Effects of pre-freeze incubation of human red blood cells with various sugars on postthaw recovery when using a dextran-rapid cooling protocol

Polymer has been used as substitute to replace glycerol for cryopreservation of red blood cells (RBCs). But polymer can not penetrate cell membrane, it can not efficiently protect the inner membrane. In this study, RBCs were incubated with glucose, fructose, galactose or trehalose and frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. The postthaw quality was assessed by RBC hemolysis, RBC morphology, PS distribution, osmotic fragility, and the 4 °C stability. The results indicated the loading efficiency of monosaccharide was significantly higher than that of trehalose. Adding trehalose and 40% dextran caused more serious hemolysis before freezing. The percent hemolysis of RBCs loaded with high concentration of trehalose was approximately 16% and significantly more than that of RBCs loaded with glucose (approximately 5%, P < 0.05). Intracellular trehalose can not increase the postthaw recovery of RBCs compared with cells frozen without sugar. However, low concentration of intracellular glucose or galactose can reduce the percent hemolysis to less than 5% and significantly less than that of RBCs frozen without sugar (P < 0.05). Finally, the ability of galactose or fructose to maintain the 4 °C stability was significantly more than that of glucose. In conclusion, the injuries caused by trehalose loading may directly lead to postthaw hemolysis and poor quality of RBCs. However, monosaccharide can enhance the recovery of frozen RBCs. The cryoprotective effect of galactose may be better than that of glucose or fructose. In the future, we will continue to look for a safe and efficient trehalose loading process and try to decrease the osmotic fragility of RBCs frozen with polymers and sugars.     

A technical bias: differences in cooling rates prevent ampoules from being a reliable index of stem cell cryopreservation in large volumes

Ampoule tests are commonly used as an index of the cryopreservation efficiency of marrow stem cells in bags. We have studied the recovery of hematopoietic progenitor cells (CFU-GM, BFUe) in 52 ampoules and compared it to the recovery in 83 standard bags. Our data showed significantly deficient CFU-GM and BFUe recoveries (respectively 47 +/- 31% and 31 +/- 30%) in ampoules when compared to bags (respectively 72 +/- 22% and 64 +/- 19%; P less than 0.001). Moreover, a good progenitor cell recovery (greater than or equal to 50%) was observed in only 46% of frozen ampoules versus 100% observed in frozen bags (P less than 0.05). We were able to relate this nonoptimal recovery to an excessively rapid freezing rate of -9 degrees C/min following the release of fusion heat which occurred in ampoules, while the freezing rate was constantly maintained at -2 degrees C/min in the corresponding bags. We therefore conclude that the cooling conditions have to be carefully controlled to ensure that the bags and ampoules are both cooled under the same conditions. Otherwise, ampoules would not be a reliable index of the true progenitor cells' cryopreservation efficiency in bags.     

Freezing injury to erythrocytes. I. Freezing patterns and post-thaw hemolysis.

The extent of hemolysis of human red blood cells suspended in different concentrations of glycerol and frozen at various cooling rates was investigated on the basis of morphological observation in the frozen state. Hemolysis of the cells in the absence of glycerol showed a V-shaped curve in terms of cooling rates. There was 70% hemolysis at an optimal cooling rate of approximately 10³ °C/min and 100% hemolysis at all other rates tested. Morphologically, a lower than optimal cooling rate resulted in cellular shrinkage, while a higher than optimal rate resulted in the formation of intracellular ice.The cryoprotective effect of glycerol was dependent upon its concentration and on the cooling rate. Samples frozen at 10³ and 10⁴ °C/min showed freezing patterns which differed from cell to cell. The size of intraand extracellular ice particles became smaller, and there was less shrinkage or deformation of cells as the rate of cooling and concentration of glycerol were increased.There was some correlation between the morphology of frozen cells and the extent of post-thaw hemolysis, but the minimum size of intracellular ice crystals which might cause hemolysis could not be estimated. As a cryotechnique for electron microscopy, the addition of 30% glycerol and ultrarapid freezing at 10⁵ °C/min are minimum requirements for the inhibition of ice formation and the prevention of the corresponding artifacts in erythrocytes.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing sucrose: relationship with posthypertonic hemolysis and solute movements

Human red blood cells were frozen at temperatures down to ?9 °C in solutions containing sucrose, and the hemolysis on thawing was measured. This was compared with the hemolysis caused by exposing the cells to high concentrations of sucrose and then resuspending them in more dilute solutions at 4 °C. The effects of the hypertonic solutions of sucrose on potassium, sodium, and sucrose movements were also investigated. It was found that sucrose does not prevent damage to the cells by very hypertonic solutions (whether during freezing and thawing or at 4 °C) but it does reduce hemolysis of cells previously exposed to these solutions if present in the resuspension (or thawing) solution. Evidence is presented that the damaging effects of the hypertonic solutions of sucrose occurring during freezing are associated with changes in cell membrane permeability but that posthypertonic hemolysis is not primarily associated with a “loading” of the cells with extracellular solutes in the hypertonic phase. It is concluded that sucrose may reduce hemolysis of red blood cells by slow freezing and thawing by reducing colloid osmotic swelling of cells with abnormally permeable membranes.     

The mechanism of cryohemolysis: by direct observation with the cryomicroscope and the electron microscope.

Blood films (3–8 μm thick) supported between two glass coverslips were frozen to ?20 °C. In the extracellular areas, ice cavities of the order of 0.2 μm separated by bands of dense plasma were evident when examined with the electron microscope; intracellular ice was not observed with the light microscope. Electron microscopy also showed the presence of intracellular ice particles of the order of 0.2–0.7 μm, these appeared as fine reticulations when observed with the light microscope. Upon gradual rewarming the following changes were observed: recrystallization in the extracellular matrix (?18 to ?8 °C), intracellular recrystallization (?13 to ?10 °C), transfer of water from erythrocytes to extracellular areas (?9 to ?7 °C), and melting and hemolysis (?6 to ?2 °C).Freezing of blood at ?3 °C and subsequent thawing did not cause hemolysis of the red cells. In blood frozen at ?3 °C and cooled to ?20 °C or frozen by abrupt exposure to 20 °C the erythrocytes hemolyzed in 7/16–11/16 of a second, whereas in blood frozen at ?3 °C and cooled to ?10 °C the cells hemolyzed in 5–15 sec even though the mode if lysis (i.e., uniform seepage of hemoglobin from the surface of the cell) was similar in all cases. This indicates that the presence of intracellular ice does not seem to play a major role in the injury to the erythrocytes. The mechanism of cryoinjury demonstrated by hemolysis has been discussed.     

In vivo circulation of mouse red blood cells frozen in the presence of dextran and glucose

Loading with monosaccharide can improve the quality of human red blood cells (hRBCs) frozen with polymer. But in vivo life span of hRBCs frozen with polymer and sugar is not determined. In this study, following incubation with glucose, mouse red blood cells (mRBCs) were frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. After thawing, hemolysis, exposure of PS, and osmotic fragility of frozen mRBCs were determined in vitro. After transfusion of fluorescein isothiocyanate (FITC)-labeled mRBCs, the 24 h recovery and half life span of frozen mRBCs were determined. The data indicated the postthaw hemolysis of mRBCs frozen with dextran and glucose were significantly less than that of cells frozen with dextran (17.23% ± 5.21% vs 25.96% ± 10.07%, P = 0.034). But freezing can also result in exposure of phosphatidylserine and increase of osmotic fragility of mRBCs. After transfusion, the 24 h recovery of mRBCs frozen in the absence or presence of glucose was similar to that of the control cells (P = 0.748 and 0.971). However, the half life span of mRBCs frozen in the absence or presence of glucose was significantly less than that of the control cells (P = 0.000). In addition, incubation with glucose can not increase the life span of frozen red blood cells (7.16 ± 0.93 d vs 7.15 ± 0.34 d, P = 0.982). In conclusion, incubation with monosaccharide could significantly increase the recovery of mRBCs frozen with polymer. Although freezing can significantly shorten the half life span of frozen cells, it can not influence the 24 h recovery of frozen mRBCs. In addition, incubation with monosaccharide before freezing can not increase the life span of frozen mRBCs. So according to the above data, to increase the life span of hRBCs frozen with polymer and monosaccharide, the osmotic fragility of the frozen RBCs must be decreased in the future.     

Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

Storage of apheresis platelets in ethylene-vinyl acetate copolymer bags: relationship between the bag size and the number of platelets maintaining aerobic metabolism

The present study was designed to determine whether a bag made from ethylene-vinyl acetate copolymer (EV) with superior flexibility at subzero temperature is suitable for a storage container of single-donor apheresis platelets. Apheresis platelets were stored with 100 ml plasma in 1-liter bags made of EV or standard polyvinyl chloride (PVC) plastic at 22 degrees C with constant agitation. The oxygen permeability of the 1-liter EV bag averaged 1447 nmol/min/atm, which was about 1.5 times higher than that of PVC bags. The partial oxygen tension (PO2) of platelet concentrates (PC) has linearly decreased to 16 mm Hg with increasing platelet counts. The level of the partial carbon dioxide was always higher in EV bags than in PVC bags. Oxygen consumption rates of platelets stored in EV and PVC bags with a sufficient oxygen supply averaged 1.25 and 1.20 nmol/min/10(9) platelets, respectively. The rates of glucose consumption and lactate production were not changed in two bags. Ninety percent of the total ATP production of about 8 nmol/min/10(9) platelets were generated through the aerobic metabolism. The platelet counts in the 1-liter EV and PVC bags, at which PO2 is 16 mm Hg, were 2.2 and 1.5 x 10(11) platelets, respectively. The study indicates that apheresis platelets stored in EV bags at 22 degrees C have no different metabolic changes when compared with those of PVC bags. In addition, the number of platelets maintaining the aerobic metabolism is 1.5 times higher than that of PVC bags.     

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells (P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.     

An experimental study on the freezing of red blood cells with and without hydroxyethyl starch

Red blood cells were frozen in small capillaries down to ?196 °C at different linear cooling rates with or without the cryoadditive HES; the thawing rate was 3000 or 6500 °C/min. Hematocrit and hydroxyethyl starch concentration varied independently. The hemolysis of red blood cells was determined photometrically after 250-fold dilution and compared to totally hemolyzed samples. The typical U-shaped curves for hemolysis as a function of the cooling rate were obtained for all cell suspensions investigated. Relative optimum cooling rates were determined for the respective combinations of HES and hct. The results show that increasing hct causes an increased hemolysis; increased HES concentration C_HES reduces the optimum cooling rate B_opt; increased hct results in higher optimal cooling rates. The findings allow one to establish a linear correlation of the HES concentration and the optimum cooling rates when the dilution of the extracellular medium by the cell water efflux during freezing is taken into account. A comparison with results from larger volumes frozen (25 ml) shows that the established relationship between hematocrit, HES concentration, and optimal cooling rate remains valid.     

Mechanism of freezing injury to erythrocytes: Effect of initial cell concentration on the post-thaw hemolysis

It has been previously reported that the post-thaw hemolysis of erythrocytes, frozen under various conditions, depends upon the initial cell concentration; increasing the cell concentration decreases the proportion of intact cells after freeze-thawing. In the present study, the effect of cell concentration upon post-thaw hemolysis, examined mainly by the morphological observation of freezing patterns in specimens with or without cryoprotectant glycerol, was most marked in concentrated cell suspensions in which the cells had become shrunken as a result of extracellular freezing. The addition of glycerol lessened the packing effect progressively as the concentration was increased. The results thus obtained may be explained by assuming that cells, deformed in the freezing process, and rigid at low temperatures, might undergo mechanical damage when subjected to compression and abnormal contact.     

Trauma to erythrocytes induced by long term in vitro pumping using a roller pump

The objective of this study is to investigate the impact of trauma on erythrocyte caused by long term in vitro pumping using roller pump. Ten bags of human blood (400 ml each) were provided by a local blood bank and they were divided into two groups with five bags in each group. Each blood bag was subject to pumping in a closed circuit, which was composed of silica gel tubes and a roller pump. Polystan and COBE pumps were used for the two groups, respectively. The blood was pumped for 16 h in vitro. Free hemoglobin (FHb), platelets (PLT), erythrocyte fragility (EF), and morphological analysis of erythrocytes observed under scanning electron microscope were measured to evaluate the impact of trauma on erythrocytes. A small amount of blood was collected for analysis before pumping, at the end of the 4th hour and then every 2 h till the end of the 16th hour. Some blood samples were also collected for electron microscope scanning before pumping and every 4 h during pumping. It was found that FHb and PLT linearly increased with the pumping time. There was a significant correlation between the two parameters (r=0.7745, p<0.001). The hemolysis indexes of the two groups were 0.296 and 0.3993 mg/L/h, respectively, with no significant difference. During the pumping process, EF changed slightly. The observation of scanning electron microscopy showed various deformed erythrocytes after pumping, including the distortion of cell membrane and the appearance of echinocytes, which increased with pumping time. This study demonstrated that long term pumping using roller pump not only caused the immediate rupture of red blood cells, i.e. the immediate hemolysis, but also caused sub-trauma to a large number of erythrocytes, which led to the delayed hemolysis. The change of erythrocyte morphology was the basis of the delayed hemolysis.     

Large-scale, high-density freezing of hybridomas and its application to high-density culture

Large-scale, high-density freezing of hybridomas was studied to apply frozen cells to start high-density culture. We showed here that hybridomas can be frozen at 1.5 x 10(8) cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large-scale freezing to store 25 mL of cell suspension with a cell density, 1.5 x 10(8)/mL. The number of cells stored in a bag (3.0 x 10(9) cells) was enough to start a high-density culture at a 10 times higher cell density (6.0 x 10(6) cells/mL) than normal inoculation, and the cells proliferated to 10(7) cells/mL within 2 days. These results indicate that the large-scale freezing method is useful for large-scale culture of mammalian cells.     

An insert for the reorienting gradient rotor for virus extraction from infected plants

A device made of nylon and which is inserted in the bowl of the reorienting gradient rotor is described. This insert acts as a container for holding infected plant tissues for the purpose of separating virus by centrifugal force from intact and fresh plants, from frozen and thawed plants and from plant tissue disintegrated by mechanical means. The extracted fluid represents 80 to 90% of that present in the untreated plant tissue. Electron micrographs taken of concentrates of the viruses prepared by the centrifugation extraction procedure indicate that extraneous materials are reduced to a low level.     

A new seed-train expansion method for recombinant mammalian cell lines

A new approach has been developed and used to minimize the timeand more carefully monitor and control the seed-train expansionprocess of recombinant mammalian cell lines. The process uses 50or 100 ml cryo-bags that contain frozen cells at high cell densities of 20 × 10⁶ ml^-1 (100 ml bags) or 40 × 10⁶ cells ml^-1 (50 ml bags). The frozen bag cell suspension is thawed and transferred directly into a bioreactorthat has been modified such that pH, DO and temperature can becontrolled at the initial volume of two liters (the working volume eventually increases to 12 l). The successful use of thesecryo-bags and the modified `inoculation' bioreactor to initiate anew seed train expansion of rBHK or rCHO cells is described herein. The interval between cell thawing and the accumulation ofsufficient cell mass to inoculate a production reactor is reducedby at least 25 to 30 days compared to the conventional method that begins with the thaw of 1–2 ml cryo-vials. This `one-step'technology leads to a much more consistent scale-up by reducingmanual operations and avoiding subjective decisions during the scale-up phase. The cell metabolic rates and product integritywere similar to the control experiments. Furthermore, it was found that it is not necessary to include a wash step to removeDMSO prior to the inoculation.     

2020年湖北省襄阳市601份冷冻食品新冠肺炎病毒检测结果分析

自新冠疫情暴发以来,食品或其包装袋不断被检测出新型冠状病毒(SARS-CoV-2)核酸阳性,均与冷冻生鲜相关。为评估SARS-CoV-2污染冷冻食品的风险,本研究采集湖北省襄阳市各大商超、批发市场和餐饮企业的601批次冷冻食品进行新型冠状病毒核酸检测,分析国内外不同品种和不同经营场所的冷冻食品的新型冠状病毒核酸检测结果。结果显示,该601批次的冷冻食品的新型冠状病毒核酸检测结果全部为阴性。本研究提示,襄阳市的冷冻食品被SARS-CoV-2污染的风险较低,其作为SARS-CoV-2传染源的可能性较小。     

An Insert for the Reorienting Gradient Rotor for Virus Extraction from Infected Plants

device made of nylon and which is inserted in the bowl of the reorienting gradient rotor is described. This insert acts as container for holding infected plant tissue for the purpose of separating virus by centrifugal force from intact and fresh plants, from frozen and thawed plants and from plant tissue disintegrated by mechanical means. The extracted fluid represents 80 to 90% of that present in the untreated plant tissue. Electron micrographs taken of concentrates of the viruses prepared by the centrifugation extraction procedure indicate that extraneous materials are reduced to low level.     

The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride

Human red blood cells, suspended in solutions of sodium chloride, have been frozen to temperatures between -2 and -14 degrees C and thawed, and the extent of hemolysis was measured. In parallel experiments, red cells were exposed to similar cycles of change in the composition of the suspending solution, but by dialysis at 21 degrees C. The tonicity of the saline in which the cells were initially suspended was varied between 0.6x isotonic and 4x isotonic; some samples from each experimental treatment were returned to isotonic saline before hemolysis was measured. It was found that the tonicity of the saline used to suspend the cells for the main body of the experiment affected the amount of hemolysis measured: raising the tonicity from 0.6x to 1x to 2x reduced hemolysis, both in the freezing and in the dialysis experiments, whereas raising the tonicity further to 4x reversed that trend. There was little difference between the freeze/thaw and the dialysis treatments for the cells suspended in 1x or 2x saline, whether or not the cells were returned to isotonic conditions. However, the cells suspended in 0.6x saline showed greater damage from freezing and thawing than from the comparable change in the composition of the solution, whether or not they were returned to isotonic conditions. Cells that were suspended in 4x saline and exposed to changes in salt concentration by dialysis showed less hemolysis when they were assayed in the 4x solution than cells that had received the comparable freezing/thaw treatment, but when the experiment included a return to isotonicity, the two treatments gave similar results. Returning the cells to isotonic saline had a negligible affect on the cells in 0.6x and 1x saline, but caused considerable hemolysis in the 2x and 4x samples, more so after dialysis than after freezing and thawing. We conclude that cells suspended in 0.6x and 4x saline behave differently from cells suspended in 1x and 2x saline and hence that cells suspended in a range of solutions of differing initial tonicity should not be treated as a homogeneous population. We argue that an effect of the unfrozen fraction of water (U) cannot be distinguished, within the framework of these freeze/thaw experiments alone, from an effect of initial tonicity, and that the biphasic nature of the correlation between haemolysis and U makes a causal connection improbable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preservation of resuspended red cell concentrate. Red cell volume and hemolysis

Sucrose in a concentration of 30 to 50 mmol/l preservation solution (10-20 mmol/l red cell concentrate (RCC) and 3-5 mmol per unit RCC) and an ionic strength of about 0.16 avoid changes of red cell volume during 6 weeks of storage. Increasing sucrose concentrations up to 80 mmol/l RCC decrease the hemolysis. But a sucrose concentration of only 10 mmol/l RCC causes an acceptable low hemolysis rate of 0.25% after 35 days of storage in PCV FENWAL plastic bags. Sucrose can be replaced by mannitol or sorbitol at the same final concentration. Changes in red cell metabolism and viability will not be expected.