intracellular light chains

Maryalice Stetler-Stevenson stetler at box-s.nih.gov
Tue Feb 18 07:18:01 EST 1997


We are trying intracellular light chain staining and not getting great
results. One problem is that we don't get many plasma cell malignancies and
we don't have a good positive control. In a report in Communications in
Clinical Cytometry, Witzig et al incubated cells with anti CD45 and anti
CD38, washed, incubated with a lysolecithin solution washed and incubated
with anti light chain antibody. We are trying proprietary lyse and
permeabilize methods to avoid the home brew aspects of the lysolecithin. We
have had negative results until now. On Friday we had a case in which the
bone marrow was packed with plasma cells. The ficolled cells were stained
for CD38 PE and CD45 PerCP, followed by Caltag's Fix and Perm reagent and
then Tago's anti-light chain reagents.  Cells were also stained without
permeabilization for a panel of antibodies. Without permeabilization we had
32% CD45 positive but with permeabilization we had 72% CD45positive. The
CD45 negative population was CD38 and light chain negative. I will get
results this afternoon on immunoperoxidase studies of the bone marrow
biopsy but I would have liked to have been comfortable with the flow
results. Obviously the flow method didn't work. Has anyone already been
down this road? I am looking for advice from the experienced.

Can any one suggest a good positive control (eg cell line) for the procedure?

What about permeabilization techniques?

Light chain reagents?

        We would appreciate any help we can get.

        Maryalice

Maryalice Stetler-Stevenson
Director Flow Cytometry Unit
Laboratory of Pathology, NCI, NIH





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