A new client recently submitted to me the following samples:
Cultured human lymphocytes (stimulation unknown; I am not always fully
informed of these experiments, nor do I have much input), aliquotted
and stained with:
1.BD Simultest isotype control (FITC/PE)
2.CD45/CD14
3. CD4/CD8
4.CD3/CD16
5.CD3/CD19
I have a Coulter Elite, no comp beads or autocomp. I requested
single-color controls for at least CD4 and CD8 so that I could adjust
compensation. This request was not honored. My response was, I could
do nothing with these samples, but spent two hours of my time
demonstrating to him the compensation issues at hand, and that, in
order to obtain reliable data, I must have controls.
Unlike a lot of you CD marker veterans, I have not had a lot of
experience with multi- CD marker panels, except in flow school. Most
of my work has been with cell lines! I do realize the need for single
color controls when using something other than a Facscan. Given the
fact that the cells are cultured, they bear little resemblance to an
in vivo patient.
I am proposing the following; give me some feedback!
1. That he narrow his markers down to those that really matter in a
culture situation, i.e, non-monocyte, non-macrophage markers, since
these are lost, anyway.
2. That he at least provide single-color controls for CD4 (FITC) and
CD8 (PE) for cross-channel comp. adjustment purposes. (Will this be
sufficient to get me in the ballpark? I do notice that CD8 is
predominating and is very bright in these samples, so other panels
must be adjusted accordingly).
3. It may be useful for me to have "normal, resting lymphocyte "
control cells, such as Coulter Cytotrols, stained in parallel. Is
this overkill, or useful information?
I am approaching this from the standpoint of a bench research person,
not a clinical person, but (perhaps) similar situations arise when
some of you are examining blood from patients with leukemias or
lymphomas. I am particularly interested in non-FACSCAN strategies.
Thanks in advance for your input!
Linda Weaver