Quality and Safety of Hematopoietic Progenitor Cell (HPC) Products Cryopreserved by Simple Passive Gradual Freezing Method

Abstract 4410

To provide quality HPC products for transplantation, harvests of bone marrow (BM), Peripheral Blood Stem Cells (PBSCs) and Cord Blood (CB) are routinely cryopreserved by gradual cooling from ambient temperature to −90°C using Controlled Rate Freezing (CRF) systems before placement in liquid/vapor nitrogen storage. CRF systems generally utilize a freezing chamber and computer-controlled freezing cycle with an established sequence of steps where timing, target temperatures and cooling rates are defined. System failures that endanger product quality may occur due to impurities in liquid nitrogen, mechanical/software problems and probe malfunction. A more convenient and economical freezing may be performed by simple “passive freezing” (PF) where products are placed directly into an ultra-low freezer (−80°C). Continuous temperature monitoring during the freeze process is achieved using individual data loggers. In the PF process, products remain undisturbed during the freeze process, and are removed for placement in liquid/vapor nitrogen storage when the target product temperature is reached (−80°C). Probe specific freezing curves are generated from downloaded data logger information.

In an internal validation study, the St. Louis Cord Blood Bank (SLCBB) investigated quality and safety of HPC products cryopreserved using this PF method. Evaluation was based on the analysis of freezing kinetics, post-thaw cell recoveries, and available transplant outcome data in reference to those obtained by the conventional CRF method. A total of twelve (12) red blood cell and plasma reduced CB products cryopreserved in 10% DMSO/Dextran by PF were thawed according to a validated albumin/dextran reconstitution thaw method. Compared to the programmed CRF system, PF product cooling rate to a temperature of −60°C was faster (1.2– 2.0°C/min compared to 1°C/min), ultimately followed by slower cooling rate to a product target temperature of −80°C (approximately 0.4°C/min compared to 10°C/min). The kinetics of PF curves resulted in longer freezing cycles with an average freeze time of 180 minutes compared to 80 minutes by CRF method. Post-thaw cell recoveries of the PF data set were comparable to those obtained from an established thaw control group of RBC and plasma reduced CB cryopreserved by CRF system (n = 25). Average percent recoveries for CB products cryopreserved by PF methodology compared to CRF system were reported as follows, respectively: viable nucleated cells (NC) by trypan blue = 84% +/− 5% compared to 71% +/− 6%, viable (7AAD) CD34+ cells = 67% +/− 7% compared to 63% +/− 15%, and colony forming unit (CFU) = 73% +/− 7% compared to 72% +/− 14%.

Subsequently, the SLCBB reviewed post-thaw cell recoveries of ten (10) PBSC harvests cryopreserved by PF and thawed at the SLCBB for clinical transplantation. Similar to the CB evaluation, results indicated a high quality HPC product post-thaw with average percent recoveries reported as follows: viable nucleated cells (NC) by trypan blue = 79% +/− 10%, viable (7AAD) CD34+ cells = 81.2% +/− 20.2, and CFU = 73.6% +/− 21.9%. All PBSC products were thawed and infused with no reports of infusion-related adverse events. These PBSC products engrafted shortly within predicted time; absolute neutrophil count (ANC) > 500/uL was reported within 12 days, while platelets > 20,000/uL was achieved within 22 days.

Retrospective review of more than 1,800 CB products distributed by the SLCBB for transplant showed that five (5) PF products were transplanted as singlet products to treat patients with myeloid malignancies. Transplant outcome data received by CIBMTR for those cases indicated 100% success of engraftment and 100% survival at 100 days post-infusion. Engraftment data reports indicated ANC > 500 was achieved within 6–30 days, and platelet count > 20,000 was achieved within 25–163 days. The transplant outcome of PF product is comparable to that reported historically for CRF CB products.