TdT caveat

Maryalice Stetler-Stevenson stetler at box-s.nih.gov
Wed Apr 23 12:00:57 EST 1997


Elizabeth,
        Why do you say it doesn't work? Too dim? False positive? scatter
bad? Obviously it works for some. Could it be critical differences in
antibodies or technique that lead to this very important diference between
labs? Anna uses 5microl MAb / 50 microl BM of the supertech monoclonal
antibody,cat no #6600. Her technique is below. What are the differences
between your two techniques?


        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 at 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





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