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We have been using Ortho's PermeaFix reagent to perform TdT by flow
on whole blood / marrow specimens for about 6 months now and have
been very pleased with the results. Check out Mark Connelly et al.,
article in Comm of Cytometry, Fall (?) 96 . Ortho's P-Fix seems sort of
expensive if you don't get a discount, but once the first bottle is
purchased, it lasts a long time... We have found it 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 TdT antibody, matched isotype control, and cell line
preparation (mix of pos and neg cells). Each patient is prepped with pos
and neg tubes; the cell line gets pos tube only. Isotype control marker is
used to compare patient positive vs. negative. Analysis of the cell line is
a comparison of pos and neg cells in the mix (internal neg control).
Note: By following Ortho's product insert, we perform surface staining
of patient pos and neg 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 i.e. logSSC
vs. CD45.
Also, we run TdT as contingency testing (it is not in our initial antibody
panel) to clarify / characterize cases as needed which enables us to
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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web