From: Alan Salkind (nepsy@fiona.umsmed.edu)
Date: Tue Mar 21 1995 - 15:01:48 EST
I am trying to analyze histograms which depict the expression of an intracellular antigen in lymphocytes. Although there is a demarcation between the overlaid histograms of the positive cells and the negative control, there is considerable overlap between the two populations, making analysis difficult. I tried subtracting the negative control histogram from the "positive" histogram using the histogram tools application in Cell Quest. I then divided (again using the histogram tools application) the number of positive cells generated from this "new" histogram by the total number of cells in the positive sample. I found this manipulation produced misleading results. Is it reasonable to place a marker at the intersection of the positive and negative histograms and consider events to the right of the marker as positive? I am concerned that this may indicate more positive cells than there really are. On the other hand, setting the marker based on 1 - 2% background fluorescence excludes the dim staining cells. I know this issue has been discussed before on this BB, but I would welcome any new (or old) thoughts on the subject. Thank you. ............................................................................ .... Alan R. Salkind, M.D. Division of Infectious Diseases, University of Mississippi Medical Center E-Mail: nepsy@fiona.umsmed.edu Tel: 601-984-5560 Fax: 601-984-5565 ................................................................................ ARS
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