Hemolysis of red blood cells during processing and storage

BACKGROUND: During processing and storage, red blood cells (RBCs) undergo changes and cell injury resulting in hemolysis. Mostly, the separation of whole blood in top‐and‐bottom quadruple bag systems with break openings takes less than 4 minutes. However, longer separation times are not uncommon. The aims were to investigate whether hemolysis is increased in RBCs with longer separation time (RBCs_{>6 min}) compared to regular RBCs (RBCs_reg), to measure hemolysis increase during storage and to study whether frequency of hemolytic donations is donor dependent. STUDY DESIGN AND METHODS: RBCs_{>6 min} (n = 172) and 172 matched controls were tested for hemolysis on Days 1 and 21 RBC units from each group were stored at 4 ± 2°C and tested again after 5 weeks. Donor dependency was retrospectively investigated for 100 hemolytic RBC units. RESULTS: RBCs_{>6 min} exhibited a higher mean hemolysis rate than RBCs_reg (0.058% vs. 0.033%). Four RBC units were hemolytic (>0.8%), all RBCs_{>6 min} (2.36%). During storage, hemolysis in both groups increased with 0.24%. Hemolysis frequency did not seem to be donor dependent. CONCLUSIONS: Increased separation time is a useful screening tool for potentially increased hemolysis rate in RBCs. Hemolysis rate increased during storage equally in both groups. Hemolysis frequency appears donor independent.     

Effects of AS-3 nutrient-additive solution on 42 and 49 days of storage of red cells

Storage of red blood cells in a nutrient-additive solution, AS-3 (Nutricel, Cutter Biological, Berkeley, CA), was evaluated after 42 and 49 days of storage by in vitro measurements of hemolysis, adenosine triphosphate (ATP) levels, glucose levels and other constituents, and in vivo study of 24-hour survival of autologous, reinfused red cells labeled with 51Cr with and without 125I human serum albumin. Two laboratories conducted the studies independently. After 42 days of storage, hemolysis was within an acceptable range (0.72 +/- 0.4% and 1 +/- 0.2%), ATP decreased to 61 percent and 56 percent in the two laboratories, and 24-hour survivals were 85.1 +/- 8.3 percent for single-label cells in one laboratory and 82.8 +/- 10 percent (single-label cells) and 84.1 +/- 13.1 percent (double-label cells) in the second laboratory. Thus, results for single- and double-label cells were similar. After 49 days of storage, ATP fell to 45 and 46 percent in the two laboratories. Twenty-four-hour recovery fell to 69.4 +/- 7.4 percent with single-label cells and to 68.2 +/- 6.7 percent with double-label cells in one laboratory. In the other laboratory, a paired study comparing AS-3 with the already approved AS-1 solution (Adsol; Fenwal Laboratories, Deerfield, IL) showed nearly identical 24-hour survivals of 71.9 +/- 8.8 percent in Nutricel and 71.8 +/- 6.5 percent in Adsol. These studies demonstrate the excellent viability of the new solution after 42 days of study. At 49 days of study, viability decreased significantly and was comparable in the two nutrient-additive solutions studied. The value of paired comparison study is demonstrated by the latter results.     

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.     

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.     

The extension of 4 degrees C storage time of frozen-thawed red cells

Blood was drawn from volunteer donors and frozen using the high glycerin, mechanical freezing procedure accepted by the United States Navy. Subsequently, the units of blood were thawed and washed. Various anticoagulants were added, and the red cells were stored in a refrigerator at 4 degrees C for periods of up to 28 days. Chemical analyses were performed periodically. These showed that the addition of the anticoagulants ACD, CPD and CPDA-1 caused the red cells to be preserved better than the currently accepted 0.9-percent NaCl, 0.2-percent glucose solution. In vivo 51Cr viability studies performed on blood stored with CPDA-1 for 14 days showed a 24-hour viability of 78.8 +/- 8.4 percent. In a subsequent study, the blood was stored for 21 days prior to freezing and then was rejuvenated and frozen. The cells were thawed, washed, and stored at 4 degrees C with CPDA-1 for an additional 14 days. The 24-hour viability of these cells was determined to be 74.0 +/- 5.1 percent. These findings show that the postthaw storage time of red cells can be increased greatly over the now-accepted 24 hours, if bacterial sterility can be assured.     

The uptake and egress of adenine from human red blood cells in vitro

The initial uptake of adenine from plasma by human red blood cells was measured at 0, 10, and 20 C. Initial uptake is completed in several minutes as distribution equilibrium is reached; however, total uptake requires several weeks at 4 C. Adenine inside the red blood cell was shown to egress to the plasma if the equilibrium shifted due to plasma dilution or exchange.     

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.     

不同保存期全血制备洗涤红细胞的超微结构变化

目的 探讨不同保存期的全血制备洗涤红细胞前后电镜下红细胞形态的变化。方法 采集CPD—A抗凝全血。实验分Ⅰ～Ⅵ组，分别于4℃保存7、10、15、20、25、30d。于制备前取全血1滴，5％戊二醛固定，经2000r／min离心10min后，分出血浆，取样检测血红蛋白。余下的红细胞再加等量生理盐水，1500r／min离心5min，连续2次，取样为制备后测定组。结果 组Ⅰ和组Ⅱ制备前后红细胞成双面凹的圆盘结构，细胞均匀混悬。组Ⅲ制备前红细胞形态正常，制备后少数红细胞出现聚集状、球形或边缘不整齐，并有棘形红细胞出现。组Ⅳ、组Ⅴ、组Ⅵ于制备前血浆微红；制备后，球形红细胞、棘形红细胞、中问漏孔的红细胞增加。结论 制备洗涤红细胞的最佳时间应在4℃保存10d内的全血，保存15d以后的全血制备洗涤红细胞形态发生异常变化，出现棘形红细胞，囊泡化后的红细胞易溶血，红细胞寿命缩短，影响洗涤红细胞的质量。     

Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage

BACKGROUND: Current additive solutions (ASs) for red cells (RBCs) do not maintain a constant level of critical metabolites such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate acid (2,3-DPG) during cold storage. From the literature it is known that the intracellular pH is an important determinant of RBC metabolism. Therefore, a new, alkaline, AS was developed with the aim to allow cold storage of RBCs with stable product characteristics. STUDY DESIGN AND METHODS: Whole blood-derived RBCs (leukoreduced) were resuspended in experimental medium phosphate-adenine-guanosine-glucose-gluconate-mannitol (PAGGG-M; pH 8.2) with and without washing in the same medium. During cold storage several in vitro variables, such as intracellular pH, 2,3-DPG, ATP, and hemolysis, were analyzed. RESULTS: During cold storage, RBCs resuspended in PAGGG-M showed a constant ATP level (approx. 6 mumol/g Hb) and a very limited hemolysis (<0.2%). The 2,3-DPG content showed an increase until Day 21 (150% of initial level), followed by a slow decrease, with at Day 35 still 100 percent of the initial level. RBCs washed in PAGGG-M even showed a continuous increase of 2,3-DPG during 35 days, with a maximum level of 200 percent of the initial value. The effect of PAGGG-M appears to be related to long-lasting effects of the initial intracellular pH shortly after production. CONCLUSION: Resuspension of RBCs in our alkaline medium PAGGG-M resulted in a RBC unit of high quality during storage for up to at least 35 days, with 2,3-DPG levels of higher than 10 mumol per g Hb, hemolysis of less than 0.2 percent, and ATP levels of higher than 5 mumol per g Hb.     

Differential profiling of human red blood cells during storage for 52 selected microRNAs

BACKGROUND: MicroRNAs (miRNAs), the negative regulators of cellular mRNAs, are present in mature red blood cells (RBCs) in abundance relative to other blood cells. So far, there are no studies aimed at identifying large‐scale miRNA profiles during storage of RBCs. STUDY DESIGN AND METHODS: RNA samples from each RBC bag stored at 4°C were collected on Days 0, 20, and 40 and subjected to miRNA profiling by using a membrane‐based array. Fifty‐two selected miRNAs of cellular apoptotic pathway represent the array. Through bioinformatics analyses, we identified potential target genes for selected miRNAs. RESULTS: Differential profiling of RBCs for 52 miRNAs revealed two distinguishable patterns during storage: Forty‐eight miRNAs demonstrated no trend at all, while four miRNAs, miR‐96, miR‐150, miR‐196a, and miR‐197, demonstrated an increase up to Day 20 and subsequently decreased during storage. We selected miR‐96 and subjected it to standard bioinformatics analyses for target gene predictions, which identified several mRNAs including the RBC proapoptotic calpain small subunit‐1 (CAPNS1) as potential targets of miR‐96. To validate these predictions, we selected CAPNS1 mRNA as an example and confirmed its presence in the RBCs. Future experimental verification would help define miR‐96–CAPNS1 interaction, if any, in the stored RBCs. CONCLUSIONS: This study for the first time provided a differential profile of stored RBCs for selected miRNAs related to cellular apoptotic pathway and opened new avenues toward identification of novel in vitro RBC biomarkers of storage lesions. Future studies focusing on target gene‐miRNA interactions in stored RBCs would also unravel underlying mechanisms of storage lesions.     

4℃长期保存洗涤红细胞体外质量研究

目的探讨延长洗涤红细胞(WRC)的保存期限。方法将悬浮红细胞均分为3份:1份不洗涤(对照组),其余2份分别用生理盐水(盐水组)和红细胞添加剂(MAP组)洗涤,终产品用MAP液悬浮,4℃保存,分6个时间段取样检测红细胞活性、功能。结果上清游离K+、Na+、Hb:盐水组0~35d,MAP组0~28d均符合标准[1];pH:MAP组<盐水组<对照组(P<0.01);ATP:盐水组与对照组下降趋势基本一致(P>0.05),35d时分别保留70.8%和66.9%,而MAP组自保存7d起低于盐水组、14d起低于对照组(P<0.01),至35d时仅保留27.8%;2-3-DPG:MAP组下降速度明显大于盐水组和对照组(P<0.01),7d时下降了92.5%;洗涤效果:盐水组红细胞回收率83.1%,白细胞清除率84.5%,血浆蛋白清除率98.9%,细菌试验阴性。结论盐水、MAP对红细胞膜基本无损伤;但较之盐水洗涤,MAP洗涤不适于WRC持续保存;盐水洗涤后WRC可在4℃继续保存21d。     

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.     

Alteration of red cell aggregability and shape during blood storage

BACKGROUND: Storage of blood units (for 35-42 days, depending on the preservative solution) has been reported to induce changes (e.g., reduction of sialic acid level) in red cells that are expected to alter their aggregability. STUDY DESIGN AND METHODS: The aggregability of stored red cells was monitored in their autologous plasma and compared to that obtained with washed cells in dextran-containing buffer throughout the storage period. Red cell aggregability was determined by using a computerized image analyzer of cell flow properties. RESULTS: Blood storage induced changes in red cells that are associated with continuous increase of their aggregability. At the same time, blood storage was associated with a reduction in the level of plasma fibrinogen, the major aggregating agent in plasma. Accordingly, the increased red cell aggregability was observed in red cells stored in dextran-containing buffer, but not in red cells stored in autologous plasma. CONCLUSION: Because blood transfusion is routinely given to patients with normal or high fibrinogen level, the transfusion of stored red cells has the potential to induce increased aggregation in vivo, depending on the storage period. This should be taken into account when blood transfusion is considered, particularly for patients with microcirculatory disorders.