In Dr. Roederer's web tutorial, which I highly recommend, he mentions that it is important to have all of the negative control off the axis when setting compensation. I am having difficulty accomplishing this goal. For example, when compensating with single color CD8 PE on normal blood, some of the negative control lymphocyte fluorescence (43% of the lymphocytes, in fact) is still on the axis, even when the positive peak is almost off scale (between the third and fourth log). The peak fluorescence for the negative control falls within the first log, but there appears to be a long tail that is offscale and includes 43% of the gated lymphocytes (using forward and side scatter gating). I am using the standard four log amplifier on a Coulter XL and post-acquisition color compensation with WinList for four color analysis: FITC, PE, PE-Cy5 and PE-Cy7. Am I doing something wrong? Suggestions? Incidentally, the scattergrams look okay on actual cases. Does that mean that it's not perfect, but close enough? Tim Singleton, MD Director, Flow Cytometry Beaumont Hospital Royal Oak, MI >>> Mario Roederer <roederer@drmr.com> 06/14/02 05:42PM >>> (OK, who's surprised that it took me this long to weigh in?) David wrote that FCS Express allows you to change compensation values with sliders "to see what happens." In fact, most current interfaces (like doing compensation on the instrument itself) allow this. This "feature" is historic in nature: the fact that instruments have let users "manually" adjust compensation since the beginning of (FACS) time has caused most people to essentially demand this "feature" in compensation software. I think it's a very bad idea. As I proved in a paper published in November's Cytometry, it is IMPOSSIBLE for people to accurately compensate based on visual estimations (like graphical displays, dot plots, etc.). In other words, unless you are relying on statistics and ignoring the graphic, you will not properly compensate by any manual (slider or other) approach. Let me back off a little and reassure you that for most applications using FITC, PE, and perhaps a 3rd color, we can come pretty close--close enough that we can consider it right. However, as soon as you start dealing with more than 3-4 colors, and whenever you are dealing with the far red colors (like Cy7PE, Cy7APC, and so forth), it is no longer possible to properly compensate visually. It must be done based on statistics (e.g., median fluorescence of bright vs. dim populations). In fact, the best approach is to let the software calculate the compensation for you--no manual interface at all. Any manual interface (letting users adjust) will lead to incorrect compensation. Now, people will tell me that they need to "tweak" the compensation because it doesn't look right. The reason it doesn't look "right" is because we don't actually know what "right" looks like! For a better explanation of this, see my web pages (particularly, <http://www.drmr.com/compensation/>, click on "Quiz") or see the manuscript in November's Cytometry, or my letter to the editor in Clinical Cytometry (Nov or Dec). Bottom line: if you are using deep-red fluorescences, or doing more than 3-color compensation, then unless you use an automated approach to calculating compensation, then you can't get the compensation exactly right--this has nothing to do with ability or intelligence, it is an result of our inability to correctly estimate central tendencies of log-transformed data! (Again, all explained in my paper in November, but not explained in the web pages). Despite users' requests, I have strongly pushed software manufacturers NOT to put manual compensation interfaces in their programs, because I'd rather users complain than for users to have incorrectly-compensated data. Compensation is a very complex subject--surprisingly so--and it is far beyond the average user to understand all of the ramifications of manual compensation setting. I published my first paper on compensation in 1986, and I'm still learning & publishing on the topic. Of course, some of you may take that to mean that I'm pretty ... um ... thick-headed... but I'd rather it be taken to mean that if even an expert won't manually compensate his data, perhaps we should all rely on automated compensation algorithms and just "believe the computer." mr
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