place but its position will have a gaussian distribution so the cells at the edge of the sort window may well go over it on re-running. In these cases either 'sort purity' is meaningless or 'bottom x%'is meaningless. What I tend to say is that we have sorted the bright from the dim but let the user know the caveats. Hope this helps (or, indeed, makes sense!) Derek On Wed, 2 Feb 2000, Carl-Magnus Hogerkorp wrote: > How does one define sort purity? Is there any consensus around how to > present purity figures? > > To exemplify: > If sorting a CD34+/CD38- "population" of 6% (based on the 34+ of the total > viable cell population) and knowing how the machine works, a population > rather corresponding to the ~25% lowest gets enriched. Within the original > sorting gate approximately 60% of the events fall and the rest will fall > above the sorting gate. These 40% are not something I would define as > impurities since they are a part of the sorted cluster of CD34+/CD38- cells. > How do I define sort purity? Is it 100% of the 25% lowest or 60% of the 6% > lowest. > > It becomes pretty obvious that, if wanting to sort these very 6%, the sort > gate should include perhaps the 1% lowest or something in that way. However, > most users of sorted fractions are not aware of the limitations of the > sorter but merely ask for the 6% lowest. This of course is a bit of a > dilemma for the person operating the sorter since there are no simple > algorithm to apply correcting for this phenomenon. How do others deal with > this? ************************************************************************ Derek Davies Voice: (44) 0207 269 3394 FACS Laboratory, FAX: (44) 0207 269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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