From: Chalmers, James (chalmersja@MSX.UPMC.EDU)
Date: Tue Mar 04 2003 - 14:01:23 EST
Hello, We are a lab in Pittsburgh, PA looking at going to four color flow panels. We use BD FACSCalibur instruments and maybe about 90-95% of their conjugated antibodies. We are discussing our antibody/fluorochrome combinations and have come up with some questions in hopes that someone has an answer or at least an opinion. The questions are as follows: 1. In reviewing BD's recommendations (something called "Pattern recognition of normal bone marrow cell subpopulations based on flow cytometric analysis of light scatter and cell surface antigen expression using four-color reagent combinations"), one of the myeloid panels uses CD117/CD34 together. I believe that is would give basically the same information. Am I correct in this assumption? 2. None of BD's tubes has HLA-DR/CD15 combination. I would think this combo would be useful in evaluating myeloid cells, especially when looking for MDS. 3. Would the addition of CD16 be useful in a MDS panel? And what about CD11b? 4. We are looking at using a tube for cytoplasmic staining: Tdt/MPO/CD3/CD34 - does this sound feasible to you. Would you recommend another combination. Also, could Tdt be used with something else other that CD3? With cytoplasmic staining, can APC be used cytoplasmically? 5. For Myeloma staining: if BD cocktailed our Ab's, I imagine that there would be two separate cocktails for this. One cocktail (which contains 2 Ab/colors) for the surface staining portion and another (contains 2 Ab/colors) for the cytoplasmic portion. Is this a correct conclusion? While talking about myeloma staining, what about the use of CD79a? Would this be useful? How specific is CD79a for plasma cells? Could the use of this CD marker be misleading? 6. In a known AML and ALL with a limited specimen (QNS for full panel), what recommendations/advice do others have to use in this situation (what 2 or 3 tube combinations are BEST to use in this situation)? 7. What is the significance of CD22 in Hairy Cell Leukemia? 8. What is the best way to use Glycophorin A in Erythroid leukemia's? What other CD markers would be used in conjunction with Glyco A? The following is possibilities of our Ab/Fluorochrome combinations, please feel free to comment on these. Lymphoma panel: 1. Kappa-FITC/Lambda-PE/CD20 PerCP-Cy5.5/CD10APC 2. FMC7/CD5/CD19/CD23 3. CD4/CD7/CD3/CD8 4. CD57/CD16+56/CD3/CD2 Acute/MDS panel: Repeat tubes 1-4 and in addition to those 5. CD22/CD10/CD20/CD34 6. CD7/CD13+33/CD19/CD56 7. CD11b/CD117/CD33/CD34 8. CD15/CD14/CD45/HLA-DR 9. Tdt-cyto/MPO-cyto/CD3-cyto/CD34-cyto Extra tubes: Myeloma Lambda-cyto/CD56/CD138/Kappa-cyto T-cell TCR-a,b/TCR-g-d/CD3/CD25 Hairy Cell CD103/CD11c/CD20/CD25 CLL additional CD5/CD38/CD19 We want to get away from repeating CD20/CD45 in the third color position so that we can better utilize the tube/panels (thus using less number of tubes - if possible). What is the opinion on this practice? Any light that can be shed on these questions would be greatly appreciated. Sincerely, James A. Chalmers Lead Medical Technologist UPMC Presbyterian Hospital (412) 624-3394 chalmersja@msx.upmc.edu - work e-mail james.chalmers@verizon.net - home e-mail
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