Altered processing of thawed red cells to improve the in vitro quality during postthaw storage at 4 degrees C

BACKGROUND: The use of a functionally closed system (ACP215, Haemonetics) for the glycerolization and deglycerolization of red blood cell (RBC) units allows for prolonged postthaw storage. In this study, the postthaw quality of previously frozen, deglycerolized RBCs resuspended in saline-adenine-glucose-mannitol (SAGM) or additive solution AS-3 was investigated. STUDY DESIGN AND METHODS: Leukoreduced RBC units were frozen with 40 percent glycerol and stored at -80 degrees C for at least 14 days. The thawed units were deglycerolized with the ACP215, resuspended in SAGM or AS-3, and stored at 2 to 6 degrees C for up to 21 days. RESULTS: The mean +/- standard deviation in vitro freeze-thaw-wash recovery was 81 +/- 5 percent. During storage, hemolysis of deglycerolized cells remained below 0.8 percent for 2 days in SAGM and for 14 days in AS-3. This difference was explained by the protective effect of citrate, which is present in AS-3. Cells stored in AS-3 showed a lower glycolytic activity and a faster decline in adenosine 5'-triphosphate (ATP) than cells in SAGM. Increasing the internal pH of cells before storage in AS-3 by use of phosphate-buffered saline (PBS) in the deglycerolization procedure resulted in elevated lactate production and better maintenance of intracellular ATP content. After 3 weeks of storage, the ATP content of PBS-washed cells amounted to 2.5 +/- 0.5 micromol per g of hemoglobin (Hb), whereas for saline/glucose-washed cells this value was decreased to 1.0 +/- 0.3 micromol per g of Hb. CONCLUSIONS: Leukoreduced, deglycerolized RBCs can be stored for 48 hours in SAGM. Improved ATP levels during refrigerated storage can be observed with thawed cells, resuspended in AS-3, when PBS is used as a washing solution.     

In vitro and in vivo measurements of gamma-radiated, frozen, glycerolized RBCs

BACKGROUND: Transfusion-associated GVHD results from the presence of viable lymphocytes in transfused allogeneic blood components. Viable immunocompetent lymphocytes have been detected in RBCs that were frozen with glycerol and washed before transfusion. STUDY DESIGN AND METHODS: The study reported here assessed the effect of irradiation on human RBCs frozen with 40-percent (wt/vol) glycerol and stored at -80 degrees C. In vitro and in vivo testing was done on human RBCs that were frozen with 40-percent (wt/vol) glycerol at -80 degrees C, with some units exposed to 2500 cGy of gamma radiation and others not irradiated, and that, after thawing and washing, were stored in a sodium chloride-glucose solution at 4 degrees C for 3 days before autologous transfusion. RESULTS: The glycerol-frozen RBCs treated with 2500 cGy before deglycerolization had a mean freeze-thaw-wash recovery of 87 percent and a mean 24-hour posttransfusion survival of 86 percent after storage for 3 days at 4 degrees C in a 0.9-percent NaCl and 0.2-percent glucose solution. For the nonirradiated units, the mean freeze-thaw-wash recovery was 85 percent and the mean 24-hour posttransfusion survival was 83 percent. CONCLUSION: These data show similar, acceptable results for RBCs frozen with 40-percent (wt/vol) glycerol at -80 degrees C and treated in the frozen state with 2500 cGy of gamma radiation and for RBCs that were not irradiated, all of which were washed and then stored in a sodium chloride-glucose solution for 3 days before autologous transfusion.     

In vivo survival of apheresis RBCs, frozen with 40-percent (wt/vol) glycerol, deglycerolized in the ACP 215, and stored at 4°C in AS-3 for up to 21 days

BACKGROUND: The FDA has approved the storage of frozen RBCs at -80 degrees C for 10 years and the postwash storage at 4 degrees C for no more than 24 hours. The 4 degrees C postwash storage period is limited to 24 hours, because the current deglycerolization systems are functionally open systems. STUDY DESIGN AND METHODS: Two units of RBCs were collected from each of 13 healthy male volunteers. The RBCs were collected in CP2D by the FDA-approved protocol for an automated apheresis device (MCS, LN8150, Haemonetics) and were stored at 4 degrees C in AS-3 for 6 days. Using a single disposable glycerolization set in an automated, functionally closed system (ACP 215, Haemonetics) each unit was transferred to a 1000-mL PVC plastic bag and glycerolized to a concentration of 40-percent (wt/vol) glycerol and frozen at -80 degrees C. A single disposable deglycerolization set in the ACP 215 was used to deglycerolize the 2 units from the same donor. The deglycerolized RBCs were stored at 4 degrees C in AS-3 for as long as 21 days. RESULTS: The mean +/- SD freeze-thaw-wash recovery value was 89.4 +/- 3 percent. The residual hemolysis in the RBCs stored at 4 degrees C in AS-3 for 21 days after deglycerolization was 0.9 +/- 0.2 percent, and the units were negative for both aerobic and anaerobic bacteria. The mean Nageotte WBC count was 9 x 10(6) per unit. When the deglycerolized RBCs were given as an autologous transfusion after storage at 4 degrees C in AS-3 for the 7- to 18-day period, the mean +/- SD 24-hour posttransfusion survival was 77 +/- 7 percent, and the index of therapeutic effectiveness was 69 +/- 8 percent. CONCLUSION: Two units of human RBCs collected from a single donor by apheresis in the MCS using an LN8150 set can be glycerolized sequentially with a single disposable set and deglycerolized sequentially with another single disposable set in the ACP 215. The previously frozen RBCs stored in AS-3 for 7 to 18 days at 4 degrees C had acceptable hemolysis and an acceptable mean 24-hour posttransfusion survival value and index of therapeutic effectiveness.     

Studies of the recovery and the cost of low-glycerol cryopreserved human red blood cells

Red blood cells were equilibrated with 28 per cent (v/v) glycerol and 3 per cent mannitol in 0.65 g/100 ml sodium chloride. The units were frozen by immersion into liquid nitrogen and stored at -160 C. After thawing, they were reconstituted and washed using the IBM 2991 Blood Cell Processor. Freeze-thaw rate curves, the effect of thawing techniques, the effect of varying postthaw washing and processing techniques, estimates of red blood cell losses because of hemolysis, and in vitro recovery were determined. In vivo recovery was determined by 51Cr techniques 24 hours after infusion and Ashby survivals and subsequent life span were measured. Metabolic, scanning electronmicroscopy, cost estimates and quality control studies were done on the reconsituted red blood cells. Recipients were evaluated before and after transfusion for metabolic erythrocyte characteristics and for evidence of hemolysis. The modified method requires less wash solution and less technician time than does the standard low-glycerol method. Two units for the same recipient could be passed through the IBM software with no alteration of cell survival or loss. Revision of the IBM 2991 processing procedure provided excellent recovery of viable previously frozen red blood cells at probably a lower cost.     

In vitro and in vivo measurements of human RBCs frozen with glycerol and subjected to various storage temperatures before deglycerolization and storage at 4 degrees C for 3 days

BACKGROUND: This study was designed to assess the effects of changes in storage temperature of frozen RBCs such as might occur during a malfunction of the -80 degrees C mechanical freezer or during shipment. STUDY DESIGN AND METHODS: Fifteen participants donated blood for autologous transfusion of RBCs; all RBCs were frozen with 40-percent (wt/vol) glycerol. Five subjects received RBCs that were stored at -80 degrees C alone before transfusion. Five subjects received RBCs that were stored initially at -80 degrees C, then at -40 degrees C for 4 weeks, and finally at -80 degrees C before transfusion. Five subjects received RBCs that were stored at -80 degrees C, then at -20 degrees C for 2 weeks, and finally at -80 degrees C before transfusion. After deglycerolization, the RBCs were stored at 4 degrees C in a sodium chloride-glucose solution for 3 days before transfusion. RESULTS: No significant differences were observed in freeze-thaw recovery, freeze-thaw-wash recovery, 24-hour posttransfusion survival, index of therapeutic effectiveness, or RBC ATP levels. Greater hemolysis and reduced RBC K+ levels were observed in the units stored at -80 degrees C/-40 degrees C/-80 degrees C and in those stored at -80 degrees C/ -20 degrees C/-80 degrees C compared with the units stored at -80 degrees C alone, but these differences did not affect the 24-hour posttransfusion survival. CONCLUSIONS: The results of this study indicated that RBCs frozen with 40-percent (wt/vol) glycerol can be stored at -40 degrees C for 4 weeks or at -20 degrees C for 2 weeks between periods of frozen storage at -80 degrees C with satisfactory results.     

A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4°C in AS-3: assessment of RBC processing in the ACP 215

BACKGROUND: The FDA has approved the storage of frozen RBCs at -80 degrees C for 10 years. After deglycerolization, the RBCs can be stored at 4 degrees C for no more than 24 hours, because open systems are currently being used. Five laboratories have been evaluating an automated, functionally closed system (ACP 215, Haemonetics) for both the glycerolization and deglycerolization processes. STUDY DESIGN AND METHODS: Studies were performed at three military sites and two civilian sites. Each site performed in vitro testing of 20 units of RBCs. In addition, one military site and two civilian sites conducted autologous transfusion studies on ten units of previously frozen, deglycerolized RBCs that had been stored at 4 degrees C in AS-3 for 15 days. At one of the civilian sites, 10 volunteers received autologous transfusions on two occasions in a randomized manner, once with previously frozen RBCs that had been stored at 4 degrees C in AS-3 for 15 days after deglycerolization and once with liquid-preserved RBCs that had been stored at 4 degrees C in AS-1 for 42 days. RESULTS: The mean +/- SD in vitro freeze-thaw-wash recovery value was 87 +/- 5 percent; the mean +/- SD supernatant osmolality on the day of deglycerolization was 297 +/- 5 mOsm per kg of H(2)O, and the mean +/- SD percentage of hemolysis after storage at 4 degrees C in AS-3 for 15 days was 0.60 +/- 0.2 percent. The paired data from the study of 10 persons at the civilian site showed a mean +/- SD 24-hour posttransfusion survival of 76 +/- 6 percent for RBCs that had been stored at 4 degrees C for 15 days after deglycerolization and 72 +/- 5 percent for RBCs stored at 4 degrees C in AS-1 for 42 days. At the three sites at which 24-hour posttransfusion survival values were measured by three double-label procedures, a mean +/- SD 24-hour posttransfusion survival of 77 +/- 9 percent was observed for 36 autologous transfusions to 12 females and 24 males of previously frozen RBCs that had been stored at 4 degrees C in AS-3 for 15 days after deglycerolization. CONCLUSION: The multicenter study showed the acceptable quality of RBCs that were glycerolized and deglycerolized in the automated ACP 215 instrument and stored in AS-3 at 4 degrees C for 15 days.     

The effects of cryopreservation on red blood cell microvesiculation, phosphatidylserine externalization, and CD47 expression

BACKGROUND: Since the 1950s, cryopreservation has been used in transfusion medicine for long-term storage of phenotypically rare red blood cells (RBCs). Recent reports have identified phosphatidylserine (PS) exposure, loss of CD47 expression, and membrane microvesiculation as important indicators of RBC storage lesion and in vivo survival. The purpose of this study was to assess the effects of RBC cryopreservation and prefreeze storage length on these novel markers of membrane injury and to correlate them to traditional RBC quality indicators. STUDY DESIGN AND METHODS: Leukoreduced RBC units were collected in citrate-phosphate-dextrose (CPD)-saline-adenine-glucose-mannitol (SAGM), hypothermically stored (1-6 degrees C) for 2 to 3 days or 13 to 14 days after collection, and then cryopreserved in 40 percent (wt/vol) glycerol. In vitro RBC quality was assessed before freeze, after thaw, and 24 hours after thaw by evaluating RBC recovery, hemolysis, sterility, residual glycerol, adenosine triphosphate, extracellular potassium, RBC indices, and morphology. RBC membrane microvesiculation, PS externalization, and CD47 expression changes were evaluated using flow cytometry. RESULTS: Leukoreduced CPD-SAGM RBCs showed acceptable in vitro quality after deglycerolization, according to conventional assays. Cryopreservation alone did not induce significant changes in PS exposure, CD47 expression, and membrane microvesiculation. Prolonged prefreeze storage, however, resulted in a significant increase in RBC PS exposure and microvesiculation after 24 hours of postthaw hypothermic storage (1-6 degrees C). No significant changes in CD47 expression were detected. CONCLUSION: High-glycerol cryopreservation does not induce microvesiculation, PS exposure, and loss of CD47 expression in RBC membranes. Since prolonged prefreeze storage can result in RBC membrane injury during the postdeglycerolization storage period, more defined criteria for this variable should be adopted.     

Posttransfusion survival (24-hour) and hemolysis of previously frozen, deglycerolized RBCs after storage at 4 degrees C for up to 14 days in sodium chloride alone or sodium chloride supplemented with additive solutions

BACKGROUND: Previously frozen human RBCs currently are glycerolized and deglycerolized by the use of open systems that limit storage of the deglycerolized RBCs at 4 degrees C to only 24 hours. STUDY DESIGN AND METHODS: Healthy male volunteers who met AABB requirements for blood donors (n = 38) were studied. A volume of 450 mL of blood was collected into CPDA-1. The RBC concentrates were stored at 4 degrees C for 3 to 6 days before being frozen with 40-percent (wt/vol) glycerol and stored at -80 degrees C. The RBCs were deglycerolized, resuspended in 0.9-percent sodium chloride and 0.2-percent glucose (SG) solution or SG solution supplemented with AS-1, AS-3, or AS-5, and stored in the resuspension medium at 4 degrees C for 14 days. RESULTS: The mean +/- SD freeze-thaw-wash process recovery was 90.0 +/- 4.0 percent for all 38 units. The mean 24-hour posttransfusion survival value was 79 percent for deglycerolized RBC stored at 4 degrees C for 7 days in SG alone, SG plus AS-3, or SG plus AS-5. Deglycerolized RBC that were stored at 4 C for 14 days in SG supplemented with AS-1, AS-3, or AS-5 had a mean 24-hour posttransfusion survival of 74 percent. After 7 days of storage of deglycerolized RBCs in SG alone, the mean hemolysis was 3. 7 percent. After 14 days of storage of deglycerolized RBCs in SG supplemented with AS-1, AS-3, or AS-5, the mean hemolysis was 2.5 percent. CONCLUSIONS: The levels of hemolysis did not correlate with the 24-hour posttransfusion survival values.     

The in vitro quality of red blood cells frozen with 40 percent (wt/vol) glycerol at -80 degrees C for 14 years, deglycerolized with the Haemonetics ACP 215, and stored at 4 degrees C in additive solution-1 or additive solution-3 for up to 3 weeks

BACKGROUND: Red blood cells (RBCs) frozen with 40 percent (wt/vol) glycerol, stored at -80 degrees C (mean temperature; range, -65 to -90 degrees C) for 14 years, deglycerolized in the Haemonetics automated cell processor (ACP) 215 with the 325-mL disposable bowl, and stored at 4 degrees C in additive solution (AS)-1 or AS-3 for 21 days were evaluated. STUDY DESIGN AND METHODS: A total of 106 units of citrate phosphate dextrose adenine-1 RBCs were frozen with 40 percent (wt/vol) glycerol in the original 800-mL polyvinylchloride plastic bag and stored in corrugated cardboard boxes at -80 degrees C for 14 years. The thawed units were deglycerolized with the ACP 215 with a 325-mL disposable bowl and stored in AS-1 or AS-3 at 4 degrees C for 21 days. RESULTS: The freeze-thaw recovery value was 94 +/- 4 percent (mean +/- SD), the freeze-thaw-wash recovery value was 80 +/- 7 percent, and there was no breakage. Thirty-eight units were processed as 19 pairs. Two units of ABO-matched units were thawed, pooled, divided equally into two units, and deglycerolized. One unit was stored in AS-1 and the other in AS-3 at 4 degrees C for 21 days. Units stored in AS-1 exhibited significantly greater hemolysis than those stored in AS-3. CONCLUSIONS: Acceptable results were achieved when RBCs frozen at -80 degrees C for 14 years were deglycerolized in the ACP 215. Deglycerolized RBCs in AS-1 exhibited significantly higher hemolysis than those in AS-3 after storage at 4 degrees C for 7 to 21 days.     

Bacteria levels in components prepared from deliberately inoculated whole blood held for 8 or 24 hours at 20 to 24 degrees C

BACKGROUND : An increase from 8 to 24 hours in the time that units of whole blood can be held at room temperature after phlebotomy would give blood centers more flexibility in component manufacturing and might allow receipt of many infectious disease test results prior to component preparation. However, the potential for bacterial growth during prolonged holding periods requires further study. STUDY DESIGN AND METHODS : In the Phase I study, 2-unit pools of ABO-identical whole blood were deliberately inoculated on Day 0 with Staphylococcus aureus or Pseudomonas fluorescens. They were then divided in half and stored at 20 to 24 degrees C. Red cells (RBCs) with additive solution, platelet concentrates (PCs), and frozen plasma were prepared after 8 and 24 hours. Bacteria levels in PCs and RBCs were monitored on Day 1; bacteria levels were measured in plasma after thawing. In the Phase II study, the same basic design as in Phase I was used, except that 10 bacterial species were studied, lower inocula were used, and RBCs prepared after a 24-hour room-temperature whole-blood hold were white cell-reduced by filtration. Bacterial growth was monitored during 42- day storage of RBCs (1 – 6 degrees C) and 5-day storage of PCs (20 – 24 degrees C) and after thawing of frozen plasma. RESULTS : For Phase I, significantly higher bacteria levels were observed in RBCs prepared after a prolonged hold (p < 0.05); higher levels were not observed in PCs and thawed plasma units. In Phase II, prior to white cell reduction by filtration, 8 of 10 organisms had significantly higher levels in RBCs prepared after a 24-hour hold than in RBCs prepared after an 8- hour hold, when both were examined on Day 1 (p < 0.05). For seven of eight organisms examined on Days 1, 21, and 42, filtration (white cell reduction) reduced the bacteria in RBCs prepared from 24-hour whole blood units to those levels found in unfiltered RBCs prepared from whole blood units held at 8 hours. A prolongation of the holding time from 8 to 24 hours resulted in significantly lower bacteria levels (p < 0.05) in PCs early in storage (Days 1, 1 – 2, or 1 – 3) for seven organisms, with no significant difference for two organisms, and a small but significant increase for one organism (Day 3, p < 0.05). There was no difference in bacteria or endotoxin levels in thawed units of plasma prepared from whole blood after 8- or 24-hour holding times. CONCLUSION : The levels of bacteria present in components after deliberately inoculated whole blood units are held for 8 and 24 hours depended on the organisms tested, the whole-blood holding period, and the blood component assayed; for RBCs, they also depended on whether WBC reduction by filtration was performed.     

Extracellular potassium concentrations in red blood cell suspensions after irradiation and washing

BACKGROUND: Extracellular potassium concentration [K(+)e] increases with duration of red blood cell storage. Sometimes red blood cells (RBC) are washed before transfusion to infants to reduce [K(+)e] of these components. AABB standards permit storage of washed RBCs at 4 degrees C for 24 hours. The [K(+)e] of washed RBCs during storage is not known. Experiments were performed to provide those data. STUDY DESIGN AND METHODS: One day after outdating, 26 RBC units were washed without irradiation or before or after irradiation (25 Gy), and [K(+)e] was measured for 24 hours. [K(+)e] was measured also immediately before transfusion of 29 nonoutdated irradiated and washed RBC units. RESULTS: After washing, [K(+)e] increased in a time-dependent fashion. [K(+)e] increased more rapidly in preparations of irradiated than nonirradiated RBCs. [K(+)e] was less after washing after irradiation (1.6 +/- 0.3, 2.4 +/- 0.3, 3.0 +/- 0.3, 3.6 +/- 0.3. 4.2 +/- 0.4, 5.3 +/- 0.5, 8.6 +/- 1.0, and 14.3 +/- 1.3 mEq/L at 0, 1, 2, 3, 4, 6, 12, and 24 hr; mean +/- SD) than washing before irradiation (p < 0.001). The increase in [K(+)e] during the first 6 hours after washing after irradiation was linear (0.61 +/- 0.08 mEq K(+)/L/hr). The probability of a unit of RBCs having a [K(+)e] greater than 5 mEq per L is 0.0 to 0.2 percent 3 hours after washing irradiated RBCs and 0.0 to 1.1 percent 6 hours after washing nonirradiated RBCs. CONCLUSIONS: [K(+)e] increases after washing irradiated and nonirradiated packed RBCs. After irradiation and washing, the [K(+)e] for the initial 6 hours can be predicted from the [K(+)e] immediately after washing. There is a low probability that a unit of RBCs would have a [K(+)e] greater than 5 mEq per L during 6 hours of storage at 4 degrees C after washing if the cells are not irradiated and for 3 hours if the cells are irradiated.     

Differences in Rat and Human Erythrocytes Following Blood Component Manufacturing: The Effect of Additive Solutions

Background

Small animal models have been previously used in transfusion medicine studies to evaluate the safety of blood transfusion products. Although there are multiple studies on the effects of blood banking practices on human red blood cells (RBCs), little is known about the effect of blood component manufacturing on the quality of rat RBCs.

Methods

Blood from Sprague-Dawley rats and human volunteers (n = 6) was collected in CPD anticoagulant, resuspended in SAGM or AS3, and leukoreduced. In vitro quality was analyzed, including deformability, aggregation, microvesiculation, phosphatidylserine (PS) expression, percent hemolysis, ATP, 2,3-DPG, osmotic fragility, and potassium concentrations.

Results

Compared to human RBCs, rat RBCs had decreased deformability, membrane rigidity, aggregability, and microvesiculation after component manufacturing process. Rat RBCs in SAGM showed higher hemolysis compared to human RBCs in SAGM (rat 4.70 ± 0.83% vs. human 0.34 ± 0.07%; p = 0.002). Rat RBCs in AS3 had greater deformability and rigidity than in SAGM. The number of microparticles/µl and the percentage PS expression were lower in rat RBCs in AS3 than in rat RBCs in SAGM. Hemolysis was also significantly lower in AS3 compared to SAGM (2.21 ± 0.68% vs. 0.87 ± 0.39%; p = 0.028).

Conclusion

Rat RBCs significantly differ from human RBCs in metabolic and membrane-related aspects. SAGM, which is commonly used for human RBC banking, causes high hemolysis and is not compatible with rat RBCs.Key Words: Red blood cells, Additive solutions, Blood manufacturing, Blood banking     

Prestorage leukoreduction and low-temperature filtration reduce hemolysis of stored red cell concentrates

BACKGROUND: Universal prestorage leukoreduction in Canada created the perception that stored red cells (RBCs) are more hemolyzed than their unfiltered predecessors. A pool-split design tested the effects of leukoreduction on hemolysis of stored RBCs. STUDY DESIGN AND METHODS: Two ABO-matched units were pooled, divided, and then processed into leukoreduced (LR) and nonleukoreduced (NLR) units with the Pall LT-WB or RC-PL systems and sampled during standard processing and storage for testing of sterility, counts, hemolysis, and osmotic fragility. RESULTS: Room temperature (RT) filtration of 10 pairs of LT-WB-LR and -NLR units showed significantly different percentage of hemolysis (0.39%) and osmotic fragility (0.643%) at 42 days. Cold-stored and -filtered units (2 days at 4 degrees C before processing) were less hemolyzed, but showed a similar proportional decrease of hemolysis in LR units (0.13% vs. 0.25% at 42 days). RBCs from RC-PL systems showed the lowest hemolysis although there was a filtration effect (0.05% vs. 0.12%, 42 days). Osmotic fragility paralleled hemolysis. Segment samples gave inaccurate results. Two-day prefiltration cold storage reduced hemolysis from 0.36 to 0.07 percent (42 days, p < 0.001). RT-LR hemolysis became significantly higher by Day 10 and 4 degrees C LR by Day 12. NLR units showed hemolysis by Day 7. LR units filtered cold were less hemolyzed (p < 0.05) than RT-LR but osmotic fragility was unchanged. CONCLUSIONS: LR-RBCs prepared by any of three methods (LT-WB, RT or cold; RC-PL), filtered at 4 degrees C, were less hemolyzed during storage than nonfiltered concentrates: 4 degrees C leukoreduction is beneficial for RBCs and does not cause hemolysis or enhance fragility.     

Infusion of P-Capt prion-filtered red blood cell products demonstrate acceptable in vivo viability and no evidence of neoantigen formation

BACKGROUND: Transmission of variant Creutzfeldt‐Jacob disease (vCJD) is a major concern in blood transfusion. The P‐Capt filter has been shown to remove around 4 log ID₅₀ prion infectivity from prion‐spiked human red blood cells (RBCs). STUDY DESIGN AND METHODS: Two independent, single‐center, randomized, open‐label studies were designed to analyze the safety of P‐Capt–filtered RBCs. RBCs prepared from leukoreduced whole blood from 43 eligible subjects were randomly assigned to P‐Capt filtration and/or storage in plasma or SAGM and stored for 28 or 42 days. Stored RBCs were analyzed for in vivo 24‐hour recovery, hemolysis, metabolic variables, blood group antigen expression, neoantigen formation, and safety after autologous infusion. RESULTS: Mean P‐Capt filtration times for leukoreduced RBCs were 41 (SAGM) to 51 (plasma) minutes. Thirteen of 14 subjects receiving P‐Capt–filtered RBCs had 24‐hour RBC recoveries of 75% or more after 42‐day storage, with a mean hemolysis of less than 0.6%. No loss of RBC antigen expression or formation of neoantigens was observed. In both studies, RBCs had white blood cell counts of less than 1 × 10⁶/unit after leukofiltration. P‐Capt prion filtration provided an additional greater than 0.8 log leukoreduction. No serious or unexpected adverse events were observed after infusion of P‐Capt–filtered full‐volume RBC units. CONCLUSIONS: P‐Capt–filtered, stored RBCs demonstrated acceptable viability and no detectable neoantigen expression, immunogenic responses. or safety issues after infusion of a complete unit. The additional filtration time and modest reduction in RBC content are within acceptable levels for implementation in countries with transfusion transmission of vCJD.     

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.     

Stability of Lipids of Erythrocytes Preserved in Liquid Nitrogen

Human erythrocytes preserved by a low-glycerol, rapid-freeze method maintained normal lipid concentrations after storage at -196 C for three to 146 weeks. In contrast to erythrocytes stored in ACD at 4 C, the frozen cells exhibited no tendency to develop progressive losses of total lipid weight, cholesterol, or lipid phosphorus. Relative distribution of individual phospholipids was similar to that of fresh control specimens. No streaking or other evidence of products of oxidative degradation was observed in thin-layer chromatograms, and assays for peroxide formation in lipid extracts were unaffected by prolonged storage of the frozen cells. These findings indicate that the structural integrity of erythrocyte membrane lipoproteins remained intact for up to three years after preservation by a low-glycerol, rapid-freeze technic and storage in liquid nitrogen at -196 C.     

Red blood cell aging markers during storage in citrate‐phosphate‐dextrose–saline‐adenine‐glucose‐mannitol

BACKGROUND: It has been suggested that red blood cell (RBC) senescence is accelerated under blood bank conditions, although neither protein profile of RBC aging nor the impact of additive solutions on it have been studied in detail. STUDY DESIGN AND METHODS: RBCs and vesicles derived from RBCs in both citrate‐phosphate‐dextrose (CPD)–saline‐adenine‐glucose‐mannitol (SAGM) and citrate‐phosphate‐dextrose‐adenine (CPDA) were evaluated for the expression of cell senescence markers (vesiculation, protein aggregation, degradation, activation, oxidation, and topology) through immunoblotting technique and immunofluorescence or immunoelectron microscopy study. RESULTS: A group of cellular stress proteins exhibited storage time– and storage medium–related changes in their membrane association and exocytosis. The extent, the rate, and the expression of protein oxidation, Fas oligomerization, caspase activation, and protein modifications in Band 3, hemoglobin, and immunoglobulin G were less conspicuous and/or exhibited significant time retardation under storage in CPD‐SAGM, compared to the CPDA storage. There was evidence for the localization of activated caspases near to the membrane of both cells and vesicles. CONCLUSIONS: We provide circumstantial evidence for a lower protein oxidative damage in CPD‐SAGM–stored RBCs compared to the CPDA‐stored cells. The different expression patterns of the senescence markers in the RBCs seem to be accordingly related to the oxidative stress management of the cells. We suggest that the storage of RBCs in CPD‐SAGM might be more alike the in vivo RBC aging process, compared to storage in CPDA, since it is characterized by a slower stimulation of the recognition signaling pathways that are already known to trigger the erythrophagocytosis of senescent RBCs.     

Extended storage of AS-1 and AS-3 leukoreduced red blood cells for 15 days after deglycerolization and resuspension in AS-3 using an automated closed system

BACKGROUND: The utilization of cryopreserved red blood cell (RBC) units had been limited by a maximum postdeglycerolization storage of 24 hours at 1 to 6 degrees C until the recent development of a closed system for the glycerolization and deglycerolization process. STUDY DESIGN AND METHODS: Sixty leukoreduced additive solution (AS), AS-1 (n = 30) and AS-3 (n = 30) RBC units from 500-mL whole blood (WB) collections were stored for 6 days, glycerolized, frozen at -70 +/- 5 degrees C for at least 14 days, thawed, deglycerolized, and stored for 15 days at 1 to 6 degrees C. Glycerolization and deglycerolization were performed with the ACP 215. In-vitro variables were tested before glycerolization, on Day 0, and Day 15 after deglycerolization storage. Forty donors were assessed for double-label 24-hour percent recovery, and T1/2 survival time was measured for 20 donors. RESULTS: Postdeglycerolization mean +/- standard deviation in-vitro RBC mass recoveries were 93 +/- 5 percent for AS-1 and 95 +/- 4 percent for AS-3. Mean hemoglobin +/- standard deviation after deglycerolization was 50.5 +/- 5.5g for AS-1 and 50.1 +/- 3.5g for AS-3. Mean hemolysis (Day 15) was 0.36 +/- 0.11 percent for AS-1 and 0.38 +/- 0.13 percent for AS-3. Double-label 24-hour in-vivo recoveries were 82.5 +/- 7.8 percent for AS-1 and 81.4 +/- 7.1 percent for AS-3. The 51Cr T1/2 value was 41.8 +/- 3.97 for AS-1 and 40.6 +/- 7.11 for AS-3. Other in-vitro variables were as expected. CONCLUSION: Leukoreduced AS-1 and AS-3 RBCs after frozen storage at -70 +/- 5 degrees C can be stored for up to 14 days when processing is performed with the ACP 215 system with resuspension of deglycerolized RBCs in AS-3.     

Effect of 24-hour storage at 25°C on the in vitro storage characteristics of CPDA- 1 packed red cells

BACKGROUND: Packed red cells (RBCs) warmed above 10 degrees C are generally discarded. Few data exist on the degree of accelerated metabolism and increased hemolysis of packed RBCs allowed to warm. STUDY DESIGN AND METHODS: Twenty-four CPDA-1 packed RBC units were combined in 3-unit pools and subdivided into 2 test units and a control unit. One test unit from each pool was warmed to 25 degrees C for 24 hours on Day 6 and the other test unit was warmed on Day 20; control units were maintained at 1 to 6 degrees C. RBC and supernatant chemistries and RBC morphology were measured weekly (Days 0, 7, 14, 21, and 28) and on the day before warming (Days 6 and 20). RESULTS: Warming CPDA-1 packed RBCs accelerated the catabolism of glucose 10-fold and produced concentrations of glucose, lactate, and ATP after 25 days of storage that were equivalent to those in unwarmed units at 35 days. Supernatant sodium and potassium concentrations were corrected partially with warming. RBC morphology transiently normalized with warming and without increased hemolysis; no bacteria growth was detected. CONCLUSION: One day of 25 degrees C storage of CPDA-1 packed RBCs accelerates essential metabolite break-down equivalent to 10 days of storage at 1 to 6 degrees C. It does not appear to matter whether the packed RBCs are warmed on Day 6 or Day 20. This information may be useful in determining the acceptability of blood allowed to warm above 10 ° C.     

Hemolysis of red blood cells after cell washing with different automated technologies: clinical implications in a neonatal cardiac surgery population

BACKGROUND: In subsets of pediatric cardiac surgery patients, red blood cells (RBCs) are often washed to reduce extracellular potassium (K) to avoid hyperkalemia, but mechanical manipulation and time delay in issuing washed products may increase hemolysis and K. This study's purpose was to evaluate the quality of washed RBCs with regard to hemolysis and extracellular K using different cell washers as a function of postprocessing time. STUDY DESIGN AND METHODS: Fresh (<4 days old) RBCs were washed on COBE 2991 blood cell processors (Model 1 and Model 2) or the Fresenius Continuous AutoTransfusion System (CATS), and K and hemolysis index (HI) were analyzed. Academic pediatric hospitals were surveyed to ascertain practice trends regarding indications for washing, washing device, and expiration time for washed RBCs. RESULTS: K concentration at 24 hours for units washed with the COBE devices met or exceeded prewash values. At 12 hours, there was a significant difference (p < 0.001) in K concentration between all devices, with the CATS maintaining the lowest K concentration. HI increased immediately after wash on all devices and showed a significant difference between the COBE devices and CATS at times of more than 6 hours (p < 0.01). At storage times beyond 4 hours, hemoglobin exceeded 100 mg/dL on the COBE Model 1. Survey of pediatric hospitals indicated that COBE devices are commonly used, and storage time after washing was 12 hours or more in blood banks queried. CONCLUSIONS: Hemolysis levels vary among different cell washers. Decreasing the expiration time of units after washing may be warranted.