Several months ago, Richard McFarland from my laboratory posted a question about cell carryover, which we had been seeing intermittently when running our FACSCalibur with an autoloader. We received many ideas and suggestions which led to some procedural changes, and I'd like to review the suggestions and our experience with follow-up. The responses suggested that this was a common problem, seen not just with the loader but also when running manually. There was a difference of opinion on whether this problem was also seen with Coulter instruments. Several people suggested that this was due to sample sticking to the outside of the SIP, and then being deposited in the next tube. We think this is likely. Since we have been more attentive to cleaning the SIP, we see carryover only rarely now. Though we don't have time to run a tube of DI water between each tube, we do run one between each case. And when we have a very limited specimen which would not allow for a re-stain, we take extra care to run it after a DI water tube or wipe the SIP. It was also suggested that a lower cell concentration would lead to less carryover. While this is true, it would also lead to longer acquisition times, so we were fortunately able to avoid doing this. It was also suggested that tube order would affect how carryover events were interpreted. We thought this was an excellent idea. So, in each of our diagnostic panels, we re-ordered the tubes to put the tube with CD45 (maximum percentage of stained cells) right before the isotypic control tube. Therefore, if carry-over starts to occur, it will be detected quickly. We also put the most sensitive tube in each panel at the beginning, right after the DI water tube, to avoid contamination there. So the bottom line is that since we've been more attentive to cleaning the SIP, the previously frequent problem with carryover has now become rare. Thanks again for all your suggestions. Deborah B. Aquino, M.D. Director, Clinical Flow Cytometry and Cellular Immunology U.T. Southwestern Medical Center
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