Automated closed volume reduction process for apheresis stem cell grafts: From development to clinical implementation.

BACKGROUND: Collection of HPC by apheresis (HPC-A) can sometimes result in higher collection volumes, increasing the dimethyl sulfoxide (DMSO) volume infused into patients and the space requirements in liquid nitrogen freezers. Volume reduction prior to the addition of cryoprotectant is an efficient means to reduce the DMSO load infused into patients and to optimize freezer storage space. STUDY DESIGN AND METHODS: To implement a closed semi-automated volume reduction process, a method was developed to produce leukocyte-rich mock apheresis products using buffy coats derived from whole blood collections. The mock HPC products were then used to measure the efficiency and reliability of the semi-automated process over a range of volumes and cell concentrations. The resulting data was used to support the implementation of the process with concurrent monitoring. RESULTS: A closed, semi-automated volume reduction process resulted in recoveries of over 93% and 91% of white blood cells and CD34+ cells with no significant loss of product viability or potency. Mean doses of CD34+ and CFU infused per kilogram recipient body weight were 4.0 ± 1.1 × 106 /kg and 4.2 ± 1.7 × 105 /kg, resulting in no delays in median time to neutrophil and platelet engraftment, significant increase in adverse reaction or nonconformances. DISCUSSION: The effectiveness outcomes of the first Canadian experience in the implementation of a closed semi-automated volume reduction system in the processing of HPC-A products for autologous transplant have met the predetermined acceptance criteria, supporting its use in a stem cell manufacturing laboratory compliant with good manufacturing practice regulations.

MSC-Derived Extracellular Vesicles in Preclinical Animal Models of Bone Injury: A Systematic Review and Meta-Analysis.

BACKGROUND AND OBJECTIVE: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are a promising treatment for bone injuries, although studies remain preclinical. A systematic review and meta-analysis can assess the efficacy of MSC-EVs and identify treatment aspects associated with enhanced bone repair. METHODS: English language, preclinical, controlled, in vivo studies identified in our systematic search (up to May 8, 2020) examining the use of MSC-EVs in bone healing were included. Risk of bias (ROB) was assessed using the SYRCLE tool. Aggregate Data Meta-Analysis was performed to determine the effect of MSC-EVs on Bone Volume/Total Volume (BV/TV) and New Bone Formation (NBF). RESULTS: Thirteen studies were included. Twelve reported either BV/TV or NBF and were included in meta-analysis. ROB was unclear in all studies. Overall, MSC-EVs displayed benefit in terms of bone healing for both BV/TV (22.2% mean difference (MD); 95% CI: 15.8-28.5%, p < 0.001) and NBF (26.1% MD; 10.3-41.8%, p = 0.001) versus controls. Substantial heterogeneity, however, was observed between studies. MSC-EVs were reported to activate multiple signaling pathways including mTOR/AKT, AMPK and BMP2. Subgroup analysis indicated no significant difference in the improvement of BV/TV when using modified EVs isolated after gene transfection, preconditioning (p = 0.61), or using EVs in combination with a tissue scaffold and/or hydrogel versus other delivery methods (p = 0.20). CONCLUSION: Use of MSC-EVs to promote healing of bone injury appears promising, however, heterogeneity between studies and the potential for reporting bias limits confidence in the extent of benefit. Reducing bias between studies and addressing aspects of potential reporting bias should augment confidence in future meta-analyses and propel the field towards clinical studies. Forest Plot analysis assessing the percentage change in bone volume (BV) / total volume (TV) in the presence (experimental) or absence (control) of MSC-EVs.

Multimedia resources to support the recruitment of committed hematopoietic stem cell donors: Perspectives of the most-needed donors.

BACKGROUND: Recruitment of committed unrelated hematopoietic stem cell donors from the most-needed demographics remains a challenge for donor recruitment organizations worldwide. Multimedia resources are gaining attention as a modality to support recruitment efforts; however, there is a lack of guidance for the development of such tools. This qualitative study explores the perspectives of eligible stem cell donors on an educational whiteboard video about stem cell donation, generating insights into how whiteboard videos and related multimedia may be optimized for donor recruitment. STUDY DESIGN AND METHODS: Eight semistructured focus groups were conducted with 38 potential donors from the most-needed demographics (young, male, and non-Caucasian) after they had watched a 3.5-minute whiteboard video explaining key concepts in stem cell donation (https://youtu.be/V4fVBtxnWfM). Constructivist grounded theory was used to identify themes and to develop a framework for understanding participants' preferred features of recruitment multimedia. RESULTS: Participants identified a range of features contributing to the effectiveness of recruitment multimedia, adding that the whiteboard video is an effective, integrated, and readily accessible format for supporting donor recruitment. Topics that participants felt are important to address include knowledge gaps regarding donation procedures, concerns about donor safety, and the particular need for specific donor demographics. Suggested avenues for improvement include the addition of donor/recipient/patient personal experiences, attention-grabbing hooks, and a call to action including opportunities for further learning. CONCLUSIONS: Several considerations were generated to inform the development of future multimedia for donor education/recruitment and are relevant to donor recruitment organizations worldwide.

Factors associated with registrant availability for unrelated adult donor hematopoietic stem cell donation: Analysis of the stem cell registry at Canadian Blood Services.

BACKGROUND: Greater use of unrelated donors to support hematopoietic cell transplantation can be hampered by unavailability of registrants when identified as potential candidates for donation. METHODS: Multivariate analysis was performed to identify donor factors associated with availability for verification of human leukocyte antigen typing (VT) needed before donor activation. All VT requests for registrants on the Canadian Blood Services Stem Cell Registry between 1 January and 31 December 2018 were reviewed (n = 1358). RESULTS: Potential donors identified by transplant centers were categorized as available at the time of VT but ineligible for medical or other reasons (n = 130 and excluded from further analysis), available (n = 622) or unavailable (n = 566) due to scheduling, loss of interest, and/or inability to contact. With multivariate analysis, registrants who previously donated blood, those recruited online or from blood donation clinics, and a shorter interval between registration and VT request were significantly correlated with increased donor availability. Donor sex and geographic location, however, displayed no correlation. CONCLUSION: Online registration and recruitment at whole blood donation centers should be enhanced to increase the availability of registrants at VT. More insight is needed to maintain registrant availability following community in-person recruitment events, especially if the interval between registration and activation is prolonged. Recruitment of male registrants who are well informed should not negatively impact availability.

Development and evaluation of stem cell collection procedure diagrams to support the education and recruitment of committed stem cell donors.

BACKGROUND: Diagrams which allow potential unrelated stem cell donors to visualize the stem cell collection process were hypothesized to support the recruitment and education of committed stem cell donors. STUDY DESIGN AND METHODS: A series of bone marrow and peripheral blood stem cell collection procedure diagrams were developed, featuring young adult male donors of varied ethnic backgrounds. Post-implementation, surveys were conducted to evaluate stakeholder perspective on the diagrams' utility. A quality improvement project was conducted at five stem cell drives from 2017 to 2018 at which recruiters did or did not show the diagrams to potential donors. Following the drives, registrants were invited to complete a survey exploring their experience, knowledge and attitude towards donation. RESULTS: The diagrams were implemented in Canada in 07/2016. Of 293 participating registrants (24·7% non-Caucasian males) recruited at five drives between 2017 and 2018, 76% (n = 197) were shown the diagrams. Participants who were shown the diagrams were significantly more likely to report that the recruiters appeared very knowledgeable (89% vs. 76%, P = 0·019) and to report improved self-reported knowledge of stem cell donation (P = 0·010) compared to participants not shown the diagram. Data are also shown demonstrating that stakeholders in donor recruitment used and valued the diagrams and that use of the diagrams was associated with improved donor recruitment outcomes in Canada. CONCLUSION: This report is the first evaluation of stem cell collection diagrams in the literature. The diagrams are relevant to donor registries, recruitment organizations and transplant centres worldwide, and their use may support efforts to educate and recruit committed, ethnically diverse donors.

Development and Evaluation of a Whiteboard Video Series to Support the Education and Recruitment of Committed Unrelated Donors for Hematopoietic Stem Cell Transplantation.

Whiteboard videos are a popular video format, allowing viewers to see drawings of concepts alongside explanatory text and speech. We hypothesized that whiteboard videos could support the education and recruitment of unrelated stem cell donors in Canada. A series of 5 sharable whiteboard videos about stem cell donation was produced and posted online in September 2018, including 1 full-length video (https://youtu.be/V4fVBtxnWfM) and 4 shorter videos titled "What Is Stem Cell Transplantation?" "How Does the Matching Process Work?" "How Are Stem Cells Donated?" and "How Can I Register as a Stem Cell Donor?" In the videos, metaphorical interpretations of stem cells as factories and genetic markers as barcode labels are employed to communicate complex concepts. The particular need for young, male, and ethnically diverse donors is reflected in the characters portrayed. Surveys demonstrated the videos (1) were used and valued by stakeholders in donor recruitment and (2) significantly improved objective and self-reported knowledge about stem cell donation and reduced donation-related ambivalence among viewers from the most-needed donor demographics. Use of the whiteboard videos was also associated with improved donor recruitment outcomes in Canada. Our work is relevant to donor registries and recruitment organizations worldwide that seek to improve their recruitment efforts.

Improved access to better HLA-matched hematopoietic cells for allogeneic transplant: analysis of donors and cord blood units selected for Canadian patients in 2018.

BACKGROUND: Patients undergoing hematopoietic cell transplantation (HCT) often require use of an unrelated donor or cord blood unit (CBU). An understanding of evolving practices in graft selection is needed for optimization of donor recruitment and cord blood collection. STUDY DESIGN AND METHODS: Each donor workup (WU) requested in 2018 involving a Canadian (CDN) patient and unique donor product or CBU was reviewed (n = 598). Degree of HLA match; product origin (domestic or international [INT]); and non-HLA factors including donor age, sex, cytomegalovirus (CMV), and ABO compatibility were analyzed for WUs that proceeded to transplant (n = 414). We also analyzed changes compared to a similar analysis performed in 2013. RESULTS: The majority of transplants used matched unrelated donors (MUDs; n = 323; 78%) and were most often young (≤35 years), male, INT donors (n = 136). The proportion of transplants involving MUDs, as opposed to mismatched unrelated donors or CBUs, increased by 12.4% compared with 2013. When young, male, CDN MUDs were identified in patient search reports but not selected, CMV mismatching and ABO incompatibility were most likely to have influenced the decision to use an INT MUD. Consistent with global trends, CBU transplants decreased compared to 2013; however, the degree of HLA matching improved significantly, and 27% of transplanted CBUs were procured from the Canadian Blood Services Cord Blood Bank. CONCLUSIONS: Access to MUDs and better HLA-matched CBUs by CDN patients has increased since 2013. Ongoing recruitment of young registrants and cord blood donors with diverse HLA haplotypes will support selection of donors with optimal non-HLA characteristics.

Obstetrical and neonatal factors associated with optimal public banking of umbilical cord blood in the context of delayed cord clamping.

PURPOSE: To assess the association of specific newborn and maternal factors with indicators of increased blood-forming capacity in umbilical cord blood to inform strategic collection strategies that could augment the quality of units in public cord blood banks. METHODS: Data regarding 268 consecutive cord blood units (CBUs) banked by Canadian Blood Services were analyzed. Multivariate analysis was performed to identify factors associated with markers of hematopoietic potency and likelihood of utilization. RESULTS: Delayed clamping of the cord beyond 60 s was associated with reduced volume collected. Any delay in clamping of the cord was associated with reduced total nucleated cell counts. Newborn weight >4,000 g was also associated with greater blood volume in the collection but not with other measures of hematopoietic potency. Cord blood acidosis at birth (pH.

Reducing ethnic disparity in access to high-quality HLA-matched cord blood units for transplantation: analysis of the Canadian Blood Services' Cord Blood Bank inventory.

BACKGROUND: Launched in 2013, Canadian Blood Services' Cord Blood Bank (CBS' CBB) has built a high-quality, ethnically diverse cord blood repository that aims to reduce ethnic disparity in accessing suitable units for transplantation. METHODS AND RESULTS: As of December 2016, 2000 units have been banked. The self-reported maternal ethnicity was 58% non-Caucasian. Overall, 26% of units were classified as multi-ethnicity with Caucasian (84%) most frequently observed in combination with Asian, First Nations (predominant indigenous peoples in Canada south of the Arctic Circle), or African ethnicity. Utilization scores that incorporate total nucleated and CD34+ cell counts in the CBS' CBB were associated with greater likelihood of utilization compared with the international inventory of units (p < 0.05). The distribution of utilization scores was similar for Caucasians compared with non-Caucasians (p < 0.05). Using HLA genotypes of cord blood units and their mothers, we determined probable ethnic assignments for each haplotype using HaploStats (National Marrow Donor Program). Significant increases in HLA-match likelihoods are predicted for all ethnicities as the inventory grows to its target of 10,000 units and the gap in HLA-match likelihoods for Caucasian and non-Caucasian patients progressively declines. CONCLUSIONS: The CBS' CBB inventory is predicted to have high HLA-matching likelihoods across a broad spectrum of ethnic groups, improving access to high-quality stem cell products for all patients.

Risk of Exposure to Zika Virus and Impact on Cord Blood Banking and Adult Unrelated Donors in Hematopoietic Cell Transplantation: The Canadian Blood Services Experience.

Zika virus has emerged as a potential threat to the Canadian blood supply system. Stem cell donors within Canadian Blood Services' Cord Blood Bank (CBB) and OneMatch Stem Cell and Marrow Network (OM) now undergo screening measures designed to reduce the risk of Zika virus transmission. The impact these screening measures have on cord blood and unrelated adult stem cell donations is currently unknown. Among 146 donor workups initiated by OM between July 2016 and May 2017, 102 were completed and 44 workups were canceled. There were 17 potential donors (11.6%) with a risk of Zika virus exposure identified by the donor questionnaire (13 completed, 4 canceled workups). None of the workups involved a donor diagnosed with confirmed Zika virus within the past 6 months. Only 1 of the 44 canceled workups (and only 1 of 4 cases with a risk of Zika transmission) was canceled because of the risk of Zika transmission, and a backup donor was selected. Canadian Blood Services' CBB identified 25 of 875 cord blood units (2.9%) from women who donated their infants' cord blood and underwent screening that otherwise met the initial cell number thresholds for banking and had at least 1 risk factor for exposure to Zika virus. No women were diagnosed with Zika virus at any point of their pregnancy. All 25 units were discarded. Unrelated donors at OM have a higher incidence of a risk of exposure to Zika virus compared with cord blood donors. Only rarely did transplant centers cancel donor workups due to potential Zika virus exposure. The impact of screening for Zika virus exposure risk on cord blood banking was minor. Continued vigilance and surveillance is recommended.

Development and testing of a stepwise thaw and dilute protocol for cryopreserved umbilical cord blood units.

BACKGROUND: It is clinically important to maintain high viability and potency of umbilical cord blood units (CBUs) for transplantation during thawing. In the absence of a standard thawing protocol, this study was designed to develop one based on the consensus practice of transplant centers and address the shortage of dextran 40 thawing solution. STUDY DESIGN AND METHODS: Frozen CBU aliquots were thawed using dextran 40 thawing solution while manipulating temperature and volume of diluent and mode of dilution. The effects of these on CD45+ and CD34+ cell viability were measured through annexin V and SYTOX green staining. The developed protocol was then used to compare dextran 40 and PLASMA-LYTE A thawing solutions and finally tested on whole CBUs. RESULTS: Step-by-step investigations resulted in the development of a protocol that thaws and dilutes CBUs with room temperature diluent to five times the original volume using two sequential dilutions separated by equilibration times. PLASMA-LYTE A diluent provided superior viability of CD45+ and CD34+ cells than dextran 40 and recovered more colony-forming units. However, both diluents were equally effective in maintaining stability of the thawed CBU for 4 hours. Moreover, the stem cell-enriched CD34+CD38- subpopulations appeared more resistant to cryoinjuries than their CD34+CD38+ counterpart. CONCLUSION: The developed thawing protocol recovers viable CD45+ and CD34+ cells above the standard thresholds and maintains CBU potency. PLASMA-LYTE A for thawing solution proved to be an efficient alternative to dextran 40. Finally, greater dilution should be avoided to maintain the viability of CD45+ cells and maximize graft cell dose.

Safety of Living Donation of Hematopoietic Stem Cells.

More than 12 000 volunteer unrelated hematopoietic stem cell donations are undertaken annually, and the World Marrow Donor Association established an expert committee to examine all reports of adverse events affecting donors globally, eventually making such reporting a necessary part of World Marrow Donor Association accreditation. The committee evaluates and responds to reported events in a nonpunitive confidential process designed to alert the community of rare events which might be missed by local follow-up. Each report is evaluated by the committee for imputability (causal link between the donation and the adverse event) and compared with that submitted by the reporting registry. In 2014, there were 50 reports received from 16 different registries in 15 countries. There were 16 reports of malignancies arising in donors including 3 hematologic malignancies. All but 2 of the 16 occurred more than a year after donation. There were 4 reports of autoimmune phenomena in donors all occurring more than a year postdonation. Of the 30 remaining events, 6 were allergic, 4 cardiac, 3 gastrointestinal, 2 infections, 2 pulmonary, and 13 miscellaneous. Causation was assessed differently to the reporting registry in 17 events with 6 thought to be less likely causally linked to the donation and 10 more likely with 1 requiring more information. Volunteer unrelated hematopoietic stem cell donation is a safe and effective altruistic contribution to the treatment of patients with life-threatening hematologic disorders. A decade of detailed examination of adverse donor events has contributed to the safety of these donations.

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.

Cord blood clinical processing, cryopreservation, and storage.

Allogeneic umbilical cord blood (UCB) hematopoietic stem cell transplantation has become a crucial advancement in the treatment for a variety of diseases including hematopoietic and non-hematopoietic malignancies, BM failure syndromes, hemoglobinopathies, and metabolic and immunodeficiency disorders. It has been well documented that the success of UCB engraftment is tied to UCB banking processes, and now there are established guidelines for standardization of collection, banking, processing, and cryopreservation for unrelated UCB units with purpose of achieving consistent production of high quality placental and UCB units for administration. In 2011, Canada's Ministry of Health has announced Canada's first national, publicly funded umbilical cord blood bank, which aims to provide altruistic donations for unrelated allogeneic hematopoietic stem cell transplant. In this chapter, we describe specific protocols for clinical processing, cryopreservation, and storage of UCB used by the Canadian Blood Services National Public Umbilical Cord Blood Bank.

Impact of ethnicity on human umbilical cord blood banking: a systematic review.

BACKGROUND: How ethnicity impacts characteristics of umbilical cord blood collected by cord blood banks remains unclear. STUDY DESIGN AND METHODS: After a systematic search, we identified 11 studies for analysis that reported on various variables of collected cord blood units. RESULTS: Non-Caucasian ethnicity of the cord blood donor was associated with a higher risk of failing to meet banking criteria, lower cord blood volume, reduced total nucleated cell count, fewer CD34+ cells, and reduced colony-forming units. CONCLUSIONS: Non-Caucasian ethnicity is associated with reductions in hematopoietic measures of collected cord blood units. Public banking efforts will have to balance issues related to building ethnic diversity within the bank and maintaining the characteristics of banked units that remain desirable by transplant centers.

A review of factors influencing the banking of collected umbilical cord blood units.

Umbilical cord blood banking efforts have increased dramatically in the past two decades in response to increasing demand for alternative sources of blood stem cells to support patients requiring hematopoietic stem cell transplantation. Transplant centres have accumulated increasing expertise in their understanding of umbilical cord blood characteristics that are associated with improved outcome following transplantation. These characteristics and factors can assist transplant centres in selecting cord blood units from the worldwide inventory of banked units. Umbilical cord blood banks, therefore, need to remain agile in adjusting the inventory of the banks to address shifts or changes in the needs of transplant centres. Public umbilical cord blood banks face the challenge of building inventory while managing limited resources and are faced with decisions regarding which units can be stored and which units that have been collected should be discarded or used for other endeavours such as research. To this end, we sought to review parameters influencing the decision to bank a collected cord blood unit. In this paper, we will address parameters associated with graft potency and address other factors that guide the decision to bank collected units.

Trehalose transporter from African chironomid larvae improves desiccation tolerance of Chinese hamster ovary cells.

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.

Osmotic transport across cell membranes in nondilute solutions: a new nondilute solute transport equation.

The fundamental physical mechanisms of water and solute transport across cell membranes have long been studied in the field of cell membrane biophysics. Cryobiology is a discipline that requires an understanding of osmotic transport across cell membranes under nondilute solution conditions, yet many of the currently-used transport formalisms make limiting dilute solution assumptions. While dilute solution assumptions are often appropriate under physiological conditions, they are rarely appropriate in cryobiology. The first objective of this article is to review commonly-used transport equations, and the explicit and implicit assumptions made when using the two-parameter and the Kedem-Katchalsky formalisms. The second objective of this article is to describe a set of transport equations that do not make the previous dilute solution or near-equilibrium assumptions. Specifically, a new nondilute solute transport equation is presented. Such nondilute equations are applicable to many fields including cryobiology where dilute solution conditions are not often met. An illustrative example is provided. Utilizing suitable transport equations that fit for two permeability coefficients, fits were as good as with the previous three-parameter model (which includes the reflection coefficient, sigma). There is less unexpected concentration dependence with the nondilute transport equations, suggesting that some of the unexpected concentration dependence of permeability is due to the use of inappropriate transport equations.