Expired But Not Yet Dead: Examining the Red Blood Cell Storage Lesion in Extended-Storage Whole Blood

Abstract

Whole blood is a powerful resuscitation strategy for trauma patients but has a shorter shelf life than other blood products. The red blood cell storage lesion in whole blood has not previously been investigated beyond the standard storage period. In the present study, we hypothesized that erythrocytes in stored whole blood exhibit similar aspects of the red blood cell storage lesion and that transfusion of extended storage whole blood would not result in a more severe inflammatory response after hemorrhage in a murine model. To test this hypothesis, we stored low-titer, O-positive, whole blood units and pRBCs for up to 42 days, then determined aspects of the red blood cell storage lesion.Compared to standard storage pRBCs, whole blood demonstrated decreased microvesicle and free hemoglobin at 21 days of storage and no differences in osmotic fragility. At 42 days of storage, ROTEM demonstrated that clotting time was decreased, alpha angle was increased, and clot formation time and maximum clot firmness similar in whole blood as compared to pRBCs with the addition of fresh frozen plasma. In a murine model, extended storage whole blood demonstrated decreased microvesicle formation, phosphatidylserine, and cell free hemoglobin. After hemorrhage and resuscitation, TNF-a, IL-6, and IL-10 were decreased in mice resuscitated with whole blood. Red blood cell survival was similar at 24 hours after transfusion.Taken together, these data suggest that red blood cells within whole blood stored for an extended period of time demonstrate similar or reduced accumulation of the red blood cell storage lesion as compared topRBCs. Further examination of extended-storage whole blood is warranted.

Introduction

Studies of civilian and military prehospital and in-hospital trauma-related deaths have demonstrated that the majority of potentially preventable deaths can be attributed to hemorrhage(1, 2). Current management of hemorrhage includes surgical control of bleeding along with resuscitation with ratio-balanced component therapy in 1:1:1 fashion (3–6). However, administration of components in the manner of reconstituted whole blood has been associated with resuscitation-associated changes such as anemia, thrombocytopenia, and coagulation factor dilution(7). Recent studies have demonstrated benefits of early administration of higher platelet and higher plasma to RBC ratios (6, 8, 9). Modifications of the component ratios in attempts to emulate whole blood have prompted implementation of whole blood programsfor massively bleeding patients in the military setting and in some civilian trauma centers (10–12).

Early studies investigating the safety and feasibility of cold stored low titer group O whole bloodadministration (13, 14) led to the recent authorization of this strategy by the American Association of Blood Banks(15). Examination of resuscitation with cold-storage whole blood as compared to component therapy demonstrated retained coagulation potential (16–19), reduced additive solution administration, reduced transfusion volumes (20), and reduced exposure of the recipient to donor plasma (7).

Unfortunately, there has beenlimited implementation of whole blood programs in the civilian setting, at least in part, due to the limitations of the storage life of whole blood. The US Food and Drug Administration-approved storage period ranges from 21–35 days based on the anticoagulant utilized. This storage duration is less than that of packed red blood cells, which have a shelf-life of 42 days. From an inventory standpoint, storage of blood products as separated components, rather that whole blood, may maximize overall availability of this precious resource to patients.

Recent data from our laboratory suggests that erythrocytes from previously stored whole blood exhibit distinct aspects of the red blood cell storage lesion when salvaged and subsequently stored as packed red blood cells for an additional 21 days(21). These data suggest that erythrocytes in stored whole blood may remain viable beyond the standard storage period. The severity of the red blood cell storage lesion during extended storage in whole blood storage is poorly understood. We undertook the present study in order to increase our understanding of the nature of the red blood cell storage lesion during extended storage of whole blood. We hypothesized that erythrocytes in stored whole blood exhibit similar or diminished aspects of the red blood cell storage lesion and that transfusion of extended storage whole blood would not result in a more severe inflammatory response after hemorrhage in a murine model.

Material and Methods

Human Blood Banking

Cold stored, low-titer, O-positive, non-leukoreduced whole blood units were obtained from six healthy male donors by our regional blood bank. The process of unused, de-identified, whole blood unit acquisition was reviewed by the University of CincinnatiInstitutional Review Board and approved as “non-human subjects research”. The whole blood was collected in citrate phosphate dextrose (CPD; 3 mg/mL citric acid, 26.30 mg/mL sodium citrate, 2.22 mg/mL monobasic sodium phosphate, and 25.51 mg/mL dextrose) and stored as 1.5ml aliquotsin 1.7ml microcentrifuge tubes at 4°C for up to 42 days. A separate aliquot of fresh whole blood from the same donors was utilized to generate standard pRBC units. Packed red blood cells units were generated via centrifugation at 300g for 7 mins with aspiration and discard of the supernatant containing platelets. Subsequently, the blood underwent centrifugation at 1000g for 15 minutes with discard of the supernatant containing the buffy coat. Additive Solution-3 (AS-3; 0.42 mg/mL citric acid, 5.88 mg/mL sodium citrate, 2.76 mg/mL monobasic sodium phosphate, 4.10 mg/mL sodium chloride, 10mg/mL dextrose, 0.30 mg/mL adenine) was added to the remaining erythrocytes in a ratio of 2:9 and stored as 1.5ml aliquots in a 1.7ml microcentrifuge tubes at4°C for up to 42 days.

Murine Animal Model

C57BL/6 male 8 to 10-week-old mice, purchased from The Jackson Laboratory (Bar Harbor, ME) were housed in a climate-controlled room with a twelve-hour light-dark cycle, fed the standard laboratory pellet diet, and provided water ad libitum. The mice were allowed to acclimate for one week prior to utilization in experiments. Murine experiments were performed after approval by University of Cincinnati’s Institutional Animal Care and Use Committee.

Murine Blood Banking

Murine blood banking was performed via a modification of our previously established murine blood banking protocol(22). Mice were anesthetized with intraperitoneal administration of pentobarbital (0.1 mg/gram body weight) and whole blood obtained via cardiac puncture. Previous characterization of murine erythrocyte aging demonstrated that murine erythrocytes age similarly to human erythrocytes, except at an accelerated rate(22).Whole blood was obtained, collected in microcentrifuge tubes with the addition of CPD in a 1:7 ratio by volume and stored at 4°C for up to 14 days.A separate aliquot of fresh whole blood was utilized to generate standard pRBCs that were placed in AS-3 and stored for 14 days. The pRBC units were generated via density gradient centrifugation at 400g for 40 minutes withaddition of AS-3 storage solution to the erythrocytesin a 2:9 ratio. Subsequently, 1.5ml of pRBCs were stored in 1.7ml microcentrifuge tubes and stored upright at 4°C.Only male mice were used in these experiments in order to minimize the variability introduced on the red blood cell storage lesion by the effect of the estrous cycle and mirror the human donor experiments(23–25). The overall design of the murine studies is presented in Figure 1.

Figure 1.

Experimental design for murine blood storage and hemorrhagic shock and resuscitation experiments.

Red Blood Cell Storage Lesion Characterization

Human and mouse pRBC units were evaluated, at intervals, for aspects of the red blood cell storage lesion. Microvesicle accumulation, Band-3 membrane protein expression, and phosphatidylserine externalization were determined using flow cytometry with protein specific antibodies for CD-235a and Ter-119 (for erythrocyte specificity in humans and mice, respectively; BD Biosciences San Jose, CA), Eosin-5-malemeide (EMA; for Band-3 binding; ThermoFisher Scientific, Waltham, MA), and Annexin V (for phosphatidylserine binding; BD Biosciences San Jose, CA), as previously described(26). Prior to analysis, microvesicles were isolated as previously described(27) using centrifugation of whole blood or pRBCs at 2,000g for 10 mins, collection of the supernatant and centrifugation at 10,000g for 10 mins, and subsequent collection of the supernatant ensuring avoidance of pelleted erythrocytes. The supernatant underwent centrifugation at 21,100g for 35 minutes to pellet the microvesicles.

The supernatant present following the final microvesicle centrifugation (21,100g for 35 minutes) was utilized to measure cell-free hemoglobin content. The cell-free hemoglobin concentration was determined by colorimetric assay (Hemoglobin Colorimetric Assay Kit, Biovision, Milpitas, CA). 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) for 30 minutes, followed by centrifugation at 10,000 × g for 10 minutes with analysis of the supernatant absorbance measured via a microplate spectrophotometer (BioTek Cytation 5, Winooski, VT). The hemolytic increment was calculated and EC50 determined by the hemolytic increment of each sample when suspended in the 0.56% NaCl solution.

Calculation of Percentage Hemolysis in Blood Units

The percentage of hemolysis in RBC units was calculated with the following formula:

$$(100-\mathrm{Hct})\times \mathrm{plasma}\mathrm{Hemoglobin}(\mathrm{g}{\mathrm{dl}}^{-1})/\mathrm{Total}\mathrm{Hb}(\mathrm{g}{\mathrm{dl}}^{-1})$$

Murine Hemorrhagic Shock and Resuscitation Model

Pressure-controlled hemorrhage and resuscitationwere carried out as previously described (28). Briefly, 8 to 10-week-old male C57BL/6 mice weighing 23 to 30g 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 skin was incised, femoral vessels exposed, and the femoral artery cannulated with a tapered polyethylene catheter. The femoral catheter was connected to pressure transducers for continuous hemodynamic monitoring of the mice (AD Instruments Lab Chart). To avoid hypothermia, the cannulated mice were placed on a circulating water blanket maintained at 41°C. After 10 minutes of equilibration, hemorrhagic shock was obtained by withdrawing blood to achieve a mean arterial pressure (MAP) of 25 ± 5 mmHg and maintained for 60 minutes (20). The volume (mL/g body weight) of blood required to achieve the desired hemorrhagic shock MAP was recorded for each mouse. Following hemorrhagic shock, mice were resuscitated with aged standard pRBCs or extended storage whole blood to achieve a MAP greater than 70 mm Hg ± 5 mm Hg. The volume (mL) of blood required to achieve the appropriate resuscitation was recorded for each resuscitation group. The mice were monitored for 15 minutes following resuscitation, the femoral artery decannulated, and mice wereeuthanized at 1-hour post procedure end. Sham mice underwent femoral artery cannulation and hemodynamic monitoring for 90 minutes, without hemorrhage or resuscitation.

Cytokine Analysis

One-hour post procedure end, resuscitated mice were sacrificed, and blood obtained. Serum samples were isolated via recipient whole blood in serum separator tube that underwent centrifugation at 8000rpm for 10 minutes. The isolated serum was immediately stored at −80°C in an upright freezer (Thermo Fisher Scientific, Waltham, MA) until analysis. 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). Cell free-hemoglobin content of the recipient serum were measured via colorimetric assay (Hemoglobin Colorimetric Assay Kit, Biovision, Milpitas, CA) and quantified via a microplate spectrophotometer (BioTek Cytation 5, Winooski, VT).

Assessment of Red Blood Cell Survival Post-transfusion

10mM aliquots of 5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester (CFSE; Thermo Fisher Scientific, Waltham, MA) were generated by diluting 25mg of CFSE in 4.48ml of dimethyl sulfoxide. 10mM of CFSE was added to 1X phosphate buffered saline (PBS) in a 1:5000 ratio. Murine whole blood and pRBCs were stored for 14 days at 4°C. The CFSE-PBS mixture was added to the stored whole blood or pRBCs in equal volumes and incubated for 30 minutes at 37°C. A 1% bovine serum albumin in PBS was added to the CFSE labeled erythrocytes and centrifuged at 450g for 10 minutes in order to bind and wash off residual CFSE. The wash was repeated for a total of 3 washes. The red blood cells were resuspended in 1X PBS to obtain a hematocrit of 30% and confirmed via the Ac•T diff hematology analyzer (Beckman Coulter, Brea, California). Mouse penile vein injection was utilized to transfuse 50μl of CFSE labelled red blood cells into recipient mice. Recipient mice blood samples were obtained via tail vein bleed at 15-minute and 24-hour post-transfusion. The samples were incubated with PE Ter 119 to label the red blood cells. Flow cytometry was utilized to quantify the remaining transfused red blood cells from stored whole blood and pRBCs.

Assessment of Coagulation Potential

Thromboelastometry

Rotational thromboelastometry (TEM Systems Inc., Durham, North Carolina) analyses were performed to investigate alterations in the coagulation status. Prior to analysis, the human blood samples were run on the Ac•T diff hematology analyzer (Beckman Coulter, Brea, California) to determine the red blood cell count, hemoglobin, hematocrit and platelet count. Non-activated rotational thromboelastometric (NATEM) assays were run on whole blood at intervals up to day 42 of storage. NATEM assays were also performed on pRBCs combined with FFP in a 1:1 ratio, which is consistent with current damage control resuscitation strategies. Subsequently, EXTEM assay tests with tissue thromboplastin activation was utilized to examine the extrinsic clotting pathway of the blood samples. FIBTEM assays, with tissue thromboplastin and platelet inhibitor cytochalasin D, were used in order to study the contribution of fibrinogen to clot formation in each blood sample(29). The viscoelastic coagulation parameters measured on NATEM, EXTEM, and FIBTEM, were clotting time (CT), clot formation time (CFT), alpha angle (α-angle), maximum clot firmness (MCF), and fibrinolysis (LI30).

Aggregation Analysis

Multiplate impedance aggregometry (Verum Diagnostics, Munich, Germany) was utilized to measure RBC and platelet aggregation as previously described(30). Analysis was performed on whole blood and pRBC units at increments up to day 42 of storage. Platelet aggregation was induced by 6.5 μM adenosine diphosphate (ADP), 0.5 mM arachidonic acid (ASPI) or 0.2 mol/L Star-TEM®20 (calcium chloride) as agonists. Contrary to Star-TEM, ADP and ASPI are standard agonists utilized to initiate platelet aggregation. In this experiment Star-TEM was utilized in order to investigate the impact of additional calcium administration on aged whole blood and pRBCs. In a massively transfusion trauma patient, administration of calcium chloride is often utilized in order to overcome resuscitation-associated coagulation changes, such as those induced by the anticoagulant effects of citrate in the storage solutions.

The agonists were prepared as dictated by manufacturer instructions (Roche Diagnostics, Mannheim, Germany; DiaPharma Group Inc., West Chester, OH). Following instrument set-up, 300 μL of diluent (0.9% NaCl or 0.9% NaCl with 3mM CaCl₂) along with 300 μL of blood sample were inserted into the test cell channels via electronic pipette and underwent a 3-minute warming/equilibration incubation period. The blood samples were eitherwhole blood or packed red blood cells in a 1:1 ratio with fresh frozen plasma (FFP). FFP was generated by centrifugation of whole blood at 1000g for 15 minutes, collection of the supernatant and storageat −80°C. Subsequently, 20 μL of the indicated agonist was pipetted into the bottom of the test cell followed by a 6-minute incubation period. Aggregation was measured by sensing the change in electrical impedance that is automatically converted into arbitrary aggregation units (AU). The area under the aggregation curve (AUC) was calculated by the total height of the aggregation curve and the slope as determined by the speed and final strength of aggregation (30).

Statistical analysis

GraphPad Prism was utilized (San Diego, CA) for statistical analysis of data via ANOVA or t-test when indicated. P<0.05 was deemed statistically significant. Data is presented as mean ± standard error of the mean.

Results

Human Red Blood Cell Storage Lesion

An important parameter for red blood cell storage is that hemolysis should not exceed 1%. When we examined the effect of storage on hemolysis, we found that the calculated percentage of hemolysis in the human blood products was 0.35% for whole blood and 0.21% for pRBCs at the end of the storage period.

Red blood cell-derived microvesicles, a consequence of storage, have been characterized as mediators of acute inflammatory response, vascular dysfunction and coagulation(31). Microvesicles have been associated with post-transfusion complications, including acute lung inflammation and venous thromboembolic events (27, 32–36). At the beginning of the storage period, there was no difference in microvesicle accumulation between whole blood and packed red blood cells. At day 21, mid-way through storage, there was a significant reduction of microvesicle accumulation in the whole blood when compared to standard packed red blood cells. By the end of the 42-day storage period, there was no significant difference in microvesicle accumulation between whole blood and packed red blood cells (Figure 2A).

Figure 2.

Aspects of the red blood cell storage lesion in human packed red blood cell units(pRBCs) as compared to whole blood (WB). (A)microvesicles, (B) cell-free hemoglobin in the supernatant of stored units, (C), Band-3 expression, and (D) phosphatidylserine externalization. N = 6. *p< 0.05 vs pRBCs at the same time point.

Cell-free hemoglobin is another fundamental aspect of the storage lesion that accumulates during storage and has detrimental consequences following transfusion, including nitric oxide depletion as well as reactive oxygen species generation with resultant vascular endothelial dysfunction(37, 38). Cell-free hemoglobin was significantly less on day 21 storage for whole blood compared to pRBCs. There was no significant difference between blood groups on day 1 and day 42 of storage (Figure 2B).

Band-3 is a transmembrane protein that plays a critical role in the function and deformability of erythrocytes. As erythrocytes age, there is a decrease in Band 3 expression that provokes the senescence pathway(39–41). There were no differences in Band 3 expression during the 42-day storage duration for whole blood can and pRBC units (Figure 2C).

Phosphatidylserine is a membrane phospholipid that is found of the inner leaflet of the red blood cell membrane. As erythrocytes age, phosphatidylserine is externalized, contributing to recognition and removal of vulnerable erythrocytes(42, 43). Similar to Band 3, there was no difference in phosphatidylserine exposure of whole blood and pRBC units at each of the timepoints (Figure 2D).

Osmotic fragility testing is utilized to identify membrane vulnerability in common hematologic disorders(44). At the beginning of storage, whole blood demonstrated higher resistance to osmotic stress (Figure 3A). At day 21 and day 42 of storage, the erythrocytes within whole blood and pRBC units demonstrated similar susceptibility to osmotic stress (Figure 3B,C).

Figure 3.

Osmotic fragility in human whole blood (WB) as compared to pRBCs at intervals during storage. Hemolysis was determined in decreasing concentrations of salt solution. N=6. *p<0.05 vs pRBCs at the same time point.

Assessment of Coagulation Potential in Human Blood Products

Rotational thromboelastometry (ROTEM) examines the viscoelastic changes that occur during coagulation and evaluates the process of clot initiation, formation, and stability(45). ROTEM is often utilized as a component of transfusion strategies to simultaneously identify coagulopathy and guide blood product resuscitation(46). NATEM, EXTEM, and FIBTEM testing were utilized to determine the coagulation potential of whole blood and pRBCs at intervals up to 42 days. On NATEM testing, the clotting time (CT) of whole blood was prolonged on day 1 but by day 42 was significantly shorter than pRBCs (Figure 4A). There was no difference in alpha angle at the beginning of the storage period, but the alpha angle was greater in whole blood by day 21 which was sustained up to day 42 (Figure 4B). The clot formation time (CFT) was significantly less in whole blood up to day 21 but by day 42, there was no difference in CFT between the expired whole blood and pRBCs (Figure 4C). Maximum clot firmness was greater when evaluating stored whole blood up to day 21. At day 42 of storage the expired whole blood demonstrated a MCF similar to the standard pRBCs (Figure 4D).

Figure 4.

Non-activated rotational thromboelastometry (NATEM) analysisofhuman packed red blood cell units (pRBCs) as compared to whole blood (WB). We analyzed (A) clotting time, (B) alpha angle, (C) clot formation time, and (D) maximum clot firmness. N=6 under each condition. *p< 0.05 vs pRBCs at the same time point.

On EXTEM analysis, the CT was prolonged for whole blood throughout the 42 day storage. The alpha angle, although greater in WB on day 1, was found to be no different by day 21 and this continued at day 42. Similar to NATEM analysis, the CFT was shorter and MCF was greater in whole blood on day 1 and 21, but was no different from pRBCs by day 42 (Figure S1). On FIBTEM analysis, there were no differences between whole blood and pRBCs in the coagulation parameters examined (Figure S2).

Multiplate analysis of platelet aggregation activated by ADP and ASPI in whole blood demonstrated gradual reduction at day 21 and day 42 (Figure S3). Platelet aggregation activated by Star-TEM demonstrated preservation of platelet aggregation at day 21, but by day 42 there was minimal platelet aggregation similar to ADP and ASPI agonists (Figure S3). This data indicates that ADP and ASPI induced platelet activity rapidly diminish in stored whole blood.

Murine Red Blood Cell Storage Lesion

In the next series of experiments, we analyzed stored murine whole blood and pRBCs for aspects of the red blood cell storage lesion. After 14 days of storage, the calculated percentage of hemolysis in the murine blood units was 0.59% and 0.51% for whole blood and pRBC units, respectively. Murine whole blood demonstrated less microvesicle and cell-free hemoglobin accumulation than did pRBC units (Figure 5A,B). We also found that there was less Band 3 expression, reduced phosphatidylserine externalization, and no difference in osmotic fragility when compared to standard pRBCs (Figure 5C,D,E,F).

Figure 5.

Aspects of the red blood cell storage lesion in 14-day old murine packed red blood cell units (pRBCs) as compared to whole blood (WB). (A) microvesicles, (B) Band-3, (C) phosphatidylserine, (D) cell-free hemoglobin in the supernatant of stored units, (E), osmotic fragility. N = 6. *p < 0.05 vs pRBCs at the same time point.

Murine Hemorrhagic Shock and Resuscitation

We next examined theeffect of extended storage of whole blood on resuscitation in a murine pressure clamp model of hemorrhage (28). We found no differences in the average start, hemorrhagic shock, and resuscitation mean arterial pressure between pRBC and whole blood treatment groups(data not shown). There were no differences in the resuscitation volumes required to achieve a mean arterial pressure of 70 mmHg. The mice receiving whole blood, as compared to mice receiving pRBCs, demonstrated a higher mean arterial pressure during the initial resuscitation period, however there was no difference in the groups by the end of resuscitation(Figure 6A,B). We analyzed serum cytokines after hemorrhage and resuscitation. Our data demonstratethat transfusion of whole blood did not result in a worsened inflammatory response in the mice (Figure 7A–E).In fact, TNF-a, IL-6, and IL-10 were decreased in mice resuscitated with whole blood (Figure 7A, C, D). There was no difference in cell-free hemoglobin content in recipient mice serum following transfusion with whole blood as compared to standard pRBCs (Figure 7F).

Figure 6.

(A) Resuscitation volume required (B) and mean arterial blood pressure (MAP) in mice undergoing hemorrhage followed by resuscitation with pRBCs or extended-storage whole blood (WB). N ≥ 6. *p < 0.05 vs pRBCs at the same time point.

Figure 7.

Murine serum cytokines (A-E) and serum free hemoglobin (F) in mice after hemorrhage followed by resuscitation with 14-day old pRBCs or whole blood (WB). N≥ 6. *p < 0.05 vs pRBCs or sham as indicated.

Post-transfusion Red Blood Cell Recovery in Mice

Post-transfusion red blood cell recovery consists of the percentage of cells that remain in circulation following transfusion(47). It is considered to be an essential marker of transfusion efficacy and is a component utilized by the U.S. FDA to establish the shelf-life of whole blood and components(48, 49). Our data demonstrated that at 15 minutes and 24 hours post-transfusion, the whole and packed red blood cells demonstrated similar red blood cell survival in recipient circulation (Figure 8).

Figure 8.

Mean fluorescence intensity (MFI) at 15 minutes or 24 hours in 14-day old whole blood of mice transfused with CFSE-labeled packed red blood cell (pRBCs) or whole blood (WB). The MFI is the average fluorescence intensity of each event and quantifies the analyzed parameter.N≥6. *p< 0.05 vs pRBCs.

Discussion

In the present study, we investigated the effect of prolonged storage of whole blood on the development of the red blood cell storage lesion. We found that human whole blood stored beyond its intended shelf life accumulated microvesicles and cell-free hemoglobin similarly to packed red blood cells. There was no difference in Band-3, phosphatidylserine expression, or osmotic fragility. Murine whole blood demonstrated reduced microvesicle and cell-free hemoglobin accumulation. There was also reduced Band 3 expression without increased hemolysis. Phosphatidylserine expression was reduced and there was no difference in osmotic fragility. Whole blood did not demonstrate an acceleration in storage lesion severitydespite being 21 days beyond its expiration date. The erythrocytes within the expired whole blood maintained membrane integrity and function during the study period. Our findings regarding the red blood cell storage lesion in pRBC units are consistent with previous data from our and other laboratories (reviewed in(26, 50, 51)). Taken together, our data suggest that the red blood cell storage lesion is no more severe in whole blood units subjected to prolonged storage than in pRBCs, and that further investigation of prolonged whole blood storage is warranted.

We utilized rotational thromboelastometry (ROTEM) and platelet aggregometry assays to assess for changes in the coagulation potential of the stored whole blood. We found that several viscoelastic parameters were superior for in-date whole blood when time-matched and compared to pRBCs tested in combination with fresh frozen plasma. When whole blood was extended beyond the standard storage period, coagulation potential, although impaired, remained similar to pRBCs with FFP.Recent studies have shown that the platelets in cold-stored whole blood retain hemostatic function(16, 52, 53). We also found that platelet aggregation was still present, albeit impaired, by day 21 in conjunction with the standard aggregation agonists, ADP and ASPI. This impairment in aggregation was overcome with the addition of calcium chloride. However, by day 42 of storage, platelet functionality was significantly impaired. Our data is consistent with (and supports) recent reports (16, 53). The viscoelastic data suggests that although impaired, the clotting factors also maintain functionality up to day 42 of storage when compared to aged pRBC:FFP in a 1:1 ratio. Previous studies from our group have demonstrated alterations in whole blood maximum clot firmness and platelet aggregation during whole blood storage (54). The present study extends these observations for whole blood and suggests continued diminution of coagulation factor activity during prolonged storage.

We utilized a murine based model of hemorrhage and resuscitation to evaluate potential in-vivo implications of transfusion with whole blood that has been stored for an extended duration. Our data suggests thatrecipient mice tolerated transfusion of extended-storage whole blood. When assessing the measures of systemic inflammation post-transfusion, there was either a reductionin the inflammatory response or similar response as compared to pRBCs. Transfusion of whole blood and pRBCs were resulted in similar cell-free hemoglobin content in the recipient circulation. Previous studies from our and other laboratories demonstrate that resuscitation with aged pRBC units leads to an increased inflammatory response as compared to the use of fresh pRBC units and that whole blood transfusion is less inflammatory than other resuscitation strategies(55–57). Data current from the current study indicate that the red blood cell storage lesion is similar during storage in AS-3 as compared to storage in whole blood.

With the re-discovery of whole blood as a beneficial component of resuscitation strategiesin massively hemorrhaging trauma patients(5, 58–61), many centers are implementing programs that focus on whole blood administration starting in the prehospital phase of care and extending until control of hemorrhage is achieved(62, 63). While the FDA has approved storage of pRBCs for 42 days in various storage solutions, the storage of whole blood is limited to 21 days in CPD, CP2Dand 35 days in CPDA-1(64). The FDA established shelf-life is based on a minimum of 75% RBC survival in recipient circulation 24 hours after transfusion(48, 65–67). Previous studies have examined some aspects of red blood cell viability in whole blood beyond the standard storage period (68, 69). The present study expands this work significantly by including additional aspects of the red blood cell storage lesion in comparison to packed red blood cell units and examining the utility of extended-storage whole blood for resuscitation after hemorrhage in a murine model. This study indicates that other parameters aside from solely RBC survival are of importance when establishing the storage shelf life of whole blood. Our data indicates that the erythrocytes, platelets, and coagulation factors within whole blood remain viable past their current shelf-life.

While this study demonstrates promising findings, there are several limitations to this study. Although the stored whole blood and pRBCs developed changes that are recognized as aspects of the red blood cell storage lesion, the blood products were stored in different materials than are utilized in blood banks. This variation from standard materials may limit the generalizability of our data. While utilization of male-only donors enabled controlling for possible confounding variables that are donor-specific such as age and gender, the human sample size was relatively limited. Further work is needed to thoroughly understand these issues as previous studies have shown that there are potentially significant clinical implications to donor factor variation (70–73). Although we found evidence that transfusion of expired whole does not worsen the recipient’s systemic inflammatory response to resuscitation, the murine in-vivo findings may not be applicable to human transfusion. In this study we utilized CPD, a storage anticoagulant that is approved for 21 days of storage. We did not investigate the impact of using CP2D or CPDA-1 on red blood cell storage lesion. Previous studies suggestthat the anticoagulant does not impact the coagulation potential during the standard storage period(53). However, the implications of the red blood cell storage lesion when utilizing CP2D/CPDA-1 beyond the FDA approved storage period were not the focus of this study and would require further investigation. Therefore, studies to further characterize in-vitro aging of erythrocytes in stored whole blood beyond the storage duration as well as in-vivo consequences of transfusion will be needed.