Buffering and dilution in red blood cell storage.

BACKGROUND: Red blood cell (RBC) storage solutions work in a narrow pH range between 7.2 and 6.4. While keeping RBC within that pH range, ATP production can be increased by buffering or dilution. STUDY DESIGN AND METHODS: In the first study, 12 units of packed CP2D RBCs were pooled in groups of four, re-aliquoted, and added to one of four additive solutions (ASs): AS-3, 110 mL; EAS-61, 170 mL; EAS-78, 170 mL; or EAS-81, 110 mL. EAS-78 and -81 contain bicarbonate. Units were sampled approximately weekly for 10 weeks for biochemical measures. In the second study, 12 volunteers donated RBCs for measures of (51)Cr in vivo recovery after 6 or 8 weeks of storage in EAS-81. RESULTS: RBCs stored in the higher-volume or buffered ASs had higher RBC ATP concentrations. The combination had an additive effect. Hemolysis was reduced in dilute ASs and less so with buffering. RBCs stored for 8 weeks (n=6) in EAS-81 exhibited 87+/- 2 percent 24-hour (51)Cr in vivo recovery and 0.4+/- 0.2 percent hemolysis. CONCLUSIONS: It is possible to store RBCs for 8 weeks in buffered conventional volume ASs. Combining buffering and increased AS volume improves stored RBC characteristics further.

Interlaboratory comparison of red-cell ATP, 2,3-diphosphoglycerate and haemolysis measurements.

BACKGROUND AND OBJECTIVES: Red blood cell (RBC) storage systems are licensed based on their ability to prevent haemolysis and maintain RBC 24-h in vivo recovery. Preclinical testing includes measurement of RBC ATP as a surrogate for recovery, 2,3-diphosphoglycerate (DPG) as a surrogate for oxygen affinity, and free haemoglobin, which is indicative of red cell lysis. The reproducibility of RBC ATP, DPG and haemolysis measurements between centres was investigated. MATERIALS AND METHODS: Five, 4-day-old leucoreduced AS-1 RBC units were pooled, aliquotted and shipped on ice to 14 laboratories in the USA and European Union (EU). Each laboratory was to sample the bag twice on day 7 and measure RBC ATP, DPG, haemoglobin and haemolysis levels in triplicate on each sample. The variability of results was assessed by using coefficients of variation (CV) and analysis of variance. RESULTS: Measurements were highly reproducible at the individual sites. Between sites, the CV was 16% for ATP, 35% for DPG, 2% for total haemoglobin and 54% for haemolysis. For ATP and total haemoglobin, 94 and 80% of the variance in measurements was contributed by differences between sites, and more than 80% of the variance for DPG and haemolysis measurements came from markedly discordant results from three sites and one site, respectively. In descending order, mathematical errors, unvalidated analytical methods, a lack of shared standards and fluid handling errors contributed to the variability in measurements from different sites. CONCLUSIONS: While the methods used by laboratories engaged in RBC storage system clinical trials demonstrated good precision, differences in results between laboratories may hinder comparative analysis. Efforts to improve performance should focus on developing robust methods, especially for measuring RBC ATP.

Biochemical and structural changes in RBCs stored with different plasticizers: the role of hexanol.

BACKGROUND: PVC containers are plasticized with di(2-ethyl)hexylphthalate (DEHP) or a related phthalate. The toxicity of DEHP has been questioned. It has been proposed to use butyryltrihexylcitrate (BTHC) as the plasticizer. The purpose of this study was to determine if hexanol, a component of BTHC, plays a role in the preservation of RBCs stored in BTHC-plasticized PVC bags. STUDY DESIGN AND METHODS: WBC-reduced RBCs of ABO- and D-matched blood groups were prepared in 1-L polyolefin (PO) bags (PL732). Six 60-g aliquots were transferred to transfer packs made of PL146 (DEHP-plasticized) and PL2209 (BTHC-plasticized) and four PO (PL732) packs. To the PL146 and PL2209 packs, 30 mL of AS-1 was added. To three of the PO packs, 30 mL of AS-1 with sufficient DEHP, BTHC, or hexanol to achieve a final concentration of 3 mM was added, and to the final PO pack, 30 mL of AS-1 only was added (control). The units were stored for 6 weeks at 1 to 6 degrees C. RBC ATP, hemolysis, morphology, membrane lipids, deformability, and fluidity were measured. RESULTS: ATP levels were not significantly different in any of the systems after 6 weeks. Compared to the PO bags, hemolysis was lowest in the PL146 containers and was also significantly lower (p < 0.006) in the PO bags with added DEHP, BTHC, or hexanol. The accumulation of vesicles was significantly less in the units stored in the PL146 and PL2209 than in the PO plastic with or without added plasticizers or hexanol (p < or = 0.004). There was no significant difference in the formation of vesicles in any of the PO units (p > 0.05). There was no demonstrable change in the membrane fluidity of the RBCs during storage in any of the systems. The decrease in deformability was the same, and the losses of cholesterol and phospholipid during storage were similar in all the studies. CONCLUSIONS: The hexanol component of the BHTC plasticizer in a concentration of 144.6 microg per mL concentration suppresses hemolysis and vesiculation of RBCs during storage. The hexanol and DEHP that are slowly leached during storage have a greater effect in suppressing hemolysis and vesicle formation than when added extraneously to AS-1 in PO containers.

The effects of polyvinyl chloride and polyolefin blood bags on red blood cells stored in a new additive solution.

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) must be stored in polyvinyl chloride (PVC) bags plasticized with di-2-ethylhexyl phthalate or a similar plasticizer to achieve their full storage life with conventional storage solutions. Improved storage solutions might remove this requirement and allow blood storage in other plastics. Experimental Additive Solution-61 (EAS-61), which maintains RBCs for 9 weeks with reduced haemolysis and satisfactory 51Cr 24-h recovery, is an appropriate candidate improved RBC storage solution. MATERIALS AND METHODS: Twenty-four units of packed RBCs were pooled in groups of four units, each pool was realiquoted into four units and stored, six pooled units per arm, in one of the following: 100 ml of EAS-61 in PVC; 200 ml of EAS-61 in PVC; 100 ml of EAS-61 in polyolefin (PO); and 200 ml of EAS-61 in PO. Haemolysis, RBC morphology indices, RBC ATP concentrations, and other measures of RBC metabolism and function were measured weekly. RESULTS: RBC haemolysis exceeded 1% by 7 weeks in PO bags containing 100 ml or 200 ml of EAS-61. In PVC bags, haemolysis was less than 1% at 11 weeks. RBC ATP concentrations were 1 mol/g of haemoglobin (Hb) higher at 2 weeks in the PVC-stored units. CONCLUSIONS: RBCs stored in PVC had markedly less haemolysis and higher RBC ATP concentrations than those stored in PO. Haemolysis would limit RBC storage in PO bags to a duration of 6 weeks, even with EAS-61.

The role of electrolytes and pH in RBC ASs.

BACKGROUND: Experimental additive solutions (EASs) containing saline, adenine, glucose, mannitol and disodium phosphate can support RBCs for 9 or 10 weeks if used in 200- or 300-mL volumes. The effects of variations in the electrolyte composition and volume of EASs were explored. STUDY DESIGN AND METHODS: In three four-arm studies, 24 RBC units were pooled in groups of 4 and realiquoted as test units to ensure that all donors were equally represented in each study arm. In Study 1, units were stored for 11 weeks in EAS containing 0, 10, 20, or 30 mmol per L of sodium bicarbonate. In Study 2, units were stored for 9 weeks in EAS containing 26, 50, 100, or 150 mmol per L of sodium chloride. In Study 3, units were stored in 100 or 200 mL of AS-3 or EAS-61. RBC ATP concentrations and hemolysis were measured weekly. RESULTS: Increasing the sodium bicarbonate content of EASs increased the pH throughout storage and increased RBC ATP concentrations in the later phases of storage, but it had no effect on hemolysis. Increased sodium chloride content of EASs led to lower RBC ATP concentrations and increased hemolysis. In EAS-61, RBC ATP concentrations were increased throughout storage, and hemolysis was lower than that of RBCs stored in AS-3. CONCLUSION: RBC ATP synthesis is highly dependent on the pH of the AS. Hemolysis is affected by the salt content and volume of the AS.

The effect of two additive solutions on the postthaw storage of RBCs.

BACKGROUND: Sterile systems for freezing and for washing thawed blood will allow the storage of RBCs for more than 24 hours after removal of the cryoprotectant glycerol. This study assessed the effect of two ASs in maintaining deglycerolized RBCs. STUDY DESIGN AND METHODS: Twenty-four RBC units were stored for 6 days, pooled in groups of 4, realiquoted, sterilely glycerolized, and frozen. One month later, the units were thawed, sterilely deglycerolized by using an automated system (H215; Haemonetics), and stored for 5 weeks in either 100 or 200 mL of AS-3 or an experimental AS (EAS-61). Sterile samples were taken weekly for chemical and morphometric analysis. RESULTS: The glycerolization and deglycerolization process produced highly comparable RBC units, but it caused a marked reduction of RBC pH, to about 6.4 at the beginning of storage. The addition of acidic AS-3 further reduced the pH, which in turn reduced glucose consumption, lactate formation, and RBC ATP concentrations. Alkaline EAS-61 increased these measures. Hypotonic EAS-61 caused increased cell swelling and hemolysis, despite better RBC morphology. CONCLUSIONS: Automation of sterile glycerolization and deglycerolization with the H215 works well, but the solutions should be reformulated for extended postthaw storage. This would best be accomplished by raising the pH of the wash solutions by the addition of disodium phosphate or sodium bicarbonate or both, by using alkaline ASs, and by matching the osmolality of the wash solution and ASs.

RBC storage for 11 weeks.

BACKGROUND: Increasing the length of RBC storage can increase both RBC availability and quality. This work addresses 11-week RBC storage in experimental ASs (EASs). STUDY DESIGN AND METHODS: Three studies were performed. In the first, 24-hour in vivo recovery of (51)Cr-labeled autologous RBCs was measured in nine volunteers after storage of their RBCs for 11 weeks in EAS 67. In the second study, 4 units of blood were divided and stored in aliquots with an EAS containing 0, 15, 30, or 45 mmol per L of mannitol; then hemolysis, RBC morphology, and microvesicle protein were measured. In the third study, 6 full units were stored for 12 weeks in the EAS containing 30 mmol per L of mannitol, with weekly sampling for morphologic and biochemical measures of RBC quality. RESULTS: RBCs stored for 11 weeks in EAS-67 had a mean 24-hour in vivo recovery of 79 +/- 5 percent, but the hemolysis was 1.35 +/- 0.68 percent. Increasing mannitol content of the EAS reduced hemolysis but increased microvesiculation. EAS-76, with 30 mmol per L of mannitol allowed 11-week storage with 0.48 +/- 0.10 percent hemolysis at 11 weeks and 0.62 +/- 0.14 percent hemolysis at 12 weeks. CONCLUSION: It is possible to store RBCs for 11 weeks in EAS with greater than 75 percent recovery and less than 1 percent hemolysis.

The effects of phosphate, pH, and AS volume on RBCs stored in saline-adenine-glucose-mannitol solutions.

BACKGROUND: RBC ATP concentrations are the most important correlate of RBC viability. Tests were performed to determine whether increased AS volume, pH, and phosphate content increased stored RBC ATP concentrations. STUDY DESIGN AND METHODS: In three studies, packed RBCs were pooled in groups of 3 or 4 units and realiquoted as combined units to reduce intradonor differences. Pooled units were stored in the licensed ASs, AS-1 or AS-5, which contain saline, adenine, glucose, and mannitol (SAGM), or in experimental ASs (EASs) containing SAGM and disodium phosphate. Ten pools were stored in AS-1 at RBC concentrations equivalent to 100, 200, or 300 mL of AS. Six pools were stored in 100, 200, 300, or 400 mL volumes of EAS-61. Ten pools were stored in 100 mL of AS-5, 200 mL of EAS-61, or 300 mL of EAS-64. RBC ATP concentration and other measures of RBC metabolism and function were measured weekly. RESULTS: RBC ATP concentrations decreased sooner with storage in increasing volumes of AS-1. In EAS-61 and EAS-64, RBC ATP concentrations initially increased and stayed elevated longer with increasing AS volume. CONCLUSIONS: The addition of disodium phosphate to SAGM AS increases the RBC ATP concentrations. Reducing storage Hct appears to have a separate beneficial effect in reducing hemolysis.

Successful storage of RBCs for 9 weeks in a new additive solution.

BACKGROUND: This study explored the effect of storing packed RBCs suspended in 200 mL of an alkaline, hypotonic, experimental additive solution (EAS 61). STUDY DESIGN AND METHODS: Packed RBC units prepared from RBCs collected from healthy donors in CPD were stored for 8 (n = 10) and 9 (n = 10) weeks under blood bank conditions after the addition of 200 mL of EAS 61 (adenine, 2 mM:; dextrose, 110 mM:; mannitol, 55 mM:; NaCl, 26 mM:; Na(2)HPO(4), 12 mM:). Standard methods were used for in vitro assays. The 24-hour in vivo autologous recoveries were measured with (51)Cr. RESULTS: Mean +/- SD recoveries at 8 and 9 weeks were 81 +/- 7 and 77 +/- 7 percent. After 9 weeks, the ATP of the RBCs was 81 percent of the initial value, hemolysis was 0.35 percent, supernatant potassium was 46 mEq per L, and the morphologic index was 94.1. CONCLUSION: Packed RBCs suspended in 200 mL of EAS 61 can be stored satisfactorily for 9 weeks. Longer RBC storage should reduce outdating, increase availability of transfusions in remote locations, and improve the efficiency of autologous donor programs.

Successful storage of RBCs for 10 weeks in a new additive solution.

BACKGROUND: The effect of storing packed RBCs suspended in 300 mL of an alkaline, experimental additive solution (EAS 64) was explored. STUDY DESIGN AND METHODS: RBC units prepared from blood collected from healthy donors into CPD were WBC reduced and stored for 10 weeks under blood bank conditions after the addition of 300 mL of EAS 64 (adenine, 2 mM:; dextrose, 50 mM:; mannitol, 20 mM:; NaCl, 75 mM:; Na(2)HPO(4), 9 mM:). For comparison, non-WBC-reduced units from the same donors were stored in a different additive solution (AS-1, Baxter Healthcare) for 6 weeks. Standard methods were used for the in vitro assays. The 24-hour in vivo recoveries were measured by using (51)Cr- and (99m)Tc-labeled RBCs. RESULTS: Mean recovery in the EAS 64 units after 10 weeks was 84 +/- 8 percent, the same as in the AS-1 units stored for 6 weeks. For EAS 64 and AS-1 units, respectively, the ATP of the RBCs was 85 percent and 64 percent of the initial value, hemolysis was 0.43 percent and 0.63 percent, supernatant potassium was 24 mEq per L and 44 mEq per L, and the morphologic index was 98 and 71. CONCLUSION: RBCs suspended in 300 mL of EAS 64 can be stored satisfactorily for 10 weeks. Longer RBC storage should reduce outdating, increase availability of transfusions in remote locations, and improve the efficiency of autologous donor programs.

A feasibility evaluation of an automated blood component collection system platelets and red cells.

BACKGROUND: The purpose of these studies was to evaluate the functional properties of blood components collected with an automated collection system. STUDY DESIGN AND METHODS: Single-donor platelets (n = 44) and packed red cell (RBC) units (n = 10) were collected. In vitro and in vivo assays were used to assess the function of single-donor platelet components stored for 5 days and of packed RBC units after storage for 42 days at 4 degrees C. RESULTS: Adverse events observed in the 44 study subjects were minor. The mean 24-hour recovery value for the packed RBC units stored for 42 days was 83.6 +/- 5.4 percent, with a mean percentage of hemolysis on Day 42 at 0.46 +/- 0.19 percent. The 25 patients receiving platelet components achieved a mean corrected count increment of 15.1 +/- 10.4 x 10(3). All platelet concentrates had less than 1 x 10(6) total white cells. CONCLUSION: Both in vitro and in vivo testing for the packed RBCs collected and stored for 42 days met the standards for both hemolysis and percentage of 51Cr 24-hour RBC recovery. The in vitro results and transfusion data on white cell-reduced platelet components transfused to thrombocytopenic patients were comparable to those on available platelet components.

Recent developments in the long-term preservation of red blood cells.

The first study to suggest the successful prolongation of the useful shelf life of red blood cells (RBCs) used a hypotonic additive solution containing glycerol. It was necessary to use twice the volume of this solution than the commercial additive solution per unit of packed RBCs. The final concentration of glycerol in the units was approximately 0.69% (75 mmol/L). A subsequent study demonstrated that the membranes of the exocytic microvesicles shed during storage had less of bands 3 and 4.1 than those in Adsol (Fenwal Laboratories, Deerfield, IL). Band 4.1 is important for strengthening the bonds between spectrin and actin in the cytoskeleton. In another study, glutamine or glutamine plus phosphate was used in a hypotonic additive solution, otherwise similar to the glycerol-containing additive. In the latter medium, phosphatidylethanolamine was less accessible to phospholipase action than in Adsol or when glutamine alone was added. Another group reported encouraging data regarding the action of L-carnitine when added to AS-3. Acylation of phosphatidylethanolamine mediated by the action of carnitine fatty acid transferase with acyl coenzyme A (acyl-CoA) occurred. Adenosine-5'-triphosphate levels and red cell recovery were better in the test units. In the last paper reviewed, the authors demonstrated that oxidant damage of erythrocytes was less if the donors were given a mixture of antioxidants for 10 days prior to donating.

The effect of hypotonicity, glutamine, and glycine on red cell preservation.

BACKGROUND: Red cells (RBCs) stored in hypo-osmolar additive solutions with the same concentrations of adenine, dextrose, mannitol, and sodium chloride and varied amounts of ammonium, phosphate, glycerol, and glutamine were better preserved than RBCs in the standard additive solution (Adsol). Cell swelling occurred in all the experimental additives. This observation prompted the evaluation of glutamine and glycine alone, as well as a combination of glutamine and glycine, all of which have been described as producing swelling of rat liver cells. STUDY DESIGN AND METHODS: Aliquots of RBCs were stored at 4 degrees C in Adsol or experimental additive solutions (EASs) all containing adenine, 2 mM; dextrose, 110 mM; mannitol, 55 mM; and sodium chloride, 50 mM. EAS 42 had, in addition, glutamine, 10 mM; glycine 5 mM, and phosphate, 20 mM. EAS 43 had glutamine, 10 mM; glycine, 10 mM; and phosphate 20 mM. EAS 44 had glutamine, 10 mM; EAS 45 had glutamine, 10 mM, and phosphate, 20 mM, and EAS 46 had only glycine, 10 mM. At intervals, measurements were made of mean corpuscular volume, mean corpuscular hemoglobin concentration, morphology, ATP, hemolysis, supernatant potassium, ammonia, pH, and microvesicles shed. RESULTS: The initial mean corpuscular volumes were larger in all EASs than in Adsol, but the greatest difference was between EASs 44 and 46 (108 fL) and Adsol (86 fL) (p < 0.001). The morphology scores were significantly better in all the EASs (p < 0.04). The ATPs were significantly greater in all the EASs (p < 0.001), and highest in those with phosphate. potassium leakage and hemolysis were less in the EASs (p < 0.001). The ammonia levels higher in all the EASs than in Adsol, with the exception of EAS 46. During storage, the extracorpuscular and intracorpuscular pH levels were essentially identical. The shedding of microvesicles was greatly reduced in all the EASs. CONCLUSION: Cell swelling induced in RBCs after collection appears to improve preservation. Ammonia and phosphate enhance RBC ATP maintenance. Glycine decrease the formation of ammonia by RBCs stored in a hypotonic medium.