Background: Cryopreserved peripheral blood stem cells (PBSC) are used following myeloablative therapy (MAT) in the autologous, and occasionally allogeneic, setting. In paediatric practice, MAT for high-risk neuroblastoma constitutes the majority of such procedures. Standard quality assurance (QA) uses post-thaw CD34+ cell counts and assessment of viability using vital dyes. In 2013, concerns were raised at Great Ormond Street Hospital (GOSH) about a series of patients who experienced delayed engraftment after MAT with cryopreserved cells, despite adequate cell viability and counts in all cases.
Methods: Root cause analysis was undertaken including all aspects of the pathway: patient factors, PBSC mobilisation, apheresis, cryopreservation, reinfusion and count recovery. Investigations suggested the freeze process itself might be a contributing factor to suboptimal engraftment. A series of experiments were undertaken in which a single PBSC product was divided into three and cryopreserved in parallel using a control-rate freezer (CRF) or passive freezing method (-80°C freezer) at GOSH, or the same passive freezing at another centre. Colony Forming Unit-Granulocyte-Monocyte (CFU-GM) assays were used to assess potency.
Results: Comparison of parallel cryopreservation methods revealed equivalent and adequate CD34+ viability in all experiments. However, although CFU-GM assays demonstrated colonies from the products cryopreserved using passive freezing (both at GOSH and at the other laboratory), products cryopreserved using the CRF did not form any colonies. The CRF device was shown to be operating within manufacturer’s specifications with freeze profile within acceptable limits. The cryopreservation process at GOSH was amended in 2014, since when there has been a statistically significant improvement in time to neutrophil and platelet engraftment and reduction in duration of admission.
Conclusions: This experience has important implications for quality assurance for all transplant programmes, particularly those using cryopreserved products. The failure of post-thaw CD34+/7AAD- counts, the most widely used routine QA test available, to ensure PBSC function is of great concern and should prompt a reassessment of protocols and QA procedures in all transplant centres.