Cord Blood Units Research Articles

Haploidentical Hematopoietic Cell Transplantation Combined with an Unrelated Cord Blood Unit for Adult Acute Myeloid Leukemia Results in Improved Survival Compared to Haploidentical Hematopoietic Cell Transplantation: Results of a Multicenter, Randomized, Phase III Trial

Introduction Haploidentical hematopoietic cell transplantation (haplo-HCT) is a potent treatment to improve the prognosis of acute myeloid leukemia (AML), but relapse and graft-versus-host disease (GVHD) related to dysregulation of alloreactivity are still the main causes of death posttransplantation. Cord blood graft usually has a promising graft-versus-leukemia effect with a lower cumulative incidence of GVHD. To improve the transplantation strategy, haplo-cord HCT combining haploidentical and unrelated cord blood (UCB) grafts (haplo-cord HCT) has been attempted. Previous studies showed significant improvement in prognosis in B-cell acute lymphoblastic leukemia or refractory acute leukemia patients with haplo-cord HCT compared to haplo-HCT. However, outcomes of haplo-cord HCT for AML patients are not yet clear. This is the first multicentric randomized trial designed specifically for the efficacy and safety of haplo-cord HCT for AML. Methods This is a multicenter, randomized, open-label, phase III study conducted in 5 centers in China (NCT03719534). Patients aged 18-60 years, diagnosed with AML (except for acute promyelocytic leukemia) with measurable residual disease, had an available haploidentical donor and were suitable for allotransplantation were enrolled. Patients were randomized 1:1 to receive UCB and haploidentical grafts infusion after Bu/Cy-based regimen or haploidentical graft infusion only after Bu/Cy-based regimen. The UCB unit, which had at least 3/6 matched HLA loci to the recipient and more than 1×10 4 CD34 + cells/kg of recipient weight, was infused immediately after resuscitation and eight hours before the haploidentical graft infusion. The primary endpoint was 3-year overall survival (OS) and secondary endpoints included progression-free survival (PFS), cumulative incidence of relapse (CIR), and non-relapse mortality (NRM). Results From June 1, 2017, to June 30, 2021, 268 patients were enrolled (Table 1). With a median follow-up of 36.00 months, the 3-year OS was 80.5% (95% CI 73.7-87.9) in the haplo-cord HCT group and 67.8% (95% CI 60.0-76.5) in the haplo-HCT group, respectively (p=0.013, figure 1A). Haplo-cord HCT group showed favorable 3-year PFS and CIR than haplo-HCT group (PFS: 70.3%, 95% CI 62.6-78.8 vs. 57.6%, 95% CI 49.6-67.0, p=0.012; CIR: 12.1%, 95% CI 12.0-12.2 vs. 30.3%, 95% CI 30.1-30.4, p=0.024, figure 1B,C). The 3-year NRM was similar in the two groups (figure 1D). Within two years posttransplantation, the most common grade 3-4 adverse events (AEs) in the haplo-cord HCT and haplo-HCT groups were infections (33.8% and 29.1%), acute GVHD (20.3% and 12.7%) and chronic GVHD (12.0% and 16.4%). AEs leading to death occurred in 8.3% of haplo-cord HCT and 14.9% haplo HCT patients. Post- hoc analysis showed similar median levels of haploidentical chimerism in bone marrow (BM) between the two groups. However, UCB microchimerism was detected in the haplo-cord HCT patients by SNP-NGS in +1m posttransplantation (BM: 0.3%, IQR 0.04-1.6; peripheral blood: 0.06%, IQR 0.03-0.4) and its chimerism percentage decreased with time (figure 1E). Compared to haplo-HCT group, haplo-cord HCT group showed a faster cumulative incidence of neutrophil recovery (p=0.026, figure 1F) and a similar cumulative incidence of platelet recovery. Monitor of immune reconstitution showed a more rapidly increasing rate of T cells in the early period after transplantation in the haplo-cord HCT group (figure 1G). Improved recovery of Th2 (p=0.042), Th17 (p=0.047), Treg (p=0.045), and Tc2 (p=0.045) cells was observed in haplo-HCT group. Conclusions Coinfusion of a UCB unit in haplo-HCT can improve OS in AML patients without excessive AEs, and affect T-cell reconstitution pattern posttransplantation. Haplo-cord HCT could serve as a suitable therapeutic option for this patient population.

Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood

Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.

The effect of caffeine intake and passive smoking on umbilical cord blood unit's quality parameters.

Today cord blood (CB) is a valuable source of hematopoietic stem cells to treat many hematological disorders. One of the limitations of CB utilization is the reduced number of nucleated cells including stem cells. Therefore, CB banks around the world have developed strategies in an attempt to improve donor selection and the quality of the CB inventory. This study aimed to determine the impact of passive smoking and caffeine consumption on CB quality. CBs were obtained from mothers who gave birth at King Abdulaziz Medical City. All mothers gave their informed consent. Personal interviews about the mother's demographics, smoking status and exposure, and caffeine consumption executed, followed by a chart review to analyze maternal and neonatal factors. Laboratory testing was performed on all collected CB units. Using descriptive statistics, maternal and newborn factors were analyzed. T-test or Mann-Whitney U Test, as appropriate, for continuous variables analysis to study the effect of second hand smoking and coffee consumption for the primary outcome. Our study demonstrated a reduction in CB MNC, including lymphocytes, in caffeine consumers among pregnant donors, as well as a reduction in cell potency activities, including total CFU and BFU-E. The effect of passive cigarette smoking on the same cohort was insignificant. Outcome of this study will help in optimizing the quality and quantity of stem cell harvesting from CB to get the maximum benefit and such knowledge will raise the awareness among pregnant women.

P‐BT‐1 | What to Expect When You Request Post‐Thaw Segment QC Testing on a Cord Blood Unit?

P‐BT‐1 | What to Expect When You Request Post‐Thaw Segment QC Testing on a Cord Blood Unit?

HLA-haplotype redundancy and rareness in Canadian Blood Services' Stem Cell Registry and Cord Blood Bank: Novel metrics for optimizing utility.

The utility of unrelated donor registries that support allogeneic hematopoietic cell transplantation could be optimized through greater understanding of redundancy and rareness of HLA phenotypes. HLA phenotype rareness was determined using known HLA haplotype frequencies. Donor redundancy was determined through pairwise comparison of donor HLA profiles within an inventory. Among 61,730 registrants in the Canadian Blood Services (CBS) Stem Cell Registry (SCR) with high resolution HLA typing at 5 loci, 6.6% of HLA phenotypes were redundant with variation across ethnic groups (8.3% of Caucasian phenotypes; 8% of Native American/First Nations, 4.4% of Asia-Pacific Islanders (API), 2.1% of Hispanic, 0.7% of African-American (AFA), and 4.5% of other ethnicities). A total of 18.5% of registrants had redundant HLA phenotypes with variation across ethnic groups. All 3716 cord blood units in the CBS's cord blood bank (CBB) had high resolution HLA typing at 5 loci and 202 units were redundant (5.4%) comprising 78 HLA phenotypes, with varying rareness. Repeated HLA phenotypes were from Caucasian donors (77%), multiple ethnicity (13%), API (9%), and AFA (1%). Registrants and CBUs with AFA ethnicity had the rarest phenotypes while Caucasian ethnicity was associated with the most common HLA phenotypes. Redundancy was greater in the SCR compared to the CBB and was most common with CAU ethnicity. Recruiting non-Caucasian registrants and continued cord blood banking should reduce redundancy. A sub-inventory of redundant donors and cord blood units could support new uses for donor-supported cellular therapies that do not require HLA matching.

Haploidentical Cord Blood Transplantation with 8 mg/kg Antithymocyte Globulin as Graft-versus-Host Disease Prophylaxis Compared to Haploidentical Transplantation with 10 mg/kg Antithymocyte Globulin in the Treatment of Acute Leukemia

Clinical outcomes of the transplantation strategy combined with a haploidentical stem cell graft and an unrelated umbilical cord blood unit (haplo-cord HSCT) with low-dose antithymocyte globulin (ATG) as graft-versus-host disease (GVHD) prophylaxis for the treatment of acute leukemia remains unclear. This study aimed to explore the clinical outcomes of haplo-cord HSCT in acute leukemia patients with the GVHD prevention strategy of 8 mg/kg ATG compared with haploidentical transplantation with 10 mg/kg ATG. A total of 130 patients with acute leukemia who underwent allogeneic HSCT between January 2016 and December 2020 were included in this study, including 70 patients who received haploidentical stem cell grafts and unrelated umbilical cord blood units (haplo-cord HSCT) with 8 mg/kg ATG (haplo-cord-ATG8 group) and haploidentical HSCT with 10 mg/kg ATG (haplo-ATG10 group) in 60 patients. Clinical data were collected and analyzed retrospectively. Patients in the haplo-cord-ATG8 group were significantly older compared with the haplo-ATG10 group (P=.000). Haplo-cord HSCT with reduced ATG to 8 mg/kg results in more rapid neutrophil recovery (P=.036). No between-group differences were observed in platelet recovery or the incidences of Epstein-Barr virus viremia, bloodstream infection, or hemorrhagic cystitis. The rate of grade II-IV acute GVHD by day 100 post-transplantation was higher in the haplo-ATG10 group (27.16% versus 11.48%; P=.033), as was the rate of chronic GVHD at 1 year (14.60% versus 3.36%; P=.048). The rate of cytomegalovirus reaction was higher in the haplo-ATG10 group (48.31% versus 26.30%; P=.022). With a median follow-up of 27.4 months for the haplo-cord-ATG8 group and 27.5 months for the haplo-ATG10 group, overall survival (OS) at 2 years was 79.4% versus 62.8% (P=.005), event-free survival (EFS) was 76.3% versus 55.9% (P=.001), the cumulative incidence of relapse was 10.11% versus 25.97% (P=.164), and nonrelapse mortality (NRM) was 14.33% versus 24.43% (P=.0040). Multivariate analysis identified Center for International Blood and Marrow Transplant Research Disease Risk Index was the sole significant predictor of relapse, NRM, OS, and EFS. Haplo-cord HSCT supported by cord blood with 8 mg/kg ATG as GVHD prophylaxis results in better outcomes compared with haplo-HSCT with 10 mg/kg ATG.

Umbilical Cord Blood Processing Techniques and Their Comparative Advantages: A Review

Background: Umbilical Cord Blood (UCB) has steadily gained prominence in haematopoietic stem cell transplantation (HSCT). Despite UCB advantages, the main disadvantage of UCB in haematopoietic stem cell transplantation (HSCT) is its limited cell dose. Initially, UCB used to be processed and then made to undergo cryopreservation as whole cord blood banking leading to the problem of storing sufficiently large number of cryoprotected UCB units which requires vast amounts of costly storage space in liquid nitrogen. The sole purpose of processing is to concentrates the stem cells and reduce the volume for storage. Different UCB processing methods have been developed. 
 Aim: This review is aimed at bringing together the literature on the different processing methods and highlighting the underlying principles of each method, the relative efficiency and advantages of the methods.
 Methodology: The work involved mainly the critical review of all available academic, professional and industry documents on cord blood processing. The relevant information was obtained from textbooks, academic journals, conference proceedings, the internet among others. The major UCB processing methods include Plasma Depletion, Density Gradient Centrifugation (DGC), Hetastarch, PrepaCyte-CB and Sepax. A study of the potential impact of Hetastarch and PrepaCyte processing methods on transplantation outcomes revealed no difference that was significant was observed between patients receiving cells after the processing regimens were compared.
 Results: A comparison of the engraftment time of PrepaCyte-CB with five other processing methods revealed a quicker engraftment time for PrepaCyte-CB processed cord blood units compared to other processing methods. PrepaCyte-CB also recovers significantly more viable stem cells than AutoXpress (AXP) and hydroxyethyl starch (HES) processing methods. Other workers demonstrated that Sepax depletion produces higher recovery of cells that are nucleated. The effect initial volume of cord blood had on the recovery of nucleated cells for the different method of processing were also compared. Recovery when using Sepax is reduced as the unit size processed increases. Hetastarch, which is a density gradient, and plasma depletion separation is also affected in like manner, however, processing done using PrepaCyte-CB was not affected by the initial volume of the collected unit. The advantage of Sepax is that it is fully automated and this allows for mass processing of samples, suitable for bigger cord blood banks. For erythrocyte removal, density gradient separation is a better method that is effective. PrepaCyte-CB is the second most efficient method for removing RBC. The result of Total Nucleated Cells (TNC) and Mono Nucleated Cells (MNC) recovery rate of Hespan and Sepax against AXP processing methods shows that both Hespan and Sepax reproducibly recover greater than 95% of the cord blood stem cells in a typical collection and result in a reduced final volume for final storage.
 Conclusion: The five most popular processing techniques are Plasma Depletion, Density Gradient, Hetastarch, PrepaCyte-CB and Automated Centrifugal Machine (Sepax). Most methods involve centrifugation, sedimentation and/or filtration for reducing the red cell content, plasma volume, or both. The different UCB processing methods each has its advantages and disadvantages.

Hospitalization and Healthcare Resource Utilization of Omidubicel-Onlv versus Umbilical Cord Blood Transplantation for Hematologic Malignancies: Secondary Analysis from a Pivotal Phase 3 Clinical Trial

A phase 3 trial (ClincialTrials.gov identifier NCT02730299) of omidubicel-onlv, a nicotinamide-modified allogeneic hematopoietic progenitor cell therapy manufactured from a single umbilical cord blood (UCB) unit, showed faster hematopoietic recovery, reduced rate of infections, and shorter hospital stay compared with patients randomized to standard UCB. This prospective secondary analysis of the phase 3 trial characterized resource utilization in the first 100 days post-transplantation with omidubicel-onlv compared with UCB. This analysis examined resource utilization, including hospital length of stay, hospital care setting, visits by provider type, rate of transfusions, and readmissions, among the 108 treated patients (omidubicel-onlv, n = 52; UCB, n = 56) from day 0 to day 100 post-transplantation. Demographics were generally balanced between the 2 arms, except a higher proportion of females (52% versus 37%) and older median age (40 years versus 36 years) were noted in the omidubicel-onlv arm. Compared with patients receiving UCB transplantation, patients receiving omidubicel-onlv had a shorter average total hospital length of stay (mean, 41.2 days versus 50.8 days; P = .027) in the first 100 days post-transplantation and more days alive and out of the hospital (mean, 55.8 days versus 43.7 days; P = .023). Fewer patients died in the omidubicel-onlv arm compared with the UCB arm (12% vs 16%) before day 100 post-transplantation. During primary hospitalization (ie, time from transplantation to discharge), fewer patients receiving omidubicel-onlv required intensive care unit (ICU) admission (10% versus 23%) and spent fewer days in the ICU (mean, .4 day versus 4.7 days; P = .028) and transplant unit (mean, 25.3 days versus 32.9 days; P = .022) compared with those receiving UCB. Patients receiving omidubicel-onlv required fewer outpatient consultant and nonconsultant visits and fewer platelet or other transfusions within 100 days from transplantation. Our findings suggest that faster hematopoietic recovery in omidubicel-onlv patients is associated with significantly shorter hospital stay and reduced healthcare resource use compared with UCB.

Abstract 16 Insights into Highly Engraftable Hematopoietic Cells from 27-Year Cryopreserved Umbilical Cord Blood

Abstract Introduction Cord blood banking has consistently outpaced the utilization of cord blood units (CBUs). Thus, the average duration of cryopreservation among banked CBUs will likely continue to increase. It remains unclear how long cryopreserved CBUs remain functional, and it is critical to determine whether duration of cryopreservation should be used as an exclusionary criterion during selection for clinical use or if alternative post-thaw metrics can identify potent cryopreserved CBUs regardless of age. Objectives Our goal was to determine whether long-term (27-year) cryopreserved CBUs retain viable and functional hematopoietic stem (HSCs) and progenitor cells (HPCs). We further sought to leverage differences in HSC/HPC function (measured by in vivo engraftment) to demonstrate the utility of using omics approaches to identify candidate genes for use as molecular potency markers. Methods We performed comprehensive ex vivo, in vivo, and molecular analyses on the numbers, viability, and function of three 27-year cryopreserved CBUs using 3-year cryopreserved and fresh CBUs for comparison. Assays included viability staining, immunophenotyping by flow cytometry, primary and secondary colony forming unit (CFU) assays, ex vivo expansion of immunophenotypic HSCs/HPCs/CFUs, limiting dilution transplantations into immune-deficient mice, secondary transplantations, and RNA-sequencing of sorted HSCs and multipotent progenitor cells. Results Compared to fresh and recently cryopreserved CBU controls, long-term cryopreserved CBUs yield statistically similar numbers of viable immunophenotypic HSCs, multipotent HPCs, and committed myeloid and lymphoid HPCs. They retain highly functional cells, demonstrating similar primary and secondary CFU numbers and expansion capacity compared to controls, as well as robust engraftment, SCID repopulating cell frequency, and secondary engraftment capacity in mouse models of transplantation. Transcriptomic modelling revealed 18 genes, including MALT1 and MAP2K1, and several gene programs, including lineage determination programs and oxidative stress responses, that are strongly enriched in high engrafting HSCs/HPCs. Discussion CBUs cryopreserved for up to 27 years retain highly functional HSCs/HPCs. Thus, duration of cryopreservation alone is not an ideal exclusionary criteria for selection of CBUs. Preserving older CBUs may help to maintain a large and diverse pool of donors for clinical selection. Further, transcriptomics can identify candidate genes associated with engraftment for elucidation of possible CBU potency markers regardless of the duration of cryopreservation.

Abstract 15 A State-Run Public Cord Blood Collection Program

Abstract Introduction The Arizona Public Cord Blood Program was created in 2011 by the Arizona Biomedical Research Centre, a sub-section of the Arizona Department of Health Services, to advance the collection and increase the number of racially and ethnically diverse cord blood units available for transplantation, as well as promote awareness of the benefits of cord blood stem cells through our educational partner, Save the Cord Foundation (STCF). Objectives Create a sustainable program for women of all racial and ethnic backgrounds to have the opportunity to donate cord blood, with the primary goal of transplantation, and secondary goal of providing non-transplantable cord blood units for research. A second objective was to educate the residents of the state of Arizona about cord blood stem cells and the need for their preservation. Methods A portion of state lottery funds supports the program monetarily. Those funds are provided to four collection hospitals who employ "cord blood consenters" whose responsibility it is to consent patients, assist delivery providers with collections, and package and ship cord blood units to our partner cord blood bank at MD Anderson Cancer Center. There are also two clinical coordinators who educate and train hospital staff on quality collection practices, with special emphasis on the importance of high volume, sterile collections. STCF provides education across the state to expectant parents, healthcare providers, schools, and the public about the need for cord blood stem cell donation for transplant and research. Results Since 2011, the Arizona Public Cord Blood Program has banked hundreds of racially and ethnically diverse cord blood units with the National Marrow Donor Program, and has had 86 life-saving cord blood units matched with patients in need around the globe. This innovative program has not only expanded cord blood awareness and promoted the preservation of cord blood; it has resulted in the creation of an economic engine for the state of Arizona that is an attractant for STEM based businesses and careers. Discussion A decade later, the Arizona Public Cord Blood Program has proven to be a sustainable model for collecting and providing suitable cord blood units for transplant to diverse patient populations.

Abstract 11 Monitoring Cord Blood Inventory Reliability by Key Quality Indicators At and After Release

Abstract Introduction As per FACT standards, Cord Blood Banks (CBB) have to verify that they provide a safe and effective product for transplant. Validated processing methods and storage conditions are the first steps towards this goal, but what are the final steps to monitor the quality of the cord blood units (CBU) released for transplant? Objectives We present here a retrospective analysis of our Program’s final verification process to meet this FACT requirement. Methods CBU shipped between January 2018 and December 2022 were reviewed for out of specification events. The following Quality Control (QC) parameters were measured on attached segments before shipment: total nucleated cells (TNC), viable CD34 cells (vCD34), and colony forming units (CFU). Confirmed date of infusion and engraftment data were obtained from CIBMTR. Results A total of 619 CBU were shipped during the study period. 550 had a segment analyzed at the time of shipment. Average post-thaw CD34 viability on the segments was 93.5% (Table 1). Average recoveries for TNC, viable CD34 and CFU were 83.3%, 64.90% and 60.86%, respectively (Table 1). 6 (1%) CBUs were found to have CD34 viability lower than FACT requirement (≥70%) (Table 1). Investigations were performed for each CBU, concluding that these were technical issues with no repercussion on the final product. No segments demonstrated absence of CFU growth. 544 CBUs had an infusion date recorded by CIBMTR (Table 1). 250 of the CBU infused had engraftment data, and 242 engrafted. 8 (3%) CBU transplants resulted in non-engraftment. All had QC results within expected range (Table 1). Discussion Our QC results confirm the continued stability of the CBU until shipment. The small subset of CBU with out of specification results were linked to technical issues and did not affect the outcome of transplant. Outcome data is another way of assessing the quality of the CBUs, even though it may be influenced by many factors out of the CBB’s responsibility (such as diagnosis, conditioning, multiple transplants, etc…). The combined analysis of the two datasets is a good way for a CBB to ensure processes are performing as expected over time and monitor the quality of the final product.

Abstract 8 Cord Blood – Defining Urgency & Delivering Speed

Abstract Introduction The ability for cord blood units (CBUs) to meet the needs of transplant patients in urgent circumstances is a key attribute of this unique cell source. CBUs are cryopreserved, fully characterized, readily available for shipment, and avoid obstacles inherent with fresh cell sources. When patient transplant timelines are pressing and cell source availability and delivery speed takes priority, cord blood is both dependable and reliable. Objectives U.S. cord blood bank (CBB) shipments over five fiscal years (FY2018 – FY2022) were analyzed. The goal of this analysis was to establish a benchmark for CBU delivery speed, volumes, and patterns to inform transplant centers and patients awaiting transplant. Methods Data were categorized by number of days between a CBU order and actual shipment date. Order and shipment categories were: Ultra-urgent (same day), urgent (1-3 days), standard (4-14 days) and routine (15+ days). Results 2,931 shipments of domestic (U.S.) CBUs took place from FY2018 – FY2022. Most shipment requests were shipped within the standard timeframe (n = 1,544 or 53%). Routine shipments (n = 643) came in at 22%, urgent shipments (n = 667) accounted for 23%, and ultra-urgent orders (n = 77) made up 3% of shipment timeframes. In the standard category (4-14 days), 65% of CBUs shipped within the first 4 to 7 days. In the urgent category (1-3 days), 46% (n = 306) of shipments were next day, 2-day shipments represented 31% (n = 209), and 3-day shipments consistently comprised 23% (n = 152) of this category year over year. In 2020, after the initial onset of the COVID-19 pandemic, 29% of all shipments fell within the ultra-urgent or urgent category. Discussion 25% of all U.S CBU shipments were distributed in 3 days or less. 59% were shipped in 7 days or less. 80% of all CBUs analyzed shipped within 14 days, with only 20% of shipped CBUs extending beyond the 14-day mark, most at the TC’s request. These data reinforce the rapid availability of cord blood for patients in need.

Abstract 9 Identification of Effective Changes to Workflow Triaging Criteria and Production Planning

Abstract Introduction The Cleveland Cord Blood Center (CCBC) analyzes fresh incoming cord blood units for the total nucleated cell (TNC) count. Units that meet the TNC criteria are processed for clinical banking and units not meeting the defined criteria are made available for research collaborations. CCBC sought to understand if adding additional pre-screening criteria would reduce post-manufacturing deferral rates and increase production of licensed units. Objectives To identify the main failure modes resulting in cord blood unit deferral for clinical banking post-manufacturing. To identify key factors resulting in unlicensed status of processed cord blood units. To implement changes to pre-screening strategies to reduce deferral rates and increase production of licensed units. Methods CCBC created four cross-departmental teams to evaluate the main reasons for deferrals post-manufacturing and to evaluate travel-risk history. Each committee had representation from collections, quality, and laboratory staff. Data was evaluated that was collected between January and June 2022. Results The committees identified that 19% of all deferrals post-manufacturing were related to low CD34 count and/or viability and that 17% of all processed units were unlicensed due to travel to Zika related areas. In March 2023, pre-screening criteria was updated to include a pre-processed CD34 count and viability and a pre-screening of the donating mother for travel history after consenting and prior to manufacturing. Units that fulfilled both the TNC and CD34 cut offs and did not have a high-risk travel history were processed for clinical banking. Data evaluated from March and April 2023 indicated that the deferral rates for low viable CD34 counts post-manufacturing went from 19% down to 0% and the percentage of high-risk travel units manufactured was reduced from 17% to 7%. Discussion Review of failure modes identified two main factors to guide improved triage efficiency. By implementing a pre-processing CD34 count, the larger TNC and CD34 cord blood units were identified for banking for clinical use. By using a pre-screening process at collection sites, high-risk travel donations were identified prior to processing. In the two-month timeframe evaluated, the overall deferral rates were reduced by roughly 7% and post-manufacturing licensure status increased from 83% to 93%.

Abstract 10 Experience with Enrollments and Releases Within a Medical Needs Program at a Large U.S. Private Cord Blood Bank

Abstract Introduction Medical Needs / Directed Donor programs provide families with access to cord blood for potential future use. In situations where a diagnosis, potentially treatable with cord blood, exists in an immediate relative of the newborn, these programs often allow families to preserve newborn stem cells at little to no cost. Objectives The objective of this study was to examine indications for enrollment and unit releases in a medical needs program at a large U.S. private newborn stem cell bank. Methods Enrollment data over a 24-year period was analyzed. Data was categorized by indications for enrollment and indications for which units were released. Results There was a total of 7,034 enrollments within the specified timeframe. Most enrollments were for indications treatable by hematopoietic stem cell transplant given the program did not include experimental indications until 2019. Approximately one-third of enrollments (33.4%) for transplant indications were for various types of leukemia. The experimental indications most frequently represented in enrollments were autism (44.3%) and cerebral palsy (38.4%). For all cord blood units stored under the medical needs program, the majority (70%) of releases were intended for use in transplant medicine. The most frequently represented transplant indication for releases was hemoglobinopathies including sickle cell anemia and beta thalassemia (44.6%). Approximately one-third (36.1%) of transplant releases were related to leukemia. Autism and cerebral palsy were the two most frequently represented experimental indications for units released at 39% and 36%, respectively. Discussion Clinical indications were mostly consistent between enrollments and releases through this cohort. This suggests the structure of the program is effective in identifying families who have a higher likelihood of utilizing their cord blood unit for a transplant or experimental infusion. Cord blood banks should continue to re-evaluate the eligibility criteria for their medical needs programs in light of relevant updates in the potential utility of newborn stem cells including changes in transplant medicine indications and treatment procedures, as well as clinical trials and experimental protocols using related cord blood units. Future research related to pathways of referral to medical needs programs may inform education gaps among women’s health providers.

Abstract 12 Evaluating the Properties of the Donor to Determine the Quality of Cord Blood

Abstract Introduction Cord blood is a valuable source of hematopoietic stem cells (HSCs) that offer advantages over other sources, but the volume and the absolute number of stem cells in cord blood are limited. Objectives The study aimed to analyze the influence of the factors relating to mothers, babies, and collection on the quality of cord blood units before cryopreservation in Vinmec Tissue Bank, Vietnam. This provides a thorough insight into available predictors that can inform the potential recipients. Methods This study, conducted at Vinmec Tissue Bank in Vietnam between January 2019 and December 2021, collected, processed, and analyzed 2,668 cord blood samples. The study evaluated the impact of donor and collection factors, including maternal age, delivery mode, gestational age, infant weight, gender, blood group, geography, season, collection volume, viability, TNC, and CD34+ cell components on cord blood characteristics. Results Results showed that maternal age, gestational age, delivery mode, and infant weight significantly affected TNC and CD34+ cells, while infant gender only has a significant impact on TNC. Additionally, the collection features such as cord blood volume, geography, the season of delivery, and waiting time associated with cord blood outcomes. Discussion In virtue of its superior matching probability, lower incidence of GVHD, and reduced recurrence rates, cord blood is considered a favorable option for HSC transplantation. However, the limited number of HSCs available from cord blood for bigger patients can be addressed through global cooperation. The possibility of using multiple cord blood units for hematologic cancers has been made feasible through inter-unit HLA matching 29,30. As global cooperation between cord blood banks expands, there is a growing opportunity to find matching samples for recipients. Our large-scale analysis of cord blood throughout Vietnam has highlighted the significance of baby weight and volume in determining cord blood quality. This report represents the first-ever study in Asia to investigate the impact of geography and demography on cord blood. Further evaluation is necessary for a comprehensive understanding of these factors, which would enable more appropriate counseling for patients. Figure 1. Elements that impact the quality of hematopoietic stem cells in cord blood.

Abstract 24 Development of an In Vitro B-Cell Differentiation Model for the Characterization of the Origin and Causation of ETV6::RUNX1+ Preleukemic Cells

Abstract Introduction Pregnant women are exposed to various carcinogenic substances that can subsequently affect their progeny. Recurrent preleukemic chromosomal translocations characteristic for childhood acute lymphoblastic leukemia (ALL) frequently emerge before birth in utero. The most common translocation t (12;21) occurs in 25% of B-cell precursor ALL and results in the formation of the chimeric oncogenic transcription factor ETV6::RUNX1. Objectives We aim to analyze factors promoting the generation of preleukemic ETV6::RUNX1 translocations, investigate the biological origin of these preleukemic cells, and develop an in vitro model of early B-cell differentiation to analyze the mechanisms underlying the persistence of ETV6::RUNX1+ preleukemic cells and leukemia evolution in children. Methods Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) (Schafer et. al., 2018) was applied to identify ETV6::RUNX1+ cord blood units (CBU). Screening results were validated by Sanger sequencing and quantified by real-time PCR (ipsogen ETV6-RUNX1 Kit, Qiagen). ETV6::RUNX1+ CBUs were thawed, sorted for CD34+ and CD19+ cells, and analyzed by qPCR. CD34+ were differentiated towards B-cell lineage on MSC feeder lines according to a modified protocol (Ichii et. al., 2008). Results We screened a cohort of 1405 cord bloods collected in 2004 and discovered that 103 (7.3%) carried the ETV6::RUNX1 fusion gene. The preleukemic translocation was detected in CD34+ stem and early B progenitor cells. For the establishment of an in vitro B-cell differentiation model, bone marrow (BM-MSCs) and CB (USSCs, CB-MSCs) stromal feeder cells were compared and the effect of different media (SCGM, DMEM/5 % FCS, MSCBM/10 % FCS) in the presence of stem cell factor (SCF), Flt-3, IL3, +/- IL7 was assessed. BM-MSCs demonstrated the highest ability to support B-lymphopoieses towards CD19+ cell differentiation in MSCBM media (~30%), primarily due to the release of IL-7 by the feeder layer. Currently, factors that may promote generation of preleukemic translocations in utero e.g. nitrosamines contained in food products, are being tested in this model. Discussion We identified the HSC/early B progenitor state as the origin of ETV6::RUNX1 translocations in CB. The establishment of an in vitro B-cell differentiation system will allow to test the impact of environmental carcinogens on the generation of ETV6::RUNX1+ pre-leukemic cells and their leukemic evolution.

Abstract 14 Omidubicel-Onlv: Robustness of the Manufacturing Process

Abstract Introduction Omidubicel-onlv is a nicotinamide (NAM) -modified allogeneic hematopoietic progenitor cell therapy derived from cord blood. NAM expansion increases the number of stem and progenitor cells, inhibits differentiation, and potentiates migration, bone marrow homing, and engraftment efficiency. A global phase 3 trial demonstrated faster hematopoietic recovery, reduced rates of infection, and shorter hospitalizations for patients randomized to omidubicel-onlv compared to standard umbilical cord blood (UCB). Omidubicel-onlv is manufactured for each patient from a single, appropriately matched cord blood unit (CBU). The inherent donor variability as well as the different processing and testing methods used by different Cord Blood Banks (CBBs) present challenges in maintaining a consistent product. Objectives This extensive study was conducted to confirm the robustness of the manufacturing process and the proven acceptable range for the critical and key process parameters. Methods Seven process parameters were evaluated in total. Of these, three parameters were related to the CD34+ expansion phase: CD34+ seeding concentration, incubation temperature, and CO2 levels. Four additional parameters were related to final product formulation and cryopreservation: hold times during harvest and formulation, cell concentration in the drug product, DMSO concentration, and storage temperature. The impact of each process parameter on product performance was assessed by a side-by-side comparison of its target value (control) versus the tested range. Each parameter was evaluated using at least three batches derived from three CBUs. Statistical analysis of the coefficient of variation or recovery against the control was applied to the data sets. Results Statistical analyses demonstrated an overall variability of ≤30% in critical attributes including fold expansion, viability, phenotyping, and recovery of cells post-cryopreservation and stability. Discussion The omidubicel-onlv manufacturing process is robust, overcoming the variability of batches derived from different donors, different processing and testing methods at CBBs, and manufacturing process parameters.

Abstract 23 HLA-Homozygous iPSC from Cord Blood for the Generation of Cardiomyocytes for Allogeneic Cell Therapy to the Heart

Abstract Introduction Based on a previous grant on HLA-homozygous (HLAh) iPS-GMP (and ATMP conform) generation from allogeneic licensed cord blood (CB) units, the Düsseldorf CBB could provide the most frequent HLA-types for reprogramming (Liedtke et al. 2020). These HLAh iPSC provide the basis for the generation of cardiomyocytes from iPSC. Patients with acute heart failure or cardiomyopathies have limited options of treatment. iPSC technology allows for de novo generation of functional heart muscle in vitro, enabling the implementation of new therapy concepts for heart regeneration. Objectives The GMP-grade iPSC-lines are the backbone for the granted EU project (HEAL (101056712) on iPSC-cardiomyocyte aggregate manufacturing for allogeneic cell therapy. Besides GMP-compliant up-scaled cell production of cardiomyocytes, the project is designed to overcome many of the scientific, safety and regulatory hurdles necessary to initiate a first-in-man clinical HLAh-based heart regeneration study. Methods After CD34+ selection/short expansion of CD34, cells were reprogrammed by GMP-conform plasmids and stored in a master cell bank with most frequent HLA-haplotypes. All iPSC lines were tested extensively for genetic stability, mycoplasma, virus markers, endotoxins and microorganisms. The differentiation into cardiomyocytes was performed in correspondence to the Wnt modulation, i.e. first activating Wnt pathway and then inhibiting the pathway with the addition of small molecules (Halloin et al. 2019). For the up-scaled production, a 3D-cardiomyocyte aggregate culture in large stirred tank bioreactors (STBR) was applied. Cryopreservation using controlled rate freezing was adapted to tissue freezing. Results Selection of the most frequent homozygous haplotypes facilitated the production of HLAh iPSC lines for differentiation into cardiomyocytes. Efficient progress had been made for the improvement of STBR platform for sufficient production of cardiomyocytes, enabling the differentiation with a purity of up to 95%, measured via cardiac troponin T (cTnT). Discussion Although high yields of cardiomyocytes in a GMP-compliant process is getting more achievable by using STBRs, the safety and regulatory hurdles are still a major problem. For safety testing, toxicology, tumorigenicity assays will be performed in mice and in pig models. This will enable the prediction of arrhythmia-related risks. For a safe distribution of the finally released cell product optimization of cryopreservation of cardiomyocytes aggregates will be performed. Disclaimer: Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them.

Abstract 13 Long-Term Stability of Dus Cord Blood Units After >29 Years of Cryopreservation: Follow-Up Data from the José Carreras Cord Blood Bank

Abstract Introduction The José Carreras Cord Blood Bank (CBB) located in Düsseldorf stores 21215 active cryopreserved cord blood units (CBUs) and has released 1476 CBUs for allogeneic transplantation. The success of transplantations, but also the generation of cells for regenerative medicine for e.g. iPSC reprogramming, significantly depends on the quality of the cryopreserved CBUs after thaw. Objectives Therefore, typical parameters like cell count, recovery and viability are analysed from thawed CBUs yearly by a stability monitoring program according to the FACT standards. The longest cryopreservation period in liquid nitrogen tested to date is 338 months (>29 years) for unseparated units and 300 months (25 years) for volume-reduced units that are licensed by the Paul-Ehrlich Institute. Methods A retrospective analysis of unseparated CBUs processed from 1993-1997, CBUs processed from 1998-2005 by manual volume-reduction and CBUs processed since 2005 by automated volume-reduction applying the Sepax100 system was performed. In consideration of the distinct processing methods, storage time and existing follow-up data of released transplants, this approach should demonstrate the high CBU quality and proof the long-term engrafting capability of CBUs cryopreserved in liquid nitrogen. Results According to the CBB stability program means of TNC count (16.32±2.61 x 108), TNC recovery after thaw (106.8±13.91%, above 100% due to earlier applied cell-count method), TNC viability (88.91±5.01%), CD34+7AAD- viability (90.73±3.69%), CD45+7AAD- viability (69.09±5.38%) and CFC count (7.10±2.45 x 106) were determined after >29 years of cryopreservation for unseparated CBUs. In volume-reduced CBUs the mean TNC count (9.37±2.14 x 108), TNC recovery (97.78±3.70%), TNC viability (84.22±10.02%), CD34+7AAD- viability (88.11±5.16%), CD45+7AAD- viability (73.67±8.31%) and CFC count (1.37±0.59 x 106) were determined after 25 years of cryopreservation. Discussion In addition, these relevant parameters were retrospectively analyzed for released transplants in correlation to the storage time. The follow-up data of recipients from CBUs demonstrate an earlier engraftment of volume-reduced CBUs as compared to unseparated CBUs in accordance to an increased TNC count over time. With regard to applied processing methods, engraftment time and patient age (children and adults) this retrospective analysis confirms the high quality of old CBUs stored in the liquid phase at -196°C.

Single laboratory evaluation of umbilical cord blood units processing methodologies for banking.

To compare the efficiency of 3 different processing methods (Sepax, AutoXpress [AXP], and manual processing with hydroxyethyl starch [HES] sedimentation) used at Stemlab during a 10-year period. Historical data were compiled and the analytical results obtained for the 3 different methods were compared. The manual processing (HES) method yielded the highest level of total nucleated cell recovery after processing, and the AXP system yielded the highest CD34+ cell number. The red blood cell reduction was also significantly higher with the HES method. Also, HES showed comparable results to Toticyte technology for umbilical cord blood (UCB) processing. These results show that the HES method is as effective as automated technologies for UCB volume reduction; hence, it is a suitable methodology for private and public UCB banks. The HES method also proved to be superior to Toticyte technology for medical applications, with higher recovery yields of total nucleated cells after thawing and equivalent CD34+ cell recovery and functionality.