Anna,
> Thanks for sharing this information. What dilution do you use of
>the Supertech monoclonal? Is it bright enough that you don't have problems
>telling dim from autofluorescence? Most people feel something works well
>when it works 90% of the time. I feel something works well when it works
>100% of the time (the curse of the truely compulsive). Your method has
>never failed that you know of?
> Thanks
>
> Maryalice
>
>>Hi!
>>We have been using the flowcytometry method for Tdt for 3years now and we
>>consider the results both reliable and reproducible both in diagnosis and
>>follow up of ALL patients.
>> We are using a monoclonal FITC-anti-HTdT-6 from Supertechs, cat no #6600,
>>and Ortho Permeafix.
>> We always do triple stainings on whole (no F/P) bone marrow
>>1. first membrane CD19TRI and CD10Pe (or CD34 Pe and CD 3 PerCP), wash
>>2. the Permeafix 30 min RT, spin down
>>3. then anti Tdt-FITC, 30 min RT , wash.
>>I can really recommend that method.
>>Best wishes
>>Anna
>>
>>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
>>>
>>>
>>>
>>>
>
>Maryalice Stetler-Stevenson
>Director Flow Cytometry Unit
>Laboratory of Pathology, NCI, NIH
>
>
>
>