Bob, Our first cytometer was an EPICS 753, and like your Ortho, all our measured signals were "integral" (i.e., "area"). The 753 let you save both integral and peak height signals from the same detector; I did this a few times and not suprisingly there was little difference for routine immunophenotyping - like CD8 and CD4 - where there is a large difference in fluorescence intensity between the negatives and positives. I think you "hit the nail on the head" when you suggest that there may be a difference between area and height measurements on signals in which a small change is expected, like DNA content/cell cycle where normally only a two-fold difference is expected. However, I think you will only see a difference if the system you are measuring has little "noise". By noise I mean that the %CV of fluorescence intensity for a given population is low, as in DNA content and microspheres. Cell-surface measurements are just too noisy in this context. The autofluorescence typically covers one decade on a log scale (a 10-fold variance) which means that in order to resolve a positive peak from autofluorescence the positives need to be at least 10-times brighter. In this system, if the positives are only 5 times brighter than autofluorescence, it will not make a difference if the cytometer measures area or height signals. (The solution, of course, as has been said many times before, is to use a better antibody/fluorochrome.) Just off the top of my head, I think the G2/G1 ratio for most intact cells for height signals is about 1.7 and for area signals is about 1.9 for our FACScans (and a perfect 2.0 when using the DNA QC kit). So, to put numbers with the above example, it doesn't matter whether the positives are 4.25-times or 4.75-times brighter if the limit for detection is a 10-fold difference. Again, I think you are on the right track when you bring up "linear measurements". It will be interesting to see if there is a difference between indo-1 [Ca]i measurements between area ratios and height ratios. The pulse processing boards in our Vantage produce a ratio signal, but it is the ratio of height signals. I may have some old files in which I saved this hardware ratio signal in addition to the blue and violet area signals, but I never had a chance to produce a software ratio of the area signals to compare to the hardware ratio. I think I tried linear FL1-Area measurements of DCF for oxidative burst in neutrophils, but later resorted to log amplification. For the few microsphere assays I've done, I think I saved both height and area measurements, but only looked at area. There are probably other situations in which I've looked at linear area signals. Bottom line: I think that in most cases the noise from biological variation wipes out any noticeable difference between area and height measurements, and those cases were a difference exists are already known about. Eric >At a local flow cytometry users group ( RTCA) on Feb 22 2001, we heard a >about the BD DIVA digital electronics. It was impressive and like all new >developments in flow cytometry it is a welcomed addition. >BD is now measuring area on all pulses instead of height that is normally >used on previously versions of BD equipment. For you BD users, there is >one board in the machines currently that can yield area measurements which >are required for DNA measurements using Doublet discrimination. Now I >remember that our old Ortho 50H yielded area measurements on all signals >and this is the way we always ran our machine. We reluctantly gave up this >measurement to use the User friendly BD FacsCalibur with their CellQuest >program. > >My questions are the following: >How is our Flow data going to change by using area instead of peak height >measurements >It should be more accurate with area but how bad is the height measurement >that we have obtained for many many years.. >This Question was raised at the meeting but it was not adequately answered >by anyone in the audience. How bad is height measurements?. Is it only bad >( inaccurate) for linear measurements like DNA??? Let's discuss this. I >think it is important. >Bob > > > >Robert M. Zucker, PhD >U.S. Environmental Protection Agency >MD 72 >National Health and Environmental Effects Research Laboratory >Research Triangle Park, North Carolina, 27711 >Tel: 919-541-1585; fax 919-541-4017 >e-mail: zucker.robert@epa.gov /\/\/\_ Eric Van Buren, aa9080@wayne.edu \ \ \ Karmanos Cancer Institute and Immunology & Microbiology \_^_/ Wayne State University, Detroit, Michigan, USA
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