Journal of Trauma and Acute Care Surgery:
EAST 2014 Plenary Papers
Cryopreserved red blood cells are superior to standard liquid red blood cells
Hampton, David A. MD; Wiles, Connor; Fabricant, Loïc J. MD; Kiraly, Laszlo MD; Differding, Jerome MPH; Underwood, Samantha MS; Le, Dinh MD; Watters, Jennifer MD; Schreiber, Martin A. MD
From the Oregon Health and Science University, Division of Trauma, Critical Care and Acute Care Surgery, Trauma Research Institute of Oregon (D.A.H., L.J.F., L.K., J.D., S.U., D.L., J.W., M.A.S.); and Portland State University (C.W.),Portland, Oregon.
Submitted: December 5, 2013, Revised: February 9, 2014, Accepted: February 10, 2014.
This study was presented at the American College of Surgeons’ Committee on Trauma Meeting, March 21–23, 2013, in San Diego, California, where it received second place in the residents’ paper competition (clinical science). The study was also presented at the 27th Annual Scientific Assembly of the Eastern Association for the Surgery of Trauma, January 14–18, 2014, in Naples, Florida. Winner of the 2014 Raymond H. Alexander, MD Resident Paper Competition.
Address for reprints: David A. Hampton, MD, MEng, Oregon Health and Science University, Division of Trauma, Critical Care and Acute Care Surgery, Trauma Research Institute of Oregon (TRIO), 3181 SW Sam Jackson Park Rd, Mail Code L-611, Portland, OR 97239-3098; email: firstname.lastname@example.org.
Liquid preserved packed red blood cell (LPRBC) transfusions are used to treat anemia and increase end-organ perfusion. Throughout their storage duration, LPRBCs undergo biochemical and structural changes collectively known as the storage lesion. These changes adversely affect perfusion and oxygen off-loading. Cryopreserved RBCs (CPRBC) can be stored for up to 10 years and potentially minimize the associated storage lesion. We hypothesized that CPRBCs maintain a superior biochemical profile compared with LPRBCs.
This was a prospective, randomized, double-blinded study. Adult trauma patients with an Injury Severity Score (ISS) greater than 4 and an anticipated 1-U to 2-U transfusion of PRBCs were eligible. Enrolled patients were randomized to receive either CPRBCs or LPRBCs. Serum proteins (haptoglobin, serum amyloid P, and C-reactive protein), proinflammatory and anti-inflammatory cytokines, d-dimer, nitric oxide, and 2,3-DPG concentrations were analyzed. Mann-Whitney U-test and Wilcoxon rank sum test were used to assess significance (p < 0.05).
Fifty-seven patients were enrolled (CPRBC, n = 22; LPRBC, n = 35). The LPRBC group’s final interleukin 8, tumor necrosis factor α, and d-dimer concentrations were elevated compared with their pretransfusion values (p < 0.05). After the second transfused units, 2,3-DPG was higher in the patients receiving CPRBCs (p < 0.05); this difference persisted throughout the study. Finally, serum protein concentrations were decreased in the transfused CPRBC units compared with LPRBC (p < 0.01).
CPRBC transfusions have a superior biochemical profile: an absent inflammatory response, attenuated fibrinolytic state, and increased 2,3-DPG. A blood banking system using both storage techniques will offer the highest-quality products to critically injured patients virtually independent of periodic changes in donor availability and transfusion needs.
LEVEL OF EVIDENCE
Therapeutic study, level II.
A packed red blood cell (PRBC) transfusion is designed to treat anemia and increase end-organ perfusion. Its efficacy is based on the red blood cells’ ability to traverse a microcapillary network and off-load oxygen.1 PRBC are stored using standard liquid preservation protocols. Donated blood mixed with citrate-phosphate-dextrose-adenine is centrifuged; the plasma is partially removed, and it is stored at 2°C to 8°C.2 The US Food and Drug Administration (FDA) has approved liquid preserved RBCs (LPRBCs) for a 42-day shelf life, after which the blood can no longer be transfused. Each year, 3% of donated blood is removed from circulation, secondary to its expiration, resulting in an estimated cost of $80 million.
While awaiting use, PRBCs undergo a collection of well-documented biochemical and structural changes known as the storage lesion. These changes adversely affect a cell’s life span, ability to off-load oxygen, and capacity to undergo conformational changes necessary for navigating the capillary network. Depletion of nitric oxide (NO) impairs vasoregulation, increasing intravascular resistance.3 Hemolysis and free hemoglobin scavenging of endothelial-derived NO further exacerbate this effect.4 Depletion of 2,3-diphosphoglycerate (2,3-DPG) impairs oxygen off-loading.5 Morphologic alterations culminate in nondeformable spheroechinocytes, which are unable to traverse capillary beds exacerbating the perfusion deficit.6 This constellation of changes results in decreased tissue oxygenation (StO2).
Transfusions of LPRBC have also been associated with prolonged hospitalization, increased rates of infection, prolonged coagulation times,7 transfusion-related acute lung injury (TRALI),8 multiorgan failure,9 multiple organ dysfunction syndrome,10 postoperative complications in cardiac surgery11 and mortality. The association of these complications is more significant with prolonged storage times.12 Currently, blood banks distribute PRBCs from a queue; the oldest cells are the first to be transfused. Therefore, critically ill patients receive PRBCs that have assumed the maximum storage lesion. Attempts to avoid these time-dependent changes by shortening the 42-day storage period could potentially deplete national or regional reserves. Even with the longer storage duration, some institutions operate with a 1-day to 2-day storage reserve; therefore, a storage method to augment or replace LPRBCs is warranted.
An alternative method, cryopreservation, pioneered by Dr. Audrey Smith in the 1950s, has the capacity to virtually eliminate the storage lesion and resolve the supply short falls.13 Although there are many techniques used, the FDA has approved cryopreserved storage at −80°C using a 40% weight/volume glycerolization. A glycerol buffer is used to prevent membrane injury from crystal formation and osmotic lysis secondary to the extreme temperature changes. When required, the samples are thawed, deglycerolized, and transfused. The FDA requires donated blood to be cryopreserved within 2 days to 6 days of donation, and it is approved for a 10-year storage life.14 Once thawed, the PRBC can be stored at 1°C to 6°C for an additional 14 days.15 Recent literature has shown that the storage lesion associated with cryopreservation is limited to the 6-day window before freezing.16 Arresting the storage lesion’s evolution and extending the PRBC’s shelf life could potentially eliminate transfusion-related complications seen with traditional storage methods.
In 1966, units of cryopreserved PRBCs (CPRBCs) were sent to the Naval Support Activity Hospital in Da Nang, Vietnam. During the South East Asian conflict, the FDA had approved a 21-day shelf life for LPRBCs. This truncated period resulted in the destruction of nearly 50% of all donated blood. CPRBCs eliminated this deficit.17 Injured service members received CPRBCs without experiencing a transfusion-related complication as compared with those receiving LPRBCs. For several decades following the Vietnam War, Massachusetts General Hospital, Chicago’s Cook County Hospital, and 30 other institutions throughout the United States developed and maintained robust CPRBC programs.18 These efforts augmented their respective LPRBCs stores. Because of advances in LPRBC storage techniques, approval of the 42-day shelf life, and concerns for improper screening methods, the daily use of CPRBCs fell out of favor. Currently, CPRBCs are routinely used for patients with rare blood types or in the military setting aboard hospital ships and during the recent Middle East conflicts.
Our institution recently performed a prospective randomized trial comparing LPRBCs and CPRBCs. Differences in outcome, correction of anemia, conventional coagulation tests, or thromboelastogram data were not appreciated; however, CPRBC transfusions were associated with a significantly increased StO2.19 This difference was noted immediately after transfusion was initiated and persisted for 180 minutes, after which it was no longer significant. A biochemical analysis comparing the two types of blood has not yet been performed. We hypothesized that CPRBCs have a superior biochemical profile compared with LPRBCs.
PATIENTS AND METHODS
This was a prospective, randomized, double-blinded study. The methods were elaborated upon in our previous study.19 Institutional review board approval was obtained. Patients admitted to the Oregon Health and Science University trauma service with an Injury Severity Score (ISS) greater than 4 and the potential need for a blood transfusion were eligible. Patients who had received a massive transfusion within the last 3 months, who had received a transfusion within the previous 24 hours, who were pregnant, or who were younger than 15 years were excluded. Enrolled patients were originally randomized into three groups (CPRBCs, LPRBCs ≤ 14 days and LPRBCs > 14 days). Because of a lack of difference between the LPRBC groups in this pilot study, they were combined.
Consent was obtained from the patient or designated medical representative. Before initiation and after completion of each transfusion, 10-mL blood samples were obtained from the patients. After 300 mL of the transfusion was completed, 12-mL test samples were obtained from the LPRBC and CPRBC units. Final laboratory samples were obtained 12 hours after completion of the last transfusion. All samples were assessed for biochemical changes. Haptoglobin (Hg), serum amyloid P (SAP), and C-reactive protein (CRP) were evaluated using the Bio-Plex Pro Human Acute Phase 4-Plex Panel (Bio-Rad Laboratories Inc.). SAP and CRP were studied because of their known anticoagulation effects. Elevated levels of SAP, in the presence of heparin, can cause a hypocoagulable state.20 Hemolysis during storage could be assessed through changes in Hg, a free hemoglobin scavenger. NO metabolites were measured with a Sievers Nitric Oxide Analyzer 280i (GE Analytical Instruments, Boulder, CO). 2,3-DPG levels were quantified with a commercially available kit (Roche Diagnostics, Indianapolis, IN). Cytokines (interleukin 2 [IL-2], IL-4, IL-6, IL-8, IL-10, granulocyte macrophage colony-stimulating factor [GMCSF], tumor necrosis factor α [TNF-α], and interferon γ [IFN-γ]) were measured using the Human Cytokine 8-Plex Assay (Bio-Rad Laboratories Inc., Hercules, CA). Coagulation data and d-dimers were measured using standard tests (Diagnostica Stago Inc., Parsippany, NJ).
All data were analyzed using SPSS version 19 (IBM, Armonk, NY). All continuous normally distributed data were analyzed using analysis of variance and paired Student’s t tests. All nonparametric data were analyzed using a Mann-Whitney U-test and Wilcoxon rank sum test. Significance was set at p < 0.05.
Fifty-seven patients participated in the study. Thirty-five received LPRBCs; 22 received CPRBCs. CPRBCs were stored for a longer time compared with the LPRBCs (Table 1, p < 0.01). Serum concentrations of Hg, SAP, and CRP were all decreased in the CPRBC units compared with LPRBC (Table 1, p < 0.01).
Regarding the patient’s biochemical profiles, there were no differences in the baseline and 12-hour posttransfusion proinflammatory cytokines IL-2, IL-6, IFN-γ, or GMCSF, in either group (Fig. 1A–D). However, IL-8 and TNF-α were elevated compared with pretransfusion values in the LPRBC group (Fig. 2, p < 0.05). These proinflammatory cytokine changes were not observed in the CPRBC group. A difference in anti-inflammatory cytokines, IL-4 and IL-10, was not seen in either group (Fig. 3). Neither group demonstrated a change in NO at any of the four measured time points (Fig. 4A). d-dimer was higher in the LPRBC group 12 hours after transfusion (Fig. 4B, p < 0.05).
Finally, a difference in 2,3-DPG was not seen between either group until after the second transfused unit when it was greater in those receiving CPRBCs (Fig. 5, p = 0.04). This difference persisted throughout the remainder of the study (p = 0.01).
Maximizing tissue oxygenation while minimizing morbidity is the goal of any transfusion. The optimization of stored blood for transfusion has been an evolutionary process that is currently ongoing. The ideal duration, temperature, and preservative solutions to store liquid blood and to combat known structural and biochemical changes have not been determined. These changes, the storage lesion, have led to numerous transfusion-related complications. They include increased mortality, longer hospitalization, increased infection rates, multiple organ dysfunction syndrome,10 inflammatory protein-related hypocoagulation,20,21 hyperkalemia-associated cardiac arrhythmias,22 tissue hypoxia,23 as well as free hemoglobin-related hypertensive crises and acute renal tubular injuries.24 This nonexhaustive list has not prevented the use of LPRBC. Our study revealed that LPRBC transfusion is associated with decreased 2,3-DPG, a proinflammatory response, elevation of potentially harmful proteins and fibrinolysis.
With a near-universal storage protocol and known storage lesion, research has targeted storage age as a metric toward improving tissue oxygenation. In rat cecal injury models, resuscitation with blood stored for 3 days or 28 days was investigated.25 Rats receiving blood stored for 3 days demonstrated improved oxygen uptake versus those receiving older blood. In a study from our institution, trauma patients receiving LPRBCs stored for 20 days or less demonstrated no change in StO2 versus patients receiving LPRBCs stored for 21 days or greater who experienced a decrease in StO2.23 The oxygenation shortfall encountered could be a product of delivery or off-loading. During liquid preservation, RBCs will metabolize glucose-generating adenosine triphosphate, lactic acid, and protons. The increasing acidic environment arrests glycolysis, adenosine triphosphate production, and promotes breakdown of 2,3-DPG.22 This could explain the LPRBC groups’ inferior 2,3-DPG profile. Conversely, cryopreservation suspends these biochemical pathways and, as evidenced by our findings, results in superior 2,3-DPG levels. In addition, the theoretical removal of nonviable and abnormally shaped RBCs during the washing and deglycerolization process may have also contributed to a greater oxygen delivery capacity and subsequent tissue off-loading.
StO2 is also limited by the RBCs’ ability to traverse the capillary network. NO’s vasodilatory capacity reduces vascular resistance and increases end-organ perfusion.26 Our study did not demonstrate a difference in NO. Previous work investigating NO has shown that increased levels of free hemoglobin secondary to hemolysis resulted in decreased NO levels and a perfusion deficit.27 Oxygen delivery is also affected by the RBCs’ deformability. Inflexible cells unable to negotiate capillary beds will not reach hypoxic tissues. It has been shown that decreased RBC deformability and prolonged storage times result in decreased oxygen delivery.28 The LPRBCs inabilities to vasodilate and change conformation are two obstacles, which will perpetuate and exacerbate end-organ hypoxia.
Our findings demonstrated that LPRBCs were also associated with an elevation in inflammatory cytokines, IL-8 and TNF-α. This response may have been catalyzed by residual donor plasma, proinflammatory proteins, and senescent white and red blood cells. TRALI, which is characterized by noncardiogenic pulmonary edema, tachypnea, fever, and hypotension, is a potentially life-threatening complication, which occurs within 6 hours of transfusion. Elevated levels of IL-8 are associated with TRALI.29 This association is strengthened by repeated LPRBC transfusion resulting in stimulation of neutrophils to express and peripheralize IL-8.30,31 Donor plasma has also been shown to incite the production of IL-1, IL-6, IL-8, and TNF-α.30–32 Numerous studies have demonstrated an association between elevated levels of TNF-α and septic shock, multiorgan failure, cardiac failure, and death.33 Massive transfusions of LPRBCs unnecessarily subject critically ill patients to the potential development of TRALI and other adverse consequences of elevated TNF-α levels. All of the aforementioned inciting factors (inflammatory proteins and senescent cells) are removed during the CPRBC washing and deglycerolization process.18,21 Patients who received CPRBC transfusions did not exhibit an increase in proinflammatory or anti-inflammatory cytokines. This absent cytokine elevation was also noted by the group of Hult et al.,34 who recently transfused 2 U of autologous CPRBC into human volunteers. A proinflammatory cytokine response was not apparent 48 hours after transfusion in any subject.
There was a significant intragroup difference in SAP and CRP concentrations. SAP levels greater than 200 mg/dL and CRP level greater than 210 mg/dL are known to impair coagulation.20 SAP’s anticoagulant activity is complemented by heparin; 100-mg/dL concentrations of SAP in the presence of heparin will arrest the coagulation pathways.20 LPRBCs concentrations were three to seven times less than these thresholds; however, during a massive transfusion, a coagulopathy related to these proteins may be apparent.
Finally, the fibrinolytic state, a known complication associated with transfusion of old blood, was evidenced by d-dimer elevation in the LPRBC group. Trauma initiates peripheralization of tissue plasminogen activator and plasminogen activator inhibitor type 1. The predominant outcome, fibrinolysis or coagulation, seems to be largely dependent on the severity of trauma and the organ system affected. Complementing the patients’ coagulation status, recent studies have shown an age-dependent relationship between LPRBC storage duration and prolonged coagulation times,7 increased d-dimer concentrations,35 and increased fibrinolytic activity.36 Hemorrhage in trauma is a preventable cause of death; complementing the fibrinolytic state with an LPRBC transfusion with similar properties exacerbates the coagulation insult. The rise in d-dimer was not apparent in patients receiving CPRBC.
The aforementioned transfusion-related complications were not seen by the US Navy during their initial trials of CPRBCs in Da Nang, Vietnam.17 Service members who received CPRBC demonstrated equivalent posttransfusion hemoglobin, platelet counts, and serum creatinine compared with those receiving LPRBCs.21 Although only 465 U of CPRBCs were documented to be transfused over a 180-day period compared with an average 600 U of LPRBCs given daily (maximum 1,200 U of LPRBCs per day, February 1969), the technology’s feasibility was demonstrated. CPRBC use segued into the civilian sector and was widely used at Massachusetts General Hospital, Chicago’s Cook County Hospital, The New York Blood Center, and numerous international organizations.18 Civilian institutions directed their efforts toward establishing reserves of rare blood types, excess supplies for chronically anemic patients, and stock piling for national disasters. With the advent of the longer storage life for LPRBCs (42 days vs. 21 days), shared resources, and concerns for inadequate pre–human immunodeficiency virus epidemic era screening techniques, the demand for frozen blood in the civilian setting diminished considerably.
CPRBCs are still a robust solution for a blood banking system whose supply may be affected by increased demand or restricted storage durations. The 2001 New York City and Pentagon and 2005 London terrorist attacks demonstrated how systems with adequate refrigerated storage infrastructures were able to endure anticipated resource strains. During the first 24 hours, London hospitals received 360 casualties, and 24 patients used 336 U of PRBCs.37 The UK National Blood Service maintains a daily supply of 55,000 U of LPRBCs. During the New York City and the Pentagon attacks, 200 hospitalized patients required 258 U of blood.38 In both instances, RBC donations dramatically exceeded demand. In the United States, more than 475,000 U of RBCs were collected, 17% were discarded because of shelf life restrictions, and only 9,500 U were cryopreserved. The immediate increased number of donations after these events underscored the public’s desire to assist during a time of crisis rather than an institutional outcry to fulfill a projected need. These major metropolitan areas have well-established resources, and a reserve blood supply deficit was not experienced or anticipated. However, a mature robust blood banking program that uses cryopreservation may have been able to salvage more than the 2% stored after the September 11 attacks.
Conversely, austere environments, where limited blood supplies are frequently encountered, present a different scenario. The 2005 Pakistan earthquake demonstrated the need for an alternative system. Four of the regions’ seven hospitals were not equipped for blood banking services. Therefore, because of poor documentation, screening, and storage facilities, the quality and quantity of available blood were uncertain. Three to five days after the earthquake, patient transfusion requirements nearly exhausted the limited LPRBC reserves. When the patient surge and increased transfusion requirement subsided, the transfusion services were established. During the initial recovery efforts, an established regional cryopreserved blood banking system would have eliminated the discontinuity between patient demand and available supply.
Currently, the US Department of Defense has prepositioned more than 50,000 CPRBC units around the world.39 In the event of an international conflict or natural disaster, these CPRBCs could augment the mobile blood banks assigned to forward-deployed units or local hospitals. Analogous to the Pakistan earthquake and the dynamic patient population being treated, the absence of blood banking services in these austere environments is uniquely addressed by cryopreservation. Even within an industrialized country with remote inaccessible populations because of inclement weather or natural disaster, the traditional means to adequately support a regional hospital may be temporarily compromised. CPRBC is a long-term solution to these short-term obstacles.
There were several limitations to our study. This was a pilot investigation, and it was not powered to assess clinical outcomes. A larger study population may have demonstrated a difference; this study is currently ongoing. The small sample size also limited our ability to determine biochemical differences between older and newer LPRBCs.19 When comparing newer versus older LPRBCs, larger populations may have demonstrated a benefit congruent with the StO2 results seen in ours and previous animal studies. The StO2 and circulating NO concentrations presented may have been related to differences in free hemoglobin between the study populations. The adverse effects of larger free hemoglobin concentrations and decreased StO2 and NO may be seen in the setting of a larger transfusion, and this is currently being investigated.
Patient laboratory data acquisition was limited to the first 12 hours; therefore, biochemical changes that developed beyond this period were not investigated. Finally, patients were limited to 2 U because we wanted to focus on the effects of blood transfusion in the absence of the shock state or other confounders seen in patients requiring larger transfusion volumes. Because of the limited transfusion volume, the beneficial or detrimental effects of a large CPRBC or LPRBC transfusion were not studied.
CPRBCs possess a superior biochemical profile compared with LPRBCs. Our study revealed the global benefits of a CPRBC transfusion: increased 2,3-DPG, removal of proinflammatory proteins, and the absence of a fibrinolytic state. These findings could potentially be translated to clinical benefits in patients receiving massive transfusions with CPRBCs. The equivalent inpatient outcomes and lack of transfusion-associated reactions make CPRBCs an attractive alternative to LPRBCs. Their 10-year shelf life can buffer national and regional blood supplies against periods of high demand, need for rare blood types, or support efforts in austere environments. CPRBCs may represent a paradigm shift from the tradition-based LPRBC to a more robust storage solution.
D.A.H., L.J.F., and M.A.S. conducted the literature search for this study. L.K., J.D., S.U., J.W., and M.A.S. contributed to study design. C.W., L.J.F., L.K., J.D., S.U., and J.W. performed data collection. D.A.H., C.W., L.J.F., J.D., S.U., and D.L. analyzed the data. D.A.H., L.J.F., and M.A.S. interpreted the data. D.A.H. and M.A.S. wrote the manuscript. D.A.H., J.D., and M.A.S. contributed to critical revision.
This study was supported by the 711th Human Performance Wing, Air Force Research Laboratory, under agreement number FA8650-09-2-6035 and FA8650-10-2-6143.
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Hampton and colleagues have presented the results of a prospective, randomized, double-blinded study comparing standard liquid preserved stored packed red blood cells (LPRBCs) to cryopreserved packed red blood cells (CPRBCs). In this study they hypothesized that CPRBCs maintain a superior biochemical profile compared to LPRBCs after transfusion.
The advantages of CPRBCs are its long storage life (up to 10 years) (Vox Sang. 2000;79:168–74), ability to serve as an immediate storage reservoir if our current liquid preservation strategies were to become unusable and/or depleted and preservation of rare phenotypes. Physiologically, CPRBCs retain pre-donation shape and function; suffer minimal storage lesion (J Thorac Cardiovasc Surg. 1960;40:611–24) and nearly 70–80% of pre-donation red blood cells are retained as viable.
There are a few disadvantages to this technology. First, the time required to prepare CPRBCs for transfusion can be up to one hour (virtually eliminating its use in the patient in shock). Second, it’s much more expensive to prepare on a unit-per-unit comparison to LPRBC, by to two- to three-fold. Finally, centers that don’t currently utilize this technique would require acquisition of equipment and processes.
The authors’ data support their hypothesis of a superior biochemical profile with CPRBCs, including increased 2,3-DPG, less pro-inflammatory proteins and the elimination of a fibrinolytic state. This study is underpowered to assess clinical outcomes, however this group has a larger study underway to address this endpoint.
Transfusion of RBCs is lifesaving but carries significant morbidity and mortality in a dose dependent manner (Arch Surg. 2012;147:49–55 & 55–56). Despite increases in oxygen content post-transfusion, the efficacy of transfusion to increase end organ oxygen consumption outside of hemorrhagic shock or symptomatic isovolemic anemia is debatable (Crit Care Clin. 2004;20:255–68).
Based on Hampton et al’s data in this and their prior studies, CPRBCs may be a more physiologically efficacious and a less immunomodulatory solution for transfusion. Combine this with restrictive transfusion practices (limiting blood draws, small volume sampling and transfusion based on clinically symptomatic isovolemic anemia) and the preparation and use of CPRBCs could become cost neutral by virtue of decreased overall need for transfusion. However, one could argue that the increased cost is acceptable for a superior product. I look forward to their ongoing work in this endeavor.
Levi Procter, MD
Department of Acute Care Surgery, Trauma and Surgical Critical Care
University of Kentucky Chandler Medical Center
Cryopreservation; transfusion; cytokines
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