Hi Ken, My initial response merely attempted to address Pal's question and was not intended to convey that we believe all autologous grafts should be assessed for viable CD34+ cells post-thaw and prior to infusion. There are many instances in which measuring the viability of a sample with 7-AAD is important and has been used by many groups. The 'elegant solution' (single platform CD34+ cell enumeration inluding the viability dye 7-AAD) you referred to was initially developed for a variety of other (but no less valid) reasons. The fact that this particular solution is the only one that can address the issue of post-thaw recovery of viable CD34+ cells is perhaps only an added bonus. Whilst we might agree that adequate collections of CD34+ cells virtually guarantees engraftment, other sample processing steps can result in lower recoveries of viable CD34+ cells that in turn can result in poor, or some unfortunate cases, no engraftment. Most previously published studies show that 5 x 10e6 CD34+ cells/Kg results in timely engraftment in the autologous setting. More recent studies including our own (based upon the results obtained from patients whose CD34+ cells were enumerated with the above methodology) suggest a 'threshold' dose of nearer 2 x 10e6/Kg is adequate. However, there are 'outliers' in many of the published studies, the existence of which can be best explained by the probability that a sufficient proportion of the infused CD34+ cells were not viable. In the autologous setting, there have been a number of instances in our own Institution (a very large transplant centre) where the ability to assess the post-thaw recovery of viable CD34+ cells was instructive. In one instance, we collected 32 x 10e6 CD34+ cells /Kg from a patient. The sample was held overnight prior to processing at which point the viable CD34+ cell count had dropped by more than 50% to about 15 x 10e6. The product was fozen in two aliquots of about 7 x 10e6/Kg and the patient given the first one a few days later. He showed no signs of engraftment by day 14 and was then given the second aliquot with similar lack of success. We were asked to assess one of the two pilot vials that had also been stored at the same time, but using the above technology, found no viable, non-apoptopic CD34+ cells to be present, providing one explanation why this patient had failed to engraft. On another occasion we were asked to assess a sample from a patient who had relapsed some 5 years after her first autotransplant. The issue obviously was whether this second apheresis pack stored at the same time would contain enough viable CD34+ cells after all these years in liquid nitrogen, to be effective for a 'tandem' transplant. Such technology is very useful in the cord blood transplant setting, where samples are collected in a variety of different locations by a variety of people with variable 'technical skills'. Samples also have to shipped from sometimes remote geographical areas to the processing centre, using different modes of shipping etc. Although not currently 'state-of-the art', we believe it will become increasingly important to be able to enumerate viable CD34+ cells in the post-thawed samples, if and when they are selected for use in either auto- or allotransplant settings. Another indication for viability measurements is in the allotransplant setting, where CD34+ cells are first selected on a CD34 selection device (eg Miltenyi, Nexell). In short, ANY manipulation that is performed on a sample (that is to be used subsequently in the transplant setting) has the potential to adversly affect the recovery of viable CD34+ cells. The single platform methodology referred to allows us to assess such affects. Cheers, Rob Sutherland Mike Keeney University Health Network, Toronto, Ontario and London Health Sciences Centre, London, Ontario. Kenneth Ault wrote: > I am sure that the reccomendations from Rob Sutherland are technically quite accurate, > however I would like to ask if there is any objective evidence that measuring either > post-thaw CD34 counts or post-thaw CD34 viability has any impact on engraftment? Our own > experience is that engraftment (and speed of engraftment) is not a problem in autologous > transplantation - if adequate numbers of CD34+ cells are collected up-front then timely > engraftment is virtually guaranteed. Do we need elegant solutions to non-problems? > > Ken Ault
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