Dear Elisabeth! I know, that is why I use the mentioned method for follow up in ALL patients only. Best wishes Anna >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 >> > >> > >> > >> > >> >> > > >
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:49:41 EST