Storage with ethanol attenuates the red blood cell storage lesion

Abstract

Background:

Current management of hemorrhagic shock relies on control of surgical bleeding along with resuscitation with packed red blood cells and plasma in a 1-to-1 ratio. Transfusion, however, is not without consequence as red blood cells develop a series of biochemical and physical changes during storage termed “the red blood cell storage lesion.” Previous data has suggested that ethanol may stabilize the red blood cell membrane, resulting in improved deformability. We hypothesized that storage of packed red blood cells with ethanol would alter the red blood cell storage lesion.

Methods:

Mice underwent donation and storage of red blood cells with standard storage conditions in AS-3 alone or ethanol at concentrations of 0.07%, 0.14%, and 0.28%. The red blood cell storage lesion parameters of microvesicles, Band-3, free hemoglobin, annexin V, and erythrocyte osmotic fragility were measured and compared. In additional experiments, the mice underwent hemorrhage and resuscitation with stored packed red blood cells to further evaluate the in vivo inflammatory impact.

Results:

Red blood cells stored with ethanol demonstrated decreased microvesicle accumulation and Band-3 levels. There were no differences in phosphatidylserine or cell-free hemoglobin levels. After hemorrhage and resuscitation with packed red blood cells stored with 0.07% ethanol, mice demonstrated decreased serum levels of interleukin-6, macrophage inflammatory protein-1α, keratinocyte chemokine, and tumor necrosis factor α compared to those mice receiving packed red blood cells stored with additive solution-3.

Conclusion:

Storage of murine red blood cells with low-dose ethanol results in decreased red blood cell storage lesion severity. Resuscitation with packed red blood cells stored with 0.07% ethanol also resulted in a decreased systemic inflammatory response in a murine model of hemorrhage.

Introduction

Current management of hemorrhagic shock relies on correction of surgical bleeding along with resuscitation with packed red blood cells (pRBCs) and blood product component therapy in a 1-to-1 ratio.^1–4 Large volume pRBC transfusion therapy is not without consequence and has been associated with harm to the recipient.^5–10 This harm, at least in part, is secondary to the accumulation of biochemical and physical changes that occur during storage and has been termed “the RBC storage lesion.”^6,11

The major causes of the RBC lesion can be linked to oxidative stress and metabolic dysfunction that occur during storage. This stress and dysfunction is then connected to organismal and physiologic effects that, in turn, result in clinical and disease-related outcomes.^5,6 Transfusion with aged pRBCs has been linked to multiple organ system failure, increased risk of thromboembolic events, failure to wean from renal replacement therapy, and increased risk of mortality.^{5,6,8–10,12} A better understanding of the RBC storage lesion and its clinical consequences allows for possible interventions during storage to decrease recipient harm.

Ethanol (EtOH) has been noted to have a positive impact on the deformability and filterability of RBCs with short-term exposure to low physiologically achievable concentrations^.13,14 Concentrations of EtOH as low as 0.02% may have a positive impact on RBCs without having negative implications on the morphology^.13,15 However, at EtOH concentrations of ≥0.5%, the cells begin to demonstrate reduced hemoglobin and hematocrit as well as increased hemolysis.^13–15

In the present study, we sought to determine the effect of storage of pRBCs with EtOH on the RBC storage lesion. We hypothesized that the addition of EtOH to pRBCs at the beginning of the storage period would lead to diminished aspects of the RBC storage lesion.

Methods

Murine animal model

The C57BL/6 8-week-old male mice (Jackson Laboratory, Bar Harbor, ME) were housed in a climate-controlled room with a 12-hour light-dark cycle and fed a standard laboratory pellet diet and provided water ad libitum for 1 week before use for experiments. The experiments were approved by the University of Cincinnati’s Institutional Animal Care and Use Committee. Male mice were used in these experiments to minimize effects of the estrous cycle on donor mice characteristics.

Murine blood banking

Murine blood banking was performed using our previously published protocol.¹⁶ The mice were anesthetized with an intraperitoneal mixture of 100 mg/kg ketamine and 16 mg/kg xylazine, and then underwent blood draw via cardiac puncture. Their blood was collected in microcentrifuge tubes with the addition of citrate phosphate double dextrose in a 1:7 ratio by volume. To prepare the pRBCs, the blood was centrifuged at 3,000 rpm for 15 minutes followed by resuspension of the erythrocytes in additive solution-3 (AS-3) storage solution in a 2:9 ratio. In additional experiments, EtOH was added to the AS-3 in pRBC units to obtain an EtOH concentration of 0.07%, 0.14%, or 0.28%. The pRBCs in AS-3 alone were used as controls. The pRBCs were then stored at 4°C for ≤14 days. We previously determined this to be similar to 42 days of pRBC storage in human units.¹⁶

RBC storage lesion

Murine pRBCs were analyzed at day 7 and day 14 of storage for aspects of the RBC storage lesion. Microvesicle accumulation, Band3 membrane protein expression, and phosphatidylserine expression were determined using flow cytometry with protein specific antibodies for TER-119 (erythrocyte specificity), eosin-5-malemeide (for Band-3 binding), and annexin V (for phosphatidylserine binding), respectively, as previously described.¹⁷ The microvesicles were isolated by centrifugation of stored pRBCs at 2,000 g for 10 minutes, collection of supernatant and then centrifugation at 10,000 g for 10 minutes, and subsequent collection of supernatant and centrifugation at 21,000 g for 35 minutes to pellet the microvesicles. The final supernatant was used to evaluate oxidative stress via the measurement of advanced oxidative protein products (AOPP) via colorimetric assay (AOPP Assay; Cell Biolabs, Inc, San Diego, CA). The RBC count and hemoglobin levels were determined using the AcT diff Hematology Analyzer (Beckman Coulter, Brea, CA). Potassium levels were determined using I-Stat (Abbott Laboratories, Princeton, NJ). The final supernatant collected from the 21,000 g spin was also used to measure cell-free hemoglobin. The hemoglobin concentration was determined by colorimetric assay (Hemoglobin Colorimetric Assay Kit; Biovision, Milpitas, CA).

Osmotic fragility

Susceptibility of the RBCs to osmotic stress was evaluated by suspending 3 μL of erythrocytes in 200 μL of a solution containing increasing concentrations of sodium chloride (0, 0.32%, 0.44%, 0.56%, 0.68%, and 0.8% NaCl) for 30 minutes. After this, the samples were centrifuged at 10,000 g for 10 minutes with analysis of the supernatant absorbance via spectrophotometer (BioTek Cytation 5, Winooski, VT). The hemolytic increment was calculated and the EC50 was determined by the hemolytic increment of each sample when suspended in 0.56% NaCl solution.

Calculation of percentage of hemolysis

The percentage of hemolysis in RBC units was calculated using the following equation ¹⁸:

$$\left(100-Hct\right)\times cellfreehemoglobin\left(gd{L}^{-1}\right)/TotalHemoglobin\left(gd{L}^{-1}\right)$$

Morphology

Red blood cell morphology was determined by evaluating RBC smears. The size and complexity of the cells were also evaluated using flow cytometry. A total of 3 μL pRBC at 14 days of storage was suspended in 500 μL of 1X phosphate-buffered saline (PBS) for analysis. The median fluorescent intensity for forward scatter was used to assess size, and the median fluorescent intensity for side scatter was used to evaluate complexity.

Endothelial cell model

Primary murine lung microvascular endothelial cells isolated from pathogen-free C57BL/6 mice were purchased from Cell Biologics (Chicago, IL). The cells were grown to confluent monolayers in cell media supplemented with 5% fetal bovine serum. Cultured cells were treated with fresh pRBCs, 14-day aged pRBCs, or 14-day aged pRBCs stored with 0.07% EtOH in a 1:2 ratio with culture medium or with culture medium alone as a negative control. Tumor necrosis factor α (TNF-α; 20 ng/mL) and LPS (lipopolysaccharide; 1 μg/mL) were used as positive controls for experimental validation. The cells were incubated for 24 hours at 37°C, and then collected and centrifuged at 8,000 rpm for 15 minutes to pellet RBCs. Supernatants were stored at −80°C until analysis by ELISA per manufacturer protocols (R & D Systems, Minneapolis, MN).

Murine model of hemorrhagic shock

Hemorrhage and resuscitation were carried out as previously described.^19,20 Briefly, the male C57BL/6 mice were anesthetized, and the femoral artery was cannulated with a tapered polyethylene catheter for continuous mean arterial pressure (MAP) monitoring (AD Instruments Lab Chart, Colorado Springs, CO). To avoid hypothermia, the cannulated mice were placed on a circulating water blanket maintained at 41°C. After 10 minutes of equilibration, blood was withdrawn to achieve a MAP of 25 ± 5 mm Hg for a total of 60 minutes. After hemorrhagic shock, mice were resuscitated with fresh (day 0) pRBCs, pRBCs stored for 14 days, or pRBCs with 0.07% EtOH stored for 14 days, until a MAP of 70 ± 5 mm Hg was achieved. The sham animals underwent induction of anesthesia, placement of femoral artery cannula, and hemodynamic monitoring for 90 minutes without hemorrhage or resuscitation.

Serum cytokine analysis

Four hours after completion of resuscitation from induced hemorrhagic shock, the mice were euthanized, and their blood was obtained via cardiac puncture. The blood was centrifuged at 8,000 rpm for 10 minutes and the serum was collected, then stored at −80°C until needed for analysis. The samples were analyzed for cytokines using flow cytometry-based cytometric bead array assays (BD Biosciences, San Jose, CA) as noted in the results.

RBC survival after transfusion

A total of 10 mmol/L 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE; Thermo Fischer Scientific, Waltham, MA) was added to 1X PBS to generate a 20 mM solution. The CFSE:1X PBS mixture was added to the pRBCs in a 1:1 ratio and incubated for 15 minutes in a 37°C water bath. The CFSE labeled erythrocytes were washed with 10 mL 0.1% bovine serum albumin in PBS and centrifuged at 400 g for 10 minutes to bind and wash off the excess CFSE. The wash was repeated a total of 3 times. The RBCs were then suspended in 1X PBS to normalize RBC count among groups and confirmed by the AcT diff Hematology Analyzer. The CFSE labeled cells were evaluated by flow cytometry to ensure >95% labeling. Mouse penile vein injection was used to transfer 100 μL of CFSE labeled RBCs into the recipient mice. The recipient mice then had blood samples collected at 15 minutes and 24 hours post-transfusion via submandibular bleed. The samples were then incubated with PE Ter-119 to label the erythrocytes, and flow cytometry was used to quantify the remaining transfused RBCs. The half-life of the RBCs in circulation was calculated using the following equation²¹:

$$T_{1/2}=T_t\times ln\left(2\right)/\left[\left(ln\right)N_0-\left(ln\right)N_t\right]$$

where ${\mathrm{T}}_{1/2}$ is the estimated half-life, ${\mathrm{T}}_{\mathrm{t}}$ is the time between transfusion and blood draw, ${\mathrm{N}}_0$ is the initial quantity of circulating erythrocytes, and ${\mathrm{N}}_{\mathrm{t}}$ is the number of viable erythrocytes after blood draw.

Statistical analysis

All data are presented as mean ± SD as noted. Two-tailed Student’s t test was used to compare the groups.

Results

Murine RBC storage lesion

Red blood cell derived microvesicle accumulation during storage is a known mediator of the acute inflammatory response system, vascular dysfunction, as well as coagulation.²² Microvesicles have been associated with acute lung inflammation and thromboembolic events post-transfusion.^9,23–28 There was no difference in microvesicle accumulation at the beginning of the storage period (data not shown). By the end of storage at day 14, each treatment group (0.07%, 0.14%, and 0.28% EtOH in AS-3) demonstrated decreased microvesicle accumulation compared to pRBCs stored with AS-3 alone (Figure 1, A). Treatment with 0.07% EtOH demonstrated decreased microvesicle accumulation compared to 0.14% EtOH, but comparable levels to pRBCs stored with 0.28% EtOH.

Figure 1.

Aspects of the red blood cell (RBC) storage lesion in 14-day-old packed RBC units stored with additive solution-3 compared to 14-day-old packed RBC units stored with ethanol (concentrations 0.07%, 0.14%, and 0.28%). (A) Microvesicles (n = 6), (B) Band-3 (n = 6), (C) phosphatidylserine (n = 6), (E) activated oxidation protein products (n = 6). AOPP, advanced oxidation protein products; AS-3, additive solution-3; EtOH, ethanol. *P < .05 vs 14-day-old packed red blood cells at the same time point.

To further evaluate the effects of EtOH on the RBC storage lesion, we evaluated Band-3 expression, phosphatidylserine (PS) externalization, and AOPP. Band-3 is in the RBC plasma membrane and functions as a major anion exchanger critical for CO₂ uptake in addition to playing an important structural role relating to deformability. Band-3 expression decreases with the age of the RBCs and stimulates the senescence pathway.^29–31 At day 14 of storage, all EtOH treatment groups demonstrated higher expression of Band-3 compared to standard storage with AS-3 (Figure 1, B).

Phosphatidylserine is a phospholipid located in the inner leaflet of the RBC membrane and becomes externalized as the cells age. External expression of PS serves as a signal for RBC removal or eryptosis. There was no difference in PS expression of pRBC treated with EtOH compared to AS-3 (Figure 1, C).

Advanced oxidation protein products are toxins generated during oxidative stress. Oxidative stress damages the RBC membrane, which decreases deformability and results in increased macrophage uptake of RBCs. Storage of pRBCs with EtOH resulted in decreased AOPP compared to AS-3 alone regardless of EtOH concentration (Figure 1, D).

The RBC count was decreased in units stored with higher EtOH concentrations (0.14% and 0.28%), but there was no difference in RBC count between the pRBCs stored with AS-3 and pRBCs stored with 0.07% EtOH (Figure 2, A). The hemoglobin level was also comparable between the pRBCs stored with AS-3 and pRBCs with EtOH 0.07% (Figure 2, B). Free potassium in the storage solution was significantly decreased at all EtOH concentrations compared to AS-3 alone. There were no differences in the potassium levels between EtOH concentrations (Figure 2, C). Based on the above results (Figures 1 and 2), 0.07% EtOH was used in the experiments moving forward as it was the lowest concentration that demonstrated decreased components of the RBC storage lesion.

Figure 2.

Properties of 14-day-old packed red blood cell units stored with additive solution-3 compared to storage with additive solution-3 with the addition of ethanol (concentrations 0.07%, 0.14%, and 0.28%). (A) Red blood cell count (n =4), (B) hemoglobin (n = 4), (C) potassium (n = 3). AS-3, additive solution-3; EtOH, ethanol; RBC, red blood cell. *P < .05 vs 14-day-old packed red blood cells at the same time point.

Cell-free hemoglobin accumulates during RBC storage and is another important aspect of the RBC storage lesion. After transfusion, cell-free hemoglobin can result in nitric oxide depletion as well as generation of reactive oxygen species, both of which can have detrimental effects on the vascular endothelium.^32,33 There was no difference in cell-free hemoglobin at day 7 or day 14 of storage between RBCs in standard storage with AS-3 and RBCs stored with 0.07% EtOH (Figure 3, A). The percent hemolysis, however, was increased in the 0.07% EtOH group compared to AS-3 alone.

Figure 3.

Aspects of the red blood cell (RBC) storage lesion in packed RBC units stored in additive solution-3 or additive solution-3 with the addition of 0.07% ethanol. (A) Cell-free hemoglobin in the supernatant of stored units at days 7 and 14 (n = 4); (B) percent hemolysis (n = 4); (C) osmotic fragility at day 14 of storage (n = 3); (D) EC50 of 14-day stored packed RBCs (n =3). AS-3, additive solution-3; EtOH, ethanol. *P < .05 vs 14-day-old packed red blood cells at the same time point.

Osmotic fragility correlates to membrane vulnerability. At 7 days of storage there was no difference in osmotic fragility between storage with 0.07% EtOH and AS-3 alone (data not shown). At day 14 of storage, AS-3 alone demonstrated higher resistance to osmotic stress compared to pRBCs with 0.07% EtOH (Figure 3, C). However, the EC50 was comparable between the 2 groups (Figure 3, D).

To evaluate the morphology, RBC smears were performed. Red blood cells stored with AS-3 alone demonstrated numerous schistocytes and acanthocytes compared with both fresh pRBCs and pRBCs stored with 0.07% EtOH (Figure 4, A–C). The cells stored with 0.07% EtOH appeared slightly smaller with a relative loss of biconcavity compared to fresh pRBCS (Figure 4, A–C). Flow cytometry was used to determine both the size and complexity of the packed RBCs during storage. The size is represented by forward scatter and complexity by side scatter. Both the size and complexity were decreased in RBCs that were stored with 0.07% EtOH when compared to storage with AS-3 alone (Figure 4, D).

Figure 4.

Morphology of red blood cell (RBC) evaluated by RBC smears and forward scatter (D) and side scatter (E) on flow cytometry. (A) Fresh packed RBCs (pRBCs); (B) 14-day pRBCs in additive solution-3; (C) 14-day pRBCs in additive solution-3 + 0.07% ethanol. n = 5. AS-3, additive solution-3; EtOH, ethanol. *P < .05.

In vitro analysis

To determine the effect of pRBC storage with EtOH on endothelial cell cytokine release, we cultured microvascular endothelial cells and then treated them with fresh pRBCs,14-day aged pRBCs in AS-3, or 14-day aged pRBCs treated with 0.07% EtOH. The proinflammatory cytokine, interleukin-6 (IL-6), was decreased in the EtOH group compared to 14-day aged pRBCs but was higher than in fresh pRBCs in AS-3 (Figure 5, A). Vascular cell adhesion molecule 1 and E-selectin (Figure 5, B–C) demonstrated decreased levels in pRBCs stored with EtOH compared with both AS-3 stored fresh pRBCs and 14-day pRBCs.

Figure 5.

Inflammatory mediator levels, including (A) interleukin-6, (B) vascular cell adhesion molecule 1, and (C) E-selectin in cell culture media after incubation of murine lung endothelial cells for 24 hours with murine fresh packed red blood cells (pRBCs; fresh), 14-day-old pRBCs in additive solution-3 (14-day), or 14-day-old pRBCs stored with additive solution-3 with the addition of 0.07% ethanol. Media was used as a control. n = 5. EtOH, ethanol. *P < .05 versus media, fresh packed red blood cells, or 14-day-old packed red blood cells as indicated.

One potential explanation for these findings is that treatment with 0.07% EtOH directly blunts the inflammatory response. To determine the direct effect of 0.07% EtOH on the endotoxin-induced inflammatory response, we treated cultured murine lung endothelial cells with media, media + 0.07% EtOH, LPS (1mg/mL), or LPS (1mg/mL) with the addition of 0.07% EtOH. After 24 hours, the cell supernatant was assayed for IL-6 and VCA protein 1. Under these conditions, the EtOH did not demonstrate any blunting of LPS-induced IL-6 or VCA on molecule production (data not shown).

Hemorrhagic shock and resuscitation

We next examined the effect of storage with EtOH on resuscitation in a murine pressure-controlled model of hemorrhage. There were no differences in the volume needed for resuscitation between the treatment groups (Supplementary Figure 1). There was also no difference in the starting mean arterial pressures or post-hemorrhage pressures. During initial resuscitation, the mice resuscitated with fresh pRBCs had a lower MAP than those resuscitated with either 14-day aged pRBCs in AS-3 alone or 14-day pRBCs aged with 0.07% EtOH. However, by the end of the resuscitation period there were no differences in MAP between resuscitation strategies (Supplementary Figure 2).

We analyzed serum cytokines 4 hours after hemorrhage and resuscitation. Resuscitation with aged pRBCs compared to fresh pRBCs was associated with increased serum TNF-α, IL-6, IL-10, macrophage inflammatory protein-1α, and keratinocyte chemokine (Figure 6, A–E). Resuscitation with pRBCs stored with 0.07% EtOH did not result in a worsened inflammatory response. IL-6, MIP-1α, and KC were decreased in mice resuscitated with pRBCs stored with EtOH when compared with those resuscitated with pRBCs stored in AS-3 alone (Figure 6, B, D, E). TNF-α and IL-10 demonstrated comparable levels (Figure 6, A, C). MIP-1β, IL-1β, granulocyte cell stimulating factor, and monocyte chemoattractant protein-1 were also evaluated and demonstrated no differences between resuscitation strategies (data not shown).

Figure 6.

Serum chemokines in mice after hemorrhage followed by resuscitation with fresh packed red blood cells (pRBCs, fresh), 14-day-old pRBCs stored in additive solution-3 (14-day), or 14-day-old pRBCs stored in AS-3 with the addition of 0.07% ethanol. The results are presented for (A) tumor necrosis factor-α; (B) interleukin-6; (C) interleukin-10; (D) macrophage inflammatory protein-α; and (E) keratinocyte chemokine. n = 5. IL, interleukin; KC, keratinocyte chemokine; MIP-α, macrophage inflammatory protein-α; TNF-α, tumor necrosis factor-α. *P < .05 versus sham, fresh packed red blood cells, or 14-day packed red blood cells as indicated.

Post-transfusion RBC recovery

In the final series of experiments, the post-transfusion recovery of erythrocytes after 14 days of storage was evaluated. All experimental units demonstrated >96% CFSE-staining efficiency. We evaluated the percentage of remaining RBCs in circulation after transfusion. The half-life of fresh pRBCs stored in AS-3 solution (T1/2 = 9.9 ± 0.4 hours) was used as a control. Post-transfusion circulation recovery was significantly blunted after 14-day storage in AS-3. However, storage with 0.07% EtOH resulted in prolonged RBC circulation compared to AS-3 alone (5.2 ± 0.3 and 7.1 ± 1.6 hours, respectively; Figure 7).

Figure 7.

Post-transfusion survival of erythrocytes from packed red blood cell units stored with additive solution-3 (AS-3) for 0 days (fresh), 14 days (AS-3), or for 14 days in AS-3 with the addition of ethanol. n = 4. AS-3, additive solution-3; EtOH, ethanol. *Indicates P < .05 by Student’s t test compared to indicated group.

Discussion

In the present study, our data demonstrated that pRBC storage with the addition of EtOH to AS-3 decreases the severity of aspects of the RBC storage lesion. We found decreased accumulation of microvesicles, increased levels of Band-3, and decreased accumulation of advance oxidative proteins during storage. This was associated with a decreased inflammatory response in cultured murine lung endothelial cells as well as in vivo after resuscitation from hemorrhage. The post-transfusion survival of transfused RBCs was also prolonged.

It has been suggested that EtOH permeates into cell membranes, causing lipid disordering and membrane stabilization,¹⁵ which in turn may decrease microvesicle formation. The Band-3 protein also plays an important structural role in the RBC membrane and naturally decreases with RBC aging. The decrease in microvesicle accumulation, as well as increased levels of Band-3 in pRBCs stored with EtOH, supports this theory of EtOH-induced membrane stabilization. Band-3 accumulation also contributes to deformability because of membrane stabilization. The increased levels of Band-3 seen in our experiments were consistent with prior data that suggested, at low concentrations, EtOH increases membrane deformability.^13,14

Oxidative damage has been identified as a major contributing factor to the RBC storage lesion. In fact, the treatment of erythrocytes with natural antioxidants has been shown to prolong RBC survival and reduce phosphatidylserine exposure.³⁴ In part, our data supported this mechanism in that we demonstrated decreased advanced oxidative protein accumulation with EtOH storage; however, we demonstrated similar levels of phosphatidylserine exposure. It is unclear why there may be a decoupling between AOPP accumulation and phosphatidylserine externalization, but this is worthy of additional study.

Red blood cells stored with 0.07% EtOH demonstrated a slightly decreased resistance to osmotic stress during storage. However, the EC50 was comparable between the pRBCs stored with AS-3 and pRBCs stored with EtOH. There was also no difference in cell-free hemoglobin. Cell-free hemoglobin increases with cell turnover and hemolysis. There was, however, a statistically significant increase in percent hemolysis among the RBCs stored with EtOH (0.91 ± 0.1 vs 0.6 ± 0.5), but neither storage condition exceeded the standard set by the Food and Drug Administration of <1% hemolysis at the end of the storage period for pRBCs.³⁵ The percent hemolysis among standard storage pRBCs is comparable to previous results from our laboratory.¹⁸ Taken together, these data suggested that the addition of EtOH to storage solutions may have a slightly detrimental effect on erythrocyte membrane fragility.

We used a murine lung endothelial model and a murine model of hemorrhage and resuscitation to evaluate any in vitro and in vivo implications of transfusion RBCs stored with EtOH. We demonstrated that both in the in vivo and in vitro model, pRBCs stored with EtOH decreased the inflammatory response compared to that seen with standard-storage pRBCs. The effects of EtOH on the immune system and inflammatory response are well documented and include a dose-dependent mechanism with acute use attenuating the inflammatory response.³⁶ Alcohol has been shown to alter many steps involved in neutrophil migration, including alteration of adhesion molecule expression, neutrophil adhesion, and proinflammatory cytokine production.^37–39 The changes seen in the inflammatory response may be 2-fold, with decreased inflammation secondary to the decreased RBC storage lesion, as well as the direct effects of EtOH. However, with the low dose used in our experiment combined with the dilutional effect when introduced into circulation, the inflammatory response seen is likely secondary to the decreased RBC storage lesion as opposed to the direct effects of EtOH itself.

The feasibility of the clinical use of pRBCs with EtOH is challenging given the negative implications of EtOH, including intoxication or even alcohol poisoning. However, we believe this to be unlikely given that the concentrations used in this experiment were low, and dilutional effects on transfusion would lead to even lower circulating EtOH levels. For example, if we take a standard 70 kg man with a blood volume of 5.5 L, a transfusion of 1 unit of pRBCs with 0.07% EtOH (assuming a 300 mL volume of pRBCs) would lead to a blood alcohol level of 0.003%. Massive transfusion with 10 units of pRBC would lead to a blood alcohol level of 0.04%. Additionally, if the transfusion used a balanced resuscitation with fresh frozen plasma, these values would be even lower and would be unlikely to be clinically significant.

Although our study demonstrated interesting findings, there were limitations to be considered. Although we found that the concentration of 0.07% EtOH was effective in mitigating aspects of the RBC storage lesion, we did not study the effect of lesser concentrations in this series of experiments, and it was possible that these concentrations may have been effective and therefore limited potential side effects. Future studies will be needed to thoroughly determine the effect of lesser EtOH concentrations during storage. Also, we used flow cytometry to evaluate microvesicles in stored pRBC units as we have described previously,^18,21,23,28 but the evaluation of microvesicles by this technique may be limited due to the size of these particles. Microvesicles ranges from 100 nm to 1,000 nm, and the lower limit of resolution for flow cytometry is around 488 nm. As such, our data can only be interpreted related to microvesicles >500 nm in size. In addition, these experiments were performed using murine models to minimize the impact of genetic variability, which is a known confounder of the RBC storage lesion.^23,40,41 Although we have previously demonstrated that many aspects of the RBC storage lesion in murine and human pRBCs are similar,¹⁶ this may have limited the impact of our findings.

In conclusion, our data indicated decreased aspects of the RBC storage lesion and decreased inflammatory response in an in vitro and in vivo model after exposure to pRBCs stored with EtOH. The storage of pRBCs with low concentration of EtOH may represent a strategy to decrease the RBC storage lesion.