Re: HSC TESTING AFTER CRYOPRESERVATION In response to Lisa and the other respondents, it is correct that very few laboratories are performing CD34+ cell enumeration on post-thawed samples. It is also true that FACHT have little if anything to say about measuring engraftment potential by flow. While there are a variety of reasons for this, the most significant one is that most flow methodologies in the past were incapable of accurately measuring viable CD34+ cells in such samples. This was due to the fact that the absolute CD34+ cell count was determined by ‘two platform’ methodologies (“%CD34+ events” from flow multiplied by the absolute leucocyte count from a hematology analyser). Both values are virtually impossible to determine with accuracy on post-thawed samples (and even some “fresh” ones). With the advent of the single platform version (Cytometry (Communications in Clinical Cytometry) 34:61-70 1998.) of the ISHAGE protocol (J. Hematotherapy 5:213-226,1996), that utilises fluorescent counting beads and the viability dye 7-AAD, it is now possible to reliably measure the absolute viable CD34+ cell count in virtually any source of hematopoietic cells, including post-thawed samples. It is essential that after thawing, samples are handled and tested in such a way that cell viability is not compromised during laboratory processing. Aliquots of packs which have been handled differently than the actual product will give little information on the product actually reinfused into the patient. At one of our institutions (MK/ICY) the thawed product is washed and resuspended in a unit of packed cells before reinfusion. This product is monitored for viable CD34+ cells to ensure there has not been unacceptable loss due to processing. In the clinical setting of peripheral blood stem cell transplantation, most studies that have correlated the CD34+ cell dose infused with engraftment have been performed on pre-cryopreserved specimens. Such studies have noted a number of ‘outliers’ in the data sets and a reason for this may be related to post thaw viability. In general, if samples are collected, processed and cryopreserved in a timely manner, using cGMP, it is doubtful whether post-thaw flow analysis will provide significant useful information to the tranplanter. However, if samples are mishandled or shipped prior to storage, or the cell concentration is too high in the pack and was not diluted immediately prior to processing, or there was a high number/concentration of neutrophils in the pack, for either ‘technical’ reasons, or due to the use of different cytokine mobilisation cocktails, recovery of viable CD34+ cells can be severely compromised. We have seen several examples of this, and it is important when there are concerns over product handling to have available a single platform flow method for the enumeration of viable CD34+ cells. In such cases, if a ‘pilot’ QC vial of frozen cells is available (or the actual product as mentioned above) it can be analysed and intelligent decisions can be taken regarding the safety of using the frozen product, as opposed to re-mobilising/collecting the patient. We have also performed many analyses on pre- and post-thawed cord blood samples for the local cord blood bank, to optimize the collection, processing, storage and recovery of viable CD34+ cells. To our knowledge, the single platform based ISHAGE protocol, that also underlies the ‘Basic Protocol’ for the enumeration of CD34+ cells for ‘Current Protocols in Cytometry (Unit 6.4), is the only flow-based method able to perform this type of analysis. At the current time, we are developing a linked web-site dedicated to the enumeration of CD34+ cells by flow cytometry with the International Society for Hematotherapy and Graft Engineering (www.ishage.org). When completed (soon) this site will contain downloadable templates and listmode files (from both Macintosh-based BDB FACS and PC-based Coulter XL instruments) of the single platform methodology used for the measurement of viable CD34+ cells. Meanwhile, we can e-mail copies of these files to any interested individuals. (For BDB platform contact rob.sutherland@utoronoto.ca and for Coulter XL files contact mike.keeney@lhsc.on.ca). Rob Sutherland, Mike Keeney, Rakash Nayar, Ian Chin-Yee
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