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