Red Blood Cell Stabilization Reduces the Effect of Cell Density on Recovery Following Cryopreservation

Cryobiology 41, 178 –194 (2000) doi:10.1006/cryo.2000.2279, available online at http://www.idealibrary.com on

Red Blood Cell Stabilization Reduces the Effect of Cell Density on Recovery Following Cryopreservation Christopher T. Wagner, 1 Michael B. Burnett, Stephen A. Livesey, and Jerome Connor Cell Biology Research, LifeCell Corporation, One Millennium Way, Branchburg, New Jersey, 08876, U.S.A. The relationship between red blood cell hematocrit and hemolysis during cryopreservation has been examined. Cells were frozen with glycerol, thawed, and deglycerolized in a model system based on the protocols used in transfusion medicine. Analysis included determination of hemolysis following thaw (Thaw) and deglycerolization (Overall) and osmotic fragility of the final cell suspensions. Results demonstrate that thaw hemolysis decreased with increasing hematocrit at all glycerol levels tested. Overall hemolysis increased with increasing hematocrit at low (15% w/v) glycerol and decreased with increasing hematocrit at high (40% w/v) glycerol levels. These results were paralleled by changes in the fragility index. Furthermore, these results indicate a distinction between freeze/thaw lysis and damage which leads to lysis during postthaw processing. To examine this further, a biochemical stabilizing solution, having no cryoprotective effects itself, was added to suboptimal glycerol concentrations. This addition resulted in hemolysis levels and fragility indices comparable to those using high (40% w/v) glycerol levels. Thus, the damage observed with increasing hematocrit is not necessarily a function of the packing on the volume of the ice-free zone, but rather an expression of cell damage. Furthermore, this damage is, in part, biochemical in nature and may be protected against through specific cellular stabilization prior to cryopreservation. © 2000 Academic Press Key Words: erythrocyte; hematocrit; cryopreservation; glycerol; biochemical stabilization.

The ability to deliver functional cells following prolonged storage has revolutionized many aspects of both clinical and investigational medicine. This is particularly true in transfusion medicine. Two methods for storing red blood cells (RBC) have been approved and are used in United States blood banks—liquid storage at 4°C in an additive solution and frozen storage at ⫺80°C with 40% (w/v) 2 glycerol as a cryoprotectant (20, 36, 37). However, frozen storage requires costly and time-consuming postthaw processing to remove the cryoprotectant prior to transfusion. Therefore, more emphasis is currently being made on finding a logistically simpler and more cost-effective method of RBC Received February 22, 2000; accepted September 19, 2000; published online December 7, 2000. This work was funded in part by the United States Department of Defense, Contract DAMD17-99-C-9018. 1 To whom correspondence should be addressed. 2 Glycerol concentrations are presented as weight per volume percentages (w/v) throughout this paper. This representation is in keeping with the blood banking notation. Glycerol concentrations from referenced works have likewise been converted to w/v for easy comparison.

cryopreservation in light of increased blood component usage in patient care. Since Lovelock (13, 14) first described slowfreezing RBC damage and methods to protect RBC during frozen storage, much debate has occurred regarding the optimal conditions for processing. Factors such as the type and concentration of cryoprotectant, freezing and thawing rates, final storage temperature and duration, and cell density have all been investigated. Several researchers have examined the specific effect that hematocrit has on RBC survival following cryopreservation. Nei (22) first reported that increased hemolysis occurred when RBC were frozen at increasing cell concentrations in the absence of cryoprotection. He argued that mechanical damage would lead to hemolysis at elevated hematocrits since there would not be enough volume for the cells as they became trapped in liquid-filled channels during freezing (23, 24). The general effect of elevated cell concentrations leading to reduced recovery has been corroborated using freezing conditions similar to those used by Nei (15–19, 27–32). Alternative mechanisms for the packing

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effect have been presented in these studies, including (1) mechanical damage due to ice crystal formation or cell– cell contact, (2) extracellular solute concentration accompanied by cellular dehydration, and (3) alterations in the unfrozen water fraction. Contrary to the above investigations, two studies independently concluded that glycerol was able to protect against enhanced hemolysis due to elevated cell concentrations (6, 34). However, methodological problems have limited the impact of their conclusions. While investigating alternative technologies for the cryopreservation of RBC, we reexamined the relationship between hemolysis and hematocrit utilizing protocols based on blood banking procedures. In particular, we use larger volumes of RBC suspensions to better model the heat transfer properties and freezing conditions present in the blood bank, as well as standard cryopreservation methodologies. Additionally, we examined differences between thaw and overall hemolysis levels and postdeglycerolized cell fragilities to distinguish between immediate freeze/thaw lysis and nonlytic cellular damage during cryopreservation. Finally, through the use of novel biochemical stabilization, we investigated the concept of a biochemical nature of cell damage and how this concept affects the relationship between cryopreservation-induced hemolysis and the hematocrit of RBC suspensions. MATERIALS AND METHODS

Blood Preparation, Freezing, Thawing, and Washing Procedures Packed RBC units in Adsol (AS-1; 111 mM dextrose, 2 mM adenine, 41.2 mM mannitol, 154 mM sodium chloride, pH 5.5) were purchased from the Gulf Coast Regional Blood Center (Houston, TX, U.S.A.) following standard testing and component isolation procedures. The packed RBC units were obtained within 72 h of donation, stored at 4°C, and used within 1 week. Prior to using for experimental purposes, the RBC were washed twice with a hypotonic solution containing 50 mM sodium

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chloride, 110 mM glucose, 10 mM glutamine, 55 mM mannitol, and 2 mM adenine and adjusted to pH 6.1. This solution, having an osmolarity of approximately 280 mOsm, was first described by Greenwalt et al. (10) to enhance the liquid preservation of RBC. Following the second wash, the cell suspension was adjusted to a 50% hematocrit and allowed to incubate for 1 h at 4°C before treatment and freezing. The model cryopreservation procedure mimics, on a smaller scale, the freezing and deglycerolization procedures described by Meryman and Hornblower (20) and Valeri (36) and used in U.S. blood banks (11, 37). The washed RBC, prepared as described above, were frozen using Glycerolyte 57 (Fenwal/Baxter, Deerfield, IL, U.S.A.) as the cryoprotectant. Each 100 ml of Glycerolyte 57 contains 57 g glycerin (glycerol), 1.6 g sodium lactate, 30 mg potassium chloride, 51.7 mg monobasic sodium phosphate (monohydrate), and 124.2 mg dibasic sodium phosphate (anhydrous) adjusted to a pH of ⬃6.8 with phosphoric acid. To generate a series of samples with various hematocrits from a single unit of donated RBC, the cells were first equilibrated with the desired final glycerol concentration. For example, if 40% (w/v) glycerol is needed, 300 ml of washed RBC at 50% hematocrit would be mixed with 700 ml Glycerolyte 57. Alternatively, for 15% (w/v) glycerol, 300 ml of RBC would be mixed with 437 ml of hypotonic solution and 263 ml of Glycerolyte 57. The Glycerolyte was added slowly while the RBC suspensions was thoroughly mixed to prevent lysis during the glycerolization process. Following equilibration for 10 min, the cells were isolated by centrifugation at 1200g. These preglycerolized RBC were mixed with a volume-adjusting solution composed of Glycerolyte 57 and hypotonic solution such that the glycerol concentration of the volume-adjusting solution is identical to the concentration used to preglycerolize the RBC. The ratio of RBC to adjusting solution was altered to generate 100-ml samples with various hematocrits. The final samples were placed in a 150-ml PVC component bag (CharterMed, Lakewood, NJ, U.S.A.). The sealed bag was placed in an alu-

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minum freezing cassette and set in a freezer at ⫺80°C to freeze. The cooling rate using this method was measured to be ⫺3.5 ⫾ 0.6°C/min. These small units were left frozen for 5 to 10 days before thawing. The frozen cells were thawed in a 40°C circulating water bath for 10 min, achieving a warming rate of 96.7 ⫾ 3.5°C/min. A 2-ml sample of the thawed cells was taken for hematologic and hemolytic analysis, as described below. The remainder of the cells were deglycerolized in a 250-ml polypropylene conical tube by serial dilutions with decreasingly concentrated saline solutions. All saline additions were added to the cell suspensions using a peristaltic pump at 10 ml/min while the cells were thoroughly mixed on an orbital shaker. Initially, 30 ml of 12% saline was added to the remaining 98-ml sample, followed by 50 ml of 1.6% saline with a 5-min rest period after each addition. The cells were then isolated by centrifugation at 1200g. This saline addition, rest, and isolation cycle was repeated first with 150 ml 1.6% saline and finally by 150 ml 0.9% saline. After the final centrifugation, the cells were resuspended to a final hematocrit of 50% with 0.9% saline/ 0.2% glucose. All supernatants from the centrifugation steps were save and combined to determine the amount of hemoglobin lost during the deglycerolization procedure. The deglycerolized RBC suspensions were checked for hematologic properties and residual free hemoglobin. These cells were also used for further testing in assays described below. Hematology Analysis and Hemolysis Determination The RBC samples from the thawed and deglycerolized suspensions were analyzed for RBC number, hematocrit, and total hemoglobin content using a Biochem Immunosystems 1099⫹ hematology analyzer. Free hemoglobin concentrations of the thawed and deglycerolized samples were determined using a cell-free sample isolated by centrifugation at 1200g for 10 min. The hemoglobin in this cell-free sample was determined following conversion to cyanmethemoglobin with Drabkin’s reagent for

spectrophotometric analysis at 545 nm (Hemoglobin Kit; Sigma Chemical, St. Louis, MO, U.S.A.). The hemoglobin content of the combined deglycerolization supernatants was determined in a similar manner. The hematologic values and free hemoglobin levels were used to calculate percentage thaw hemolysis, percentage overall hemolysis, and residual free hemoglobin. The thaw hemolysis value was calculated as the ratio of free hemoglobin concentration (H free) to the total hemoglobin (H total) concentration, weighted by the postthaw suspension hematocrit (Hct) as an indication of extracellular volume. Thus, thaw hemolysis ⫽ H free 䡠 (1 ⫺ Hct)/H total and represents an immediate lysis event due to the freeze/ thaw cycle alone. The overall hemolysis value was calculated as the fraction of hemoglobin in the combined deglycerolization supernatant compared to the total hemoglobin at the time of thaw. It should be noted that the thaw hemolysis is a subset of the overall hemolysis since the free hemoglobin at the time of thawing is removed during the first wash step. The difference between the overall hemolysis and the thaw hemolysis indicates the amount of cell lysis that occurred during the deglycerolization procedure and represents, in part, a damaged condition not associated with immediate lysis. Thus, by determining the difference between these two hemolysis values, a distinction can be made between an immediate freeze/thaw lysis event and a level of damage which leads to delayed lysis during the deglycerolization procedure, whether ice-induced or biochemical in nature. Finally, the percentage residual free hemoglobin in the deglycerolized RBC suspension was determined in a manner similar to the thaw hemolysis level. Osmotic Fragility Analysis A standard osmotic fragility method utilizing saline solutions ranging from 0.1 to 0.9% was employed to determine the ability of the cells to withstand hypotonically induced water flux which leads to hemoglobin leakage and cell lysis (3). RBC suspensions were diluted 1:100 in each saline solution within the range, mixed,

RBC STABILIZATION REDUCES CELL DENSITY EFFECT

and allowed to incubate for 30 min at room temperature. Following the incubation period, the samples were mixed again to resuspend any settled RBC and the percentage hemolysis was calculated by determining the ratio of free hemoglobin to the total hemoglobin content using a spectrophotometric measurement of cyanmethemoglobin as described above. Free hemoglobin introduced as part of the original RBC suspension was subtracted prior to the hemolysis calculation so that the osmotic fragility curves represent only hemolysis due to dilution in the saline solutions. The concentration of saline necessary to induce 50% hemolysis was determined from these curves. This value, termed the fragility index of the cell suspension, was used to quantify the degree of fragility. Using this method, a larger fragility index corresponds to a more fragile cell.

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5°C/min from room temperature to ⫺60°C. Once cooled to ⫺60°C, the sample was held for 5 min and then warmed to room temperature, also at a rate of 5°C/min. This slow freezing profile was used to match the freezing rate of between 1 and 5°C/min of RBC units placed at ⫺80°C. Statistical Analysis Results are expressed as the mean value ⫾ standard error. Each experiment was performed using different donated units of RBC. To the best of our knowledge, no single donor contributed more than one unit of blood to this study. Significant differences were computed using a two-tailed Student’s t test with the significance threshold defined as P ⬍ 0.05. Implicit in this type of analysis, the values are assumed to be normally distributed.

Biochemical Stabilization We developed a solution to enhance recovery of RBC following cryopreservation at reduced cryoprotectant levels. The solution is prepared in the hypotonic buffer described above, such that the final RBC unit contains 5 mM pentoxifylline, 25 ␮g/ml flurbiprofen, 500 ␮M nifedipine in 1% (v/v) nikethamide, and 400 mM nicotinamide prior to freezing. Addition to the RBC was done slowly, similar to the glycerolization procedure. All biochemical reagents were purchased from Sigma Chemicals. Differential Scanning Calorimetry (DSC) Differential scanning calorimetry was used to monitor the freezing and thawing characteristics of RBC suspensions of various hematocrit and cryoprotectant conditions. DSC was performed using a Shimadzu calorimeter and accompanying analysis software to determine enthalpy changes and the onset temperatures of the freezing and thawing events. The enthalpy change during freezing was used to gauge the amount of ice formed during the freezing process. Fifteen microliters of RBC suspension with the desired experimental cryoprotectant and hematocrit condition was placed in an aluminum DSC pan, covered, and then slowly cooled at a rate of

RESULTS

In standard blood banking, glycerolization yields a final glycerol content of ⬃40% (w/v) and a hematocrit of between 25 and 35%. The deglycerolization procedure outlined under Materials and Methods is based on this glycerol content. Experiments were initially performed to verify that the model mimics the full-scale blood banking procedure and to identify suboptimal glycerol conditions. Additionally, the experiments were used to demonstrate that the glycerolization and deglycerolization procedures do not harm the RBC at any of the glycerol concentrations tested. RBC glycerolized with 15% (w/v) or 40% (w/v) glycerol and at 30% hematocrit were either immediately deglycerolized or cryopreserved, thawed, and then deglycerolized. Results of this experimental series are shown in Table 1. In the absence of freezing, glycerolization to either 15% (w/v) or 40% (w/v) glycerol concentration did not cause lysis (thaw levels of 0.4 ⫾ 0.1 and 0.5 ⫾ 0.1%, respectively). Deglycerolization of the nonfrozen RBC induced less than 5% overall hemolysis, presumably due to water and glycerol flux across the cell membrane, and completely removed all free hemoglobin from

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WAGNER ET AL. TABLE 1 Hemolysis Analysis of Glycerolization and Deglycerolization Procedures with and without Cryopreservation (mean ⫾ SE, n ⫽ 3, Hematocrit ⫽ 30%) Not cryopreserved

Cryopreserved

Hemolysis

15% Glycerol

40% Glycerol

15% Glycerol

40% Glycerol

Thaw Overall Residual

0.4% ⫾ 0.1% 4.2% ⫾ 0.5% 0.2% ⫾ 0.1%

0.5% ⫾ 0.1% 4.9% ⫾ 0.1% 0.3% ⫾ 0.1%

47.8% ⫾ 5.1% 63.1% ⫾ 2.2% 1.7% ⫾ 0.3%

3.1% ⫾ 0.2%† 8.7% ⫾ 0.9%† 0.6% ⫾ 0.1%†

† Denotes significant difference with respect to 15% Glycerol-cryopreserved condition.

the RBC suspension, as denoted by the residual values. Using the model procedures and freezing RBC with 40% (w/v) glycerol yielded results that are equivalent to full-unit-scale results when deglycerolizing with a Haemonetics 115 cell washing system (not shown). The overall and residual values, 8.7 ⫾ 0.9 and 0.6 ⫾ 0.1%, respectively, are well within maximum allowable limits for a transfusable cryopreserved and deglycerolized RBC unit (11). Since the model glycerolization and deglycerolization do not harm RBC in the absence of freezing and yield acceptable results when used to freeze RBC using the standard 40% (w/v) glycerol concentration, the increased hemolysis observed at 15% (w/v) glycerol is exclusively a function of suboptimal protection at this glycerol concentration. This lack of protection results in increased cellular damage during the freeze/thaw cycle, which is observed as increased levels of thaw and overall hemolysis. The high amount of hemolysis observed under this condition also causes an elevation in the residual free hemoglobin. Although the washing procedure does not completely clear this free hemoglobin, the residual level is still less than the maximum allowable transfusion limit and the resulting RBC are no more fragile then RBC processed using 40% (w/v) glycerol at 30% hematocrit, as seen in Fig. 3. The residual free hemoglobin levels were similar for all experiments and are, therefore, not shown in the results below. Figure 1 shows that using suboptimal concentrations of glycerol reveals a packing effect similar to that described previously (17, 22, 27).

The overall hemolysis of RBC suspensions frozen with 15% (w/v) glycerol increases significantly as the hematocrit of the suspension is elevated above 40%. Although not statistically significant, the overall hemolysis obtained with the highest hematocrit range is elevated compared to the lowest range, having a P value of 0.060. In contrast, the hematocrit and thaw hemolysis are inversely proportional. As the hematocrit of the RBC suspension is increased, the level of lysis during the freeze/thaw cycle is significantly reduced. The inset of Fig. 1 shows the individual data points from the experimental series using 15% (w/v) glycerol and varying the hematocrit. The mean regression lines of the thaw (solid) and overall (dashed) hemolysis fit the data with correlation coefficients (R 2 ) of 0.65 and 0.39, respectively. This plot shows more clearly the increasing amount of hemolysis that occurs during the deglycerolization procedure (distance between regression lines) as the hematocrit is increased. Increasing the hematocrit of the RBC suspension when using 40% (w/v) glycerol for cryoprotection does not have the same effect on cell recovery as does the lower glycerol concentration. Shown in Fig. 2, the thaw hemolysis levels decrease with increasing hematocrit, similar to those of the 15% (w/v) glycerol condition. However, as opposed to the effect observed using 15% (w/v) glycerol, increasing the hematocrit from 21 to 95% had no detrimental effect in the overall hemolysis levels. Interestingly, both the thaw and the overall hemolysis levels obtained with the lowest hematocrit range are significantly elevated compared to all other he-

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FIG. 1. RBC were frozen with 15% (w/v) glycerol at various hematocrits. Following storage, the cells were thawed and washed to remove the cryoprotectant. As described under Materials and Methods, hemolysis was determined following the thawing (Thaw) and washing (Overall) procedures. The bar graph represents mean ⫾ SE for n ⫽ 5 different donor experiments with groupings of hematocrit in 20% ranges. † Denotes significance (P ⬍ 0.05) with respect to the lowest hematocrit range. Inset: Scatter plot of values for n ⫽ 5 experiments is shown. The solid (R 2 ⫽ 0.65) and dashed (R 2 ⫽ 0.39) lines are derived from linear regression of the thaw and overall hemolysis values, respectively.

matocrit ranges and would not meet current criteria for transfusion-quality RBC, despite using the approved cryopreservation protocols with 40% (w/v) glycerol. The inset of Fig. 2 shows the individual data points from the experiments using 40% (w/v) glycerol. The 20% hematocrit threshold for optimal cell recovery is further exemplified in this plot. The regression lines for the thaw and overall hemolysis data points were calculated excluding data below a 20% hematocrit and have R 2 values of 0.88 and 0.10, respectively.

A standard saline osmotic fragility assay was used to test the quality of the deglycerolized RBC following cryopreservation. Figure 3 shows representative fragility curves for RBC frozen with either 40% (w/v) or 15% (w/v) glycerol (Fig. 3A) and the cumulative plot of fragility indices (Fig. 3B), as defined under Materials and Methods. RBC frozen with 15% (w/v) glycerol have a statistically significantly reduced ability to respond to osmotic changes at the highest hematocrits compared to 15% (w/v) glycerol at the lowest hematocrit. Furthermore,

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FIG. 2. RBC were frozen with 40% (w/v) glycerol at various hematocrits. Following storage, the cells were thawed and washed to remove the cryoprotectant. As described under Materials and Methods, hemolysis was determined following the thawing (Thaw) and washing (Overall) procedures. The bar graph represents mean ⫾ SE for n ⫽ 8 different donor experiments with groupings of hematocrit in 20% ranges. † Denotes significance (P ⬍ 0.05) with respect to the lowest hematocrit range. Inset: Scatter plot of values for the same n ⫽ 8 experiments is shown. The solid (R 2 ⫽ 0.88) and dashed (R 2 ⫽ 0.10) lines are derived from linear regression of the thaw and overall hemolysis values, respectively, excluding hematocrits less than 20% in both cases.

the 15% (w/v) glycerol sample had statistically significantly higher fragility indices compared to 40% (w/v) glycerol once hematocrits reached 60%. Since the conditions of the RBC samples tested were identical except for the hematocrit, the deviations observed are exclusively the result of the increased cell concentration. In contrast to the effect observed with 15% (w/v) glycerol, there is no increase in deglycerolized RBC fragility when 40% (w/v) glycerol is used during cryopreservation. A biochemical stabilizing additive was used

to determine whether the ability to overcome the packing effect with increased glycerol is a function of increased cryoprotection or overall cellular stabilization. This solution was designed to biochemically protect the RBC and was tested for its ability to maintain RBC integrity at suboptimal glycerol concentrations. Figure 4 shows the results of adding the biochemical stabilizing solution to RBC suspensions at a 40% hematocrit. Both the thaw and the overall hemolysis values obtained with cryopreservation of RBC with 15% (w/v) glyc-

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FIG. 3. (A) Representative osmotic fragility curves are shown for postwashed RBC cryopreserved with either 40 or 15% (w/v) glycerol over a range of hematocrits. The hematocrits for both panels of this figure are 10.6 ⫾ 2.5, 22.6 ⫾ 5.8, 37.5 ⫾ 6.8, 60.0 ⫾ 4.6, 77.6 ⫾ 2.3, and 93.1 ⫾ 1.0% from lowest to highest. (B) Using osmotic fragility curves as shown in (A) the fragility of each cell population was quantitated by determining the saline percentage at which 50% hemolysis was observed. Each bar represents the mean ⫾ SE of n ⫽ 3 different donor experiments. † Denotes a significant (P ⬍ 0.05) elevation of fragility with respect to the same hematocrit level using 40% (w/v) glycerol as the cryoprotectant; ‡ denotes a significant (P ⬍ 0.05) elevation of fragility with respect to the lowest hematocrit level within each glycerol condition.

erol and the additive solution were statistically significantly reduced compared to 15% (w/v) glycerol alone. However, the hemolysis results with the additive solution were still significantly elevated compared to the levels using 40% (w/v) glycerol. Addition of this solution to RBC prior to freezing allows significant reductions in glycerol concentration while maintaining the reduced hemolysis obtained with 40% (w/v) glycerol. Studies were performed to determine whether the additive solution could protect against the packing effect observed at reduced glycerol

concentrations using osmotic fragility as an indicator of cellular damage. The results are shown in Fig. 5. As expected, RBC cryopreserved with 15% (w/v) glycerol and elevated hematocrits are statistically significantly more fragile than either RBC similarly cryopreserved at lower hematocrits or RBC cryopreserved with 40% (w/v) glycerol at the same hematocrits. However, RBC cryopreserved with 15% (w/v) glycerol and the additive solution had reduced fragility indices with respect to 15% (w/v) glycerol alone, independent of the hematocrit. Furthermore, use of the additive solution

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FIG. 4. RBC were frozen at a 40% hematocrit with either 40% (w/v) glycerol, 15% (w/v) glycerol, or 15% (w/v) glycerol plus the additive solution. This graph represents the mean ⫾ SE thaw and overall hemolysis values from n ⫽ 3 different donor experiments. † Denotes a significant (P ⬍ 0.05) increase in thaw or overall hemolysis with respect to the 40% (w/v) glycerol condition; ‡ denotes a significant (P ⬍ 0.05) reduction in thaw or overall hemolysis compared to 15% (w/v) glycerol-only condition.

yielded indices lower than 40% (w/v) glycerol, but only at low hematocrits, for which 40% (w/v) glycerol appears to be less protective (see Fig. 2). Despite the ability of the additive solution to protect the RBC with respect to both the hemolysis levels and the fragility indices, increasing the hematocrit does cause an increase in the fragility index. The degree of increase in fragility is significantly reduced with the additive compared to 15% (w/v) glycerol alone. Furthermore, the fragility index at the highest hematocrit using 15% (w/v) glycerol with the additive solution (the highest index of this condition) is comparable to the value using 40% (w/v) glycerol. To determine whether the additive solution was serving as a substitute cryoprotectant in the cryopreservation system, DSC was used to determine the freezing characteristics of glycerol with and without the additive solution. Table 2A shows that increasing the concentration of glycerol alone reduces the enthalpy change upon freezing, a result caused by a reduction in the amount of ice formed. Additionally, the higher glycerol content causes a lowering in the onset temperatures of the freezing and thawing

events. In contrast, there are no differences in the measured values for 15% (w/v) glycerol with or without the additive solution. This indicates that the additive does not protect the cells in the same manner as the glycerol cryoprotectant. The lack of a difference in the freezing characteristics for 15% (w/v) glycerol with and without the additive solution is consistent across a range of hematocrits, as shown in Table 2B. The only discernable effects are a decrease in the enthalpy change and a depression in the freeze and thaw onset temperatures as the hematocrit is increased. These characteristics, in conjunction with the results above, distinguish between the cryoprotective effect of glycerol and the biochemical effect of the additive solution and indicate that the additive solution is not serving as the former. The additive solution in combination with 15% (w/v) glycerol reduced the levels of thaw and overall hemolysis and the fragility indices compared to 15% (w/v) glycerol alone, but did not achieve results equivalent to 40% (w/v) glycerol. To determine whether the additive solution could be used with suboptimal concentrations of glycerol to achieve results equivalent

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FIG. 5. Using osmotic fragility curves as described in the legend to Fig. 3, the fragility of postwash cell populations cryopreserved with 40% (w/v) glycerol, 15% (w/v) glycerol, or 15% (w/v) glycerol plus the additive solution were quantitated by determining the saline percentage at which 50% hemolysis was observed. The hematocrits are 9.4 ⫾ 0.3, 17.6 ⫾ 0.4, 27.1 ⫾ 0.6, 38.4 ⫾ 1.1, 52.4 ⫾ 1.6, and 71.2 ⫾ 1.5% from lowest to highest. Each bar represents the mean ⫾ SE of n ⫽ 3 different donor experiments. § Denotes a significant (P ⬍ 0.05) elevation of fragility with respect to the same hematocrit level using 40% (w/v) glycerol as the cryoprotectant; † denotes a significant (P ⬍ 0.05) decrease of fragility with respect to the same hematocrit level using 15% (w/v) glycerol as the cryoprotectant; ‡ denotes a significant (P ⬍ 0.05) elevation of fragility with respect to the lowest hematocrit level within each cryoprotectant condition.

to 40% (w/v) glycerol, the cryoprotectant concentration was increased to 20% (w/v) glycerol. Figure 6 shows the thaw (Fig. 6A) and overall (Fig. 6B) hemolysis levels of 20% (w/v) glycerol with and without the additive solution compared to 40% (w/v) glycerol. The hematocrit range is the same as that reported for Fig. 5. The results show that 20% (w/v) glycerol does not provide optimal protection during cryopreservation. Furthermore, using 20% (w/v) glycerol alone reveals the same decrease in thaw hemolysis as a function of hematocrit, as was observed with 15% (w/v) glycerol. Despite this similarity, the overall hemolysis values do not show a consistent trend dependent upon the hematocrit. As with 15% (w/v) glycerol, the additive solution statistically significantly reduced both hemolysis levels at all hematocrits with respect to 20% (w/v) glycerol alone. Additionally, these levels were not significantly elevated compared to 40% (w/v) glycerol.

Figure 7 displays the osmotic fragility of the cells cryopreserved using 20% (w/v) glycerol with and without the additive solution compared to 40% (w/v) glycerol. The hematocrit range is the same as that reported for Fig. 5. Figure 7A shows representative fragility curves of the three different cryopreservation conditions. The curves of the higher hematocrit levels are right-shifted when using 20% (w/v) glycerol alone. This shift is reversed upon addition of the additive solution, resulting in a profile that appears similar to RBC cryopreserved with 40% (w/v) glycerol. In Fig. 7B, the fragility index is used to quantify this effect. RBC cryopreserved with 20% (w/v) glycerol are indeed statistically significantly more fragile than RBC frozen with 20% (w/v) glycerol with the additive solution. Furthermore, using the additive solution in conjunction with 20% (w/v) glycerol yielded indices lower than 40% (w/v) glycerol for a broader range of hematocrits. The increase in the fragil-

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WAGNER ET AL. TABLE 2 DSC Analysis of RBC Suspension Freezing Characteristics (mean ⫾ SE, n ⫽ 3) A Condition (30% Hct)

⌬H (J/g)

Freeze (°C)

Thaw (°C)

40% Glycerol 15% Glycerol 15% ⫹ Additive

92.2 ⫾ 0.3 180 ⫾ 0.0‡ 170 ⫾ 5.8‡

⫺36.9 ⫾ 1.3 ⫺18.9 ⫾ 0.1‡ ⫺22.0 ⫾ 4.1‡

⫺26.0 ⫾ 0.4 ⫺12.9 ⫾ 0.3‡ ⫺13.1 ⫾ 0.6‡

B 15% Glycerol Hematocrit (%)

⌬H (J/g)

Freeze (°C)

Thaw (°C)

0.0 16.0 ⫾ 2.5 29.0 ⫾ 1.3 41.4 ⫾ 1.2 50.7 ⫾ 3.3 67.3 ⫾ 2.8

206 ⫾ 3.3 186 ⫾ 3.3† 183 ⫾ 3.3† 180 ⫾ 0.0† 166 ⫾ 3.3† 160 ⫾ 5.8†

⫺14.0 ⫾ 1.8 ⫺17.3 ⫾ 0.5 ⫺19.5 ⫾ 2.8 ⫺18.9 ⫾ 0.1 ⫺23.1 ⫾ 0.9† ⫺21.9 ⫾ 1.0†

⫺12.0 ⫾ 0.4 ⫺12.8 ⫾ 0.4 ⫺12.5 ⫾ 0.5 ⫺12.9 ⫾ 0.3 ⫺13.9 ⫾ 0.8 ⫺13.9 ⫾ 0.3†

15% Glycerol ⫹ additives Hematocrit (%)

⌬H (J/g)

Freeze (°C)

Thaw (°C)

0.0 15.8 ⫾ 1.2 29.8 ⫾ 0.5 41.8 ⫾ 1.8 52.5 ⫾ 0.0 64.7 ⫾ 3.6

196 ⫾ 3.3 183 ⫾ 3.3† 180 ⫾ 5.8 170 ⫾ 5.8† 166 ⫾ 3.3† 156 ⫾ 3.3†

⫺15.7 ⫾ 0.9 ⫺17.9 ⫾ 0.3 ⫺17.3 ⫾ 0.8 ⫺22.0 ⫾ 4.1† ⫺22.7 ⫾ 1.8† ⫺25.1 ⫾ 1.0†

⫺10.9 ⫾ 0.4 ⫺12.5 ⫾ 0.7 ⫺12.3 ⫾ 0.8 ⫺13.2 ⫾ 0.6† ⫺13.2 ⫾ 0.7 ⫺13.5 ⫾ 0.4†

‡ Denotes significant difference with respect to 40% glycerol; No significant difference between 15% glycerol and 15% glycerol with additive. † Denotes significant difference with respect to RBC free condition; No significant difference between 15% glycerol and 15% glycerol with additive.

ity index with respect to the hematocrit, using 20% (w/v) glycerol with the additive solution, is not statistically significant between the lowest and the highest hematocrits and is never greater than the index for 40% (w/v) glycerol. DISCUSSION

In the cryopreservation of cells, the characteristics of the freezing protocol are paramount to successful cell recovery. Included in these characteristics is the cell concentration of the frozen suspension. In this work, we have presented results reexamining the relationship between hematocrit and cell recovery following

cryopreservation with approved blood bank protocols (37). The first observation that increased hematocrit induces hemolysis during cryopreservation was made by Nei (22) in 1967. Several investigators have postulated mechanisms to explain this “packing” phenomenon. Mazur and his colleagues (17, 18) hypothesized that the dominant predictor of cell damage is the unfrozen water fraction. This is particularly true for low unfrozen fractions, such as suspensions with high hematocrits. The decreased available space for cells to occupy as the water freezes results in increased cell–ice and cell– cell contact, which

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FIG. 6. RBC were frozen with either 40% (w/v) glycerol, 20% (w/v) glycerol, or 20% (w/v) glycerol plus the additive solution. The graph represents mean ⫾ SE thaw (A) and overall (B) hemolysis levels from n ⫽ 3 different donor experiments. The hematocrits are 9.4 ⫾ 0.3, 17.6 ⫾ 0.4, 27.1 ⫾ 0.6, 38.4 ⫾ 1.1, 52.4 ⫾ 1.6, and 71.2 ⫾ 1.5% from lowest to highest. † Denotes a significant (P ⬍ 0.05) decrease in hemolysis with respect to the lowest hematocrit range within each cryoprotectant condition. All hemolysis levels using 20% (w/v) glycerol plus the additive solution are significantly decreased compared to 20% (w/v) glycerol alone.

in turn may lead to increased damage and hemolysis. This concept fits with the SEM observations showing shrunken cells with membrane distortions that may interact with each other or ice formations in a semilocking manner (25, 32). Pegg and Diaper’s (30, 31) later hypothesis stresses the initial tonicity of the solution prior

to freezing as the primary predictor of survival—shrunken cells surviving better than swollen cells. This hypothesis returns to the solutebased concept of cell damage first postulated by Lovelock (13, 14) and provides an explanation of the observations to date without utilizing the concept of unfrozen fraction. Two other investigations, by Rapatz and

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FIG. 7. (A) Representative osmotic fragility curves are shown for postwashed RBC cryopreserved with either 40% (w/v) glycerol, 20% (w/v) glycerol, or 20% (w/v) glycerol plus the additive solution over a range of hematocrits. The hematocrits for both panels of this figure are 9.4 ⫾ 0.3, 17.6 ⫾ 0.4, 27.1 ⫾ 0.6, 38.4 ⫾ 1.1, 52.4 ⫾ 1.6, and 71.2 ⫾ 1.5% from lowest to highest. (B) Using osmotic fragility curves as shown in (A), the fragility of the postwash cell populations were quantitated by determining the saline percentage at which 50% hemolysis was observed. Each bar represents the mean ⫾ SE of n ⫽ 3 different donor experiments. § Denotes a significant (P ⬍ 0.05) elevation of fragility with respect to the same hematocrit level using 40% (w/v) glycerol as the cryoprotectant; † denotes a significant (P ⬍ 0.05) decrease of fragility with respect to the same hematocrit level using 20% (w/v) glycerol as the cryoprotectant; ‡ denotes a significant (P ⬍ 0.05) elevation of fragility with respect to the lowest hematocrit level within each cryoprotectant condition.

Rapatz in 1973 (34) and Dalgliesh in 1976 (6), reported that glycerol (23 and 26% (w/v), respectively) was able to reverse the effects of increased cell concentration during cryopreservation. In both studies, only the thaw hemolysis level was measured and the concentrations of glycerol used were lower than optimal concentrations. Due to these and other limitations (27), neither study demonstrated that the lack of a packing effect was due to the protected nature

of the cell, as opposed to a nonspecific effect of the penetrant glycerol. We have shown that a packing effect similar to that previously described does occur when overall hemolysis is assayed. That is, this packing effect is apparent only following deglycerolization. Analysis of the thaw hemolysis levels reveals a decrease in hemolysis as the hematocrit is raised. Despite being intact, the increased overall hemolysis at elevated hematocrits is in-

RBC STABILIZATION REDUCES CELL DENSITY EFFECT

dicative of a degree of damage to these “intact” cells. This distinction between thaw and overall hemolysis has not been made in previous reports. Furthermore, the packing effect was observed only by using suboptimal cryoprotective conditions. With optimal glycerol concentrations, there is in fact a beneficial effect of increasing the hematocrit of the sample in both the thaw and the overall hemolysis levels. This result indicates that the protected nature of the cells determines the effect of the cell density. Osmotic fragility analysis further supports this conclusion. Cell samples that had been frozen at high hematocrits and low glycerol concentrations had increased osmotic fragility indices. In contrast, cells frozen at 40% (w/v) glycerol showed normal fragility curves that were independent of the frozen suspension hematocrit. These results further substantiate the concept that the degree of cellular protection influences the effects of elevated hematocrit. As we have shown here, the thaw hemolysis level is not elevated in response to RBC packing during cryopreservation and it is necessary to analyze either the overall hemolysis or the postdeglycerolized RBC fragility indices to discern an effect. Thus, it is unclear whether the previous investigations by Rapatz and Rapatz (34) or Dalgliesh (6) would have come to similar conclusions, since their freezing conditions were suboptimal. The model deglycerolization procedure is based on the standard system used when RBC are cryopreserved with 40% (w/v) glycerol. Table 1 demonstrates that this procedure does not damage cells when 15% (w/v) glycerol is used. In particular, the initial dilution of thawed RBC with 12% saline does not cause hyperosmotic damage to the RBC, resulting in posthypertonic hemolysis, as described by Zade-Oppen (39). In addition to the results shown in Table 1, other experiments were performed in which 6% saline was substituted for 12% saline. The osmolarity ratio of 12% saline to 40% (w/v) glycerol and 6% saline to 15% (w/v) glycerol are both ⬃0.75. The results of this experimental series (not shown) demonstrated no difference in either the hemolytic or the fragility levels of RBC

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deglycerolized with 6% saline compared to 12% saline. Thus, the increased hemolysis at elevated hematocrits observed at low glycerol concentrations and the increased osmotic fragilities brought about by these conditions are a function of damage that occurs during the freeze/thaw cycle. Although reports regarding cell packing and hemolysis are well documented in the literature, there are significant differences between those reports and our study. Principally, we have examined the relationship between hematocrit and cell lysis under large-volume conditions similar to those found in blood banking. Our observations have shown that small volumes tend to underestimate the amount of hemolysis that takes place in larger volumes under identical conditions. Furthermore, the freezing characteristics are different with respect to the concentration of glycerol and we did not seed or alter the freezing and warming rates, as was done in the majority of the previous works (15, 25, 28). More recently, De Loecker et al. (7) reported results using dimethyl sulfoxide (Me 2SO) as the cryoprotectant. Although their results showed a significant packing effect, the baseline hemolysis at low hematocrits was nearly 20%. In these experiments, the RBC exhibited significant damage independent of the hematocrit. Based on our conclusions here, the damage due to inadequate protection may explain the increased lysis under packed conditions. Whereas ice formation and extracellular rises in solute concentration do occur during the freezing process, they are not necessarily the exclusive methods of cell damage. The biochemical status of the RBC has gone largely ignored with respect to cryopreservation. The theories developed to explain the packing effect are based on the currently accepted theories of cell damage during freezing at normal hematocrits. We have shown that the cell packing phenomenon does not occur, or is at the least significantly attenuated, if the cells are adequately protected and that this protection does not need to be provided by agents considered cryoprotective. Furthermore, our results concur with those of Pegg (27) in that the packing

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effect is not observed until the hematocrit is elevated beyond a threshold level, although we observed the threshold to be closer to a hematocrit of 40% for both the hemolysis levels and the fragility indices. Prior explanations stress that new effects of ice formation and solute concentration arise when cell densities exceed this threshold value, thereby resulting in damage which reduces recovery. However, an alternative hypothesis is possible—that cell damage occurring under the suboptimal conditions used is enhanced at high cell concentrations. It was considered that the benefit of the additional glycerol was to reduce the amount of ice formed and decrease the amount of cell damage. Glycerol has been shown to permeate and coat RBC, presumably protecting them from ice- and solute-induced damage in that manner (14). However, we show that the positive effects of additional glycerol can be accomplished through the use of an additive solution, that itself does not have traditional cryoprotective properties. That is, the solution does not alter the thermodynamic freezing characteristics of the suspension. Thus, the damages that occur during cryopreservation under suboptimal conditions may be protected against through biochemical stabilization. The biochemical additive solution was developed based on analysis of the RBC biochemistry and observed damage to the RBC during cryopreservation at reduced glycerol concentrations. Each reagent incorporated into the biochemical additive solution has been used clinically. Pentoxifylline increases the fluidity of the lipid membrane and has been used to prevent or reduce sickle cell crisis and to improve acrosome reaction in cryopreserved spermatozoa (1, 8). Flurbiprofen is a nonsteroidial antiinflammatory agent and blocks oxidative reactions catalyzed by cyclooxygenase (4). Cyclooxygenase and lipoxygenase are both active in the mature erythrocyte and may contribute to membrane lipid alterations (9, 12, 33). Nifedipine is a dihydropyridine-type Ca 2⫹ channel blocker and has been shown to enhance RBC stability (2, 26). Nicotinamide is a precursor of NAD(H), a significant source of reducing potential and

requirement of glycolysis, the only energy-producing cycle in the RBC (21, 35). Finally, nikethamide is an analeptic agent used for respiratory stimulation (38). In this formula, nikethamide was used to solublize nifedipine and flurbiprofen prior to addition to RBC suspensions. While the definitive mechanism for this type of protection is still under investigation, this concept has been shown previously. Previous work has demonstrated that the addition of reagents targeted to specific storage lesions of platelets enhances the recovery following cryopreservation compared to conventionally cryopreserved platelets (5). More importantly, the biochemical stabilization reduces the Me 2SO cryoprotectant requirement for cryopreservation of platelets threefold. Together with the results presented here, this concept may establish a general protection scheme for the cryopreservation of other cell types by substituting nonspecific cryoprotection with noncryoprotective, cell specific stabilizing reagents. The results presented in this study yield two distinct concepts. The first is that the packing effect associated with freezing RBC is a function of both the hematocrit and the degree of protection. This concept allows for a distinction to be made between freeze/thaw lysis and biochemical damage. Furthermore, it questions the relevance of the “packing” effect with respect to conditions which provide proper protection during cryopreservation. The second concept is that stabilizing RBC with specific biochemical stabilization agents reduces the need for nonspecific cryoprotection with glycerol. Classic theories on the effects of cryopreservation place the majority of emphasis on ice formation, mechanical cellular damage, and extracellular solute concentration. Whereas these events do occur, we show that there exists a significant second biological component to cryopreservation-induced damage. Heretofore, there have been no conditions used to cryopreserve RBC for which increasing the density of the cells has had a beneficial effect on recovery. Ultimately, this work (1) indicates that cryopreservation-induced cell damage results in part

RBC STABILIZATION REDUCES CELL DENSITY EFFECT

from RBC instability which can be reversed by specific stabilization strategies, (2) demonstrates the ability of noncryoprotective stabilization treatment to enhance cell recovery following freezing, and (3) shows that biochemical stabilization can negate the effects of high cell densities. REFERENCES 1. Ambrus, J. L., Reddington, T. M., Meky, N. N., and Conway, J. “Stiff red cell syndrome”: A review of the treatment of sickle cell disease with pentoxifylline. J. Med. 24, 1–9 (1993). 2. Beresewicz, A., and Karwatowska-Prokopczuk, E. Erythrocyte membrane stabilization by calcium channel blockers, calmodulin antagonists and scavengers of oxygen free radicals. Pol. J. Pharmacol. Pharm. 42, 355–364 (1990). 3. Brown, B. A. “Hematology: Principles and Procedures,” 6th ed. Lea & Febiger, Philadelphia, 1993. 4. Cryer, B., and Feldman, M. Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am. J. Med. 104, 413– 421 (1998). 5. Currie, L. M., Livesey, S. A., Harper, J. R., and Connor, J. Cryopreservation of single-donor platelets with a reduced dimethyl sulfoxide concentration by the addition of second-messenger effectors: Enhanced retention of in vitro functional activity. Transfusion 38, 160 –167 (1998). 6. Dalgliesh, R. J. Effect of an interaction between haematocrit and cryoprotectant concentration on freeze–thaw haemolysis of bovine erythrocytes. Cryobiology 13, 254 –257 (1976). 7. De Loecker, W., Koptelov, V. A., Grischenko, V. I., and De Locker, P. Effects of cell concentration on viability and metabolic activity during cryopreservation. Cryobiology 37, 103–109 (1998). 8. Esteves, S. C., Sharma, R. K., Thomas, A. J., Jr., and Agarwal, A. Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate. Hum. Reprod. 13, 3384 –3389 (1998). 9. Grasselli, S., Guerciolini, R., Iadevaia, V., Parise, P., Gresele, P., and Nenci, G. G. In vitro and ex vivo effects of indobufen on red blood cell deformability. Eur. J. Clin. Pharmacol. 32, 207–210 (1987). 10. Greenwalt, T. J., Rugg, N., and Dumaswala, U. J. The effect of hypotonicity, glutamine, and glycine on red cell preservation. Transfusion 37, 269 –276 (1997). 11. Klein, H. G. “Standards for Blood Banks and Transfusion Services,” 17th ed. Am. Assoc. Blood Banks, Bethesda, MD, 1996. 12. Kobayashi, T., and Levine, L. Arachidonic acid metabolism by erythrocytes. J. Biol. Chem. 258, 9116 – 9121 (1983).

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13. Lovelock, J. E. The haemolysis of human red bloodcells by freezing and thawing. Biochim. Biophys. Acta 10, 414 – 426 (1953). 14. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim. Biophys. Acta 11, 28 –36 (1953). 15. Mazur, P., and Cole, K. W. Influence of cell concentration on the contribution of unfrozen fraction and salt concentration to the survival of slowly frozen human erythrocytes. Cryobiology 22, 509 –536 (1985). 16. Mazur, P., and Cole, K. W. Roles of unfrozen fraction, salt concentration, and changes in cell volume in the survival of frozen human erythrocytes. Cryobiology 26, 1–29 (1989). 17. Mazur, P., Rall, W. F., and Rigopoulos, N. Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes. Biophys. J. 36, 653– 675 (1981). 18. Mazur, P., and Rigopoulos, N. Contributions of unfrozen fraction and of salt concentration to the survival of slowly frozen human erythrocytes: Influence of warming rate. Cryobiology 20, 274 –289 (1983). 19. Mazur, P., Rigopoulos, N., and Cole, K. W. Contribution of unfrozen fraction and of salt concentration to the survival of slowly frozen human erythrocytes: Influence of cell concentration. Cryobiology 19, 679 (1982). 20. Meryman, H. T., and Hornblower, M. A method for freezing and washing red blood cells using a high glycerol concentration. Transfusion 12, 145–156 (1972). 21. Micheli, V., Simmonds, H. A., Sestini, S., and Ricci, C. Importance of nicotinamide as an NAD precursor in the human erythrocyte. Arch. Biochem. Biophys. 283, 40 – 45 (1990). 22. Nei, T. Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures. I. Microscopic observation of hemolyzing erythrocytes during the freezing and thawing process. Cryobiology 4, 153– 156 (1967). 23. Nei, T. Mechanisms of hemolysis of erythrocytes by freezing at near-zero temperatures. II. Investigations of factors affecting hemolysis by freezing. Cryobiology 4, 303–308 (1968). 24. Nei, T. Mechanism of haemolysis of erythrocytes by freezing. In “The Frozen Cell” (G. E. W. Wolstenholme and M. O’Connor, Eds.), pp. 131–142. Churchill, London, 1970. 25. Nei, T. Mechanism of freezing injury to erythrocytes: Effect of initial cell concentration on the post-thaw hemolysis. Cryobiology 18, 229 –237 (1981). 26. Oonishi, T., Sakashita, K., and Uyesaka, N. Regulation of red blood cell filterability by Ca 2⫹ influx and cAMP-mediated signaling pathways. Am. J. Physiol. 273, C1828 –C1834 (1997).

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27. Pegg, D. E. The effect of cell concentration on the recovery of human erythrocytes after freezing and thawing in the presence of glycerol. Cryobiology 18, 221–228 (1981). 28. Pegg, D. E., and Diaper, M. P. The packing effect in erythrocyte freezing. Cryo-Letters 4, 129 –136 (1983). 29. Pegg, D. E., and Diaper, M. P. On the mechanism of injury to slowly frozen erythrocytes. Biophys. J. 54, 471– 488 (1988). 30. Pegg, D. E., and Diaper, M. P. The “unfrozen fraction” hypothesis of freezing injury to human erythrocytes: A critical examination of the evidence. Cryobiology 26, 30 – 43 (1989). 31. Pegg, D. E., and Diaper, M. P. The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride. Cryobiology 28, 18 –35 (1991). 32. Pegg, D. E., Diaper, M. P., Skaer, H. L., and Hunt, C. J. The effect of cooling rate and warming rate on the packing effect in human erythrocytes frozen and thawed in the presence of 2 M glycerol. Cryobiology 21, 491–502 (1984). 33. Rajeswari, P., Natarajan, R., Nadler, J. L., Kumar, D., and Kalra, V. K. Glucose induces lipid peroxidation

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