(Sorry for the lengthy entry...) 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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