Timothy, you are kind of between the proverbial rock and a hard place here. However, rest a little easier in knowing that the least important part of compensation is getting your "negatives" on-scale. You are likely miscompensating only a little bit. However, there is a relatively straightforward solution that will allow you to correctly compensate your sample no matter where your "negatives" are! First, realize that compensation does NOT require a positive and a negative sample. It only requires a bright (hopefully brightest that you have) sample and a "less-bright" sample. In general, the greater the separation between these two populations, the more accurate is your estimate of proper compensation. Nonetheless, it is perfectly accurate to compensate using a bright and a dim population. For example, if your brightest sample is so bright that you have to decrease the PMT voltage to keep it onscale (and please, keep it well onscale--the last half-decade of intensity scale is notoriously nonlinear), then simply stain another sample with a different conjugate (of the same dye) so that those cells are a few decades duller. It doesn't matter if the negatives are below scale--you are now just lining up the bright and the "not-so-bright" populations to compensate properly. If the bright and "not-so-bright" populations now have the same medians (after compensation) in the other channels, then your compensation is properly set! You will now find that your negatives, although being completely on the axis for the comp color, should have the same medians as your two populations. mr At 9:09 AM -0400 6/18/02, Timothy Singleton, M.D. wrote: >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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