Hi Carl-Magnus, You always have some interesting questions that I am tempted to answer. First of all, you should keep in mind that when you analyse sorted cells you are actually adding another variation of measurement on the same population. You should expect to get 2 SD increase in population spread and therefore always need to expand the gate with reanalysis. Even if you run, theoretically, one cell two times it can end up once (during the sort) in your gate and second time (reanalysis) just outside the gate and still belonging to the same population. Especially, if you split one population arbitrary, like you did, you should expect this effect. 6% was just used for orientation and if all cells are CD38- negative after sort you should consider it 100% successful. Sticking with the same gate is always to cut some of them off! Purity should be defined comparing populations set to be sorted and what you got at the end. If they want 6% of absolute number of cells in some population at the end, estimate your yield and divide 6% with it (if it's e.g. 70%, 6/0.7=8.5% should be the size of your gate). That was my input. Cheers, Sasha Dr Sasha Sreckovic Dept Path & Micro University of Bristol University Walk Bristol, BS8 1TD, UK Sasa.Sreckovic@bristol.ac.uk 0117-928-8606 ---------- >From: Carl-Magnus Hogerkorp <carl-magnus.hogerkorp@molmed.lu.se> >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >Subject: Purity?? >Date: Wed, Feb 2, 2000, 10:27 am > > > Dear all, > > 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? > > > Thanks for your input > > > Carl-Magnus Hφgerkorp > Stem Cell Laboratory > University Hospital of Lund > Sweden >
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