From: Darren Hickerson (DHICKERSON@brody.med.ecu.edu)
Date: Tue Feb 10 1998 - 16:15:27 EST
Several have written in response to the latest inquiries conerning how to properly ratio data. I still have unanswered questions, though. I don't have all the letters in one place, so I can't quote exactly who said what (sorry). I do greatly appreciate the feedback, so please pardon my humble ignorance on this topic. One comment stated that resolution was too poor in the upper decades of the log axis scale since numbers were so far apart, so using these values to calculate ratios was less accurate. Another writer stated the mathematical problems involved in dividing true logarithmic values. Here's my question from actual data. I run a sample and get mean 2000 on FL3, and mean 50 on FL1. After treatment, I get mean 200 on FL3 and mean 1000 on FL1. This is all visualized on the "log" axes. If I divide the numerical values given for the means, I get ratios of 0.025 pre and 5.0 post, for a fold change in ratio of 200. First question: do I invoke the mathematics of dividing log values when what I'm really dividing are "linear" values presented on a log scale? Is the above division appropriate? Second question: The above plots show a tight, homogenous population moving from lower right to upper left on the FL1(Y) v FL3(X) plot. The same data acquired on the evenly spaced 1024 channel scale would have a population scattered all over the place rather than a tight population, and the movement would be a smear rather than a precise relocation of the distinct population. Furthermore, some particles would be off scale to one end of the spectrum pre-tx, and then off scale on the opposite side post treatment. I guess the question is, are the events really any "closer" together if the population is scattered all over the non-compressed axis (as many as 1000 channels apart), as opposed to the log axis, where the data may be numerically further apart, but much more visually concise? Furthermore, even if greater numerical spacing occurs, is that a real problem considering the number of events that will be squashed against the axes, giving false replication of 0's (or 1's?) and 1024's? Third question: even if the "log" scale is not completely accurate in giving true relative linear fluorescence values (i.e., 10^2 may or may not really be 10 times brighter than 10^1), it is pretty close, is it not? After all, some companies are selling fluorescence quantification reagents under the idea that the numbers are accurate representations of actual relative fluorescence intensities. If the log value is considered somewhat accurate (relatively), and data is collected with this assumption, why is it acceptable to calculate ratios from a falsely overlaid 1024 channel scale? If something gets 10 times brighter, it should be recorded as such, and changes in ratio will reflect this. If the 10 fold change is converted to 1024 scale, the change will be greatly underestimated. Is this acceptable? If so, why? Any expert advice, or references to publications will be greatly appreciated. Thanks, Darren Hickerson dhickerson@brody.med.ecu.edu Core Flow Cytometry Facility, Brody 4W37 Department of Microbiology and Immunology East Carolina University School of Medicine Greenville, NC 27858 Phone: (919) 816-2799 Fax: (919) 816-5018
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