Prolonged storage of red cells at 4 degrees C

One of the events associated with red cell storage at 4 degrees C is the development of an increasing proportion of echinocytes. Vesicles also may bud off the spicules, presumably leading to a decreased surface-to-volume ratio and decreased deformability. Pursuing the hypothesis that increasing the surface tension of the cells by increasing their volume might reduce the tendency toward echinocytosis and extend refrigerated storage time, packed red cells were resuspended in a solution hypotonic (210 mOsm) with respect to solutes that do not penetrate the cell. Since a reduced ionic concentration results in increased membrane permeability for cations, normal ionic concentration was maintained by the addition of NH4C1, which readily penetrates red cells and therefore contributes no osmotic support. Adenine, glucose, mannitol, citrate, and phosphate also were included. Unexpectedly, the predominant effect of red cell storage in this solution was a remarkable elevation of adenosine triphosphate (ATP). At 4, 8, and 10 weeks, (ATP) levels averaged 165, 135, and 110 percent of initial values, respectively. At 16 weeks, ATP still averaged 50 percent of initial values. Twenty-four-hour in vivo survival of red cells measured at 12 to 18 weeks ranged between 70 and 80 percent, and hemolysis ranged from 0.3 to 7.1 percent. Both the hypotonicity and the ammonium salt appear to be necessary for the high ATP.     

Liquid storage at 4 degrees C of previously frozen red cells

Fresh human blood was collected in citrate-phosphate-dextrose, frozen by a high-glycerol technique, and stored at -80 degrees C. The red cells were thawed, deglycerolized, and resuspended in a final wash solution, ADSOL (Fenwal Laboratories), or an additive solution (AS) containing glucose, adenine, mannitol, and phosphate. The cells were then stored at 4 to 6 degrees C for 21 days and assayed weekly for adenosine triphosphate and 2,3 diphosphoglycerate, pH, glucose use, and lysis. AS and, to a lesser extent, ADSOL produced metabolic profiles similar to or better than profiles of cells not frozen and stored in commercially available additive solutions. AS offers a potential post-thaw preservative solution for red cells that would greatly increase the flexibility and reduce the expense of using frozen blood. A sterile post-thaw storage capability will make the stockpiling of frozen red cells a practical concept for both military and civilian blood banks.     

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.     

Lack of success with a combination of alanine and phosphoenolpyruvate as an additive for liquid storage of red cells at 4 degrees C

Red cells were stored at 4 degrees C in a storage solution containing alanine or alanine plus phosphoenolpyruvate (PEP). The intention was to investigate whether alanine and PEP might act synergistically to maintain a normal level of both red cell ATP and 2.3 diphosphoglycerate (2.3 DPG) under normal liquid storage conditions. Storage in the presence of alanine kept the red cell concentration of 2.3 DPG higher than the reference solution for an extended period of time, provided the initial pH was about 7.0 (37 degrees C). When the pH of the storage solution containing alanine plus PEP was lowered to facilitate the transport of PEP into the red cells, the concentration of 2.3 DPG was lowered to a rate equal to that in the reference solution. The level of ATP was also about the same as in the reference solution. The majority of the added PEP was continuously converted to 2 phosphoglycerate and 3 phosphoglycerate in the extracellular fluid. A small amount of unconverted PEP penetrated the red cell membrane when the pH went below 6.5; this occurred after 3 weeks of storage. The intracellularly located PEP, however, was not metabolized to 2.3 DPG to any significant extent within the first 6 weeks of storage. These findings indicate that PEP is not suitable as an additive for liquid storage of red cells at 4 degrees C. The combination of alanine and PEP that theoretically could be a suitable additive for liquid storage of red cells was not satisfactory in practice.     

The distribution and utilization of adenine in red blood cells during 42 days of 4 C storage

The fate of adenine in CPD whole blood (17.3 mg/500 ml, or 0.25 mM) was evaluated during 42 days of 4 C storage. In whole blood, 95 per cent of the adenine was removed from the plasma by 42 days while the cellular adenosine triphosphate (ATP) levels remained above 60 per cent of the initial concentration. Packed red blood cells (concentrates) were stored with the same relative quantity of adenine (0.1 mg/ml red blood cells) used in whole blood units by the addition of adenine after packing and were shown to take up adenine in a similar manner. Calculations of the initial adenine distribution indicated higher intracellular adenine concentrations than predicted from distribution equilibrium based on volume considerations. The presence of inorganic phosphate has marginal effects on adenine incorporation but does elevate ATP levels, while contributing to the reduction of 2,3-diphosphoglycerate (2,3-DPG) content. The free adenine equilibrium between plasma and red blood cells favors the red blood cells, suggesting adenine binding by red blood cell membranes as shown by initial distribution studies with ¹⁴C-adenine and equilibrium dialysis.     

The effect of platelets and red cells on granulocytes stored at 22 degrees C

Granulocyte concentrates contain varying numbers of platelets and red cells depending upon the method of collection. Either platelet or red cell concentrations may be as high as 2.0 × 10(12) per I. Studies were done on unwashed and washed granulocyte concentrates and on pure granulocyte suspensions with known numbers of platelets or red cells added. These suspensions or concentrates were stored for 72 hours at 22 degrees C. In both experiments, the following were measured: leukocyte and absolute granulocyte counts, dye exclusion, chemotaxis, plasma glucose, plasma pH, and osmolality. Red cell contamination did not adversely effect granulocyte storage. Platelets, however, did contribute to the functional deterioration of stored granulocytes. In the presence of high concentrations of platelets, both granulocyte dye exclusion and chemotaxis were adversely affected at 48 hours of storage. In another experiment, fresh autologous granulocytes incubated for 18 hours in hydroxyethyl starch-citrated-plasma obtained from stored granulocyte concentrates showed a progressive decrease in chemotaxis related to the age of the stored plasma. Glucose supplementation of the spent plasma maintained chemotactic activity. Contamination of granulocyte concentrates with other cells and the availability of glucose to granulocytes are variables affecting the short-term liquid storage of granulocytes at 22 degrees C.     

红细胞冰冻前的最佳保存期探讨

背景:既能适当延长冰冻红细胞在冰冻前的保存期,又能保证冰冻红细胞的质量,是十分有意义的研究课题。目的:适当延长稀有血型红细胞冰冻前在(4±2)℃的保存期,满足急症稀有血型患者的输血,降低血液成本,减轻工作强度。方法:将冰冻前(4±2)℃保存1~6d和冰冻前(4±2)℃保存7~12d制备的冰冻红细胞进行质量比较;根据血液保养液的红细胞保护原理、美国AABB相关标准及质检结果将含添加剂的红细胞在冰冻前的保存期设定为自采血之日起14d内,对采取此措施前后2001/2009本中心的Rh(D)阴性供血情况进行统计分析。结果与结论:质量检测结果达到国家标准,两组质量检测数据比较差异无显著性意义;将含添加剂的红细胞在冰冻前的保存期设定为自采血之日起14d内,本中心Rh(D)阴性血年供血量虽逐年增加,但冰冻红细胞的供血量却未增加,其占供血的比例还在逐年下降,(4±2)℃保存血的供血比例则逐渐增加。冰冻前在(4±2)℃保存14d再制备的冰冻红细胞发往临床均未发现因此而产生的血液质量问题。结果可见增加Rh(D)阴性(4±2)℃保存血供血量,减少冰冻红细胞供血量,能及时满足急症Rh(D)阴性患者的输血,降低制备冰冻红细胞导致的高血液成本,减轻员工制备冰冻红细胞的工作强度。     

Contractile properties of rat skeletal muscles following storage at 4 degrees C.

The purpose of this study was to assess the potential of preservation solutions for protecting skeletal muscle function during storage at 4 degrees C. The soleus and the cutaneus trunci (CT) from the rat were stored for 2, 8 or 16 h at 4 degrees C in University of Wisconsin solution (UW), HTK-Bretschneider solution (HTK) or Krebs-Henseleit solution (KH). After storage, muscles were stimulated electrically to analyse the isometric contractile properties, such as the maximum tetanic tension (P(0)). Histological analysis was also performed. In separate experiments, the effect of the diffusion distance on muscle preservation was studied by bisecting the soleus. After 8 h of storage in UW or HTK, the contractile properties of the CT were similar to those of the control, whereas those of the soleus were reduced (P(0) values of 16% and 69% of control in UW and HTK respectively). At 16 h, the contractile properties of the CT (P(O) 28%) were again better preserved than those of the soleus (P(0) 9%). Muscle function deteriorated most after storage in KH (P(0) at 16 h: soleus, 3%; CT, 17%). The bisected soleus was equally well preserved as the CT (P(O) of bisected soleus at 8 h in UW and HTK: 86%). The functional data corresponded well with the histological data, which showed increasing muscle fibre derangement with increasing storage time. For both muscles and all solutions, the threshold stimulus current increased with increasing storage time (control, 0.1 mA; 16 h, 1.2 mA) and was strongly correlated with the deterioration in contractile properties. It is concluded that, at 4 degrees C, muscle is preserved better in UW and HTK (intracellular-like solutions) than in KH (extracellular-like solution). The soleus and CT were best protected in HTK. The diffusion distance is a critical factor for successful preservation of muscle function at 4 degrees C. The reduced function after 16 h of storage at 4 degrees C was caused by hypercontraction and necrosis of about 25% of the muscle fibres, and by deterioration of the electrical component of excitation-contraction coupling of the remaining fibres.     

A comparison of storage viability of nonmeshed and meshed skin at 4 degrees C

Skin stored in nutrient medium at 4 degrees C produces acceptable short-term viability. This study compared the storage viability of nonmeshed v meshed skin stored at 4 degrees C in nutrient medium. Skin specimens from six human donors were stored for up to 35 days in RPMI 1640 tissue culture medium at 4 degrees C. Skin specimens (1 cm in diameter) were transplanted to surgically created defects on the thorax of nude mice at fixed intervals during the storage period. Gross and microscopic techniques were used to determine the graft viability at 10 days postgraft. Skin was divided into two storage groups as nonmeshed or meshed 1.5/1. The storage configuration was free-floating, 10 cm x 2 cm sheet grafts. The ratio of skin surface area to volume medium was 300 cm2/100 mL. There was no significant difference between the viability of the nonmeshed group compared to the meshed group. Prior meshing of human allograft does not adversely affect the viability of banked skin. Therefore, skin can be stored in a meshed configuration. This eliminates operating room time spent preparing allograft for application, which is cost-effective.     

血液储存时间对红细胞免疫功能的影响与临床安全输血的研究

目的探讨血液离体后在4℃保存过程中红细胞免疫功能的变化及对临床安全输血的影响。方法随机抽取50份常规CPD保养液充分混匀的全血,从细管小辫端取少量血样作为新鲜血样供当天检测,其余热合封闭成5段小样4℃保存备用(与血液相同条件保存);每次各取1个小样测定,分别于1、2、3、4和5周,对上述标本分别做红细胞C3b受体(RBC-C3b)和红细胞免疫复合物(RBC-IC)检测,同时在d0、d14和d28任取其中25份标本检测RBC超氧化物歧化酶(RBC-SOD)水平和酵母菌激活全血细胞免疫反应产生白细胞介素8(IL-8)的含量。结果血液保存时间在2周以内红细胞免疫功能有所下降,但不明显,随着保存时间的延长,RBC-C3b、RBC-SOD明显降低,d0、d14和d21 RBC-C3b RBC-SOD分别为18.84±2.23、16.54±2.42、14.28±2.15;为16222±4322、14111±4213、10459±389;而RBC-IC、IL-8则增高,在d0、d14和d28 RBC-IC分别为5.15±1.56、5.97±1.19、6.72±1.37;IL-8则为65.51±46.32、80.34±39.25、109.67±26.89。结论血液离体后在4℃保存过程中,随时间的延长,红细胞膜有所受损,表现为红细胞(部分)免疫功能降低。在大手术、老年人、严重的心脑血管疾病及因补体系统导致溶血性贫血等患者需要输血治疗时,尽可能输注较年轻(<14d)的血液有利于输血安全。     

The importance of warming at 37 degrees C for removal of leukocytes and platelets from reconstituted red cells

Red cells suspended in sodium phosphate buffer (R-RBC) were incubated at 37 degrees C, and the distribution of the leukocytes and platelets in the suspension was compared to a similar suspension incubated at 22 degrees C. Following centrifugation, a more dense packing of the red cells was observed in warm R-RBC, and less leukocytes and platelets remained in the red cells below the buffy coat, than was observed in R- RBC packed at room temperature. These experiments indicate that incubation at 37 degrees C prior to separation improves the quality of leukocyte- and platelet-depleted red cells.     

Immunosuppressive effects of red blood cells on monocytes are related to both storage time and storage solution

BACKGROUND: Reduced monocyte function is associated with adverse outcomes from critical illness. Red blood cells (RBCs) are thought to impair monocyte function but relationships between RBC storage solution and monocyte suppression are unknown. This study was designed to test the hypothesis that immunosuppressive effects of RBCs on monocytes are related to both storage time and preservative solution. STUDY DESIGN AND METHODS: Monocytes from healthy adult donors were co‐cultured with RBCs that had been stored in AS‐1, AS‐3, or CPD only for 7, 14, or 21 days. Cells were then stimulated with lipopolysaccharide (LPS) and their supernatants assayed for tumor necrosis factor (TNF)‐α and interleukin (IL)‐10. Transwell experiments were performed to evaluate the role of cell‐to‐cell contact. Monocyte mRNA expression was quantified by real‐time–polymerase chain reaction. RESULTS: LPS‐induced TNF‐α production capacity was reduced compared to controls for all groups, but CPD‐only RBCs suppressed monocyte function more than RBCs stored in AS‐1 (p = 0.007) and AS‐3 (p = 0.006). IL‐10 production was preserved or augmented in all groups. A longer storage time was associated with reduced TNF‐α production capacity for AS‐1 and AS‐3 groups but not CPD. Preventing cell‐to‐cell contact did not eliminate the inhibitory effect of RBCs on monocyte responsiveness. RBC exposure was associated with decreased LPS‐induced TNFA mRNA expression (p < 0.05 for all groups). CONCLUSIONS: CPD‐only RBCs suppressed monocyte function more than RBCs stored with additive solutions. TNF‐α production was reduced even in the absence of cell‐to‐cell contact and was impaired at the mRNA level. Further work is needed to understand the role of preservative solutions in this process.     

保存人红细胞的新策略一抗氧化剂保养液保存效果的研究

塑料血袋盛义务献血员的静脉血（含适量保养液）并置4℃冰箱保存。在保存前、后不同时间检查各项指标,旨在研究抗氧化剂保养液延长人血红细胞在4℃条件下的保存时间,以缓解血液保存供应的压力,为输血抢救创造有利条件。实验分3组：ACD组,GMA组和抗氧化剂（SOD）组）。研究结果表明,在4℃条件下保存75天后SOD组红细胞血红蛋白回收率为87．2％,血浆血红蛋白（mg/L)为193．2,P50（mmHg)为34．0（正常值为33．1）,最大变形指数（DImax）为0．2413,即为正常值的74．3％,外观检查无明显溶血、变色、气泡和凝块。结论提示,用抗氧化剂保养液保存红细胞在4℃条件下可延长75天,其红细胞血红蛋白和血浆血红蛋白回收率附合国家《血站基本标准》规定要求。     

Studies on 4 C stored frozen-reconstituted red blood cells. II. Chemical and cytological changes during storage

In order to determine the feasibility of 4 C storage of frozen-thawed red blood cells beyond the 24 hours now allowed by the Food and Drug Administration (BoB), chemical and cytological measurements were made of blood units processed by different methods. The only significant findings were a progressive increase in the plasma hemoglobin and plasma potassium. These were most pronounced when the storage medium was the 0.8% NaCl-0.2% dextrose and storage was continued past five days. Storage in autologous plasma produced less pronounced changes, but the changes were still about twice that found in a unit of whole blood stored for the same amount of time. Storage in 5% albumin or Plasma Protein Fraction did not seem to provide conditions much superior to the dextrose-saline solution. Contamination of the unit produced somewhat more hemolysis than in its sterile control but not enough to be readily detectable. From these studies it seems logical that the 4 C storage time could be extended to at least three, and in all probability five days, with no significant harm to the recipient, and with a great increase in the useable life of the blood unit, thus increasing the availability of a very precious commodity.     

Short-term skin preservation at 4 degrees C: skin storage configuration and tissue-to-volume medium ratio

This study was designed to examine the effect of the storage configuration of skin and the ratio of tissue-to-storage medium on the viability of skin stored under refrigeration. Human skin specimens were stored in four physical configurations in RPMI 1640 tissue culture media at 4 degrees C. Skin was transferred to surgically created defects on nude mice after specific storage intervals. Grafts were examined grossly and microscopically after ten days. In the rolled configuration, on storage day 15, the viability of the outside of the roll was significantly better than the inside (P less than 0.01). The graft viability of the outside of the skin rolls was similar for both tissue-to-media ratios as well as for both free-floating configurations (P = 0.27). These findings suggest the optimum cold storage configuration is free floating, and 300 cm2/100 mL is an appropriate skin surface area to volume media ratio. This proportion of tissue to media is in agreement with the minimum ratio currently recommended by the Skin Council of the American Association of Tissue Banks.