Cryopreserved hematopoietic stem/progenitor cells stability program-development, current status and recommendations: A brief report from the AABB-ISCT joint working group cellular therapy product stability project team.

BACKGROUND: The AABB-ISCT Joint Working Group Stability Project Team (SPT) was assigned to roadmap a path toward standardization of cryopreserved hematopoietic stem/progenitor cell (HSPC) stability programs. HSPC stability encompasses a broad scope of conditions including non-frozen ("fresh") and cryopreserved cell products, and varying methods for storage, thaw, and administration. This report assessed current practices and focused solely on cryopreserved HSPC cell therapy products to establish preliminary recommendations for a stability program roadmap. METHODS: A survey was prepared by the SPT and distributed to ISCT and AABB members. Survey results were summarized and recommendations were outlined based on the responses from the survey. This report highlights current practices for cryopreserved HSPC stability programs, including additional considerations and recommendations. RESULTS AND DISCUSSION: Eighty-two (82) centers worldwide participated in the survey. Survey results indicate variability across programs. HSPC stability depends on multiple factors within the processing facility (e.g., cryopreservation techniques, reagents used, and storage temperature) and independent variables (e.g., donor-related factors and starting material variability). While retention of hematopoietic engraftment potential is the primary goal for cryopreserved HSPC stability, engraftment results should not be used as the sole metric for stability programs. Based on the survey results, the SPT provides recommendations for consideration. CONCLUSIONS: The SPT recommendations for best practices are not intended to replace existing standards. The survey results emphasize the need for the community to optimize best practices and consider initiating collaborative projects to improve the standardization of cryopreserved HSPC stability programs for cell therapy products.

Cryopreserved hematopoietic stem/progenitor cells stability program-development, current status and recommendations: A brief report from the AABB-ISCT joint working group cellular therapy product stability project team.

BACKGROUND: The AABB-ISCT Joint Working Group Stability Project Team (SPT) was assigned to roadmap a path toward standardization of cryopreserved hematopoietic stem/progenitor cell (HSPC) stability programs. HSPC stability encompasses a broad scope of conditions including non-frozen ("fresh") and cryopreserved cell products, and varying methods for storage, thaw, and administration. This report assessed current practices and focused solely on cryopreserved HSPC cell therapy products to establish preliminary recommendations for a stability program roadmap. METHODS: A survey was prepared by the SPT and distributed to ISCT and AABB members. Survey results were summarized and recommendations were outlined based on the responses from the survey. This report highlights current practices for cryopreserved HSPC stability programs, including additional considerations and recommendations. RESULTS AND DISCUSSION: Eighty-two (82) centers worldwide participated in the survey. Survey results indicate variability across programs. HSPC stability depends on multiple factors within the processing facility (e.g., cryopreservation techniques, reagents used, and storage temperature) and independent variables (e.g., donor-related factors and starting material variability). While retention of hematopoietic engraftment potential is the primary goal for cryopreserved HSPC stability, engraftment results should not be used as the sole metric for stability programs. Based on the survey results, the SPT provides recommendations for consideration. CONCLUSIONS: The SPT recommendations for best practices are not intended to replace existing standards. The survey results emphasize the need for the community to optimize best practices and consider initiating collaborative projects to improve the standardization of cryopreserved HSPC stability programs for cell therapy products.

Transient warming affects potency of cryopreserved cord blood units.

BACKGROUND AIMS: Cryopreserved cord blood units (CBUs) can be exposed to transient warming events (TWEs) during routine banking operations, which may affect their potency. NetCord-FACT guidelines recommend removal of these CBUs from inventory. The objective of this work was to evaluate warming kinetics of frozen CBUs in different settings to determine the optimal working environment and define the impact of different TWE scenarios on CB post-thaw quality and potency. METHODS: The warming kinetics of frozen CBUs was influenced by both working surfaces and ambient working temperature, with cold plates providing better protection than vinyl or metal surfaces. Measurement of time for required operational activities revealed that CBUs are probably exposed to core temperatures greater than -150°C even when cold plates are used to reduce warming rates. RESULTS: On the basis of the warming kinetics and observed operational activities, three TWE causing scenarios (control, typical, worst case) were investigated using a pool-and-split design and cell viability, recovery and potency (colony-forming unit [CFU]) assays were performed. TWEs were found to have little impact on the recovery of total nucleated cells or on the viability of CD34+ cells. In contrast, the viability and recovery of CD45+ cells in the smaller CBU compartments were reduced by TWEs. Moreover, the worst-case TWE reduced CFU recovery from CBUs, whereas the typical-scenario TWE had little effect. CONCLUSIONS: Our results demonstrate that the distal segment underestimates the viability and potency of CBUs and that TWEs can affect the post-thaw viability and potency of CBUs. Although TWEs are almost inevitable during cord-blood banking operations, their effects must be diminished by reducing exposure time, using cold plates and strict operational protocols, to prevent worst-case TWEs.

Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.

BACKGROUND AIMS: In 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards. METHODS: Twelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures. RESULTS: Excellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells. CONCLUSIONS: The current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.

Overcoming the deceptively low viability of CD45+ cells in thawed cord blood unit segments.

BACKGROUND AND OBJECTIVES: There is no standard methodology for post-thaw sample preparation for viability analysis of umbilical cord blood units (CBU). A common challenge faced by CB bank is for their product to meet the post-thaw cell viability threshold for CD45+ cells set at 40% by NetCord-FACT. The objective of this work was to improve the post-thaw staining method to maximize CD45+ cell viability so that clinically valuable samples meet the NetCord-FACT threshold criteria for CD45+ and CD34+ cell viabilities. MATERIALS AND METHODS: Samples of CBU buffy coats and CBU segments were thawed and taken for staining. Various parameters were evaluated on CD45+ and CD34+ cell viability as measured by 7-actinomycin D (7-AAD) staining. RESULTS: The results revealed that initiating the staining at 20 min post-thaw instead of 30, shortening the red cell lysis treatment, or performing lysis on ice and removing this step all together, all improved the viability of CD45+ cells. Using CBU segments, it was shown that the most effective approach in increasing the viability of CD45+ cells was the complete omission of red cell lysis step. However, removal of the lysis step can create technical artefacts during flow cytometry acquisition that results in an underestimation of the viability of CD34+ cells. This can be avoided and CD34+ cell viability restored with additional thresholding on CD45 signal. CONCLUSION: CB CD45+ cells are sensitive to red cell lysis treatment post-thaw; omission of this step provides the best viability and ultimately better reflects the quality of cells used for transplantation.

Development of a proof of principle for universal neutralization of antibiotics in cord blood by-products used for sterility testing.

BACKGROUND: Bacterial contamination of cord blood (CB) represents a safety risk for transplantation patients. CB sterility testing at Canadian Blood Services' Cord Blood Bank is performed using a 1:1 mix of CB-derived plasma and red blood cells (RBCs). Culture bottles of an automated culture system, which lack antimicrobial neutralization properties, are used for bacterial screening of CB. This process is unsuitable for CB-containing antibiotics, potentially resulting in false-negative results. This study was aimed at developing a protocol for antibiotic neutralization in CB used for sterility testing. STUDY DESIGN AND METHODS: Phase 1: four neutralizers-penicillinase, ion exchange resins L and A, lecithin + Tween80, and activated charcoal (AC)-were individually tested to neutralize penicillin or gentamicin in cultures of Staphylococcus epidermidis or Klebsiella pneumoniae, respectively, adjusted to 100 colony forming units/mL, in Müller-Hinton broth (MHB). Phase 2: combinations of penicillinase plus resin L or penicillinase plus AC were assayed for the simultaneous neutralization of both antibiotics in MHB. Phase 3: penicillinase plus resin L was used to neutralize both antibiotics in CB sterility testing samples (plasma + RBCs). RESULTS: Phase 1: penicillin was neutralized by penicillinase and resin A, while gentamicin was neutralized by resin L and AC. Phase 2: the antibiotics were simultaneously neutralized by the two neutralizer combinations tested. Phase 3: neutralization of both antibiotics in CB was achieved with penicillinase and resin L. CONCLUSION: A protocol for antibiotic neutralization in CB sterility testing samples has been successfully developed at Canadian Blood Services' Cord Blood bank. This in-house assay applies to any culture-based CB bacterial screening method.

Assessing opportunities and challenges for establishing a national program to distribute cord blood for research.

BACKGROUND: Research is needed to enhance cord blood (CB) transplantation outcomes and to develop new clinical applications. Based on quality criteria for transplantation, CB collected by public CB banks (CBBs) is often unsuitable for banking, but may still be valuable for research. Canadian researchers have described a need for a centralized program providing ethically sourced CB for research projects. To meet this need, Canadian Blood Services (CBS), in partnership with The Ottawa Hospital, launched the Cord Blood for Research Program (CBRP) in 2014. STUDY DESIGN AND METHODS: The CBRP developed processes for donor research consent and research project approval with oversight from CBS's CBB and appropriate research ethics boards. The CBRP distributes deidentified CB products to research projects across Canada. RESULTS: Since its inception, the CBRP has distributed more than 525 CB units to researchers, supporting 11 research projects. Of the mothers who donate their baby's CB, 77% have chosen to consent to its use for research if it is not bankable. The number of CB units currently available for research via the CBRP exceeds the requests from researchers. CONCLUSION: The CBRP reliably distributes quality CB products that do not qualify for banking to investigators across Canada in an ethical, legal, and transparent manner. This provides an opportunity for the public to directly support research, helps meet the need expressed by Canada's research community, and maximizes the donor's gift. More research is needed to clarify the factors influencing donor and researcher participation in the CBRP.

Immunoablation and autologous haemopoietic stem-cell transplantation for aggressive multiple sclerosis: a multicentre single-group phase 2 trial.

BACKGROUND: Strong immunosuppression, including chemotherapy and immune-depleting antibodies followed by autologous haemopoietic stem-cell transplantation (aHSCT), has been used to treat patients with multiple sclerosis, improving control of relapsing disease. We addressed whether near-complete immunoablation followed by immune cell depleted aHSCT would result in long-term control of multiple sclerosis. METHODS: We did this phase 2 single-arm trial at three hospitals in Canada. We enrolled patients with multiple sclerosis, aged 18-50 years with poor prognosis, ongoing disease activity, and an Expanded Disability Status Scale of 3.0-6.0. Autologous CD34 selected haemopoietic stem-cell grafts were collected after mobilisation with cyclophosphamide and filgrastim. Immunoablation with busulfan, cyclophosphamide, and rabbit anti-thymocyte globulin was followed by aHSCT. The primary outcome was multiple sclerosis activity-free survival (events were clinical relapse, appearance of a new or Gd-enhancing lesion on MRI, and sustained progression of Expanded Disability Status Scale score). This study was registered at ClinicalTrials.gov, NCT01099930. FINDINGS: Between diagnosis and aHSCT, 24 patients had 167 clinical relapses over 140 patient-years with 188 Gd-enhancing lesions on 48 pre-aHSCT MRI scans. Median follow-up was 6.7 years (range 3.9-12.7). The primary outcome, multiple sclerosis activity-free survival at 3 years after transplantation was 69.6% (95% CI 46.6-84.2). With up to 13 years of follow-up after aHSCT, no relapses occurred and no Gd enhancing lesions or new T2 lesions were seen on 314 MRI sequential scans. The rate of brain atrophy decreased to that expected for healthy controls. One of 24 patients died of transplantation-related complications. 35% of patients had a sustained improvement in their Expanded Disability Status Scale score. INTERPRETATION: We describe the first treatment to fully halt all detectable CNS inflammatory activity in patients with multiple sclerosis for a prolonged period in the absence of any ongoing disease-modifying drugs. Furthermore, many of the patients had substantial recovery of neurological function despite their disease's aggressive nature. FUNDING: Multiple Sclerosis Scientific Research Foundation.

Validation of sterility testing of cord blood: challenges and results.

BACKGROUND: Sterility testing for cord blood (CB) products is mandatory to prevent transplantation-transmitted microbial infections. Here, the automated BacT/ALERT (bioMérieux) culture system was validated to detect microbial contamination in CB units processed at the Canadian National Public Cord Blood Bank. STUDY DESIGN AND METHODS: A three-phase validation was developed. CB units were prepared with pentastarch (Phases 1 and 2) or hetastarch (Phase 3). In Phase 1, CB was spiked with approximately 100 colony-forming units/mL of Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Bacteroides fragilis, and Candida albicans. Plasma (8 mL) and buffy coat (BC; 0.5 and 8 mL) were inoculated into culture bottles. In Phases 2 and 3, a mix of red blood cells (RBCs) and plasma (4 mL each) was used as the inoculant. In Phase 3, Aspergillus brasiliensis was added as a test organism and microbial concentrations in the by-product RBCs and plasma were determined. The BC fractions were cryopreserved and tested 3 months later. RESULTS: In Phase 1, bacteria failed to grow in CB units containing antibiotics. Thus, antibiotic-free units were used for the other phases. C. albicans was not always captured in plasma, but using a mix of RBCs and plasma, all organisms were detected. The use of pentastarch or hetastarch did not affect microbial recovery. C. albicans and A. brasiliensis were preferentially recovered in RBCs and BC. Cryopreservation did not affect microbial survival during CB processing. CONCLUSIONS: A mix of plasma and RBCs is appropriate for CB sterility testing. Interestingly, fungi preferentially segregate to cellular fractions. The clinical significance of the bactericidal /or bacteriostatic effect of antibiotics in CB merits further investigation.

Evaluation of the sterility testing process of hematopoietic stem cells at Canadian Blood Services.

BACKGROUND: Sterility testing of hematopoietic stem cells (HSCs) at The Canadian Blood Services Stem Cell Laboratory is performed using BacT/ALERT aerobic (SA) culture bottles. This study was conducted to verify the efficacy of this method and to assess the use of the BacT/ALERT aerobic (BPA) and anaerobic (BPN) culture bottles for microbial testing of HSCs. STUDY DESIGN AND METHODS: HSC products, including cryopreserved apheresis peripheral blood, marrow, and cord blood and fresh cord blood, were spiked with four aerobic organisms including Staphylococcus epidermidis, Bacillus cereus, Pseudomonas aeruginosa, and Candida albicans, and the anaerobe Bacteroides fragilis at a target concentration of 100 colony-forming units (CFUs)/mL. One to 2 mL of pre- and postspiked samples was inoculated into SA, BPA, and BPN bottles in duplicate and incubated for 5 to 10 days. The presence of the testing organisms in positive culture bottles was confirmed by plating on blood agar. RESULTS: The BacT/ALERT system detected the aerobic organisms in all HSCs in SA and BPA bottles within 34.1 hours while B. fragilis was detected only in BPN bottles within 68.6 hours. The mean recovered concentration of microorganisms in the HSC products ranged from 55 to 352 CFUs/mL with the exception of B. cereus, which was greater than 10(3) CFUs/mL. CONCLUSION: This study shows that the current sterility testing process at the Canadian Blood Services Stem Cell Laboratory detected the tested aerobic but not the anaerobic microbial contaminants in HSCs. The ability of the BacT/ALERT system using BPA and BPN bottles to detect bacterial contamination in HSCs was also demonstrated.

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy.

BACKGROUND AIMS: The ability of hematopoietic progenitor cells-apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented. METHODS: In this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2-7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage). RESULTS: The doses of nucleated and CD34(+) cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10(8) and 6.8 ± 4.3 × 10(6), respectively) and long- (4.0 ± 4.9 × 10(8) and 6.1 ± 3.4 × 10(6), respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10(9)/L (median 11 days for both short- and long-term storage) and platelets >20 × 10(9)/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10(6)/kg CD34(+) cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets). CONCLUSIONS: Cryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.