Post‐thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes

Abstract

Standard quality assurance (QA) of cryopreserved peripheral blood stem cells (PBSC) uses post-thaw viable CD34(+) cell counts. In 2013, concerns arose at Great Ormond Street Hospital (GOSH) about 8 patients with delayed engraftment following myeloablative chemotherapy with cryopreserved cell rescue, despite adequate post-thaw viable cell counts in all cases. Root cause analysis was undertaken; investigations suggested the freeze process itself was a contributing factor to suboptimal engraftment. 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 laboratory. Viable CD34(+) counts were equivalent and adequate in each. Granulocyte-monocyte colony-forming unit assays demonstrated colonies from the products cryopreserved using passive freezing (both laboratories), but no colonies from products cryopreserved using the CRF. The CRF was shown to be operating within manufacturer's specifications with freeze-profile within acceptable limits. This experience has important implications for quality assurance for all transplant programmes, particularly those using cryopreserved products. The failure of post-thaw viable CD34(+) counts, the most widely used routine QA test available, to ensure PBSC function is of great concern and should prompt reassessment of protocols and QA procedures.

Cryopreservation of Hematopoietic Stem Cells: Emerging Assays, Cryoprotectant Agents, and Technology to Improve Outcomes

“…In 2016, Morgenstern et al [88] found several instances of patients with delayed engraftments despite sufficient post-thaw viability of CD34+ cells. Determining post-thaw functionality is thus an important step before moving into clinical trials.…”

The value of the post‐thaw CD34+ count with and without DMSO removal in the setting of autologous stem cell transplantation

“…Both techniques should be considered complementary, and we don't know if the observed loss of CD34+ cells in some of our grafts would also have translated into a decreased stem cell potency, as it would be measured by functional assays . This problem has been addressed recently, following a detailed analysis of patients with delayed engraftment after ASCT …”

“…The latter effect could be due to the widespread supportive use of granulocyte colony‐stimulating factor (G‐CSF) after transplantation, facilitating ANC reconstitution, as well as to G‐CSF administration at time of apheresis, which “primes” CD34+ cells toward granulocyte differentiation . Although CD34+ cell counts are considered a reliable surrogate marker for engraftment, they do not provide any information on functional activity . Even though different studies have shown a good correlation of postthaw CD34+ counts and engraftment, assessment of CD34+ cell counts is exclusively performed before freezing in most centers.…”

Applications and optimization of cryopreservation technologies to cellular therapeutics

“…Post-thaw culture for 24 h was found to be effective in reparation of many of the cryo-induced abnormalities. A recent review of cryopreservation practices across UK centres applying slow cooling cryopreservation for peripheral blood stem cells identified differences in practices which were reported under the umbrella term 'cryopreservation' [63]. These combined studies highlight an important point for all cell therapy cryopreservation activities; this is that cryopreservation is not a simple 'standardised one option' process, the various protocol steps need to be well documented in standard operating procedures, and the equipment involved should be applied with care and monitored frequently against documented performance criteria.…”