controlling autofluorescence red cells/whole blood

From: Steve Woodard (steve.woodard@ibb.gatech.edu)
Date: Tue Feb 08 2000 - 15:06:32 EST


Does anyone have experience in performing analysis of red cells in whole
blood. I am encountering problems with the amount of autofluorescence
associated with whole blood samples. When I look at FL1 VS FL2  plots of
"Cells Only",  the majority of cells are in the lower left quadrant. There
are some cells that are found at a 45 degree angle to the lower left
quadrant. It reminds me of a "comet". The problem is that the
autofluorescence is interfering with stained cells that are double
positives.  In order to complicate matters, we are looking for 50 events
out of 50,000 in the whole blood ( 0.1%). How is this type of problem
usually solved ? I tried using trypan blue but there was only a minor
reduction in autofluorescence. I came across crystal violet as a quencher
but fixation of the whole blood is not an option. 

thanks in advance for your responses,

Steve



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