Dr Mike Watts of UCL Hospital London is using a Planer Kryo 560-16 to evaluate the association between viability and function of cryopreserved HPCs according to freeze rate used. Non-viable cells are routinely detected by an increase in cell permeability and resultant staining with dyes such as trypan blue or 7AAD.
These tests however cannot be informative on the functional activity of the remaining unstained viable cells. The expression of the CD34 antigen of the HPC population allows specific viability measurement of this target cell population and flow cytometric dual CD34+/7AAD staining is widely used to assess the integrity of HPC post cryopreservation.
This test has been shown in at least one clinical study to be associated with functional activity measured by colony forming cell (CFC) assays in culture (Yang et al 2005 BMT 35:88). Mike is looking specifically at the quality issues in clinical use in relation to a poor engraftment incident where this was not the case and there was complete dissociation between viability and function, consistently high thaw CD34+/7AAD viability results failing to detect catastrophic losses in CFC activity. Further study showed that poor CFC growth was related to a more rapid freeze rate than is more normally used.
When the same HPC samples were cryopreserved using two different freeze rates, the faster rate failed to preserve CFC activity resulting in cells cryopreserved in one of two states, viable and functional (normal freeze rate) or viable but non-functional (faster freeze). The inability of high thaw CD34+/7AAD viability to distinguish between major losses in CFC function according to the freeze rate used was also confirmed recently in experimental cord blood studies reported by Yang et al 2015 (Yang et al 2015 Transfusion 55:70-78)
Thaw colony assays are a useful research tool but have proved difficult to standardise in inter-laboratory QC exercises for routine use. However, transplantation laboratories maintain high quality, well standardised flow cytometry for routine CD34+ cell counting and Mike believes that flow cytometric viability tests that rely on metabolic functional activity rather than simple permeability could represent an attractive alternative. The flow cytometric detection of high aldehyde dehydrogenase (ALDH) activity in HPC for example, has been shown in several studies to correlate with thaw clonogenic activity. The first question for such a test is whether it can distinguish between HPC samples cells with high viability but where occult loss of CFC activity has been induced by the freeze rate used. Mike is planning to prepare batch control samples using defined faster or slower freezing rates. If successful, this will permit inter-laboratory standardisation studies and ultimately improve quality assurance in clinical cryopreservation practice for HPC.
