More on Flow Tdt

Tue, 22 Apr 1997 18:34:20 -0400


We also use Ortho's PermeaFix reagent to perform TdT by flow
on whole blood / marrow specimens. Check out Mark Connelly et al.,
article in Comm of Cytometry, Fall (?) 96 . We have found Ortho's P-Fix
works well for cytoplasmic Ig's and cCD3, too. And it doesn't require a
separate lysing reagent. We haven't tried Caltag's reagent and so can't
comment on comparisons.

We use Supertech's TdT antibody, matched isotype control, and a cell
line preparation of mixed TdT pos and neg cells. Each patient is prepped
with a TdT and an isotype control tube; the cell line gets a TdT tube only.
The isotype control marker is used to "compare" patient positive vs.
negative. Analysis of the cell line is a comparison of TdT pos and neg
cells in the mix (internal neg control).

Note: By following Ortho's product insert, we perform surface staining
of patient TdT and control tubes with CD45-PerCP and blast-specific
PE-conjugated antibodies before permeabilization and staining with
TdT-FITC. This helps with target gating / selection of cells (we normally
use log SSC vs. CD45 most often).

Also, we run TdT as contingency testing (it is not in our initial antibody
panel) to clarify / characterize cases as needed. Because we do not
have a 4-color instrument, we must tailor the choice of PE antibody used.
Of course, running TdT after the rest of your markers are complete adds
another two hours or so to your turn around time. Not a very popular
approach on Friday afternoon...

Cathy Fritschi
Immunodiagnostic / DNA Analysis Lab
Sentara Norfolk General Hospital
Norfolk, Virginia

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