Just to add to the comments of David and Jens . Apart from the fact that a few things can happen to the fluorescet molecules of a cell between sorting and reanalysing, the key thing to consider is indeed the problems of distributions. The simplest test indeed is to sort the top 1/2 of a bead population of high and very low intensity. In cases were the variation of the signal is based on the distribution of the fluorochrome, indeed the sorting will reflect that. However, at low signal intensities one starts playing with statistics. This gets even worth when sorting compensated populations as the compensation increases the variation. Jens refers here to the formation of doublets in primary samples. Another related problem comes from coincidence events. Platelets can serve as a nice example. When coinciding with leukocytes (or even being attached to them) they tend to give fluorescent signals but do not show up in changes in light scatter or signal pulse. Again after a high dilution this coincidence will vanish. Regards Gerhard -----Original Message----- From: Jens Fleischer [SMTP:jfleischer@tesionmail.de] Sent: Monday, November 05, 2001 6:01 PM To: Cytometry Mailing List Subject: Re: Sort purity In my humble opinion we have to consider various effects: 1. As David McFarland correctly described, our populations are most often (always?, don't nail me on this, this would be another endless thread...) gaussian distributions, thus if you sort a "histogram shoulder" you will always get again a gaussian distribution and nothing between "goal posts". Recently I saw a software package that nicely calculated the possible gaussain distributions under an immunfluorescence curve, similar as the modelling for cell cycle analysis. I do not know when and how this will be commercially available. 2. Als Volker Eckstein wrote, loss of fluoresence and bleaching can have a serious impact, for example PerCP is quite sensible for this. He also mentioned the gaussian distribution by telling that a cell never falls into the same fluorescence channel if you measure it several times. 3. We should also not forget the formation of doublets in the primary sample which fall apart after sorting and washing the sorted fractions. Ideally, possible aggregates should be excluded from the sorting process. However, this is often not possible using the traditional scatter height signals. Depending on the orientation while going through the beam intercept you cannot resolve all aggregates by comparing the height. Optimally, one should use the area signals also for scatter AND fluorescence. 4. Electronics: I do not think that there are really bugs in the sorting electronics of any vendor (we would definitely have realized it before). If your machine can sort beads 100% pure there is no reason why it should not be able to do this with cells. So sample preparation and sorting conditions (sample/sorted fractions warm/cool, flow rate above threshold, sort rate, abort rate, sorted drops, coincidence) play the most important role. And by the way, don't forget to do some extensive washing before you reanalyse your sorted cells again on the very same machine you sorted them. The sample tubing can be a steady fountain of cells after three hours of sorting... Jens Fleischer
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