> Mikayla Kob wrote: > > Dear cytometry community, > > We have been establishing a panel to phenotype lymphocytes from > clinical peripheral blood samples. We have been using a whole blood > staining and lysis procedure. The samples that I have received so far > have had an unusually large percentage of CD45- cells (over 50%) in > the lymphocyte gate as determined by forward and side scatter. These > cells are also CD3, 4, 8, and 19 negative, appear to be CD43 negative, > and are not NK cells. It may be helpful to know that these samples had > very poorly defined populations in the forward vs. side scatter plot. > I have not yet tried erythrocyte or platelet stains to rule out > aggregates or nucleated red blood cells, nor have I tried staining > isolated mononuclear cells. Lyse/No Wash and light scatter gating indeed can bring some serious trouble. Depending on the vendor not every lysing solution is useful for this. BD FACSLyse is definitely not working with this approach, this is why all their clinical assays use CD45 or CD3 on the threshold. Self-made ammonium chloride however should do the job. I haven't tried Beckman-Coulter or the old OrthoLyse, so I cannot comment on these. >From your description it looks like there are a lot of unlysed erythrocytes left, which will in FSC/SSC pretty much look like lymphocytes. Glycophorin-A should tell you the truth. I don't think that it is a question of nucleated erythrocytes (50% would be a real clinical case). Incomplete lysis sounds more familiar to me. BTW, can you resolve the neutrophils in the light scatter? Or are they virtually absent? Hope this helps Jens Fleischer
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