Dear Rob, First of all thanks for your extensive help. My step by step answers to your questions are: "Currently, the only accurate way to measure the absolute CD34+ cell content of a sample, both before AND after cryopreservation, is to perform single platform analyses, i.e., use a counting bead-based flow assay." I totally agree with you and that's why I introduced it in our laboratory. "Incidentally, why are you enumeratiing CD34+ cells in marrow to begin with? Graft adequacy for marrow transplants was not traditionally assessed by flow techniques, but more usually by 'weighing the bag' and performing a crude leukocyte count thereon. Later, CFC assays were performed and an adequate dose was established based upon the numbers of Colony-Forming-Cells/Kg patient weight." I'm a biologist not a medical doctor and I think this question should have been adressed to our haematologists in the hospital and I will ask for their opinion surely. "To better help you, we need to know what methodology you are using to count CD34+ cells." I use our own non-ProCount protocol. Briefly: we stain the blood, PBSC, or bone marrow with CD45 and CD34 in the TruCount tube. After 20 minutes we add 0,5 ml FACS Lysing Solution and incubate for 15 minutes. Finally we measure on a FACSort ( without any washing steps of course). We acquire and analyse with CellQuest. In order to count most accurately the CD34 pos. cells I use a complex gating strategy combining blast gates from the FSC/SCC, CD45/SSC and CD34/CD45 dot plots. "If you are using ProCount to enumerate CD34+ cells in (even fresh) marrow samples, I would advise you to stop doing so. This kit was developed for the enumeration of CD34+ cells in fresh PB and apheresis samples only. Even in these limited circumstances, the automated software that acquires/analyses these samples will sometimes 'flag' them, especially if there are debris/platelet aggregates in the smaple. This usually requires a manual re-acquisition and analysis of the sample and even then analysis may still be problematic. I know that it's not recommended to use TruCount tubes for BM. Of course BM is more complex to analyse than PB but in my opinion with sufficient routine it may not be problem to analyse. We don't use automated software. "This product is not recommended for use on post-thawed samples (PB, CB, marrow, etc) because it does not include a viability dye (only a nucleic acid dye for 'gating' nucleated cells)." In our studies of recovery of viable CD34+ cells in cord blood samples using the single platform ISHAGE protocol, that contains the viability dye 7-AAD, we have noted a wide range of viable CD34+ cell recoveries, ranging from less than 50% to over 85%. While there may be a variety of other factors that determine recovery, methods of collection, time between collection and processing/storage, and the mode of transport used to get the samples from the collection point to the processing laboratory are no doubt important factors. " I agree, our method does not include viability stain but it can be done separately. Because usually the fresh PB or PBSC samples have only very small dead cell population (1-5% in my experience) I think it's not very important to count with it. In the case of thawed samples there is another situation. Yesterday before I made viability stain on two thawed BM MNC samples and the viability was only about 50% so I think there is good correlation with the findings that the CD34 pos. cell number decreased to the half of the original fresh samples. If I understood right our haematologist the goal of these post-thawed measurements is only for the controlling purposes. Thanks again, Pal Jakso University of Pecs Faculty of Medicine Dept. of Pathology 7643, Hungary 12. Szigeti str.
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