Hi Pal, not totally sure what all your questions are, but I have a couple of observations/questions. 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. If you use a combination of '%CD34+ cells' (from flow) multiplied by the absolute white cell count from a hematology analyser to generate your absolute CD34+ cell count, your data, particularly for the post-thawed sample will be seriously flawed. Hematology analysers were not developed to accurately measure the absolute WBC in marrow samples even before crypreservation, let alone after thawing. In the latter case, most of the granulocytes are lost and much debris is generated that in turn can masquerade as 'real' leukocytes. The same problem arises when post-thawed PB stem cell or cord blood samples are analysed by two-platform methods. 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. To better help you, we need to know what methodology you are using to count CD34+ cells. You indicated the use of TruCount tubes and the fact that you get the same results with such tubes as when you use lyse-no-wash sample preparation. If the TruCount tubes used are part of the ProCount kit from BDB, then you should already be using lyse-no-wash sample processing. Indeed, even if you use TruCount tubes with your own non-Procount method, you still have to practise lyse-no-wash processing. Otherwise, (as with other bead based methods currently available), you will 'wash out' the beads and your data will be seriously flawed/useless. 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. 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. Many of these issues have been discussed in earlier 'threads' in this forum (search for 'CD34') and there are a number of demonstration dot-plots and a 'Frequently Asked Questions' section pertaining to CD34+ cell enumeration by flow cytometry on the ISHAGE web-site (www.ishage.org/committees/Committees/Graft_Evaluation/graft.htm). Cheers, Rob Sutherland University Health Network, Toronto Jáksó Pál wrote: > Dear Flowers, > > Anybody has an idea why can I measure approximately 50 % or more > decrease in the CD34+ cell concentration of bone marrow mononuclear > cell fraction or PBSC after thawing ? I use TruCount tubes from BD, but > I have almost the same results using "lyse no wash" method and > calculation the CD34+ concentration from the percentage and white cell > count. > Thanks, > > Pal Jakso > > University of Pecs > Faculty of Medicine > Dept. of Pathology > > 12. Szigeti str. > Pecs > 7643, Hungary
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