Re Sharon's questions and the responses of Scott and William, it is our experience that an increasing number of laboratories are using only CD34+ cell enumeration to routinely assess auto- and allo-graft quality. As Scott indicated at least in part, the reasons for this are many and varied but in our view, they nevertheless remain valid at this time and perhaps bear restating. Colony-forming assays have been described in the ongoing dialogue as 'functional assays', but in fact only measure relatively 'late' or single, lineage-committed progenitors of the myeloid and erythroid lineages. Typically, the only colonies 'scored' in these assays for autograft assessment are colony-forming units for granulocytes and macrophages (CFU-GM). Unfortunately, due to differences in the way these assays are performed between different centres, including widely differing culture conditions (leucocyte-conditioned medium versus recombinant growth factors etc.), and the subjectivity involved in identifying various colony types, it is difficult to make meaningful comparisons on data from different institutions. Except in the hands of a few specialist laboratories, CFC assays are not used to measure what many consider the most relevant progenitor subset, namely megakaryocyte progenitors (CFU-MK, BFU-MK). Similarly, most labs do not measure lymphoid-lineage progenitors and because of 'subjectivity' issues, most labs still performing graft assessment by CFC assays do not even score so-called 'mixed' lineage progenitors (CFU-mix, or CFU-GEMM). In vitro assays for precursors of the lineage-restriced CFCs such as CFU-Blast, Long Term Culture-Initiating Cells, Cobblestone Area-Forming Cells, etc., rely upon technologies that are even more esoteric and generally beyond the technical capabilites of all but a few dozen laboratories worldwide. Finally, there exists in 1998, no in vitro assay capable of identifying and quantitating the tiny fraction of marrow leucocytes that are responsible for long term, multilineage engraftment in vivo. As Scott already indicated, an additional limitation of colony-forming cell assays for hematopoietic progenitors is the 10-14 day interval required for assay read-out. This facet alone precludes the assay any role in optimising the timing of apheresis collections, a potentially useful thing to do for heavily pre-treated cancer patients in whom obtaining an adequate does of PBSC may be problematic. Finally, CFC assays cannot be used to monitor 'on-line', the yield of PBSC in the apheresis collection. Thus, while all of the above assays have played and will continue to play a vital role, primarily in the research laboratory environment, we feel that their potential utility in the routine measurement of engraftment potential is limited. Studies performed in Seattle in the late 80s have clearly shown that reinfusion of purified marrow CD34+ cells results in long-term, multilineage hematopoietic recovery following myeloablative therapy. Thus the impetus to develop flow-based protocols for enumerating CD34+ cells began. This mode of assessment takes less than 1 hour, is less susceptible to subjectivity, 'operator', and (hopefully) nowadays, 'reagent' issues. The CD34+ population encompasses the earliest stem cells and maturing, lineage-committed progenitors of all lineages, and can be used to 'time' apheresis collections and monitor the yield of PBSCs 'on-line'. It is also suitable for the determination of optimal timing for apheresis collections. Finally, the development of both 'positive' and 'negative' selection techniques to enrich for CD34+ cells has driven the need for rapid, accurate and reliable flow techniques to assess the purity of such supensions in both research and clinical laboratory settings. Despite the above, and it is easy to tell which side of the fence we sit on, both CFC assays and CD34+ cells are merely surrogate markers that either alone or even in combination, are incapable of answering all of our questions. Hematopoietic cell transplantation is unlikely to become a risk-free therapy any time soon, particularly in the allogeneic setting. This despite the fact that the best flow techniques for measuring CD34+ cells, such as the ISHAGE protocol, even when properly performed, can occasionally fail, or generate flawed data. Recent experience has indicated that flow techniques are more likely to generate flawed or inaccurate data on purged or otherwise manipulated samples of the type eluded to by William. One potential reason for this is that while the gating strategy published in the original version of the ISHAGE Guidelines (J. Hematother. 5:213,1996) is at least able to distinguish non-CD34+ dead cells from true CD34+ cells, it is not capable of distinguishing dead CD34+ cells from viable CD34+ cells. Ultimately it is the viable CD34+ cells reinfused which will ensure engraftment. By incorporating 7-AAD and fluorescent counting beads into the basic method, one can delineate dead from viable CD34+ cells and generate an absolute viable CD34+ cell count using only flow cytometery. Experience with this modification has clearly indicated that purged/manipulated marrow and PB/apheresis samples and post-thawed cord blood samples have the greatest potential to generate seriously flawed data. The simple inclusion of this viability stain renders the method highly reliable at quantitating viable CD34+ cells (Keeney et al Comm. Clin. Cytomery, in press 1998). It should be noted, however, that CFC assays done pre-cryopreservation/manipulation will also suffer the same limitation, i.e. may have little bearing on actual number of functional stem cells infused to the patient. So whilst those proclaiming the virtues of functional assays may feel vindicated by Scott and William's comments if, as we believe, the quality of the post-cryopreservation stem cells is crucial, then the addition or replacement of CD34 assays with CFC assays before storage is unlikely to solve the problem. We all love anecdotes and over the years they have played a major role in advancing the 'state of the art' in many fields, including this one. However, we could do with a little more specific information on the example William cited to support his continued committment to routine CFC assays. Allografts purged of T lymphocytes have been shown to fail on many occasions and the reasons for this have little or nothing to do with the presence of sufficient viable CD34+ cells in the graft. Questions arising from his correspondence include, but are not necessarily limited to: 1. What was the clinical diagnosis in this case and how much prior therapy had the recipient received. 2. How closely matched were donor and recipient? 3. Which protocol did the flow lab use and was a viabilty dye included so that only viable CD34+ cells were enumerated. 4. Which method of T cell purging was used and did it involve cytotoxicity. When cytotoxic regimens are used it common for 'innocent bystanders' including CD34+ cells to be killed also. 5. Did any other CFC assays set up by the same operator at the same time using the same assay media generate poor colony growth? Rob Sutherland, Mike Keeney, Ian Chin-Yee, Rakash Nayar. Oncology Research, The Toronto Hospital, and London Health Sciences Centre, London Ontario Canada.
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