Bob Zucker wrote- >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. The area or integral of a pulse is always an accurate representation of the total signal, i.e., the intensity of fluorescence or scattering. When the height of the illuminating laser beam (i.e., the axis dimension in the direction along the flow stream) is substantially greater than the diameter of the cell or particle being analyzed, so that the whole particle is within the illuminated region during transit, the pulse height or peak is proportional to the area. When the illuminating beam height is close to or smaller than the cell or particle diameter, passage through the beam produces a "slit-scan"; the pulse height is now proportional to the density of fluorescent or scattering material in the cell, but not to the area. The height is thus sensitive to shape; the area is not, and that is why plots of peak vs. integral can be used to remove data from many (but usually not all) doublets in DNA analysis in instruments with small beam heights. The Ortho 50H had a very tightly focused beam, with a height supposedly around 5 um; the pulse integral provided accurate measurements of fluorescence, while the peak height did not. In B-D's earliest stream-in-air instruments, the focused laser spots were originally round; while later models and the FACScan, Calibur, etc. use elliptical beams, I believe that the heights are larger than what Ortho used. If the beam height is 20 um or more, pulse height will probably be proportional to integral and thus provide an equally accurate measurement for particles 10 um or less in diameter. In instruments which do not use high-resolution analog-to-digital conversion, both pulse height and integral are derived using analog electronic circuits. Peak height is detected by peak detector circuits, but there are two ways to get an integral. One can use an integrator circuit, or one can pass the pulse through a low-pass filter (electronic, not optical), which also effectively integrates it, and use a peak detector to get the peak height of the filtered pulse, which is proportional to the integral of the original pulse. When the pulses are log amplified, the second method should be used, because the desired quantity is the log of the integral, not the integral of the log. This problem goes away when you use digital electronics. B-D's DIVA electronics compute the pulse integral or area from 32 14-bit samples or "slices" of a pulse, and this is almost certainly more accurate than analog circuits would be; since log amps are dispensed with, their associated inaccuracies disappear from the measurement. Digital pulse processing is potentially less accurate than peak detection for capturing peak height, because the highest point of a pulse may come between samples. Thus, while one can get an integral accurate to better than 1 per cent from only 8 samples of a pulse (as Luminex does), more samples are needed to get an accurate peak height. I would imagine that accuracy on peak height measurements with 32 slices as used in the DIVA electronics is within a few per cent. For what it's worth, pulse width measurements are also less accurate than integral measurements with digital processing, because the pulse may rise above and/or fall below threshold between slices. So, I wouldn't worry about the accuracy of the digital measurements in the DIVA; the integral (analog or digital, provided it is taken properly) is always a good measurement. As far as your old data goes, one would not expect manufacturers to use heights instead of integrals if the heights were not accurate. This shows up in linearity; look at a fluorescence distribution from beads or from nucleic stained for DNA. If the modal value of the peak [the peak of the distribution, not the pulse] from bead doublets isn't within a channel or so of twice the value for the peak from singlets (and/or if the peak from G2/M cells and doublets isn't within a channel or so of twice the value for the peak from G0/G1 cells), there's a problem. And, if you can use the peak-vs.-integral method for doublet discrimination, you can assume that the integral is an accurate measurement of total intensity and that the peak is not. B-D is to be congratulated for implementing digital pulse processing at a high enough speed to be compatible with sorter operation. But I should point out that the high speed processing is essentially happening in a black box; I have only seen the software briefly (at Montpellier), and I can't comment on what it's like for the user (I'll try to get more hands-on experience with the commercial instruments in time for the 4th Edition). -Howard
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