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______________________________ Reply Separator _________________________________
Subject: Re: Friday night flow special
Author: steven_micko@email.eushc.ORG at INTERNET
Date: 17/05/96 00:17
Does anyone have experience with leaving paraformaldehyde fixed stained
bone marrow cells for more than 18 hours? We got a specimen late on friday
from a patient with small non-cleaved lymphoma treated aggressively, now
for post treatment evaluation. We stained and fixed with 1%
paraformaldehyde. Unfortunately, due to a number of problems, acquisition
could not be performed the following day. In fact, acquisition was delayed
until Monday morning. The scatter looks good but there are basically no
B-cells (89% T-cells, 10% NK cells in lymphoid CD45/SSC gate). Previous
specimens also had a profound B-cell lymphopenia. Does anyone know if
B-cells are lost with time in paraformaldehyde?
Maryalice
Maryalice Stetler-Stevenson
Director Flow Cytometry Unit
Laboratory of Pathology, NCI, NIH
We try very hard to run the cells (lymphomas, leukemias, breast
cancers) the same day they were stained and fixed. However, because of
the times they are delivered to us, we sometimes have to stain and fix
around 5 or 6 p.m. and then acquire on the FACScan the next morning.
Under these conditions, we see no significant increase in background
(auto-?) fluorescence in the controls. When specimens are delivered
late on a Friday and acquired the following Monday (Tuesday in the
case of a holiday) we begin to see significant background fluorescence
on the controls. This fluorescence increases when the cells sit in the
refrigerator longer, say a week or two. This throws doubt upon the
validity of the percent positive calculations. Our conclusion from
this experience is to run the cells *as soon as possible* after
staining and fixation, but no longer than *one day* later, even if it
incurs overtime at the hospital. Also, we encourage our sources to get
samples to us no later than 3 p.m.
Steve Micko
Emory University Hospital
Atlanta, GA
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