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 > > > > > > > > > >
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