[NOTE: I'm reposting this because it apparently got scrambled somewhere ] [in our system. Steve ] Dear Andrea, In response to your questions: 1. which procedure is recommended these days? There are several methods of enumerating CD34 commercially available and the one you choose depends on your own specific requirements. The IMAGN 2000 from BD is a microvolume fluorimeter (non flow based) method which is perhaps the simplest to use. Recent report show good C.V.s on blood and apheresis. Limitations are high C.V.s in low CD34 count range (<10/ul)due to the limited volume analysed and as it is a single color system the effect of low viabilty is undocumented. The ProCount from BD is a 3 color single platform method utilizing a vital dye on FL1, CD34PE and CD45PerCp. The addition of TruCOUNT fluorospheres allows absolute CD34 numbers to be derived directly from the flow cytometer. Published studies also show good C.V.s for this method and the availabilty of an automated algorithm may improve reproducibilty between sites. Limitations are the inabilty to measure viabilty and because all 3 PMT's are used in this method it is not possible to do CD34 subset analysis on a 3 color laser. Apheresis packs with many platelet clumps may cause a problem for the automated software, and a manual gating strategy using either ProCOUNT or ISHAGE is available. The StemKIT from Immunotech/Beckman-Coulter is a 2 color CD45FITC CD34PE method that uses FlowCOUNT fluorospheres to allow single platform CD34 absolute counts to be determined. Currently gating is performed manually using the ISHAGE guidlines. An advantage of this method is the ability to include 7-AAD in FL3 (or FL4) to assess viabilty. Subsets of CD34 can also be determined by the addition of a 3rd antibody. Recent reports from the European Working Group on Clinical Cell Analysis show very good inter and intra laboratory C.V.'s with this method. 2. which antibody is one supposed to use? Are the ISEHAGE guidelines still recommending CD34-PE? For 2 color analysis CD34PE is the antibody of choice because of its excellent signal/noise ratio. As long as class III antibodies of CD34 are used FITC conjugates also work well. Other fluorochromes can be used but each fluorochrome/antibody combination should be evaluated before implementing in a clinical lab. 3. are most people just doing the single parameter CD34 percentage or is the lineage-restricted vs non-restricted %CD34 analysis becoming more important. >From the research viewpoint there is tremendous interest in subsets of CD34. However most clinical data published to date confirms that the single most important factor predicting both short and long term engraftment is related to the total number of CD34+/kg body weight infused into the patient. Back to your initial problem. Do you wash your samples after lysing? Most experts in the field now recommend no wash techniques. Are the absolute numbers as well as the percentages different? One or other method may be causing a selective loss of a specific cell type. How does the other lab measure their CD34+ cells? It is safe to measure CD34 cells without lysing in apheresis packs as the number of contaminating rbc's is usually low. Simply resuspend the stained sample in 1ml of PBS before analysis. This is not recommended for whole blood or cord blood due to the very high number of red blood cells. I hope this helps. Some excellent references can be found in the Journal of Hematotherapy, Transfusion and Clinical Cytometry. Many of the points you raised have also recently been covered in Current Protocols in Cytometry by Jan Gratama, Rob Sutherland and myself. Feel free to contact me directly if you wish followup on any of the points. Good Luck!! Mike Keeney <<< "Andrea Illingworth" <dcdsflow@mint.net> 1/ 3 1:01p >>> Dear flow group, We are currently in the process of evaluating a reliable procedure for CD34 enumeration. We have been sharing samples and comparing results with another institution and have been surprised to find out in the process that lysed specimens yield consistently higher percentages than unlysed specimens. We have been using the Coulter Stemkit which uses CD34-PE/CD45- FITC and their designated lysing solution. The other lab is using a no-lysis procedure and a CD45-FITC/CD34-PerCP combination. I am sure I am not the first one to observe this finding and my questions are : 1. which procedure is recommended these days? 2. which antibody is one supposed to use? Are the ISEHAGE guidelines still recommending CD34-PE? 3. are most people just doing the single parameter CD34 percentage or is the lineage-restricted vs non-restricted %CD34 analysis becoming more important. Thanks for your help and have a happy and safe change into the new millennium! Andrea Illingworth, MS, H(ASCP) Dahl-Chase Diagnostic Services Flow Cytometry 333 State Street Bangor, Maine 04401
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