Dear Sir, I have visited your interesting page for rules obtaining high quality DNA histograms analysis. Regarding the range of debris I set the gap inside the diploid Go/G1 peak (under the mode value). I think that the nuclei fragments have to be smaller than the mode value corresponding to the complete nuclei. What do you think about this? Ana Salas Cytometry Service University of Oviedo, Spain Donald Herbert -Tech Support escribió: > DNA Tetraploids: > > I might be able to provide some more insight into the current discussions > regarding DNA tetraploid tumors. The data behind my comments was recently > published in the reference sited below: > > Bagwell CB, Clark GM, Spyratos F, Chassevent A, Bendahl P-O, Stål O, > Killander D, Jourdan ML, Romain S, Hunsberger B, and Baldetorp B: > Optimizing Flow Cytometric DNA Ploidy and S-Phase Fraction as Independent > Prognostic Markers for Node-Negative Breast Cancer Specimens, Communications > in Clinical Cytometry, Vol 46:3, pps 121-135, 2001. > > I must also add that the data was from node-negative breast cancer patients > and although the conclusions may be relevant to other tumor systems, it is > dangerous to overly generalize. > > The first questions to ask is a practical one: how do we define a DNA > tetraploid histogram? This question is harder to answer than it initially > appears. There are two basic problems in assessing whether a histogram is > from a DNA tetraploid tumor or not. The first question to answer is > whether the peak at approximately the 4C location is a diploid G2M or is it > another DNA ploidy population's G0G1. Many investigators have tried to > tackle this question by creating arbitrary cutoff values for the G2M > percentage. For example, if the 4C peak represents more than, say, 20% of > the cells between 2C and 4C, it is assumed to be another G0G1 peak. A > better technique is to look for other evidence that it is a separate > population such as a S-phase or a valid G2M downstream from the 4C location. > One of the efforts we made in publishing the above article was to come up > with a set of rules that would guide operators to making the same conclusion > of whether this peak was an additional peak or not. Of all the strategies > tested, the one that worked the best was to look at the relative heights of > the 6C peak versus the 8C peak. If the 8C peak appeared to be greater or > equal than the 6C peak, the peak at approximately the 4C location was deemed > to represent the G0G1 of an additional cell cycle. For those interested in > these rules, we have posted them on our web site, > http://www.vsh.com/products/mflttraining/mflttrainhm.htm. > > The above paragraph deals with the issue of whether the 4C peak is a G2M or > an additional G0G1 peak, but does not distinguish between a near DNA > tetraploid and a "true" DNA tetraploid ploidy histogram. At least for the > above node-negative breast cancer study, we found this distinction was of > critical prognostic importance. Patients with "true" DNA tetraploid > histograms had a prognosis indistinguishable from patients with DNA diploid > histograms; whereas, patients with near DNA tetraploid histograms had a > prognosis indistinguishable from patients with DNA aneuploid histograms. At > least some of the controversy concerning the prognostic significance of DNA > ploidy probably can be traced to this issue. Part of the complexity in > separating "true" DNA tetraploids from DNA aneuploids with DI's close to 2 > is that our flow cytometers and staining procedures are generally not > perfectly linear. We don't always find a diploid G2M at exactly twice the > diploid G0G1. For a given flow cytometer running a given staining > procedure, this ratio can typically range from around 1.94 to 2.02. In the > above study, we found that we could define DNA tetraploid DI range using > population statistics. The details of the method can be found in the > reference. It's also now possible to calculate the underlying linearity of > a DNA histogram through a modeling technique and that should also allow one > to better define a DNA tetraploid DI range. > > There were other of other issues addressed in the above paper, but the DNA > tetraploid question was one of the more important ones. I hope this > discussion answers more questions than it raises. > > Bruce > > C. Bruce Bagwell MD., Ph.D. > President > Verity Software House, Inc. > PO Box 247 > Topsham, ME 04086 > > Tel: (207) 729-6767 x102 > FAX: (207) 729-5443 > EMAIL: cbb@vsh.com
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