I'd like to weigh in on this one because I disagree with most of what has been said. Correct me if I'm wrong. Intuitively, I would expect the variance to increase when reporting measurements of bound antibody or number of molecules precisely because you subtract the background. We do not usually get something-for-nothing; when maKing two measurements to obtain one calculated value, presumably you have two independent sources of error, so the calculated value should have more error associated with it. I did some quick modelling to see if my intuition might be correst. By randomly sampling from two normal distributions with equal CV's and then subtracting the sampled value of distribution with the lesser mean from a sampled value from the distribution with the greater mean, a large sample of such calculated values was created and the mean, CV calculated of this distribution. The CV of the subtracted value distribution decreases and approaches that of the two parent distributions as a function of the distance between the distribution means. The decrease fits an equation something like CV = a * separation distance^b, therefore, when the two means are close, the error in making the calculation is substantial, supporting Calmun's observations and my intuition. However, if you choose to not make the calculation, but instead report out the number of bound antibodies or molecules of antigen as the fluorescence of the positively stained sample, you are choosing precision over accuracy (presumably the reason one would like things in molecules is to get closer to the truth). To me, it is more important to make more repeated measures, get the CV down to something you can live with, and shoot for accuracy. Why would anyone bother to use beads that convert fluorescence to numbers of bound antibodies when (say) half the measurement is not antibodies? You would be better off sticking to fluorescence in AU. in table form: background equivalent to 10 molecules, and variable #of bound antibodies, background 10 10 10 10 molecules 10 20 30 40 total 20 30 40 50 if you report out that there are 20 molecules when there are really 10, you are in error by 100%. If you measure the background (and I think a perfectly good way to do that is with an isotype control - if you can't use a positively stained negative cell equal in size etc. to your positive population) and subtract it, then you report the truth, 10, 20, 30, 40 molecules - of course subject to errors of making measurements. If you are using the antibody beads solely to get better interlaboratory and inter-machine consistency, and you do not subtract the background, then the instrumental variation that you would like to eliminate is still there. I believe you would have accounted for staining variation, though. In this case, it would be misleading to report the answer in molecules or bound antibody, and in my opinion, that practise should be avoided. James W. Jacobberger, PhD Associate Professor Director Flow Cytometry Case Western Reserve Univ Cancer Research Center & Dept. Genetics Ph. 216-368-4645 -----Original Message----- From: Steve Perfetto [mailto:sperfetto@pasteur.hjf.org] Sent: Friday, January 07, 2000 2:34 PM To: cyto-inbox Subject: Re: MESF and correcting for isotype matched controls Calman, I absolutely agree. The same type of bias can be introduced in subtracting out instrument noise since this is essentially instrument specific it would allow for huge interlaboratory variation. For this reason we always report the raw values without subtraction based on the Quantum Simply Cellular system. However, if a low cell control can be standardized to reference the low end as well as a high end cell control than these values should be valid for subtraction. Currently such a control does not exist. Stephen P. Perfetto, MS.,MT. (ASCP) Walter Reed Army Institute of Research Department of Molecular Diagnostics and Pathogenesis 1600 East Gude Drive Rockville, MD. 20850 ____________________________________________________________________________ ___ Subject: MESF and correcting for isotype matched controls From: Calman Prussin <CPRUSSIN@niaid.nih.gov> at Internet_Gateway Date: 1/6/00 11:42 AM We are quantitating cell associated antigens using standardized beads and then generating an MESF from the standard curve generated from the beads. The question has come up whether or not to "correct" the MESF values obtained from the specific antibody by subtracting the MESF values obtained for the isotype matched controls. The problem with correcting the values is that it has little effect on the samples with high density of Ag, but has a large effect on those with low expression. As such, if we run replicate samples on the low expressing samples we find a larger amount of variation in the corrected MESFs. My bias is not to use it, as it seems to introduce more "noise" into the system. Your thoughts? Thanks! > _______________________ > Calman Prussin > Laboratory of Allergic Diseases > NIAID/ National Institutes of Health > Calman, I, personally, agree entirely with your logic. We wrestled with the same issue when we were quantifying TCR's on stimulated T cells (which has now fallen out of popularity). I always argued against normalizing against isotype quantitative values (for the reason you stated), and I used to use a silly analogy in the attempt to make this logic inescapable: If you and I were sitting in the stands at Veterans Stadium at night when the lights were off and you had your flashlight on, then I accidently turned mine on, the increase in the intensity of light would be two-fold. In this case, you and I are the background, and my turning on my flashlight represents a (two-fold) fluctuation in background due to noise. Now, if you again had only your flashlight on and we were joined by 998 fans, each with their flashlights on, and then I turned my flashlight on, does the intensity of light which fills the stadium go up two-fold? Of course not. It goes up by 1/1000th of the intensity before I turned my light on. But the former is what one would be implying by normalizing samples with high fluoresence/MESF to the fluoresence/MESF of background, and thus one introduces the potential for tremendous artifact. Now, as you imply, this analogy/argument begins to break down when the antigen density decreases (say to 3- to 5-fold above background. My opinion is that is that if you can show that the variance in the MESF of the specific stain from sample-to-sample is significantly less than the variance of the isotype control values, then it would be most appropriate to NOT normalize against isotype. These rationalizations make me feel better, I hope they help you also. AW Andrew D. Wells, Ph.D. University of Pennsylvania Department of Medicine 728 Clinical Research Building 415 Curie Boulevard Philadelphia, PA 19104 (215) 573-1840 (office) (215) 898-1951 (lab) (215) 573-2880 (FAX) adwells@mail.med.upenn.edu Calman, I've spoken out against using isotype "matched" controls many times, and won't go into that again, except to say, don't use them for antigen density measurements! Instead, use a parallel aliquot of cells that are stained with everything EXCEPT for the color on which you are doing the measurement (and leave that one unstained). It is very important to include the other antibodies, not just isotypes for them--to fully correct for problems with compensation, spectral shifts, etc. Eventually, you should subtract the background value. But this assumes that you are only interested in a single value, and single values are not good for representing distributions (unless the distribution happens to be normal, or, more precisely, log-normal). Our approach was to calculate a series of values, including not only the median (which is far better than the mean when you are using log-scale), but also the 10th, 25th, 75th, and 90th percentiles of fluorescence. This allowed us to build up a picture of the distribution of fluorescences. Correcting these values by background, however, is not simple. i.e., from the 10th percentile, do you subtract the median of the unstained? the 10th percentile of the unstained? Once you start considering questions like this, you realize that subtracting the MESF of the control from the MESF of the sample has significant pitfalls of its own! Our approach now is to use the calibration platform in FlowJo--this platform allows you to rescale any fluorescence channel based on a standard fluorescence measurement (either by fitting to a bead set or just by entering the appropriate conversion value). At this point, all graphs & statistics are shown in terms of absolute molecules rather than relative fluorescence. Thereby you can easily calculate the above statistics (or show histograms etc) in units which are meaningful for your final desired answer, and you can then decide what type of analysis is appropriate. So, while the simplistic answer to your question is "Yes, subtract background values", there is an enormous complexity underlying the process which really requires considerable more thought. mr
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