Re: TdT caveat
Maryalice Stetler-Stevenson (stetler@box-s.nih.gov)
Fri, 18 Apr 1997 13:43:49 -0400
Walter,
What is your method? I hate TDT and never have found a method that
works well all the time with all technicians. We had a tech years ago that
did the best TDT on cytopreps using an immunoperoxidase technique. After
she left, no one could do it reliably well-too many false negatives. So we
moved on to other methods. I find our flow method on rare occasions
(actually only with one CAP test specimen) is false positive. We have used
IFA on slides (works well but we have no capability for permanent record of
result) plus flow to do TDT and whenever possible just not done it (e.g.
never done on repeat specimens from a patient or get it done by ipox-
immunoperoxidase lab has it working well on paraffin embedded tissues).
Luckily, we rarely have lymphoblastic/leukemia specimens and don't need it
often. I am glad you are opening up a discussion on this topic. Others talk
as if they never have problems with this technique. We have used the
polyclonal and one monoclonal antibody by Supertech. Judging from what has
been on the list lately, maybe a cocktail of clones would work better. We
used Ortho's reagent to permeabilize. I use an internal negative control
and we have a cell line positive control. I know I am extremely compulsive
but TDT methods have never fulfilled all of my criteria. If someone could
just summarize the secret to always being happy with TDT (if that actually
is possible) I would be grateful.
>
>
>
> Maryalice
>
>>Following earlier discussions re: Caltag and, perhaps, tying in to the more
>>recent TdT neg ALL exchanges we also had an apparently negative T-ALL
>>(2/cy3/5/7/8/34) the other day.
>>I say apparent because a repeat of the staining using our in house method
>>gave a clear (admittedly dim) TdT positive result where the HT6 clone in
>>Fix & Perm was unequivocally (0%) negative.Both methods were whole blood
>>using identical concentrations & timings.
>>The strength of the cytoplasmic CD3 staining was the same by both
>>techniques so it appears that the nuclear membrane may be a little too
>>tough for An der Grubb's stuff.
>>As I said way back when, I have always been suspicious of the strength of
>>Caltag TdT staining but I didn't expect such a big disagreement between
>>methods.
>>In future all our TdT staining (tho' not MPO!) will be by our own method -
>>we just have to sort out the higher autofluorescence seen with AML's after
>>fixation (must re-read the postings on this).
>>
>>Since I'm on about TdT, what do other contributors feel about setting the
>>positive region ?
>>Matched isotypic, unstained control, TdT staining on normal lymphs or what
>>?
>>
>>Wal Sharp
>>SQU
>>Oman
>
Maryalice Stetler-Stevenson
Director Flow Cytometry Unit
Laboratory of Pathology, NCI, NIH
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