Hi, Yes, we have much experience with the system you describe. It took us a while to figure out how to deal with it, but it works well for us now. The issues for us were: 1. Each ECD antibody we have used (Coulter) has a different amount of PE leakage, so the amount of compensation required is different for each ECD antibody. CD8 has had the most leakage (which we initially tried), and results in significant overcompensation of the PE signal when other ECD antibodies are used. CD45 has tended to be next, followed by CD3. CD19 has had the least leakage. CD45, CD3 and CD19 are close enough that one can approximate the comps using any one of them for compensation. Ideally one needs to have a separate compensation matrix set up for each ECD antibody used. 2. Use median values, not arithmetic or geometric means, for compensation. The fluorescence distributions of the population, in particular the negative population, is not normally distributed and incorrect compensation will result. Unfortunately, the Coulter automatic compensation software uses a mean for its calculations and results in suboptimal compensation, most noticeable as marked PE overcompensation. If one attempts to use medians, often the median for negative populations in the higher wavelength PMTs lies very close to the axis and makes accurate determination of the comps difficult. The best solution for us has been to use off-line software compensation with WinList. The resulting comps are consistent and reliable, and the amount of time required to perform the compensation is dramatically reduced. However, one does have to run the samples through the instrument uncompensated. Since we do all our data analysis off-line anyway, this is not a problem. 3. Do not expect to use quadstats to analyze the data. Due to the significant spectral overlap of ECD with both PE and PE-Cy5, the true negative does not lie parallel with the axis (unlike orthogonal quadstats). This effect can be quite marked and can best be seen on histograms displaying PE vs. ECD and ECD vs. PC5. Hope this helps, Brent Wood MD PhD Associate Director of Hematology Laboratory University of Washington Medical Center EM: woodbl@u.washington.edu Phone: (206) 548-6199 Fax: (206) 548-6189 ---------- >From: "robert gniadecki" <rgniadecki@hotmail.com> >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >Subject: 4 color staining and Argon laser >Date: Fri, Jan 22, 1999, 6:57 AM > > >Hello! >We are planning immunophenotyping with 4 colours: FITC, PE, Pe-Texas red >(ECD) and Pe-Cy5. We have however experienced some severe problems with >compensation between PE and PE-TR. > >Could you share your experience with me? Is that fluorochrome >combination possible to use? > >We have 4 photomultipliers and a 488 nm argon laser. >Thanks, > >Robert Gniadecki >Bispebjerg hospital >Copenhagen, Denmark > >______________________________________________________ >Get Your Private, Free Email at http://www.hotmail.com >
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