Cord Blood Processing Research Articles

Abstract 20 A Rapid and Sensitive IL-3-Based Assay Reveals How Various Pre-Processing Conditions Affect the Potency of Hematopoietic Stem Cells in Fresh Cord Blood Units

IntroductionOptimized procedures to collect, process, and store cord blood (CB) units are crucial to ensure stem cell product quality; however, current methods to assess hematopoietic stem cell (HSC) potency are time-consuming or lack sensitivity. Therefore, developing methods that rapidly and accurately portray HSC potency in fresh blood is important to help optimize clinical outcomes.ObjectiveWe sought to adapt to fresh CB a potency assay previously developed for HSCs contained in cryopreserved CB [Simard et al. Transfusion. 2019;59(6):2074-2083.]. We next used the assay to evaluate the impact of various CB processing conditions on HSC potency.MethodsFresh CB units were exposed to various pre-processing storage conditions (ie, temperature changes and various anticoagulants). Units were then analyzed at various time points up to 96 hours using traditional iShage and colony-forming assays, in parallel with the IL-3-based potency assay.ResultsFor CB units maintained at room temperature (RT), the potency measured with the IL-3-based assay (ie, the proportion of IL-3-responsive CD34+ cells as measured by pStat5 activation) linearly decreased over time. A potency of 69% was observed when tested within 0-24 hours, compared with 56% and 40% in the 24-48–hour and the 48-72–hour groups, respectively. In addition, the IL-3-based assay showed that pre-processing storage at 4°C prevented this loss of HSC potency. By contrast, the HSC count by iShage and the CFU numbers remained unchanged with time or temperature variations.DiscussionThe newly developed IL3-based assay appears reliable and sensitive to characterize the potency of CD34+ cells in fresh CB. In addition, this assay indicated that HSC potency linearly decreased with time when CB units were maintained at RT but remained stable when units were stored at 4°C pre-processing. This temperature-related difference was not observed with traditional assays, indicating that the test could be a complementary tool to characterize CB units in research and operational settings.

Evaluation of Microbial Contamination Rates in Umbilical Cord Blood Collection and Processing for Biobanking in Vietnam

The umbilical cord blood (CB) has recently been considered an abundant source of hematopoietic stem cells (HSCs) for transplantation compared to bone marrow. However, the collection and processing of CB have a high risk of microbial contamination. Hence, the procedures to collect and process the UCB are carefully considered. This study evaluates the microbial contamination rate to find the frequency and distribution of bacterial organisms among CB sampling and processing in Vietnam. In addition, this study correlates with contamination rates between the delivery method, cesarean section, and vaginal delivery. The results create best practices to avoid a high level of contamination of UCB during collection and processing for biobanking.

Enhanced collection of phenotypic and engrafting human cord blood hematopoietic stem cells at 4°C.

The number of hematopoietic stem cells (HSCs) collected in cord blood (CB) at the birth of a baby is a limiting factor for efficacious use of CB in hematopoietic cell transplantation (HCT). We now demonstrate that collecting and processing of human CB at 4°C within minutes of the baby's birth results in significantly enhanced numbers of rigorously defined phenotypic HSC and self-renewing NSG immune-deficient mouse engrafting and SCID-repopulating cells. This was associated with decreased numbers of hematopoietic progenitor cells (HPC), as noted previously for hypoxia collected/processed cells blocking ambient air induced differentiation of HSC to HPC. We have thus defined a simple, cost-effective, means to collect increased numbers of CB HSC, of potential use for clinical CB HCT.

Cord blood processing by a novel filtration system.

ObjectivesAvailability of cord blood (CB) processing has been limited by the need for electrically aided centrifugal techniques, which often produce only low final cell product yield. Here, we describe development and characterization of a novel filter device aimed at allowing CB processing, using gentle gravity‐led flow.Materials and methods CB was processed with a novel filter device (CellEffic CB, consisting of non‐woven fabric), without any centrifugation. Cells were harvested by flushing the filter with either HES or physiological saline solution (SALINE). Differential cell counts and viability analysis, combined with Fluorescence‐Activated Cell Sorting (FACS) (total nucleated cells [TNC], mononuclear cells [MNC], CD45+ CD34+ cells, hematopoietic precursor cells [HPCs]) and clonogenic assay, were employed for analysis of CB pre‐ and post‐processing, and after freeze/thawing.ResultsProcessing using the novel filter yielded high quality RBC depletion while maintaining good recovery of TNC, MNC, CD34+, HPCs and colony forming unit (CFU) output. The filter performed equally well using HES or SALINE. Gravity‐led flow provided gentle cell movement and protection of the stem cell compartment. Post‐thaw CFU output was maintained particularly, an important indicator for CB banking.ConclusionsGeographical limitations of CB transplantation and banking have required a non‐electrical, non‐centrifugal solution. This novel filter CellEffic CB device revealed rapid yet gentle cell processing while maintaining the stem/progenitor cell compartment required for both haematological and regenerative medicine therapies.

Influence of maternal, infant, and collection characteristics on high-quality cord blood units in Guangzhou Cord Blood Bank.

The operation of cord blood banks (CBBs) requires immense labor, material, and financial resources. Thus, increasing the ratio of high-quality cord blood units (HQCBUs) in storage that are qualified for clinical use is critical for the efficient use of limited resources. Understanding the factors that contribute to HQCBUs, including maternal, fetal, and processing conditions, may improve the number of HQCBUs in storage. The maternal, fetal, and processing conditions of 4613 CBUs at the Guangzhou Cord Blood Bank were analyzed retrospectively to determine their effect on HQCBUs. All CBUs were obtained following strict standard operation procedures. Several factors may contribute to HQCBUs: fetal age older than 37 gestational weeks, female fetus, large cord blood (CB) volume (>80 mL), high birthweight (>3500 g), vaginal delivery, and a shorter amount of time between CB collection and processing (12 hr). We report for the first time that α-thalassemia carriers exhibit a postprocessing total nucleated cell count (p-TNCC) increase to at least 1.25 × 10(9) and an increase of the CD34+ cell count to at least 6.01 × 10(6) . Meconium-stained amniotic fluid and mothers younger than 25 years of age exhibited increased p-TNCC to at least 1.25 × 10(9) , and colony-forming units increased to at least 23.24 × 10(5) . We identified several factors that affect HQCBUs. These results may be used as a reference for updating CB collection strategies, with priority given to collecting CBUs from female fetuses older than 37 gestational weeks, at high birthweight, and born by vaginal delivery from mothers younger than 25 years of age, especially newborns with one parent carrying the trait or with meconium-stained amniotic fluid. The collected CBUs should be sent to the laboratory as soon as possible for priority processing, which will help to increase the number and ratio of HQCBUs and the effective use of CBB resources.

Current status of cord blood banking during first two years of 'National Government-Assigned Public Cord Blood Banks Operation' in Korea.

Cord blood (CB) has become a viable stem cell source for hematopoietic stem cell transplantation (HSCT), and public cord blood banks (CBBs) have been established to manage donated cord blood units (CBUs) for unrelated HSCT. As the potential uses for CB continue to grow, there is a global tendency to encourage public CBBs. The aim of this study was to investigate the current status of public CBBs that were designated and supported by the Korean national government. We analyzed 6,168 CBUs preserved at the 3 Government-Assigned Public CBBs during their first two years of operation (2012-2013) and the standard operating procedures for CB processing at each CBB. CBU inventories at ALLCORD, Catholic HSC Bank and Fatima Public CBB were 4,022, 1,207 and 939 units, respectively. Total nucleated cell (TNC) counts per unit were 11.0 × 10(8) at ALLCORD, 10.7 × 10(8) at Fatima and 9.8 × 10(8) at Catholic, and all far exceeded the requirement for cryopreservation under the law (7.0 × 10(8)). CD34(+) cell counts and % were as follows: 3.5 × 10(6) (0.31 %) in ALLCORD, 2.2 × 10(6) (0.20 %) in Fatima and 2.2 × 10(6) (0.22 %) in Catholic. All the three banks observed the 'CB Act' in dealing with CBUs, including collection, processing, laboratory tests and cryopreservation. Government supported and strictly law-abiding public CBBs in Korea have considerable CBU inventories of high quality in terms of efficacy and safety. Legislation and accompanying government-support will be helpful for establishing CB standardization, vitalizing CBT and improving clinical outcomes.

Sterility testing of minimally manipulated cord blood products: validation of growth‐based automated culture systems

The St Louis Cord Blood Bank submitted a biologics license application for cord blood (CB) products processed by PrepaCyte-CB (BioE), supported with a validation study of a microbial detection system for product sterility testing (BACTEC-FX, Becton Dickinson). This article provides the validation approach followed to fulfill Food and Drug Administration requirements pertinent to sterility testing method. System qualification, culture media quality verification, and validation of CB processing by-product (CB-BP) sample as surrogate to final product for sterility testing were followed by studies evaluating method sensitivity, specificity, reproducibility, ruggedness or robustness, and bacteriostatic or fungistatic effect of CB-BP sample. CB-BP cultures and control samples were formulated using BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Myco F/Lytic media. Samples were seeded with selected test organisms (n = 13 at 10-100 colony-forming units [CFUs] per vial) and cultured for 14 days (bacterial) and 30 days (fungal). Under testing conditions, no stasis effect of test sample on microbial growth and no false-positive or false-negative results were reported. Although a 7-day culture was sufficient to detect all validation test organisms seeded at ≤ 26 CFUs/vial, growth in actual product sterility testing practice may require a 10- to 14-day culture. Assay reproducibility was uncertain at very low bioburden (< 10 CFUs/vial). Growth time to detection neither varied between different media lots nor prolonged in culture vials with loading delay (6-8 hr at room temperature). BACTEC-FX culture and detection system and BACTEC media formulae have high detection capability and can be effectively validated for sterility testing of CB products.

A novel filtration method for cord blood processing using a polyester fabric filter

Cord blood (CB) is being increasingly used as a source of hematopoietic stem cells for transplantation to treat diseases of the blood and immune systems, and there is an urgent need to expand CB banking worldwide. CB processing requires costly machinery or a clean room that hampers wider application of CBT particularly in the developing countries. We developed a novel filtration system using a nonchemical-coated and nonwoven polyester fabric filter, which traps cells through affinity and does not require centrifugation or potentially toxic chemicals. Cell processing with the device resulted in minimum cell loss of total cells and CD34⁺ cells, without impairing the ability of CD34⁺ cells to engraft and differentiate both in vivo and in vitro. CB processing with this device is simple, cost-effective, and nontoxic without requiring costly equipment will thus facilitate international CB banking, which helps in meeting the increasing worldwide demand for CB for allogeneic hematopoietic stem cell transplantation.

Cell recovery comparison between plasma depletion/reduction- and red cell reduction-processing of umbilical cord blood

Limited cell dose has hampered the use of cord blood transplantation (CBT) in adults. One method of minimizing nucleated cell loss in cord blood (CB) processing is to deplete or reduce plasma but not red blood cells - plasma depletion/reduction (PDR). The nucleated cell loss of PDR was studied, and determined to be less than 0.1% in the discarded supernatant plasma fraction in validation experiments. After testing and archival sampling, the median nucleated cell recovery for PDR processing was 90%, and median CD34(+) cell recovery 88%. In a CB bank inventory of 12 339 products with both pre- and post-processing total nucleated cells (TNC), PDR processing resulted in median post-processing TNC recoveries of 90.0% after testing and archival samples removal. Using the same 10 CB units divided into two halves, we compared directly the recovery of PDR against hydroxyethyl starch red cell reduction (RCR) for TNC, CD34(+) cells and colony-forming units (CFU-GM, CFU-E, CFU-GEMM and total CFU) after parallel processing. We also compared the loss of very small embryonic-like stem cells (VSEL). We demonstrated significantly higher recoveries using PDR for TNC (124%), CD34(+) cells (121%), CFU-GM (225%), CFU-GEMM (201%), total CFU (186%) and VSEL (187%). The proportion of high TNC products was compared between 10 912 PDR and 38 819 RCR CB products and found to be 200% higher for products that had TNC ≥150 × 10(7) (P = 0.0001) for the PDR inventory. Our data indicate that PDR processing of CB provides a significantly more efficient usage of this valuable and scarce resource.

The Mannheim Cord Blood Bank: Experiences and Perspectives for the Future

Background and Methods: As a source of hematopoietic stem cells, cord blood (CB) is an alternative to bone marrow or peripheral blood stem cells (PBSC). The Mannheim Cord Blood Bank has currently stored about 1,750 allogeneic CB units. Here we report our experiences and discuss future perspectives of CB banking. We analyzed CB units for nucleated cell (NC), mononucleated cell (MNC) and CD34+ cell count, volume, colony-forming units (CFU-GM) as well as ethnic background of the donor. Transplanted CB units were analyzed for patient and transplant characteristics and compared to stored CB units. Results: Only 25% of all collected CB units met storage criteria. Main reasons for exclusion were: i) insufficient volume (57.7%), ii) delayed arrival at the processing site (19.2%) and iii) little cell count (7.2%). Up to now 36 CB units have been released for transplantation mainly to children (62%). Transplant indications were hematological diseases, immune deficiencies and metabolic diseases. Transplanted CB units showed significantly higher cell counts compared to stored units (NC: 12.5 vs. 7.2 × 10⁸, MNC: 4.7 vs. 2.9 × 10⁸, CD34+ cells: 3.3 vs. 1.8 × 10⁶, mean; p < 0.001 each) and were found more often in ethnic minority groups (36 vs. 20%; p = 0.04). Conclusions: Even though cell count and volume are key parameters for the eligibility of CB units, our data indicate that the ethnic background of the donor also plays a major role. Collection and processing of CB should be optimized in order to gain maximum volume and cell counts.

Red blood cell depletion of cord blood using an automated system-Evaluation of the AXP system

As a cord blood (CB) bank, we routinely reduce the volume of each CB unit by manually sedimenting red blood cells (RBCs) using hydroxyethyl starch (HES). This is a demanding technique, and an automated volume reduction of CB is expected to standardize the procedure while complying with the requirements of the quality control system. We compared the manual HES sedimentation method with the AutoXpressTM system (AXP, Thermogenesis, New York) without HES. Preparing identical units, we assessed the total nucleated cell (TNC) count, mononuclear cell (MNC) count, CD34+cells, and colony-forming unit (CFU) content from both methods in 24 pairs. Recoveries of TNC, MNC, CD34+and total CFU for the AXP group were 88.4%, 97.3%, 93.4% and 101.2%, respectively, which were significantly better than the manual HES sedimentation group. The time required for CB processing with the AXP system was significantly shorter than that for our HES sedimentation method. The main advantage of the manual HES method was its higher RBC depletion. The recovery of cells after a freeze and thaw was comparable between the manual and AXP groups for TNC, CD34+and CFU. Use of the AXP system to enable CB banks to automate and simplify the processing procedure is feasible.

Cord blood: from garbage to life-saving source.

Cord blood: from garbage to life-saving source.

Evaluation of four methods for processing human cord blood and subsequent study of the expansion of progenitor stem cells isolated using the best method

The application of cord blood (CB)-derived hematopoietic cells in allogeneic transplantation has been restricted because of the unavailability of adequate cell numbers from one unit of CB and their delayed engraftment in recipients. The main purpose of this study was to develop an economic process for isolating nucleated cells from CB and expanding enriched CD133(+) cells. Four protocols for processing CB, using different combinations of density-gradient centrifugation, hydroxyethyl starch (HES) and NH4Cl treatment, were compared regarding the yields of CD45(+), CD34(+)/CD133(+) and colony-forming cells. CD133-enriched cells were expanded for 10 days in the presence of hematopoietic growth factor-supplemented medium. The performance of the culture was evaluated by analyzing colony-forming potential, fold-expansion of primitive hematopoietic stem cells (HSC) and lineage commitment by flow cytometry. A two-step treatment of CB with HES followed by NH4Cl resulted in the highest recovery of CD45(+) (84.3%) and CD34(+) (69.1%) cells compared with that present in umbilical CB. A suspension culture of CD133-enriched cells resulted in an average 150-fold increase in nucleated cells, of which CD133(+), CD34(+) and CD34(+) CD38 cell expansions were 17.2+/-1.3, 40+/-4.6, and 6.9+/-1.1 fold, respectively. The total myeloid colony-forming cell count was almost 3800-fold higher than that present in the processed CB. The highest yields of nucleated and progenitor stem cells were obtained with a two-step processing of CB. The CD133(+) cells obtained by this method are expected to yield enough hematopoietic progenitors for potential allogeneic transplantation.

Cord Blood (CB) Unit Mononuclear Cell (MNC) Dose: Effect on Transplantation Outcome and Relevance to Processing Method and CBU Selection.

Among the CB graft characteristics that affect post-transplant engraftment and survival TNC dose has become a critical variable in the selection of CB units. However, the cryopreserved TNC depends on the method of CB processing. The AXPTM system, used by the NYBC-NCBP since August 2006, automatically reduces the volume and separates the components of a CB unit in a closed system, resulting in >95% recovery of MNCs, CFU and CD34+ cells and excellent viability, consistently, while the average recovery of TNC is 80%. MNCs represent 47% (±0.07) of the pre-processing TNC. Thus, when processing has no effect on type of cells recovered, a CB unit with a TNC of 900x10^6 (cell number criterion used for inclusion in the National Cord Blood Inventory) would have an average of 425x10^6 MNCs. As a result of the differential recoveries, however, of all CBUs with a pre-processing TNC >1100x10^6, 58% will have a post-processing TNC count ≥ 900x10^6; in contrast, 81.5% will have MNC ≥ 425x10^6 (N=11012 units processed with the AXP system during the period: 2006–2008). To evaluate whether the MNC dose has an impact on transplant outcome, and compare it to that of the TNC dose, we evaluated the effect on time to engraftment (time to ANC ≥ 500) and transplant related mortality (TRM) in all patients with leukemia or myelodysplasia that received single unit CB grafts from our Bank following myeloablative cytoreduction, during the period 1993–2006. A total of 1044 patients (mean age: 13.9 years, median: 9.2 years, range: 0.2–64 years, 22% >20 years) were included, 87% had follow-up data. CBUs had 0 (5%), 1 (33%) or ≥ 2 (61%) HLA mismatches with the recipient; the median cryopreservation TNC/kg was 4.1x10^7. Pre-freezing complete counts were obtained by automated hematology analyzers (H*1, Technicon, Bayer Corporation or Sysmex XE-2100, Roche Diagnostics). Until July 2006, CBUs were processed manually [PNAS 94(22);1995]. For this study, MNC included nucleated RBC. In a multivariate Cox regression analysis TNC/kg, HLA mismatch level, GvHD prophylaxis, transplant center and year were independently predictive of time to ANC ≥ 500. When MNC/kg was added to the analysis with TNC/kg, only the MNC dose was a significant predictor (RR:1.5, p:0.03), and the TNC dose was no longer predictive (p:0.7). Low MNC/kg (<1x10^7) was associated with delayed engraftment; time to ANC ≥ 500 shortened (showed by increasing RR of engraftment) with higher doses of MNC. Similar variables: HLA mismatch level, TNC/kg, race, transplant center and year affected TRM independently, and again, the significant effect of MNC/kg on TRM, when included in the multivariate analysis, removed the significance of TNC dose. As shown, low MNC/kg (<1x10^7) was associated with increased TRM (RR:1.8, p<0.001).Time to ANC ≥ 500Transplant-related mortalityMNC/kg x(107)N=944RR(95%CI)p valueN=831RR(95% CI)p value<1.01540.6(0.5–0.7)<0.0011591.8(1.5–2.2)<0.0011.0 – 1.9291Reference group309Reference group2.0 – 2.91441.0(0.8–1.1)0.5591510.9(0.7–1.1)0.2643.0 – 3.9921.2(0.98–1.4)0.085990.8(0.6–1.1)0.192≥ 4.01501.7(1.4–2.0)<0.0011580.7(0.6–0.95)0.02MNC dose, therefore, emerges as a better predictor of time to engraftment and TRM than the TNC dose, and thus, as a better measure of graft quality to be used in selecting CBUs for transplantation. In this context, because AXP processing retains more of the MNCs originally present in the CBU than other systems, this processing maybe used as a criterion of CBU quality when both the TNC and MNC are used as indices of CBU cellular contents.

CD45−/ALDHlow/SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin− Very Small Embryonic-Like (VSEL) Stem Cells Isolated from Umbilical Cord Blood as Potential Long Term Repopulating Hematopoietic Stem Cells

Recently, we identified a population of very small embryonic-like (VSEL) stem cells in umbilical cord blood (CB) (Leukemia 2007; 21:297–303) These VSELs are:smaller than erythrocytes;SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−;responsive to SDF-1 gradient; and iv) possessing large nuclei that contain unorganized chromatin (euchromatin).Data obtained in a murine model indicate that a similar cell population isolated from bone marrow (BM) does not reveal hematopoietic activity after isolation. However, in appropriate models (i.e., in vitro co-culture over OP-9 cells or in vivo after intra bone injection), these cells contribute to hematopoiesis and thus possesses potential of long term repopulating hematopoietic stem cells (LT-HSCs). To investigate the hematopoietic activity of CB-derived, CD45 negative VSELs, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. We sorted CD133+/CD45−/ALDHhigh and CD133+/CD45−/ALDHlow sub-fractions of VSELs from CB samples and established that both freshly sorted CB-derived populations did not grow hematopoietic colonies in vitro. However, when activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and initiate hematopoietic colonies composed of CD45+ cells when replated into methylcellulose cultures. Furthermore, while CD133+/CD45−/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD133+/CD45−/ALDHlow VSELs was delayed. The data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD133+/CD45−/ALDHlow cells (Fig. 1A). In parallel, real time PCR analysis revealed that freshly isolated CD133+/CD45−/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference) while CD133+/CD45−/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference) as compared to total CB mononuclear cells (Fig. B). Furthermore and somewhat unexpectedly, we found that because of their unusually small size, these important cells may be partially depleted (in 42.5±12.6%) during standard preparation strategies of CB units for storage that employ volume reduction. In conclusion, our data suggest very small CB mononuclear cells expressing VSEL markers that are CD133+/CD45−/ALDHlow are highly enriched for the most primitive population of LT-HSCs. These cells may be responsible for long term CB engraftment and be a population of cells from which HSCs should be expanded. We are currently testing this in an in vivo model by performing heterotransplants of CD45− ALDHlow VSELs into immunodeficient mice. It is important to stress that currently employed, routine CB processing strategies may lead up to ~50% loss of these small cells that are endowed with such remarkable hematopoietic activity. [Display omitted]

Stem Cell Recovery Following Implementation of an Automated Cord Blood Processing System in a High Volume Laboratory.

Background: Many cord blood (CB) processing facilities are transitioning to automated systems to standardize methods and decrease human error. The AutoXpress Platform™ (AXP™) is an automated, functionally closed, sterile CB processing system. When placed within the AXP device and centrifuged, the whole blood product is separated into its composite cell populations and the TNC fraction of the CB unit is separated and automatically delivered into a blow-molded freezing bag at a uniform volume of 21 mL. Objective: This study evaluated the use of the AXP system in a directed donor (DD) family cord blood bank, targeted at producing consistently high TNC and MNC recovery rates regardless of broad variability in collection volume.Methods: Cord blood units were collected between 12/5/2006 and 2/24/2007 from 1414 consenting mothers who elected to preserve and bank CB at Cord Blood Registry (CBR). Collection kits were provided at the time of enrollment, and after delivery, CB was collected from the umbilical cord and transported to CBR's processing facility in Tucson, Arizona. During the study period, all units arriving at the CBR laboratory were allocated to either ficoll or AXP processing based on the volume, age, and the degree of clotting in each unit. Units processed using AXP had a volume of 40–130 mL, an age of less than 48 hours since collection, and a clotting score of 0 to 2+ (based on an internal scale). TNC and MNC counts were measured both pre- and post-processing, using the Sysmex analyzer.Results: The mean age of cord blood units arriving at the laboratory was 23.59 hours, and the mean collection volume was 72.93 mL (±18.10mL). The mean TNC count post-processing was 9.94x108, the TNC percent recovery was 96.19%, and the mean MNC percent recovery was 98.65%.Volume (mL)Mean Post-Processing TNC (x108)TNC Percent Recovery (%)Mean Post-Processing MNC (x108)MNC Percent Recovery (%)Mean72.939.9496.194.0598.65Standard Deviation18.104.4311.871.599.16Conclusions: AXP automated cord blood processing provides consistently high TNC and MNC recovery rates, which has important implications for stem cell dose if the sample is used in transplant. Because limited cell dose is frequently cited as an obstacle to CB transplantation, processing results could impact the usability of each unit. Because DD family CB banks process all units, regardless of collection volume, percent recovery becomes particularly important in evaluating the differences between processing centers. The AXP system yields the highest published cell recovery rate to date and can be easily integrated into a CB processing center such that it decreases the labor and time required for processing while maintaining MNC recovery of greater than 98%.

Red blood cell depletion with a semiautomated system or hydroxyethyl starch sedimentation for routine cord blood banking: a comparative study

The major problem with long-term cord blood (CB) banking is the required storage space. In this sense, many studies have been performed to establish techniques for volume reduction of CB units. We compared two different methods for CB volume reduction in both development and routine phases: hydroxyethyl starch (HES) sedimentation and top-and-bottom fractionation with the Optipress II (Baxter Healthcare). Monitoring the total nucleated cell (TNC) count, lymphocytes, CD34+ cells, and colony-forming unit (CFU) content in both preprocess and postprocess CB units assessed the volume reduction process. The CB units processed in both groups had comparable volume and cells counts before and after volume reduction, except for number of red blood cells (RBCs), which was significantly greater for the Optipress II group. Recoveries of CD34+ and RBC depletion were significantly better for the HES group. For routine processing, TNC and lymphocyte recoveries were significantly better for CB units processed by the Optipress II system. There was, however, significantly less depletion of RBCs for this group. The time required for CB processing with the Optipress II was significantly shorter than the time needed for volume reduction by addition of HES (25+/-5 min vs. 55+/-10 min). The volume reduction method with the Optipress II is a closed time-saving system that allows good cell recoveries. In contrast, the main advantage of the HES method is the higher RBC depletion that influences CFU content. Reducing RBC content must be the object of further improvements for volume reduction using the Optipress II method.

Cord Blood Derived CD34+ and AC133+ Progenitor Cells Ex-Vivo Expanded in the Presence of Tetraethylenepentamine: Reproducibility among Cord Blood Units.

Ex-vivo expansion strategies of cord blood (CB) derived human progenitor cells (HPC) have been developed to provide an answer to the delayed time to engraftment and to the extended periods of neutropenia and thrombocytopenia encountered. These problems occur in transplants of CB products performed in adults due to the low yield of HPC. Reports correlating the clinical outcome with the number of CD34+ cells suggest that the transplantation of ex vivo expanded CD34+ cells may shorten the time to engraftment. The use of copper chelators such as tetraethylenepentamine (TEPA) has been shown to prolong expansion of HPC by inhibiting cell differentiation and thus allowing self-renewal of primitive HPC (Exp Hematol. 2004; 32:547). The variability observed in the expansion results, caused by the intrinsic differences among the various sources of CB units and processing methodologies, complicates the interpretation of published results. In the present report we summarize our results of CD34+ cell ex-vivo expansion of over 100 units in the presence of IL-6, TPO, Flt-3 ligand and SCF with and without TEPA. After 3 weeks, the total nuclear cell (TNC), colony forming unit (CFU), and the total CD34+ cell fold expansion of TEPA-treated cultures were 424±10.5 (n=230), 104±7 (n=112) and 19±3.2 (n=113), respectively, with no significant differences compared to controls. However, the percentage of the primitive subset of HPC, CD34+/38− cells, significantly (p<0.0001) increased in the TEPA-treated cultures (3.2%±0.2, n=59) vs. controls (1.6%±0.27, n=147). In contrast, after 5 weeks in culture, the TNC fold expansion was significantly (p<0.05) higher in TEPA-treated cultures compared to the controls, 1471±63.5 (n=89) vs. 1270 ±240 (n=55), respectively. The increase in TNC in TEPA-treated cultures did not result in increased HPC differentiation, but was accompanied by an increased self-renew capacity of CD34+ cells as represented by a 57±5.9 fold (n=47) vs. a 32±3.5-fold (n=38) amplification in the controls (p<0.0009). The overall fold expansion in culture analyzed by a Kaplan-Meier survival curve function demonstrate that the TNC, CFU, CD34+ and CD34+/38− cells derived from TEPA-treated cultures have higher in vitro survival probabilities than controls (p<0.0014). Cumulative values of all parameters were calculated and a transformation performed using the rank procedure. The results underline that TEPA increases CFU potential and CD34+ and CD34+/38− content during the ex-vivo expansion (p<0.01). The TEPA supplemented expansion technology was further tested after up-scaling of the processing and culturing procedures. AC133+ cells isolated by the CliniMACS device from frozen CB units obtained from 6 different banks. The expansion results of TNC, CD34+ cells, CFU and %CD34/38- were 337±23 (n=57), 21±4.3 (n=19), 133±27.5 (n=19) and 2.7% ±0.7 (n=19) fold, respectively. A clinical trial with TEPA expanded cultures for treatment of leukemia patients is currently ongoing at MD Anderson Cancer Center, USA.

Aspects of donation and processing of stem cell transplants from umbilical cord blood

In 1996, the Institute of Transfusion Medicine and Immunology started the banking of unrelated cord blood (CB) transplants in collaboration with the Department of Gynecology and Obstetrics at the Mannheim University Hospital. Up to now, more than 2000 placental blood samples have been cryopreserved. We analyze the results of CB collection and processing, and describe the legal background of CB collection in Germany. CB is only collected with informed consent of the mothers, and without evidence of neither maternal infection nor deformity of the baby. Units with a volume of > 60 ml or > 5 x 10(8) total nucleated cells (NC) are processed as potential transplants. All specimens are evaluated for cell count, HLA typing, sterility testing, ABO/Rh blood groups and clonogenic capacity of hematopoietic progenitor cells. The maternal blood is screened for various infectious disease markers. To reduce the volume of the transplants, we perform buffy coat processing by centrifugation. Finally, we cryopreserve the transplant using a 5.5 percent final concentration of DMSO, and store it in the vapour phase of liquid nitrogen. Results We collected on average 86 +/- 21 ml CB containing 8.87 +/- 3.41 x 10(8) NC (mean +/- SD). 45 percent of the collected units had to be discarded for various reasons. We achieved a mean mononucleated cell recovery by the buffy coat preparation method of 93.4 percent. Defining a threshold dose of 3.0 x 10(7) NC per kg body weight (BW) for transplantation, the mean BW of patients to be sufficiently treated with the stored transplants showed to be 29.6 kg. Conclusions In a very close collaboration between transfusion medicine and obstetricians, a routine collection program for unrelated and related allogeneic CB transplants was established. The CB donation needs to be in accordance with the German drug law. On the other hand, in terms of evidence based medicine, it seems not to be reasonable to collect autologous placental blood for a future transplantation.

Processing of Human Cord Blood by Three Different Procedures for Red Blood Cell Depletion and Mononuclear Cell Recovery

Background and Objectives: Human cord blood (CB) is an important source of stem cells which may be used for hematopoietic reconstitution as an alternative to bone marrow transplantation. Banking of CB would be accomplished by removing red blood cells (RBC) and plasma from CB collections. Our aim was to compare three different procedures for CB processing. Materials and Methods: Poligeline, hydroxyethyl starch gel (HES) and gelatin were used as separation media in processing 79 CB units for RBC depletion and mononuclear cell (MNC) recovery. Results: The best MNC recoveries were obtained performing the HES– and the gelatin–based procedures (80.9 and 84.7%, respectively), but the gelatin procedure allowed us to obtain the highest RBC depletion (96.4%); CD34+ cell recovery was higher using HES or gelatin as separation media (85.6 and 85.9%, respectively). Conclusion: The best results, as far as RBC removal and MNC recovery are concerned, were obtained by using gelatin as RBC sedimentation medium. Gelatin is a low–cost, animal–derived reagent, which has been successfully used for CB transplantation; the procedure is simple to perform and appears to be suitable for large–scale banking in view of CB transplantation.