Dear All, HELP!!! This is probably a very basic problem, but I hope someone out there can offer me some advice. I am studying minimal residual disease in acute leukaemias using triple colour flow cytometry and a stain and lyse technique. My problem is with lysing my specimens adequately, in that several specimens have not lysed well recently resulting in them being unanalysable on the flow. This has happened once or twice previously and I put it down to something I was doing wrong in my early days in flow cytometry! However, over the past 2 days I have had problems with ALL of my specimens. I have not changed my lysing reagent or technique in any way, and the problem occurs with both commercial and home-made lysing reagent. It is also occurring with both peripheral blood and bone marrow specimens. I have several questions: 1. Has anyone had similar experience, and if so how have they overcome it? 2. Any tips or pointers to getting these stubborn cells to lyse? 3. I often have specimens with low cell counts, and so usually add a larger volume of the specimen to the antibodies. This obviously means that there are more red cells present to lyse. Is it okay to just add a greater volume of lysing reagent? If not, how does one overcome the problem of low counts? I would be most grateful for any advice anyone can offer. Speedy advice would also be appreciated as I have more specimens tomorrow!!. Thanks in anticipation. Sarah Lawson (isaac.lawson@btinternet.com)
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