Washing Packed Red Blood Cells Decreases Red Blood Cell Storage Lesion Formation

Abstract

Background

Transfusion of blood products is the ideal resuscitative strategy after hemorrhage. Unfortunately, older packed red blood cells (pRBCs) have been associated with increased morbidity and mortality after massive transfusion. These pRBCs accumulate biochemical and structural changes known as the red blood cell storage lesions. The effect of washing on the formation of red blood cell storage lesions is unknown. We hypothesized that washing pRBCs during storage would decrease the development of the red blood cell storage lesions.

Methods

Blood from 8–10 week old male mice donors was stored as pRBCs for 14 days. A subset of pRBCs were washed with phosphate –buffered saline (PBS) on storage day 7 and resuspended in AS-1 solution for an additional 7 days as washed pRBCs. Subsequently, the pRBCs were analyzed for microvesicle release, band-3 erythrocyte membrane integrity protein (Band-3), expression of phosphatidylserine (PS), cell viability (calcein), accumulation of cell-free hemoglobin, and osmotic fragility (EC50).

Results

In the washed pRBC group, there was less microvesicle accumulation, greater Band-3 expression, less PS expression, a decrease in cell-free hemoglobin accumulation, and a decrease in osmotic fragility, but no differences in RBC viability.

Conclusions

Washing pRBCs during storage decreases the accumulation of red blood cell storage lesions. This strategy may lessen the sequelae associated with transfusion of older pRBCs.

TOC Statement- 20-csa-26

We examined the impact of washing stored, packed red blood cells on the formation of storage lesions in the red blood cells. The importance of this study is to offer a potential method to decrease the deleterious consequences of storage of packed red blood cells and its impact during blood transfusion.

Introduction

Trauma remains one of the leading causes of preventable death among individuals less than 45 years of age (1). After initial injury, trauma-related complications are often aggravated by acute traumatic coagulopathy, a state associated with activation of the coagulation cascade, depletion of fibrinogen, and platelet dysfunction requiring aggressive resuscitation (2). Strategie of damage control resuscitation often beginning in the prehospital phase of care involve transfusion of packed red blood cells (pRBCs) as an essential aspect of resuscitation in traumatic hemorrhage (3). Current observational studies support balanced transfusion of pRBCs, fresh frozen plasma, and platelets, in a 1:1:1 approach as one of the means to decrease complications and improve survival rates among massively transfused patients (4).

Unfortunately, the administration of stored pRBCs is not without a substantial risk. The available pRBCs for transfusion are often of varying ages of storage. As a result of storage at subphysiologic pH and temperature, these RBCsundergo metabolic, oxidative, and physiologic changes known as the red blood cell storage lesions (5). The accumulation of this storage lesion has clinical implications after transfusion of pRBCs, including increased complications and increased mortality (6). Aspects of the RBC storage lesions appear to directly contribute to these effects. For example, microvesicles released from RBCs as a consequence of storage have been shown to be pro-inflammatory and procoagulant in transfusion recipients (7).

Previous studies have attempted to decrease the complications associated with transfusion through modifications in processing, storage, and administration of pRBCs (8, 9). Washing of units of pRBCs immediately prior to administration in specific patient populations has served to decrease complications associated with the proinflammatory components of residual plasma in the unit (10). The current indications for washing pRBCs include, but are not limited to, severe/recurrent allergic transfusion reaction, IgA deficiency, and individuals at risk for hyperkalemia. The impact of washing units of pRBCs during the storage period on the accumulation of the red blood cell storage lesions has not been studied previously. Therefore, we hypothesized that washing the RBCs during the storage period would result in mitigation of formation of RBC storage lesions.

Methods

Murine Blood Banking

To study the effects of washing on the formation of RBC storage lesions, we utilized our well-established model of murine blood banking (11). All procedures were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati. Briefly, 8–10 week old C57BL/6 male mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in standard environmental conditions with a pellet diet and water ad libitum allowing acclimation to the environment for one week prior to experimentation. Male mice were utilized due to known variable effects of sex and the hormonal environment on the formation of RBC storage lesions (12). Whole blood was obtained after anesthetization with intraperitoneal pentobarbital (0.1 mg/g body weight) and cardiac puncture and placed into citrate phosphate double dextrose (CP2D). Packed BRCs were generated from whole blood via density gradient centrifugation at 400g for 45 min, followed by resuspension of the pelleted erythrocytes in AS-1 in a 2:9 ratio. Standard pRBCs were stored at standard storage conditions for 14 days. A thorough evaluation and characterization of murine red blood cell aging has shown that murine erythrocytes age in a similar fashion to human erythrocytes but at an accelerated rate (11). Subsequently, storage day 14 for mice RBCs was determined to be similar to storage day 42 for human RBCs. On day 7 of storage, pRBCs underwent washing with a 1:1 (vol:vol) wash with 1X phosphate –buffered saline (PBS) followed by centrifugation and resuspension in AS-1 in a 2:9 ratio. Washed pRBCs were stored for an additional 7 days prior to analysis.

Characterization of the Erythrocyte Storage Lesion

The RBC storage lesions include microvesicle release, accumulation of cell-free hemoglobin, decreased expression of Band-3 erythrocyte membrane integrity protein (Band-3), increased expression of phosphatidylserine, and worsened osmotic fragility (EC50). To determine microvesicle accumulation, microvesicles were isolated as described previously (13). Aliquots of stored pRBCs underwent centrifugation at 2,000g for 10 min, and the supernatant was discarded. Samples then underwent centrifugation at 10,000g for 10 min, and the supernatant was discarded. Samples were then subjected to centrifugation at 21,100g for 35 min to pellet the microvesicles. Microvesicles were quantified via flow cytometric analysis on an Attune Flow Cytometer (Life Technologies, Foster City, CA) after incubation with Ter-119 (BD Biosciences San Jose, CA - mouse) antibody. The supernatant that was obtained from the final centrifugation at 21,100g for 35 min was utilized to measure cell-free hemoglobin, which was quantified via a hemoglobin colorimetric assay (BioVision Inc., Milpitas, CA) and read at 575nm on microplate spectrophotometer (BioTek Cytation 5, Winooski, VT).

The percentage of RBCs expressing Band-3 membrane integrity protein (Band-3) and phosphatidylserine on the erythrocyte surface were quantified via the fluorescent antibody Eosin-5-malemeide (EMA) and Fluorescein isothiocyanate (FITC) Annexin V (BD Biosciences), respectively, via flow cytometry. Murine RBCviability was determined via a flow cytometry utilizing an acetixymethyl ester of calcein (Calcein-AM) marker (Millipore Sigma, St. Louis, MO).

An osmotic fragility assay was utilized to determine the susceptibility of the stored RBCs to osmotic stress. Osmotic fragility was determined by suspending aliquots of RBCs in solutions containing increasing concentrations of sodium chloride (0, 0.32, 0.44, 0.56, 0.68, and 0.8% NaCl) for 30 min, followed by centrifugation at 10,000 × g for 10 min with analysis of the absorbance of the supernatant measured via a microplate spectrophotometer at 575nm. The hemolytic increment was calculated, and the EC50 determined by the hemolytic increment of each sample when suspended in the 0.56% NaCl solution.

Biochemical Evaluation

The i-STAT handheld blood analyzer (Abbott Laboratories, Chicago, IL) was utilized to obtain blood gas, electrolyte, and hematologic information.

Statistical Analysis

Data is presented as mean ± standard error of the mean. Groups were compared using Student’s t-test. p<0.05 was determined to be statistically significant.

Results

In an initial series of experiments, we determined the RBC count, lactate concentration, hemoglobin level, and hematocrit of the standard and washed pRBCs(Table 1). The RBCcount, a measure of the total count of RBCs present within each sample, were the same between the standard and washed pRBCs. While there was no difference in the number of RBCs present, there was a statistically significantly greater intracellular hemoglobin content within the washed pRBCs compared to those units stored under standard conditions.

Table 1.

Biochemical and Hematologic Evaluation

	Standard pRBCs	Washed pRBCs
RBC count (×1012/L)	6.9 ± 0.2	7.0 ± 0.2
Hemoglobin(g/dL)	10.2 ± 0.1	12.8 ± 0.1*
Hematocrit (%)	30.0 ± 0.6	37.3 ± 0.4*
Lactate (mmol/L)	16.4 ± 0.3	7.8 ± 0.1*

^*indicates p<0.05 by t-test. N=6 under each condition.

Lactate, a byproduct that accumulates during cell storage, has been associated with inhibited metabolism of RBCs as well as interference with ATP production (5). The washed pRBCs demonstrated statistically significantly decreased lactate accumulation at the end of the storage duration.

Studies from our and other laboratories demonstrated previously that microvesicle formation is a substantial aspect of the RBC storage lesion, with increased lung injury, activation of endothelial cells, and formation of microthrombi after transfusion (13). We found that washing pRBC units during the storage period was associated with a statistically significant decrease in microvesicle accumulation in stored units (Figure 1).

Figure 1.

Microvesicle accumulation in standard-storage as compared to washed pRBC units. The washed pRBC demonstrated a decrease in microvesicle release. N=11 for each group. * p<0.05 by t-test.

We next evaluated the effect of washing on the accumulation of free hemoglobin. Previous studies examining the RBCstorage lesion have focused on potential harm resulting from increased, cell-free hemoglobin in the pRBC units. Cell-free hemoglobin in the transfusion recipient plays a role in scavenging of nitric oxide and subsequent vascular impairment (14). Our data demonstrated that washed pRBC units contained less free hemoglobin at the end of the storage period (Figure 2).

Figure 2.

Hemoglobin concentration in supernatant of standard-storage as compared to washed pRBC units. At the end of the storage period, there was decreased accumulation of cell-free hemoglobin in the washed pRBC group. N=8 for each group. * p<0.05 by t-test.

Band-3 is an erythrocyte transmembrane integrity protein on erythrocytes that plays an important role in the structure, flexibility, and function of the red blood cell membrane. During storage, there is typically a breakdown and/or aggregation of the Band 3 protein, which in turn acts as a senescence antigen for aged RBCs (15). We found that there were no differences in Band 3 expression when comparing standard pRBCs to washed pRBCs (Figure 3).

Figure 3.

Band-3 protein expression in erythrocytes of standard-storage as compared to washed pRBC units. N=6 for each group. * p<0.05 by t-test.

Phosphatidylserine is a phospholipid component of erythrocyte membranes which typically faces the cytosol of red blood cells. During aging and degradation of the RBC membrane, phosphatidylserine becomes outward facing, and serves as signal for removal of the erythrocytes from the circulation (16). Utilizing flow cytometry, we determined the effect of washing on the expression of phosphatidylserine expression. Our data demonstrates that washing pRBCs during storage results in a statistically significant decvrease in in externalization of phosphatidylserine (Figure 4).

Figure 4.

External membrane expression of phosphatidylserine on RBCs of standard-storage as compared to washed pRBC units. N=6 for each group. * p<0.05 by t-test.

Acetoxymethyl calcein (calcein AM), utilized as an indicator of cell viability, is a nonfluorescentdye that becomes fluorogenic when acted on by esterases present within functional cells. Our data indicate that there is similar cell viability in standard and washed pRBCs (Figure 5).

Figure 5.

Erythrocyte viability as determined by calcein assay in erythrocytes of standard-storage as compared to washed pRBC units. N=5 for each group. * p<0.05 by t-test.

The testing of osmotic fragility t is often employed when evaluating for deficiencies in RBCmembrane proteins in hematologic disorders such as hereditary spherocytosis (17). In this experiment, we utilized osmotic fragility to evaluate aspects of the RBC storage lesion that impact the RBC membrane proteins and increases their susceptibility to osmotic stress. We found that RBCs from units that were previously washed demonstrated increased resistance to osmotic stress (Figure 6A,B).

Figure 6.

Osmotic fragility of erythrocytes as determined by hemolysis in salt solutions. The washed pRBCs were less susceptible to osmotic stress (A,B). N=4 for each. * p<0.05 for washed vs standard for each concentration as determined by t-test.

Discussion

In the present study, we examined the effect of washing pRBC units mid-storage on the severity of the formation of RBCstorage lesions. We found that a single wash resulted in decreased accumulation of microvesicles nd a cell-free hemoglobin, decreased expression of phosphatidylserine, and increased resistance to osmotic stress. While some of the measured decreases in different aspects of the storage lesion such as the accumulation of free hemoglobin may simply reflect dilution or replacement, other aspects such as the expression of phosphatidylserine reflect measurements directly from the erythrocytes and thus are indicative of an alteration of the storage lesion. Taken together, our data indicate that washing RBCunits during storage attenuates the formation of the RBCstorage lesions. In addition, washing pRBCs may represent a strategy to decrease post-transfusion sequelae, especially in the setting of massive transfusion after traumatic hemorrhage.

The utilization of washed pRBCs is not novel in transfusion medicine. Washed pRBCs are used commonly used for patients with history of recurring and/or severe allergic transfusion reactions refractory to medical management, because studies have shown that washing pRBCs Mrkedly decreases the incidence of allergic transfusion reactions (18). In each of these situations, washing occurs at the conclusion of the storage period as units are prepared for transfusion. Our data suggest that the indications for washing may be extended and included as a strategy to attenuate the formation of the storage lesions.

The RBCstorage lesion is a collection of pathophysiologic changes that occur during pRBC storage. These changes impair RBCmetabolism, membrane structure and functionality, as well as viability (5). By the end of the FDA approved 42 days storage duration, cold storage of pRBCs result in acidosis, the accumulation of metabolic byproducts, electrolyte imbalance, oxidative stress, membrane damage, and eventual RBCdeath (5, 9). The mechanism of the RBCstorage lesion is poorly understood. Some studies indicate that extracellular calcium may drive the process (19), while others suggest that the pH of the storage solution may play a role (20). Our findings suggesting that washing mid-storage may alter the development of the RBCstorage lesion are important in this context in that they are supportive that the mechanisms responsible for this process are related to the extracellular environment, rather than within the erythrocytes. Further studies will be needed to understand and mitigate these potential contributors to the severity of the storage lesion.

Microvesicles, a byproduct of pRBC storage, have been shown previously to play an important role in the in-vivo sequelae after resuscitation from hemorrhagic shock. Microvesicles that are pro-inflammatory in nature activate neutrophils and contribute to post-transfusion lung inflammation (21). They have also been shown to be prothrombic in nature via various mechanisms, contributing to post-transfusion venous thrombosis (13, 22). An investigation of the clinical implications of these in-vivo findings suggested that the administration of increasing quantities of older pRBCs have been associated with an increased likelihood of 24-hour mortality in massively transfused trauma patients (6). Our data support the concept that a decrease in the severity if the pRBC storage lesion is associated with less microvesicle shedding.

At the completion of our study, we found that murine pRBCs that were washed midway through storage resulted in substantial mitigation of the formation of the RBC storage lesion. The murine model of the murine erythrocyte storage lesion was utilized in order to control for donor-specific factors that impact pRBC aging, such as donor age and sex. The detrimental pathophysiologic changes incurred by stored RBCs were diminished in the RBCs that underwent a wash with 1X PBS on day, mid-way through storage. The process of washing likely resulted in the removal of microvesicles, cell-free hemoglobin, and fragile RBCs that accumulated during the first 7 days of storage. The process of washing did not appear to damage nor accelerate the biochemical and structural changes, because the integrity of the RBC membrane remained intact, there were less RBCs marked for eryptosis, and there was decreased osmotic fragility when compared to the standard pRBCs. In the light of our previous study on washed pRBCs, these findings suggest that utilization of pRBCs that have undergone a wash during storage, could mitigate the systemic inflammatory reaction in massively transfused trauma patients (23).

There are limitations that exist within this study. We utilized a murine model of blood banking in order to control for recognized confounders in the secerity of RBC storage lesions, including donor age and sex. Although the murine model demonstrated strong evidence of a decrease in the RBC storage lesions, this may not translate into human pRBC units. In addition, the pRBC units in this study were hand-washed via density gradient centrifugation. Previous studies have demonstrated that the specific type of washing device utilized has an impact on erythrocyte aging and could even cause additional damage (24, 25). In a previous study, utilization of the Haemonetics Cell Saver Elite device to wash stored pRBCs resulted in greater hemolysis when compared to the Terumo Cobe 2991, a FDA-cleared device for washing stored RBCs (24). Finally, the current recommendation for washed pRBCs is to discard the units after 24 hours or 14 days for the open and closed washing systems, respectively, due to increased risk for bacterial contamination (25), which may limit future implementation of this strategy.

In conclusion, we demonstrated that washing pRBCs during storage resulted in mitigation of the RBC storage lesion, including a decrease in the accumulation of microvesicles, release of cell-free hemoglobin, and expression of phosphatidylserine when compared to unwashed pRBCs. This study shows promise with regard to the potential use of washed pRBCs in resuscitation of hemorrhage. Future studies are required to further understand the molecular mechanism, the optimal strategy of washing, and to investigate the potential role of modified RBCstorage solutions in decreasing the storage lesion.