Artur Plett writes: > >This is another question about ambiguity and the question itself may be >ambiguous: > What can one do with a stain in which the whole population shifts into >a higher fluorescence and no discernible separation of a subpopulation >ocurrs? (The population leaves a gap in the left hand side) > Say, for example in the case of activation markers CD69 and CD25. After >activation all the cells are on their way of expressing it, but after >short term incubation only a percentage will shift away from the >"background fluorescence", "isotype fluorescence" or "negative" >quadrant. > Can the change in MFI for the whole population be considered relevant >(if indeed they are all positive?), or is the only right way to look at >this by cutting off the "negative" and looking at the MFI of the >"positive" population? > > Could a possible explanation be that cells who were initially in the >low "negative" population have actually shifted into a higher "positive" >fluorescence, but this shift is being masked by the background >fluorescence? > When cells acquire or upregulate an antigen in the same compartment in which they are measured, as is the case in in vitro activation, it is typical to see distribution shifts rather than "positives" and "negatives". From the statistical point of view, it may be more legitimate to compare shifts in median fluorescence intensity (the median being a more robust statistic than the mean for distributions which deviate substantially from normal) than to attempt to define "positive" and "negative". The latter terms make sense when you look at cells which acquire or modulate antigen outside the compartment in which they are observed, as happens with peripheral T-cells. In they thymus, they acquire both CD4 and CD8; one of these antigens is lost before the cells migrate into peripheral blood, and one can therefore observe clear positives and negatives in distributions of CD4 and CD8. -Howard
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