An investigation of red blood cell concentrate quality during storage in paediatric-sized polyvinylchloride bags plasticized with alternatives to di-2-ethylhexyl phthalate (DEHP).

BACKGROUND AND OBJECTIVES: Di-2-ethylhexyl phthalate (DEHP) is a blood bag plasticizer. It is also a toxin, raising concerns for vulnerable populations, for example, neonates and infants. Here, the in vitro quality of red cell concentrates (RCC) stored in paediatric bags formulated with alternative plasticizers to DEHP was compared. MATERIALS AND METHODS: RCC were pooled and split into polyvinylchloride (PVC)/DEHP, PVC/1,2-cyclohexanedicarboxylic acid diisononyl ester (DINCH) or PVC/butyryl trihexyl citrate (BTHC) bags. Quality was assessed on storage days 5, 21, 35 and 43. RESULTS: Metabolism differed among the bags: pCO2 levels were lowest and pO2 were highest in BTHC bags. Glucose consumption and lactate production suggested higher metabolic rates in BTHC bags. ATP levels were best maintained in DINCH bags (day 43 mean level: 2·86 ± 0·29 µmol/g Hb). RCC in BTHC bags had the greatest potassium release (54·6 ± 3·0 mm on day 43). From day 21, haemolysis was higher in BTHC bags (P < 0·01) and by day 43 had exceeded 0·8% (0·85 ± 0·10%). RCC in BTHC bags showed more microparticle formation than RCC in DEHP or DINCH bags. CONCLUSION: The results suggest that the BTHC formulation used was detrimental to RBC quality. DINCH bags could be a viable alternative to DEHP: they outperformed DEHP bags energetically, with better maintenance of ATP levels.

The effect of timing of gamma-irradiation on hemolysis and potassium release in leukoreduced red cell concentrates stored in SAGM.

While irradiation of red cell concentrates (RCC) prevents graft-versus-host disease in susceptible transfusion recipients, it also damages red blood cells (RBC). To understand the ability of irradiation regulations to prevent transfusion of inferior units, we irradiated 980 RCC in saline-adenine-glucose-mannitol (SAGM) using various combinations of pre-irradiation age and post-irradiation storage times, and measured hemolysis and extracellular potassium levels. We observed unacceptably high hemolysis (>0·8%) in some RCC and elevated extracellular potassium levels in all gamma-irradiated RCC. This suggests that more restrictive storage times should be considered for RCC in SAGM.

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood.

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.

Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment.

In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34(+) cells, and granulocyte-macrophage colony-forming units (CFU-GM) cryopreserved in quality control vials. The median (range) post-thaw recovery of viable CD34(+) cells and CFU-GM was 66.4% (36.1-93.6%) and 63.0% (28.6-85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P=0.136 and 0.417, respectively) or platelets (P=0.88 and 0.126, respectively). However, the reinfused viable CD34(+) cells/kg of patient weight pre- or post-cryopreservation showed significant correlation to engraftment of neutrophils (P=0.0001 and 0.001, respectively) and platelets (P=0.023 and 0.010, respectively), whereas CFU-GM pre- or post-cryopreservation was significantly correlated to neutrophils (P=0.011 and 0.007, respectively) but not to platelets (P=0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34(+) cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34(+) cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage.

Evaluation of red blood cells stored at -80 degrees C in excess of 10 years.

BACKGROUND: RBCs frozen in 40 percent (wt/vol) glycerol are currently approved by the FDA and the AABB for storage at -80 degrees C for up to 10 years. STUDY DESIGN AND METHODS: This study examined 20 RBC units that had been cryopreserved in 40 percent (wt/vol) glycerol and stored at -80 degrees C for up to 22 years. Measures of the freeze-thaw-wash (FTW) recovery, ATP, 2,3-DPG, methemoglobin, RBC indices, morphology, and osmotic fragility were made immediately after deglycerolization and after 24 hours of storage at 4 degrees C. RESULTS: RBCs frozen for longer than 10 years had acceptable mean FTW recovery, normal oxygen transport function, RBC morphology, RBC indices, methemoglobin, and osmotic fragility. Statistical analysis indicated that the in-vitro viability and function of cryopreserved RBCs was not dependent on the length of frozen storage or postthaw storage at 4 degrees C but did correlate with the storage length at 4 degrees C before cryopreservation. CONCLUSION: The data reported in this study demonstrate that RBCs can be stored at -80 degrees C beyond 10 years with acceptable in-vitro quality and suggest that more defined criteria for the cryopreservation process be adopted.

Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation.

Three widely used viability assessments were compared: (1) membrane integrity of nucleated cells using trypan blue (TB) exclusion and a fluorometric membrane integrity assay (SYTO 13 and propidium iodide), (2) enumeration of viable CD34+ cells, and (3) clonogenic assay (granulocyte-macrophage colony-forming units, CFU-GM). Post thaw peripheral hematopoietic progenitor cells (HPC) were incubated at 0, 22, and 37 degrees C for 20-min intervals before assessment. The recovery of viable nucleated cells assessed by TB and SYTO/PI decreased significantly with time at incubation temperatures of 22 and 37 degrees C (P<0.05), and correlated with the concentration of mononuclear cells (MNC) (r=0.936, P<0.05). The decrease in recovery of viable nucleated cells was slower when thawed cells were incubated at 0 degrees C compared with 22 degrees C or 37 degrees C. The recovery, measured by absolute viable CD34+ or CFU-GM, was not affected by 2 h post thaw incubation (P>0.05) at 0, 22, and 37 degrees C (P>0.05). There were no significant differences in the measured recovery of viable CD34+ cells and CFU-GM at all incubation times (P>0.05) and temperatures (P>0.05). Both CFU-GM and absolute CD34+ cells can be used as post thaw viability assays for HPC cryopreserved for transplantation.

High-efficiency volume reduction of cord blood using pentastarch.

Human umbilical cord blood (UCB) has been used successfully to treat a variety of genetic, hematological, and oncologic disorders. However, the low number of hematopoietic progenitor cells available in donated cord blood samples limits transplantation of cord blood to children and small adults. Reduction of the volume of umbilical cord blood is widely used in cord blood banking to reduce the storage requirements in large-scale UCB banks. Unfortunately, during the volume reduction process, up to 40% or more of the progenitor cells are lost using current reduction methods. This study describes a highly reproducible, double collection technique using Pentaspan to reduce UCB volume by red cell depletion. This results in the preservation of critical hematopoietic progenitor cells. The final volume of the leukocyte concentrates (LC) was 19.8 +/- 0.4 ml with 95% red cell depletion. The recovery of nucleated cells (NC), mononuclear cells (MNC), CD34(+) cells and colony-forming units (CFU) following double collection was 89%, 94%, 96%, and 106%, respectively. This is significantly higher than the recovery from single collections, where recovery was 74%, 77%, 84%, and 91% for NC, MNC, CD34(+) and CFU, respectively. The double collection technique provides an efficient and highly reproducible method for the preparation of UCB for long-term storage and transplantation.

Damage and protection of UC blood cells during cryopreservation.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.