Steve- Bruce H. Davis, M.D. Maine Medical Center Research Institute 125 John Roberts Rd., Suite #8 South Portland, Maine 04106 207-761-9090 ext. 294 FAX: 207-761-2130 Email: davisb@mail.mmc.org >>> Steve Woodard <steve.woodard@ibb.gatech.edu> 02/08/00 03:06PM >>> 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 viI imagine what you are seeing (assuming you are gating on RBCs and are not including leukocytes) is the reticulocyte population. They have higher autofluoresence, which I assume is due to the higher RNA content and hence Trypan Blue will not work. I suggest getting a birghter signal or alternative marker on your population of interest as the only solution. Good luck. olet as a quencher but fixation of the whole blood is not an option. thanks in advance for your responses, Steve
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