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Published in final edited form as: Surgery. 2021 Dec 30;171(3):833–842. doi: 10.1016/j.surg.2021.11.020

Improving Packed Red Blood Cell Storage with a High Viscosity Buffered Storage Solution

Kasiemobi E Pulliam 1,‡, Bernadin Joseph 1,‡, Amy T Makley 1, Charles C Caldwell 1, Alex B Lentsch 1, Michael D Goodman 1, Timothy A Pritts 1
PMCID: PMC8887606  NIHMSID: NIHMS1762083  PMID: 34974917

Abstract

Background:

Massive transfusion with older packed red blood cells (pRBCs) is associated with increased morbidity and mortality. As pRBCS age, they undergo biochemical and structural changes known as the storage lesion. We developed a novel solution to increase viscosity in stored pRBCs. We hypothesized that pRBC storage in this solution would blunt storage lesion formation and mitigate the inflammatory response following resuscitation.

Methods:

Blood was obtained from 8–10 week old C57BL/6 male donor mice or human volunteers and stored as pRBC units for 14 days for mice or 42 days for humans in either standard AS-3 storage solution or EAS-1587, the novel pRBC storage solution. pRBCS were analyzed for microvesicles, cell-free hemoglobin, phosphatidylserine, band-3 protein, glucose utilization, and osmotic fragility. Additional mice underwent hemorrhage and resuscitation with pRBCs stored in either AS-3 or EAS-1587. Serum was analyzed for inflammatory markers.

Results:

Murine pRBCs stored in EAS-1587 demonstrated reductions in microvesicle and cell-free hemoglobin accumulation as well as preserved band-3 expression, increase glucose utilization, reductions in phosphatidylserine expression and susceptibility to osmotic stress. Serum from mice resuscitated with pRBCs stored in EAS-1587 demonstrated reduced pro-inflammatory cytokines. Human pRBCs demonstrated a reduction in microvesicle and cell-free hemoglobin as well as an increase in glucose utilization.

Conclusions:

Storage of pRBCs in a novel storage solution mitigated many aspects of the red blood cell storage lesion as well as the inflammatory response to resuscitation following hemorrhage. This modified storage solution may lead to improvement of pRBC storage and reduce harm after massive transfusion.

Two Sentence Article Summary

In the present study, the authors demonstrate that a novel red blood cell storage solution leads to decreased storage lesion formation. This modified storage solution may lead to improvement of pRBC storage and reduce harm after massive transfusion.

Introduction

Hemorrhage remains a leading cause of early and potentially preventable death in patients following traumatic injury1. Resuscitation strategies such as early implementation of a massive transfusion protocol and blood product transfusions in a balanced ratio have been shown to have survival benefits. Administration of packed red blood cells (pRBCs), plasma, platelets, and other hemostatic adjuncts are utilized to treat acute post-injury anemia, volume depletion, and coagulopathy2–4. However, the use of packed red blood cells is not without risk5. During storage, pRBCs undergo a progressive series of physical and biochemical alterations collectively termed the “red blood cell storage lesion.” These changes include, but are not limited to, decreases in pH, metabolic activity, membrane deformability, and viability, as well as increases in the release of extracellular vesicles and free hemoglobin from the stored erythrocytes.6,7 The red blood cell storage lesion alters the quality and function of pRBCs and is associated with post-transfusion complications such as acute pulmonary inflammation, thromboembolic events, and increased mortality.7–10

Previously, storage solutions have been modified in order to reduce the development of the red blood cell storage lesion. We have demonstrated that altered pH affects the severity of the red blood cell storage lesion.11 Additional alterations to the composition of storage solutions have included removal of citric acid/sodium citrate and/or sodium chloride, use of mannitol, introduction of sodium bicarbonate, and use of various other additives.12–14 We recently examined the concept of salvaging packed red blood cells from previously stored whole blood 15. Our data showed that red blood cells from previously stored whole blood demonstrated attenuated aspects of the red blood cell storage lesion. As one of the key differences between the storage conditions for erythrocytes in whole blood as compared to standard storage conditions is related to the viscosity of the storage medium, we hypothesized that storage of pRBCs in a solution with increased viscosity and a more alkaline pH would lead to a reduction in the red blood cell storage lesion and a reduced inflammatory response to resuscitation following hemorrhagic shock. Our data demonstrate that this solution, termed EAS-1587 (Experimental Additive Solution 1587), has a significant effect on the onset and severity of the red blood cell storage solution.

Methods

Murine Blood Banking

Murine experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Cincinnati. Male 8–10 week old C57BL/6 mice, obtained from the Jackson Laboratory (Bar Harbor, ME), were acclimated for two weeks in a climate-controlled room with a 12 hour light/dark cycle and fed with standard pellet diet and water ad libitum. Murine blood banking was performed using a modification of our previously characterized protocol and was developed based on processing protocols utilized by our regional blood bank.16 The mice were anesthetized with intraperitoneal pentobarbital (0.1 mg/g body weight) and whole blood was obtained via terminal cardiac puncture. Packed red blood cell units were generated via centrifugation of whole blood at 1,000g for 15 minutes. The plasma was discarded and the erythrocyte pellet was resuspended in a 2:9 ratio of storage solution, as outlined in the results, under sterile conditions in a laminar flow hood, and stored for 14 days. Our laboratory has previously investigated and characterized murine blood banking and storage and determined that the 42-day storage duration of human red blood cells is similar to the 14-day storage duration of murine red blood cells.16

Human Blood Banking

Using a protocol approved by the University of Cincinnati Institutional Review Board, human blood was donated at our regional blood bank and collected into a preservation solution system (PL 2209, Fenwal, Lake Zurich, Illinois). Units were centrifuged to separate the plasma, which was discarded, from the red blood cells, which were then leukoreduced using the integrated leukoreduction filter. The erythrocytes were resuspended in storage solution, as outlined in the results, under sterile conditions in a laminar flow hood, in a 2:9 ratio, and stored for the 42-day Food and Drug Administration (FDA) approved storage duration at 4°C.

Evaluation of Red Blood Cell Storage Lesion

Several aspects of the red blood cell storage lesion were evaluated. Red blood cell derived microvesicles were isolated by centrifugation procedures as previously described.8 Briefly, pRBCs underwent centrifugation at 2,000g for 10 minutes with collection of the supernatant, further centrifugation at 10,000g for 10 minutes, with collection of the supernatant and final centrifugation at 21,100g for 35 minutes to pellet the microvesicles. Microvesicle accumulation was quantified via flow cytometric analysis (Invitrogen Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA) in mice via phycoerythrin (PE) conjugated rat, anti-mouse Ter-119 antibody (BD Biosciences San Jose, CA) binding and in humans via PE conjugated mouse anti-human CD235a (Glycophorin-A) antibody.

Band-3 erythrocyte membrane protein (Band-3) expression and phosphatidylserine externalization expression were assessed via eosin-5’-maleimide (EMA) fluorescence (ThermoFisher Scientific, Waltham, MA), and Fluorescein isothiocyanate (FITC) Annexin-V (BD Biosciences San Jose, CA) antibody binding respectively. The percentage of red blood cells expressing Band-3 and phosphatidylserine on the membrane surface were quantified via flow cytometry.

To determine cell-free hemoglobin content, a marker of erythrocyte lysis, pRBC samples were centrifuged at 21,100g for 35 minutes, then supernatant cell-free hemoglobin quantified via a hemoglobin colorimetric assay (BioVision Inc., Milpitas, CA) on a microplate spectrophotometer (BioTek Cytation 5, Winooski, VT).

In order to evaluate oxidative stress, a commercially available advanced oxidative protein products (AOPP) assay (Cell Biolabs Inc., San Diego, CA) was utilized, then analyzed on a microplate spectrophotometer.

Susceptibility of red blood cells to osmotic stress was determined by suspending aliquots of erythrocytes in solutions containing increasing concentrations of sodium chloride (0, 0.32, 0.44, 0.56, 0.68, and 0.8% NaCl) as previously described.9

Viscosity Assessment

The viscosity of samples was measured via a viscometer (Microvisc, Rheosense Inc., San Ramon, CA) at a shear rate of 480 s−1. The samples were assessed at varying temperatures via a temperature control chamber (Microvisc TC, Rheosense Inc., San Ramon, CA). 100μL of AS-3 alone, EAS-1587 alone, pRBCs stored in AS-3, and pRBCs stored in EAS-1587 were analyzed to determine their viscosity at 4°C and 37°C in order to determine the effect at (a) storage and (b) body temperature.

Erythrocyte Structural and Biochemical Evaluation

Blood smears were utilized to assess the stored erythrocytes for morphological changes that occurred during storage. Findings on the blood smear were confirmed via forward scatter (FSC) and side scatter (SCC) findings on flow cytometry. FSC increases with increased cell size while SCC increases with increased membrane complexity. An i-STAT handheld blood analyzer (Abbott Laboratories, Chicago, IL) was utilized to obtain blood gas, electrolyte, and hematologic information. The percentage of glucose metabolized over time was quantified by calculating a percentage change utilizing the glucose concentrations.

Murine Model of Hemorrhagic Shock

Hemorrhage and resuscitation were carried out as previously described.10 Briefly, 8–10 week old male C57BL/6 mice were anesthetized with intraperitoneal pentobarbital (0.1 mg/gram body weight) followed by groin clipping and sterile preparation with povidone-iodine solution and alcohol. The femoral artery was cannulated with a tapered polyethylene catheter. The catheter was connected to pressure transducers for continuous hemodynamic monitoring of the mice (AD Instruments Lab Chart). Hemorrhagic shock was initiated by withdrawing blood to achieve a mean arterial pressure (MAP) of 25 ± 5 mmHg and was maintained for 60 minutes. Following hemorrhagic shock, mice were resuscitated with pRBCs to achieve a MAP greater than 70 mm Hg ± 5 mm Hg. The mice were monitored for 15 minutes following resuscitation, femoral artery decannulated, and euthanized at 1-hour post procedure end. Sham animals underwent femoral artery cannulation and hemodynamic monitoring for 90 minutes, without hemorrhage or resuscitation.

Serum Cytokine Analysis

One hour after hemorrhage and resuscitation, mice were euthanized, and blood obtained via cardiac puncture in a serum separator tube (SST). After 30 minutes, samples were centrifuged at 8,000 rpm for 10 minutes in order to isolate the serum. Serum samples were analyzed for inflammatory chemokines and cytokines as described in the results utilizing a flow cytometry-based cytometric bead array assay (BD Biosciences, San Jose, CA).

Statistical Analysis

GraphPad Prism was utilized (San Diego, CA) to perform statistical analysis of data via ANOVA or Student’s t-test as noted in the results. P<0.05 was deemed statistically significant. Data is presented as mean ± standard error of the mean. The sample size noted refers to pRBC units from each individual murine or human donor.

Results

Development of a modified murine red blood cell storage solution

Hydroxy-propyl-methyl-cellulose (Hypromellose) is a biocompatible cellulose ether that is utilized in the processing and production of food, cosmetics, and pharmaceutical products.17–19 It increases the viscosity of solutions by forming a hydrophilic matrix that absorbs and retains water 19. Hypromellose is commonly utilized in nasal sprays, ophthalmic solutions, and oral tablets20 and has been safely utilized for injection in endomucosal resection of large colonic polyps21 as well as experimentally for preparation of injectable nanoparticle hydrogels,22 and rivaroxaban.23 Based on its biochemical properties and biocompatibility, we chose to utilize hypromellose as a solution constituent in order to increase the viscosity of the modified storage solution.

In preliminary experiments, hypromellose was added to additive solution 3 (AS-3), a standard solution for pRBC storage. Initial experimentation with hypromellose in murine packed red blood cells stored in AS-3 demonstrated that there was a dose dependent reduction in microvesicle release (FIGURE 1A), cell-free hemoglobin accumulation (FIGURE 1B), and advanced oxidative protein products (FIGURE 1C). There was greater retention of Band-3 erythrocyte membrane integrity protein expression in a dose dependent fashion (FIGURE 1D). There was no difference in phosphatidylserine externalization (FIGURE 1E). Taken together, these data indicate that the addition of hypromellose to AS-3 standard storage solution significantly ameliorated aspects of the red blood cell storage lesion.

Figure 1.

Figure 1.

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Murine pRBC units stored in additive solution 3 (AS-3) or with different concentrations of hypromellose (HPM) were analyzed for aspects of the red blood cell storage lesion. (A) Microvesicle concentrations; (B) cell free hemoglobin concentration; (C) advanced oxidation protein product (AOPP) accumulation; (D) Band 3; (E) phosphatidylserine surface expression; (F) Viscosity analysis of AS-3 and experimental additive solution 1587 (EAS-1857) storage solutions alone at storage (4°C) and physiologic (37°C) temperatures. N=4 per group for A-E and n=5 per group for F. *p<0.05 as compared to indicated group by t-test. mPA.s = millipascal seconds. PM, AOPP, EAS

During storage, the pH of pRBC units steadily decreases16. Studies from our and other laboratories have demonstrated that use of a more alkaline storage solution for packed red blood cells attenuates aspects of the red blood cell storage lesion.11,24 Based on these findings as well as our preliminary findings with hypromellose, we developed a modified storage solution with 2% (weight/volume) hypromellose to increase the viscosity of the solution and with increased sodium bicarbonate to create a more alkaline pH. Citric acid was not utilized in the modified storage solution in order to minimize acidic components that contribute to a lower pH as the hydrophilic matrices of hypromellose rapidly dissolve when placed in a medium with pH less than or equal to 5.8.25 The components of AS-3, a standard storage solution, as well as the buffered high-viscosity storage solution, termed experimental additive solution-1587 (EAS-1587) are presented in TABLE 1. We next determined the viscosity of the AS-3 and EAS-1587 storage solutions. This analysis showed that the EAS-1587 solution has an increased viscosity compared to AS-3 at both 4°C and 37°C (FIGURE 1F).

Table 1.

Components of AS-3 and EAS-1587

Components (mM) AS-3 EAS-1587
Citric acid 2 --
Sodium citrate 20 20.0
Sodium bicarbonate -- 12.5
Sodium phosphate 23 9.5
Dextrose 55 55
Adenine 2.22 2.22
Sodium chloride 70 50
Other parameters
Hypromellose (%) -- varies – see text
pH 5.6 8.4
Osmolarity (mOsmol/L) 326 291
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AS-3: additive solution 3; EAS-1587: experimental additive solution 1587.

EAS-1587 is associated with decreased severity of the murine red blood cell storage lesion

Previous studies from our and other laboratories have demonstrated the key role that microvesicle accumulation in pRBC units during storage plays in subsequent lung injury and increased inflammatory response after resuscitation.8,10 In order to determine the effect of pRBC storage in EAS-1587 on microvesicle accumulation, murine pRBCs were stored in either AS-3 or EAS-1587 for up to 14 days. Microvesicles were then isolated and quantified. Erythrocyte storage in EAS-1587 was associated with decreased accumulation of pRBC-derived microparticles at days 7 and 14 of storage compared to storage in AS-3 (FIGURE 2A).

Figure 2.

Figure 2.

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Effect of storage of murine pRBCs stored in additive solution 3 (AS-3) or experimental additive solution 1587 (EAS-1587) for up to 14 days. (A) Microvesicle counts as determined by flow cytometry. (B) Cell-free hemoglobin accumulation in storage solution. (C) Band-3; (D) Phosphatidylserine externalization expression; (E) Hemolysis in response to osmotic stress. N=4 per group. *p<0.05 as compared to AS-3 by t-test.

Another potentially harmful aspect of pRBC storage is the accumulation of free hemoglobin, which has been associated with end organ damage after transfusion.26 When supernatants of pRBCs stored in AS-3 were analyzed, we found increased free hemoglobin during the duration of storage (FIGURE 2B). This increase was blunted by storage in EAS-1587 (FIGURE 2B).

When we investigated additional parameters of the red blood cell storage lesion, we found that storage in EAS-1587 was associated with greater expression of the membrane protein Band-3 (FIGURE 2C), reduced phosphatidylserine externalization (FIGURE 2D), and reduced susceptibility to osmotic stress (FIGURE 2E) as compared to murine pRBCs stored in AS-3.

Red blood cell storage results in decreased glucose metabolism.27 In order to assess the effect of the novel storage solution on glucose metabolism, we determined the percentage of glucose metabolized after 14 days of storage. There was a higher percentage of glucose metabolized in pRBCs stored in EAS-1587 as compared to pRBCs stored in AS-3 (FIGURE 3A). We also found that there were no differences in intracellular hemoglobin content (FIGURE 3B) after EAS-1587 storage. There were also reduced accumulation of advanced oxidative protein products for the pRBCs stored in EAS-1587 (FIGURE 3C).

Figure 3.

Figure 3.

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Effect of storage of murine pRBCs stored in additive solution 3 (AS-3) or experimental additive solution 1587 (EAS-1587) for up to 14 days. (A) Percentage of glucose metabolized during storage period; (B) Erythrocyte (RBC) hemoglobin content; (C) Advanced oxidative protein products (AOPP). N=4 per group. *p<0.05 as compared to AS-3 group by t-test.

As pRBCs age, the membrane of the erythrocytes undergo structural changes, including loss of discocytic shape and a discocytic-to-spherocytic transformation28. Upon examination via peripheral smear, the murine RBCs stored in EAS-1587 had less spheroechinocytic membrane transformation after 14 days of storage (FIGURE 4A, B). The peripheral smear findings were confirmed with increased forward scatter and reduced side scatter complexity as determined by flow cytometry for pRBCs stored in EAS-1587 (FIGURE 4C). Evaluation of pH, sodium, potassium, ionized calcium, hemoglobin, and hematocrit demonstrated no differences in these parameters (data not shown).

Figure 4.

Figure 4.

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Murine pRBC microscopy smear comparison of murine pRBCs stored in additive solution 3 (AS-3; A) or experimental additive solution 1587 (EAS-1587; B) for up to 14 days. (C) Forward (FSC) and side (SCC) scatter evaluation of erythrocytes morphology utilizing flow cytometry. N=4 per group. *p<0.05 as compared to indicated group by t-test.

pRBC storage in EAS-1587 is associated with altered inflammatory response after hemorrhage and resuscitation in mice

Resuscitation with aged stored pRBCs after hemorrhage is associated with an increased inflammatory response.29 To determine the effect of resuscitation with pRBCs stored in EAS-1587, mice underwent hemorrhage followed by resuscitation with pRBCs stored in either AS-3 or EAS-1587 for 14 days. The recipient serum demonstrated a reduction in the pro-inflammatory cytokines TNF-α and MIP-1α (Figure 5A, B). There was an increase of the pleiotropic cytokine IL-6 when compared to resuscitation with AS-3 stored pRBCs (Figure 5C). The amount of serum-free hemoglobin was lower in mice resuscitated with pRBCs stored in EAS-1587 as compared to those resuscitated with pRBCs stored in AS-3 (Figure 5D). Taken together, these data indicate that resuscitation with pRBCs stored in EAS-1587 resulted in a decreased inflammatory response.

Figure 5.

Figure 5.

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Inflammatory markers and serum free hemoglobin from mice that underwent hemorrhagic shock and resuscitation with pRBCS stored in additive solution 3 (AS-3) or experimental additive solution 1587 (EAS-1587) for up to 14 days. (A) Tumor necrosis factor-alpha (TNF-α); (B) macrophage inflammatory protein 1-alpha/CCL3 (MIP-1α); (C) Interleukin-6 (IL-6); (D) Cell-free hemoglobin. N=6 per group. *p<0.05 as compared to indicated group by t-test.

Evaluation of a modified red blood cell storage solution in stored human pRBCs

Our data indicate that storage of murine pRBCs in EAS-1587 resulted in decreased severity of the red blood cell storage lesion as well as decreased inflammation after hemorrhage and resuscitation. We have previously demonstrated that the red blood cell storage lesion in murine and human pRBCs is similar but not identical 15,16. Initial evaluation of hypromellose in storage of human pRBCs suggested that the optimal concentration of hypromellose was 4% (data not shown). Therefore, this concentration was utilized going forward.

EAS-1587 attenuates aspects of the red blood cell storage lesion in human pRBCs

In order to determine the effect of pRBC storage in EAS-1587 on human pRBCs on the red blood cell storage lesion, human pRBCs were stored in either AS-3 or EAS-1587 for up to 42 days, the FDA limit for storage duration. Microvesicles were then isolated and quantified. Erythrocyte storage in EAS-1587 was associated with decreased accumulation of pRBC-derived microparticles at days 21 and 42 of storage compared to storage in AS-3 (FIGURE 6A). When supernatants of pRBCs stored in AS-3 were analyzed, we found increased free hemoglobin at day 21 and 42 of storage (FIGURE 6B). This increase was blunted at 21 days by storage in EAS-1587 (FIGURE 6B). There was no difference at 42 days of storage (FIGURE 6B). There was a higher percentage of glucose metabolized in pRBCs stored in EAS-1587 as compared to pRBCs stored in AS-3 (FIGURE 6C). There were no significant differences in susceptibility to osmotic stress, expression of Band-3 or phosphatidylserine, accumulation of advanced oxidation protein products, or intracellular erythrocyte hemoglobin content (Supplemental FIGURE 1) between pRBC storage in AS-3 compared to EAS-1587, indicating that the storage solutions were similar in these aspects of storage lesion formation. Evaluation of pH, sodium, potassium, ionized calcium, hemoglobin, and hematocrit demonstrated no differences in these parameters (data not shown). Taken together, these data demonstrate that storage in EAS-1587 mitigates aspects of the red blood cell storage lesion in human pRBCs.

Figure 6.

Figure 6.

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Aspects of the red blood cell storage lesion in human pRBCs stored in additive solution 3 (AS-3) or experimental additive solution 1587 (EAS-1587) for up to 42 days. (A) Microvesicle counts as determined by flow cytometry. (B) Cell-free hemoglobin accumulation in stored units. (C) Percentage of glucose utilized. N=5 per group. *p<0.05 as compared to indicated group by t-test.

Discussion

In the present study, we examined the impact of a novel storage solution on the formation of the red blood cell storage lesion in stored pRBC units. We found that storage of pRBCs in EAS-1587 resulted in attenuated aspects of the red blood cell storage lesion in human and murine pRBC units as well as a decreased systemic inflammatory response in mice undergoing hemorrhage and resuscitation. Modifications of red blood cell storage solutions have been previously utilized to reduce the detrimental effects of erythrocyte lesion accumulation.13,14,30 Our work extends previous findings by examining a storage solution with increased viscosity. Our study demonstrates that increasing the viscosity of the pRBC storage solution is associated with decreased severity of the storage lesion as well as a diminished inflammatory response after resuscitation. Taken together, our data show that a novel storage solution with increased viscosity and pH is a potentially useful strategy to improve the quality of, and recipient response to, stored pRBCs.

Blood transfusion is the preferred treatment of hemorrhagic shock in order to combat the detrimental effects of ongoing RBC and volume depletion.31 However, transfusion of pRBCs is not without risk. Pre-transfusion microvesicle and cell free hemoglobin accumulation not only affects the stored pRBCs, but also impacts recipient red blood cell viability, end organ microvasculature, and inflammatory status.7,32–36 Following transfusion, the vulnerable RBCs undergo extravascular and intravascular hemolysis.7 The toxic components that accumulated during storage and are released following hemolysis in the recipient are cleared via macrophage endocytosis.37,38 Unfortunately, when there is a large amount of microvesicle and cell-free hemoglobin content in circulation, the mechanism of removal can be overwhelmed, resulting in reduced clearance of these pro-oxidant and pro-inflammatory components. Previous studies have shown that elevated concentrations of cell-free hemoglobin in the recipients’ circulation scavenges endothelial nitric oxide, a key component of maintaining vascular perfusion, and may lead to end-organ damage.39 Our data indicate that storage of pRBCs in EAS-1587 resulted in a reduction in microvesicle and cell-free hemoglobin accumulation in vitro as well as a reduced inflammatory response in vivo. Recipient mice demonstrated a reduction in systemic TNF-α and MIP-1α. This demonstrates that pRBC storage with EAS-1587 can reduce the systemic inflammatory response compared to resuscitation with pRBCs stored in AS-3.

The relationship between storage duration of pRBCs and recipient harm after transfusion is controversial and complex. Although lifesaving in the acute setting, the liberal use of blood products during resuscitation may be harmful to patients, particularly due to the duration of pRBC storage prior to transfusion40. The current FDA approved shelf life for pRBC units is 42 days. In practice, blood banks often release the oldest pRBC units first in order to avoid wastage41. This can be an especially severe problem in trauma patients, who may receive large numbers of pRBC units that are near the end of their shelf life. Recent clinical studies have begun to investigate this issue and appear to demonstrate that transfusion of older pRBCs is not harmful in non-trauma patients42–44. Unfortunately, no prospective randomized study to date addresses the issue of pRBC storage duration given for massive transfusion of trauma patients. Thus, the application of this data to the severely injured trauma patient in hemorrhagic shock is limited, as many of these studies vary widely in the definition of “old” and “new” pRBCs, focus on transfusion of a limited number of pRBC units, or did not include hemodynamically unstable patients45. Previous studies in trauma patients demonstrate that transfusion of older pRBC units is associated with increased mortality, delirium, renal dysfunction, pneumonia, sepsis, and MOSF 41,46–53. The time of storage at which pRBCs become potentially harmful may be as early as day 28, but this remains an unanswered question 50,53–55.

The results of these studies are, in part, attributed to the erythrocyte storage lesion that accumulates in pRBCs over their storage period.7 During cold storage, erythrocytes in pRBCs have reduced metabolic activity resulting in a decrease in ATP production.7 In our study, we found that storage in EAS-1587 resulted in a significantly increased RBC glucose metabolism, suggesting that metabolic activity was improved in these cells. This is a somewhat unexpected finding and the implications of this are unclear. We plan to explore this further in future studies by performing a more in depth analysis of this finding, including the impact of this on ATP and 2, 3 DPG in the stored pRBC units. Also during storage, the mechanisms that protect again oxidative stress are impaired, resulting in an accumulation of reactive oxygen species that contribute to RBC membrane damage.56 We found that storage in EAS-1587 resulted in reduced accumulation of advanced oxidative productive products in murine pRBCs, with a trend toward a reduction in human pRBCs. With increased membrane damage during storage, erythrocytes are unable to maintain their normal biconcave disc shape and demonstrate reduced membrane stability, increased susceptibility to osmotic stress, and increased expression of the senescence signal, phosphatidylserine, on the cell surface.57 When compared to the standard storage solution, AS-3, pRBCS stored in EAS-1587 demonstrated a greater maintenance of Band 3, reduced phosphatidylserine expression, and reduced osmotic fragility, in mice, while similar in humans. Over the duration of storage, the ability of the red blood cells to maintain homeostasis deteriorates, resulting in acidosis, progressive and persistent microvesicle release, as well as RBC hemolysis. Red blood cells stored in the EAS-1587 solution demonstrated attenuation of these deleterious changes, resulting in reduced microvesicle accumulation and reduced RBC lysis with less free hemoglobin release in mice and humans.

There are a number of limitations to this study. Although our novel storage solution demonstrated increased viscosity, our data do not demonstrate that decreased red blood cell storage lesion is due to this change in viscosity. Due to the complex and multifactorial nature of the red blood cell storage solution as well as the response to hemorrhage and resuscitation, it is unlikely that viscosity is the sole mechanistic driver of our findings, and additional potential mechanisms, including free radical and electron absorption capabilities may play an important role in our findings. We plan to investigate these factors as we move forward with evaluating the novel storage solution. In addition, leukoreduction may alter aspects of the red blood cell storage lesion. While the human pRBC units utilized in the current study were leukoreduced by filtration, the murine units were not. Murine packed RBCs were generated from only male donors, subsequently avoiding the influence of donor factors such as sex on the formation of the red blood cell storage lesion.58–60 Murine hemorrhagic shock and resuscitation experiments, while promising, may not be translatable to in-hospital resuscitation which would involve patients of varying age and gender. In addition, while our data regarding inflammatory mediators suggest that transfusion with pRBCs stored in EAS-1587 results in an attenuated inflammatory response to resuscitation with older pRBCs, we did not carry out a comprehensive analysis of potential harm from the utilization of hypromellose in storage solutions, study concentrations greater than those present, or examine additional timepoints after hemorrhage and resuscitation. These experiments will be necessary as further investigation of this storage solution proceeds. Finally, human erythrocytes, vasculature, and end-organs may have differing susceptibilities following transfusion. These factors need to be considered when planning for on-going and future evaluation of EAS-1587.

In conclusion, we have demonstrated that EAS-1587, a novel buffered high viscosity storage solution, leads to reduced accumulation of the red blood cell storage lesion during storage as well as attenuated inflammatory cytokines following resuscitation with aged pRBCs. Our data suggests that EAS-1587 is as a promising alternative to the current standard storage solutions. This strategy has the potential to attenuate the aging of red blood cells during storage and reduce the sequelae that results from systemic inflammation following transfusion of stored packed red blood cells.

Supplementary Material

1

Supplemental Figure. Effect of storage of human pRBCs in stored in additive solution 3 (AS-3) or experimental additive solution 1587 (EAS-1587) for 42 days. (A) Hemolysis in response to osmotic stress; (B) Phosphatidylserine expression; (C) oxidative stress as determined by advanced oxidation protein products (AOPP); (D) Erythrocyte (RBC) hemoglobin content. N=5 per group.

Acknowledgments

Funding/Financial Support Statement

This study was supported by the following grants from the NIH/NIGMS:

T32 GM008478 (ABL).

K08 GM126316 (ATM)

R01 GM124156 (MDG)

R01 GM107625 (TAP)

Biographies

DR. MARY-MARGARET BRANDT (Oklahoma City, Oklahoma): Good morning. Thank you for allowing me to discuss this important paper. I'm Maggie Brandt from the University of Oklahoma in Oklahoma City.

This paper I think is important not only in trauma, but iťs important for anybody who transfuses patients. Blood's a precious commodity and a very limited resource. And we donť have any good synthetic replacements. Congratulations to the presenter and authors for their excellent work.

What effect does the hypromellose have on the recipient for the transfused blood? Is that something that needs to be washed away? Is that something that needs to be diluted? Is there any kind of negative side effect? Is there a better way to store blood, maybe? Should we be storing whole blood and then maybe dividing it into different components when iťs needed rather than dividing it and then trying to combine it back into whole blood as we go? Those are really -- oh, one sort of silly question. Is this the 1,587th iteration of this system, like WD-40? Anyway, I really congratulate you on an excellent presentation, and thank you for your time.

DR. PULLIAM: Thank you. During the preliminary experimentation we first injected the EAS 1587 in mice to make sure that there were no detrimental effects. Now, do I know whether there's a threshold that we reached that it could be harmful? We donť know that information yet, and thaťs something that we want to study, but the current percentage and concentration of hypromellose that we've used in a mouse seemed to be inert and not harmful, which is howwe chose hypromellose sensitive biocompatible and use it in products in humans currently. With regards to utilizing whole blood versus breaking it up into components, there's been significant data showing the benefits of utilizing whole blood versus individual components. Unfortunately, by the powers that be, iťs been deemed economically unfeasible. So we're hoping that with presenting persistent positive data for utilizing whole blood, that it will be implemented in national programs worldwide. And then, this is not the 1,587th iteration of the storage solution. We just wanted to come up with kind of a generic name, so iťs the room number of our lab.

DR. DAVID R. FARLEY (Tower, Minnesota): How do you plan to go forward with evaluating this clinically in trials since you've shown some benefits in human whole blood as well?

DR. PULLIAM: I think we're going to need to do a little bit more research with regards to utilizing it as a storage solution in humans, because unlike the mice that we use that are purely male and are genetically similar, humans have female versus male differences. How those packed red blood cells age in this storage solution given the differences in hormone levels that are present is unknown. The storage solution is going to have a different impact on human packed red blood cells than they would in mice. (Applause)

Footnotes

Conflict of Interest/Disclosure Statement

The University of Cincinnati has applied for intellectual property related to the novel red blood cell storage solution (US provisional application 63/050904). The authors declare that they have no other potential conflicts of interest related to this study.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental Figure. Effect of storage of human pRBCs in stored in additive solution 3 (AS-3) or experimental additive solution 1587 (EAS-1587) for 42 days. (A) Hemolysis in response to osmotic stress; (B) Phosphatidylserine expression; (C) oxidative stress as determined by advanced oxidation protein products (AOPP); (D) Erythrocyte (RBC) hemoglobin content. N=5 per group.

NIHMS1762083-supplement-1.tiff (3.6MB, tiff)

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