Anna Porwit-MacDonald
Haematopathology lab.,
Department of Pathology
Karolinska Hospital
Stockholm, Sweden
anpo@mb.ks.se
fax:+46-851775843
>
>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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