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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
Since there are no cells which are both helper and suppressor (in theory,
anyway), you might try to use this sample to set your compensation. But I grant
that it may be a little easier to have the single-stained cells.
4.CD3/CD16
5.CD3/CD19
This sample also might be used to set compensation, since a cell cannot be both
T- and B-cell (in theory). The point is, you should use a sample to set up
compensation which has no dual-stained cells in it.
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.
Sounds like somebody was butting heads. In fact, you might be able to
do quite a lot with these tubes. It is true that you must have
controls, but you may already have them in the three tubes above:
Simultest, CD$/CD8, and CD3/CD19. Whatever setting you use for PMT
voltages and compensation are going to be relative anyway, that is, you
are going to define what is negative (by running your Simultest), and
therefore the positives are, by definition, everything which is not
negative.
One additional control that might be useful (in addition to the
Simultest control, is a tube with unstained cells. This would let you
see if there is any fluorescence associated with the cells
(autofluorescence) which is not due to non-specific binding of the
fluorochromes.
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.
The particular brand or type of flow cytometer does not matter when it
comes to routine compensation settings. The most rigorous controls for
setting compensation on any two-color (or more) instrument is still
single-color controls.
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).
It would be nice to have the single-color controls to set up
compensation, but that is not always possible due to insufficient
numbers of cells, unavailability of antibody, etc. However, just
remember to set up your PMT voltages before you start to set
compensation, since the compensation amounts (percents) depend on the
particular high voltage settings of your fluorescence detectors.
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.
The technology of flow cytometry is the same for both clinical and
research issues. The differences arise mainly in the routine-ness of
the clinical work.
I am particularly interested in non-FACSCAN strategies.
The strategies for setting PMT voltages, compensations, and using
proper negative controls are the same for any brand of flow cytometer.
There may be differences between the different manufacturers, but
these are mostly cosmetic and convenience-oriented differences rather
than fundamental differences in the principles that make flow
cytometers work. In other words, if you've seen one flow cytometer,
you've seen 'em all!
Hope this helps. If not, either let me know how I can help further, or
ignore this message.
Steve Micko
Emory Univesity Hospital
Thanks in advance for your input!
Linda Weaver
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