UC Blood Research Articles

Microbial screening of UC blood units by an automated culture system: effect of delayed testing on bacterial detection

Microbial screening is a mandatory test for banked UC blood (UCB) to comply with the code of good manufacturing practice (GMP). Cord blood banks (CBBs) are not always closely located to a GMP-licensed microbiology laboratory, resulting in time delays for transport of specimens prior to microbiological testing. This study investigated the influence of >/=24 h delays in initiating automated microbial screening on the detection of bacteria in UCB, by analysis of specimens deliberately spiked with bacteria and the recovery of bacteria from cryopreserved spiked-UCB. UCB was processed according to standard CBB procedures and spiked with Staphylococcus epidermidis or Escherichia coli [2-2000 colony forming units (CFU)/mL]. Spiked-UCB (0.5 mL) was (1) held at room temperature (RT) and inoculated into pediatric BacT/Alert bottles (bioMérieux) at Days 1, 4 and 7 (delayed inoculation); and (2) inoculated directly (Day 0) into replicate BacT/Alert bottles and held at RT for 1, 4 or 7 days before loading onto the BacT/ALERT system (delayed loading). Spiked-UCB samples were cryopreserved. Bacterial counts were quantitated on horse blood agar plates. Bacterial growth in UCB spiked with a single bacterium was capable of detection by the BacT/ALERT system. S. epidermidis grew in all conditions of delayed testing (ie. delayed inoculation and delayed loading). E. coli failed to grow under conditions of delayed inoculation but grew at all time points of delayed loading. S. epidermidis and E. coli were recovered from cryopreserved spiked-UCB. Inoculation of culture bottles as soon as possible after sample preparation is preferable. Bacteria can maintain viability in BacT/ALERT bottles inoculated and held at RT for up to 7 days prior to automated culture testing. Bacteria can be successfully recovered from cryopreserved UCB.

Kinetics of quiescent cord blood stem/progenitor cells with high proliferative potential in stem-cell expansion culture.

The most primitive engrafting hematopoietic stem cell (HSC) resides mainly in a tumor growth factor-beta (TGF-beta)-dependent quiescent phase of the cell cycle. In this study, ex vivo expansion of UC blood (UCB) HSCs has been investigated, with the aim of showing whether quiescent HSCs can be recovered from expansion culture. AC133(+) stem/progenitor cells from six full term-pregnancies UCB-samples were immunomagnetically selected, followed by ex vivo expansion culture in the presence of thrombopoietin (TPO), c-kit ligand (KL), flt-3 ligand (FL) and IL-6. Quiescent HSCs were detected by a clonogenic assay that allows the detection of multipotent and committed single- lineage quiescent stem/progenitor cells, named mHPP-Q and cHPP-Q, respectively, by means of a TGF-beta blocking Ab. Expansion culture of fresh selected AC133(+) cells for 1 week caused maintenance rather than expansion of mHPP-Q cells and a 1-fold increase in cHPP-Q cells. A further week culture initiated with 7-day expanded AC133(+) cells resulted in an additional 1.5-fold expansion of cHPP-Q while no mHPP-Q cells could be detected. Amplification of cHPP-Q cells in long-term expansion cultures initiated with 14-day expanded AC133(+) cells was observed for at least a further 4 weeks. A small proportion of HPP-Q cells recovered from 7-day expansion cultures retain their multilineage potential: longer culturing of these cells results in the loss of multilineage potential while they maintain quiescent behavior and high proliferative potential.

Long term cryostorage of UC blood units: ability of the integral segment to confirm both identity and hematopoietic potential

With the maturation of UC blood banking, cord blood (CB) units stored for years prior to use in transplantation present a new set of issues in clinical transplantation, including interval improvements in immune typing and confirmation of product identity and viability. A preliminary analysis of the transplants supported by the St Louis Cord Blood Bank, looking for an impact of length of CB storage time and transplant outcome was performed. We evaluated the utility of an integral segment containing an aliquot of cryopreserved product that has been exposed to the same post-processing storage conditions as the unit as a quality control tool for CB banking. Engraftment and survival following unrelated donor UC blood transplant were evaluated based on length of CB product storage at the St Louis Cord Blood Bank. A strategy of routine testing of the contiguous segment for high-resolution HLA typing (also confirming identity) and CFU analysis was tested in 283 consecutive CB searches. Comparison between CB unit and contiguous segment viability and hematopoietic potential was performed on 30 research CB units that had been stored up to 5 years. There was no statistical difference in engraftment or survival following unrelated donor cord blood transplant employing units banked < 1 year or > 3 years. Confirmatory HLA typing, CFU and viability testing was successfully performed from the same segment as part of a strategy for product release evaluation. When comparing the segment with its corresponding CB unit, the total colony-forming units (CFU) measured in the two was similar (P = 0.51, paired t-test). Three research units purposely sabotaged by an overnight thaw and refreeze had no CFU growth, but viability as measured by Trypan was still 68-98%. No deterioration of hematopoietic potential has been detected with storage up to 5 years. The contiguous segment CFU is representative of the product, and thus is a useful tool for quality control and confirmation of product viability. Viability, as measured by Trypan blue dye exclusion may be falsely reassuring.

Long term cryostorage of UC blood units: ability of the integral segment to confirm both identity and hematopoietic potential

Long term cryostorage of UC blood units: ability of the integral segment to confirm both identity and hematopoietic potential

Generation of Ag-specific cytotoxic T lymphocytes by DC transfected with in vitro transcribed influenza virus matrix protein (M1) mRNA.

Application of DC transfected with tumor Ag RNA is promising for DC-based tumor immunotherapy. In this study, Ag-specific cytotoxic T lymphocytes (CTL) were generated by priming lymphocytes with DC transfected with in vitro transcribed (IVT) influenza virus matrix protein M1 (M1) mRNA. Human UC blood-CD34+ cell-derived DC were transfected with IVT mRNA encoding either the enhanced green fluorescence protein (EGFP), or M1 by square-wave electroporation. DC were confirmed to have typical morphology and phenotype. DC transfected with IVT EGFP mRNA were analyzed with the FACScan flow cytometer, to confirm the efficiency of this transfection method. On Days 7, 14, 21 and 28 after the start of DC culture, DC were harvested and electroporated with M1 mRNA. The transfected DC were co-cultured with autologous UC blood CD34- cells. One week after the fourth priming of autologous CD34 negative cells with M1 mRNA electroporated DC, Ag-specific CTL activity was evaluated. To prepare target cells, M1 mRNA was added to autologous DC 48 h prior to CTL assays. Our CTL assays results indicate that UC blood CD34+ cell-derived DC transfected with M1 mRNA by electroporation stimulated Ag-specific CTL responses that are capable of recognizing and lysing autologous DC loaded with M1 mRNA. M1 mRNA transfected DC-primed CTL showed a significant cytotoxic activity against M1 mRNA loaded autologous DC, while nearly baseline cytotoxic activity was recorded for the M1 mRNA unloaded DC. Our results showed that mRNA-transfected DC are potent stimulators of T-cell immunity in vitro. In addition, mRNA-loaded DC can function as targets in CTL cytotoxicity assays, which offer a practical substitute for tumor cells in assays to test the immunological effects of specific Ags.

Implication of maternal-cell contamination in the clinical banking of umbilical cord blood.

The increasing utilization of human UC blood (UCB) in transplantation has drawn attention to the need for rationalization of selection, collection, processing, testing, banking and release of UCB. However, the issue of maternal blood contamination has not been well addressed. There are concerns that maternal T cells might elicit GvHD post-UCB transplant. Maternal T cells in 58 male UCB allografts were enumerated using fluorescent in situ hybridization and flow cytometry. Obstetric factors, preceding labor, multi-parity and gestational age, were also analyzed. Levels of maternal cells of 0.75-5.25% were found in 15.5% (9/58) UCB. There was no association of maternal-cell contamination with preceding labor [25% (2/8) with previous delivery versus 35.4% (17/48) first born, P = 0.702], nor any correlation with multi-parity [37.5% (3/8) para > or = 3 versus 16.7% (8/48) para < 3, P = 0.181]. Gestation age of newborns also exhibited no association with maternal-cell contamination (39.47 weeks in newborn UCB with maternal cells, versus 39.58 weeks without: P = 0.674). The extrapolated maternal T cells/kg in nine UCB transplants were 1.05 x 10(5) +/- 1.12 x 10(5) (3.40 x 10(4) - 3.18 x 10(5)). In relation to the arbitrary threshold of 1 x 10(5) T cells/kg in HLA-mismatched transplants utilizing T-cell depleted BM, 22.2% (2/9) of UCB transplants having maternal-cell contamination might be at risk of GvHD. Data support the need for testing for maternal blood in UCB, and evaluating the clinical relevance of GvHD in patients post-UCB transplant. The establishment of guidelines and standards for release of such UCB collections would be advisable in evidence-based UCB transplantation.

A simple and reliable procedure for cord blood banking, processing, and freezing: St Louis and Ohio Cord Blood Bank experiences

In UC blood banking, volume and RBC reduction of the collected UC blood allows more efficient long-term storage and decreases infusion-related hemolysis and DMSO toxicity. However, high cell yields are imperative. At the St Louis Cord Blood Bank, we have systematically addressed processing/freezing and have developed a simple processing/freezing procedure. The methodology is a modification of the hetastarch sedimentation and volume reduction approach of Rubinstein at the New York Placental Blood Program. Cord blood is mixed with a 1:5 v/v ratio of hetastarch. The product is incubated for 45 min in an inverted position in a refrigerated centrifuge (4 degrees C), and then is spun for 5 min at 50 g. RBC concentrate is drained from the bottom. The volume drained is calculated to remove 80% of RBC. The UC blood unit is then resuspended and spun for 13 min at 420 g. Plasma is expressed from the top. A final product volume of 27 mL (range 16-58 mL) was obtained from an original 50-200 mL of UC blood collected. The average yield of total nucleated cells pre- and post-processing was 90% for the first 4055 UC blood units banked. Pre- and post-processing CFU and CD34 yields were tested in a cohort and were similarly conserved. With a processing time of 3 h for a single cord, this process is time efficient and lends itself well to processing several units at the same time. The technique has been exported to other laboratories with similar yields. This simple methodology results in reliable yields and is well suited to larger scale banking.

Damage and protection of UC blood cells during cryopreservation

Current procedures for the cryopreservation of umbilical cord blood (UCB) progenitor cells, which are based on techniques used for BM, have had varying degrees of success (survival 9-118%). Improving the effectiveness of UCB cell therapies demands a more comprehensive understanding of freezing injury during cryopreservation. Leukocyte concentrates from UCB, with or without 10% DMSO were cooled at 1 degrees C/min to different subzero temperatures (-5 to -50 degrees C), then either thawed directly (thaw) or plunged into liquid nitrogen before thawing (plunge). Single-platform flow cytometry with 7-amino-actinomycin D was used to directly quantify survival of CD34(+) cells. Fluorescent microscopy was used to examine plasma membrane integrity of nucleated cells. Without DMSO, recovery of nucleated cells was approximately 80% for both thaw and plunge. Survival was 9%, indicating damage to the plasma membrane. With 10% DMSO, nucleated cell recovery was also approximately 80%, indicating that DMSO does not improve recovery of nucleated cells. Survival, however, was much higher with DMSO, > 60% for nucleated cells thawed directly, and 30-55% for cells thawed from plunge, demonstrating cryoprotection conferred by DMSO. With DMSO, survival of CD34(+) cells was higher than that of nucleated cells, indicating that CD34(+) cells with 10% DMSO are more tolerant to cryopreservation than the total nucleated cell population. This study provides the necessary data on the low temperature response of UCB progenitor cells that are critical for the development of standards for the cryopreservation of UCB.

Human UC-blood banking: impact of blood volume, cell separation and cryopreservation on leukocyte and CD34+ cell recovery

UC blood represents an increasingly useful source of hematopoietic stem cells for BMT, although, currently, low cell numbers generally limit its use to pediatric patients. We have determined parameters that influence the recovery of viable cells during processing and cryopreservation, in an effort to set guidelines for determining whether a sample will yield sufficient cells to be of use in the transplant setting. UC blood was collected from donors from January 1996 to December 1999. Volume was reduced using Ficoll, followed by cryopreservation under liquid nitrogen. Total leukocyte and CD34(+)-cell counts were determined prior to processing and a subset of samples was also assessed post-processing and post-cryopreservation. Outcomes for 3816 samples were analyzed to determine the correlation between cell number, cell type, volume, and time between collection and processing. A positive relationship was observed between volume and cell number for both leukocytes and the CD34(+) cells. This correlation allowed us to determine the number of leukocytes and CD34(+) cells expected from a sample based on volume, and to set guidelines for determining the practicality of storing any given sample prior to processing and cryopreservation. Measuring blood volume gives a very useful indication of the total leukocyte and CD34(+) cell number. The majority (75%) of cord-blood samples contain sufficient leukocytes for a pediatric transplant, and the number of cells available can be determined prior to processing by measuring blood volume.

Umbilical cord blood processing with the Optipress II blood extractor

We have previously shown that UC blood (UCB) units can be volume-reduced manually, in a closed system, without major losses of nucleated and CD34(+) cells and without the addition of exogenous material. Our aim was to use an automated method for the separation of the UCB components using the Optipress II, extractor, with the 'buffy-coat' collection in a standardized volume. After centrifugation, the 51 UCB units were separated into the three blood components, plasma, buffy coat (BC) and red cells, using the Optipress II. The final volume of the BC fraction, rich in nucleated and progenitor CD34(+) cells, was set at 30 mL. The nucleated and CD34(+) cell content of the UCB collections and the resulting BC were evaluated. The UCB units were grouped according to the volume collected: Group I < 80 mL and Group II > or = 80 mL. Standardization of the BC at 30 mL resulted in significant volume reduction for both groups, with median values of 51% in Group I and 70% in Group II. The nucleated and CD34(+) cell recoveries in the BC from Group I were 88% and 99% respectively; for Group II they were 80% and 97%. This semi-automated method of volume reduction efficiently reduces low, as well as high volume UCB units, with good nucleated- and progenitor-cell yields. Being a closed system and free of external material, the risk of contamination is minimized. The resulting fractions are then available for validation studies of the unit, effectively fulfilling the main requisites for UCB banking.

Intradermal immunization by paternal and non-paternal cells in patients with infertility and sterility

We have previously shown that UC blood (UCB) units can be volumereduced manually, in a closed system, without major losses of nucleated and CD34+ cells and without the addition of exogenous material. Our aim was to use an automated method for the separation of the UCB components using the Optipress II, extractor, with the ‘buffy-coat’ collection in a standardized volume.After centrifugation, the 51 UCB units were separated into the three blood components, plasma, buffy coat (BC) and red cells, using the Optipress II. The final volume of the BC fraction, rich in nucleated and progenitor CD34+ cells, was set at 30 mL. The nucleated and CD34+ cell content of the UCB collections and the resulting BC were evaluated.The UCB units were grouped according to the volume collected: Group I < 80 mL and Group II ≥80 mL. Standardization of the BC at 30 mL resulted in significant volume reduction for both groups, with median values of 51% in Group I and 70% in Group II. The nucleated and CD34+ cell recoveries in the BC from Group I were 88% and 99% respectively; for Group II they were 80% and 97%.This semi-automated method of volume reduction efficiently reduces low, as well as high volume UCB units, with good nucleated- and progenitor- cell yields. Being a closed system and free of external material, the risk of contamination is minimized. The resulting fractions are then available for validation studies of the unit, effectively fulfilling the main requisites for UCB banking.