Post Thaw Recovery of CD34+ Cells > 100%
Question: As part of our stability program we have been thawing cryopreserved products to determine total nucleated cell counts (TNC) and CD34 recovery, as well as CD34 viability. We have noticed that our recoveries for CD34 cells frequently exceeds 100%. TNC recoveries are in the ange of 90%. Our clinical labs cannot offer possible explanations for the unexpectedly high CD34 cell recovery.
Answer: First of all, there is no evidence that stem cells proliferate during freezing so the counts that you have observed reflect different factors. Secondly, what you have observed is not unusual and has been observed (and published) by a variety of other investigators. This issue has not been studied rigorously and should be. We believe that two factors may account for the measurements. Clearly additional studies must be performed to confim these hypotheses.
Improper gating: Flow cytometric analysis of fresh hematopoietic stem cell products has been extensively studied and there have been attempts to standardize sample preparation and analysis. Fresh cell and frozen cells are significantly different. Frozen cells have been subjected to high levels of dehydration and it is highly likely that forward and side scattering of the cells post thaw differs from that of non-frozen cells. Studies of changs in cell volume and morphology post thaw (compared to pre-freeze) have been published for other cell types (not HSCs) and it is highly likely that HSCs exhibit similar differences. Therefore, gating and analysis for frozen and thawed cells may need to be adjusted to get more accurate results.
Accounting erros: Please keep in mind that flow cytometric analyses express CD 34 as a function of intact cells. During freezing, there are three different populations: intact and viable cells, intact cells that are not viable and cells that are neither intact nor viable. That third population is frequently ignored. Cell lysis is one of the most common manifestations of cell damage during freezing. If you do not correct your counts to account for cell lysis, you will bias your post thaw assessment measures higher (and in some cases over 100%).
