I thought it might be useful to add a little more information on this subject. I presented a poster at the last ISAC meeting that discussed several of the parameters often associated with determining histogram quality. One of these parameters was the number of events, or to be more exact the number of events in the cycling population. I found that it was important to maintain an average of 200 events per channel over the cycling population to obtain good S-phase results. The DNA Consensus Conference Guidelines (1) indicated that 10,000 events (excluding debris and aggregates) were required for proper S-phase analysis. According to our work, this value is correct but only as long as the number of channels between the first G0G1 and the last G2M is 50 or less. This would only be true for DNA Diploid samples with the DNA Diploid G0G1 set at channel 50 (normally only used when collecting at 256 channel resolution). My experience is that operators frequently use "10,000" as a general guide for any collection resolution. Instead users should adjust the number of events collected to insure that a minimum of 200 events per channel in the cell cycle when analyzed . For example, when collecting at 1024 channels with the DNA Diploid G0G1 mean set at channel 200 you would need a minimum of 40,000 events just in the cell cycle (remember to exclude the debris and aggregate events). Based on extensive DNA Cell Cycle analysis and correlating the results with known outcome, we have found that in the majority of cases, 256 channels are sufficient. However collecting at 1024 channel resolution and analyzing using a 256 channel histogram has a lot of merit. You can keep the number of events needed lower and while being able to go back to the higher resolution if needed. I have attached some of the panels of my poster in a zipped PowerPoint file. If you wish more information or a copy of the full poster I would be happy to send it to you. Best regards Don Donald J. Herbert Technical Support Manager Verity Software House, Inc. PO Box 247 45A Augusta Road Topsham, ME, USA 04086 Phone: (207) 729-6767 ext.190 Fax: (207) 729-5443 email: tech@vsh.com web: www.vsh.com 1. T. Vincent Shankey, Peter S. Rabinovitch, Bruce Bagwell, Kenneth D. Bauer, Ricardo E. Duque, David Hedley, Brian H. Mayall, and Leon Wheeless: Guidelines for Implementation of Clinical DNA Cytometry. Cytometry 14:442-447, 1993
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