Maryalice
>Be careful - Ortho Permeafix does not work for TdT in AML. We have
>published on that (Leukemia 9:226, 1995).
>E. Paietta
>
>On Mon, 21 Apr 1997, Anna Porwit-MacDonald wrote:
>
>>
>> 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