Hi Mikayla,
The lysing procedure takes practice. We have our new people practice
with their own normal blood(no antibodies) until they get the hang of it.
Nucleated RBC do not lyse and some bloods from sickle cell anemia patients
will have to be lysed twice. A normal blood should have no CD45 negative
populations detectable at PB settings. So you could practice on a normal
until there is just a small amount of debris. Let me know which lysing
reagent you are using and I will give you specific tips.
Paula
At 04:40 PM 6/6/2001 -0400, 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.
>
>Does anyone have an idea what these cells may be? Could anither cell
>fraction with these scatter qualities potentially be large enough to make
>up over 50%? Has anyone had any experience with a pathological state of
>which this may be symptomatic or for which whole blood staining and lysis
>may yield erroneous results? Any information or advice would be
>appreciated. Thank you in advance.
>
>Sincerely,
>
>Mikayla Kob
><mailto:kob@cbr.med.harvard.edu>kob@cbr.med.harvard.edu
Paula Fukushima
Flow Cytometry
LP, NCI, NIH
10 Center Drive MSC-1500
Bethesda, Md. 20892-1500
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