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> Does one need to run a positive (and a negative?) control with TdT? We used
> to have a + and a - control for slide based TdT. Somehow I donot think we
> need those controls, now that we have switched to flow TdT.
Bakul:
I think controls are important for flow TdT for a number of reasons:
Depending on how you are permeabilizing the cells, granular cells (eg
early grans) may be non-specifically FITC avid-this does vary between
antibodies and so is not necessarily detected merely by using a
non-specific-FITC reagent; if your permeabilization method does not
retain side scatter well, you can't necessarily tell by gating. Two
color analysis using lymphoid markers (if you're looking for ALL) may be
helpful. Also, the intensity of TdT staining can be variable, and a good
negative control may be especially important.
MOLT-3 is a good positive control, although its TdT expression tends to
be lower than normal TdT+ bone marrow precursors, although comparable to
many cases of ALL. HL60 is a good negative control. Even better
negative control would be normal grans, so that the issue of nonspecific
adherence to myeloid cells can be directly addressed.
I have also found that directly conjugated TdT-FITC antibody gives more
non-specific fluorescence than does indirect5 labeling.
We dealt with these methodologic issues in the following papers:
Gore, SD, Kastan, MB, Goodman, SN, and Civin CI. Detection of minimal
residual T cell acute lymphoblastic leukemia by flow cytometry. J.
Immunological Methods 132: 275-286, 1990.
Gore, SD, Kastan, MB, and Civin CI. Normal human bone marrow precursors
that express terminal deoxynucleotidyl transferase include T-Cell
precursors and possible lymphoid stem cells. Blood 77: 1681-1690, 1991.
Hope this helps.
Steve Gore
Johns Hopkins Oncology Center
Oncology 2-109
600 N. Wolfe Street
Baltimore, MD 21287-8963
sdgore@welchlink.welch.jhu.edu
410-955-8781 (phone)
410-614-1005 (fax)
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