Narrative review of strategies for blood product shelf‐life extension

Over 90% of potentially survivable United States (U.S.) battlefield fatalities are associated with severe hemorrhage, underscoring the importance of early prehospital blood transfusion to improve survival outcomes. Future near-peer conflicts, such as Large-Scale Combat Operations (LSCO), will introduce new challenges, including increased severity of injury patterns, delayed evacuations, and limited resupply of blood products. In such environments, finding innovative ways to prolong the shelf-life of blood products is critical to ensuring the distribution of an adequate blood supply across operational theaters. This narrative review focuses on current and future methods to extend the shelf-life of blood products. Many of the highlighted blood products are still in the development pipeline pending regulatory approval and may help support future blood banking constraints or augment whole blood use during times of surge. Massive hemorrhage remains the leading cause of potentially preventable death in trauma patients in both civilian and military sectors.1, 2 These deaths often occur within 1 h due to exsanguination, coagulopathy, and shock.1, 3, 4 Restoring blood volume, neutralizing coagulopathy, and re-establishing oxygen and hemostatic function within the first few minutes are crucial to preventing mortality.5 Thus, trauma resuscitation guidelines emphasize early whole blood or component therapies targeting a 1:1:1 ratio of red blood cells (RBCs), plasma, and platelets.6, 7 Around the world, military and civilian institutions are developing Walking Blood Bank programs to facilitate urgent resuscitation efforts in resource-constrained environments.8 While observational evidence supports the association of low titer group O whole blood (LTOWB) with improved early mortality, logistical benefits, and reduced product utilization versus component therapy, ongoing randomized controlled trials, such as the Low-Titer Group O Whole Blood or Products (TROOP) Trial, are needed to solidify its superiority.9, 10 Nevertheless, whole blood has limited availability, thus requiring additional blood products during times of constraint or surge. Future LSCO are expected to have increased rates and severity of injuries, straining blood product inventories.11 It is anticipated that inventory, resupply, and operational mobility will be challenged due to a highly mobile battlefield, lack of air superiority, and interruption of communications, with modern estimates of anticipated blood requirements rising to 10,000 units per month.12-14 Civilian health systems have also had challenges maintaining blood inventories during mass casualty events, exemplified by poor donor turnout resulting in shortages during the COVID-19 pandemic.15 The relatively short shelf-life of most blood products adds complexity to maintaining adequate blood inventories,16 and storage results in physiological reductions in function, often referred to as ‘storage lesions’.17, 18 Maintaining blood inventories during LSCO or mass casualty events will require leveraging all existing blood products through advancements that improve shelf-life.19, 20 Here, we review the current methodologies for improving the shelf-life and efficacy of blood products, including anticoagulants, additives, deoxygenation/anaerobic storage, cryopreservation, and lyophilization of various blood components. Table 1 summarizes the major blood product shelf-life extension strategies discussed in this review, highlighting their mechanisms and potential advantages in disaster settings (Table 1). Yoshida, T., et al., Vox Sang, 2007. p. 22–3160 D'Alessandro, A., et al., Transfusion, 2020. p. 786–79866 Rabcuka, J., et al., Blood Adv, 2022. p. 5415–542864 Dumont, L. J., et al., Transfusion, 2016. p. 392–40363 Cap, et al., Military Medicine, 2018. p. 52–5467 Chang et al., Blood Transfusion, 2017. p. 341–34768 Strandenes, G., et al., Anesthesiology, 2020. p. 1173–118355 Zantek, N.D., et al., Clin Trials, 2023. p. 36–4656 Apelseth, T.O., et al., Science Series, 2017. p. 488–49557 Valeri, C.R. et al., Transfusion, 2005. p. 1890–881 Cohn, C.S., et al., Vox Sang, 2017. p. 684–68883 Cid, J., et al., Transfusion, 2016. p. 580–684 Noorman, F., et al., PLoS One, 2016. p. e016840187 Reade, M.C., et al., BMJ Open, 2022. p. e06893382 Whitman, G. et al., JTCVS Open. 202291 Zaza, M. et al., Damage Control Resuscitation, 2019. p. 145–6295 Pusateri, A. E., et al., Anesthesiology, 2022. p. 327–335105 Shlaifer, A., et al., J Trauma Acute Care Surg, 2017. p. 675–682102 Cuenca, et al., J Spec Oper Med, 2020. p. 43–45103 Benov, A., et al., Injury, 2016. p. 993–1000101 Zaza, M. et al., Damage Control Resuscitation, 2019. p. 145–6295 Shuja, F., et al., J Trauma, 2011. p. 664–71112 Pusateri, A.E., et al., Transfusion, 2016. Suppl 1 p. S16-2399 Bynum, J.A., et al., Transfusion, 2019. p. 1490–1498118 Kuhn, B.J., et al., J Thromb Haemost, 2024. p. 686–699119 Ohanian, M., et al., Am J Hematol, 2022. p. 256–266120 Goodrich, R.P., et al., Proc Natl Acad Sci USA, 1992. p. 967–7122 Inducing transient pore formation on RBC membranes to facilitate trehalose loading and allow for lyophilization of RBCs, and increase recovery of viable RBCs The primary challenge in maintaining a supply of blood is its inherent ability to clot ex vivo, prompting the development of anticoagulants to temporarily inhibit this process. Citrate is the primary component of anticoagulant solutions for transfusable blood, serving as a safe, reversible calcium chelator to inhibit the calcium-dependent coagulation cascade.21 Human blood transfusion with citrate began in 1914 but was standardized by Richard Lewisohn's work in 1915, which established an optimal dosing regimen of sodium citrate and confirmed its biocompatibility in both canine and human subjects; a follow-on publication reported 22 successful clinical transfusions.22 His work enabled widespread, reproducible, large-volume blood transfusions. Building on this foundation, in 1918, U.S. Army physician Oswald Hope Robertson was credited with developing the “world's first blood bank.”23 Using the “Robertson Blood Jar,” a single-operator blood collection apparatus prefilled with 3.8% citrate preservative for the collection of universal donor blood, units could be stored for 10 to 14 days on average before transfusion24 and transported to forward casualty clearing stations for critical transfusions.22 Modern anticoagulant solutions are still primarily reliant on citrate but also incorporate nutrients and buffers such as dextrose and phosphate to preserve cellular function. Table 2 provides a comparative overview of commonly used anticoagulant and additive solutions, including their approved storage durations, key features, benefits, and selected challenges relevant to blood product storage (Table 2). WB is currently stored in citrate–phosphate-dextrose (CPD), citrate–phosphate-double dextrose (CP2D), and citrate–phosphate-dextrose-adenine (CPDA-1). CPD and CP2D are permitted to be stored at 4°C for up to 21 days, while CPDA-1 allows for 35 days.16, 25-28 Results from a comparative in vitro analysis of WB stored in CPD, CP2D, or CPDA-1 for 35 days demonstrated no significant declines in mean platelet volume, hematocrit, or hemoglobin by day 35, although there were significant decreases in platelet count, coagulation function, and platelet aggregation in CPD and CP2D, with higher levels of lactate observed in CP2D by day 14.26 Interestingly, the decline in function of older or expired WB was mitigated in a simulated massive transfusion by mixing fresh blood with old at ratios of 1:4 or 1:5.26 This serves as a proof-of-concept for extended CPD-/CP2D-stored WB in resource-limited environments, especially in concert with a fresh unit from a walking blood bank. Additive solutions (AS) serve as nutrients for packed RBCs during storage at 1–6°C.29 AS maintain the levels of 2,3-diphosphoglycerate (2,3-DPG) in RBCs, which are critical for optimal hemoglobin-oxygen affinity in post-transfusion peripheral tissue oxygenation.30 AS also maintain intracellular adenosine triphosphate (ATP) levels integral to RBC morphology, post-transfusion recovery, and survival.30 There are currently four FDA-approved RBC AS: AS-1 (Adsol®, Fenwal Inc.), AS-3 (Nutricel™, Cutter Biological), AS-5 (Optisol™, Terumo Blood and Cell Technologies), and AS-7 (The SOLX™ System, Hemerus Medical), all of which currently allow for a 42-day shelf-life.16 AS-7 is an alkaline solution designed to support RBC metabolism, reduce the storage lesion, and provide buffering capacity.29, 31, 32 While AS-7 is FDA-approved for 42-day storage, Cancelas et al. discovered that 82% ± 3% of 56-day AS-7 stored RBCs remained viable at 24 h post-transfusion during autologous in vivo studies, aligning with FDA guidelines that require at least 75% RBC viability with <1% overall hemolysis.29 Dumont et al. also demonstrated AS-7's preservation of RBC ATP maintenance in CPD WB over 56 days when initially held for up to 18 h at room temperature (20–24°C) prior to RBC processing and refrigeration.33 In the 42-day storage arm, all 27 human recipients in the in vivo post-transfusion arm also met FDA criteria.33 The development of AS that allows for delaying WB processing while still permitting up to 42 days of cold storage is advantageous, as it operationally accommodates improved inventory management, extended transport windows, and blood collection outside of high-risk areas. RBC rejuvenation is an FDA-approved process in which aged RBCs are briefly incubated in a solution (such as pyruvate, inosine, phosphate, and adenine) to restore the generation of 2,3-DPG and ATP, improving oxygen unloading capability and reducing RBC storage lesions.34, 35 Decades of studies have been conducted, with the first clinical report of the administration of rejuvenated RBCs to humans in 1972.36, 37 Cardiac surgery patients receiving rejuvenated, frozen, deglycerolized cells (stored for 1 year) exhibited higher 2,3-DPG levels and improved oxygen utilization and cardiac index compared to patients receiving stored allogeneic or autologous RBCs.38 Interestingly, the rejuvenation process also reduced potassium levels in stored blood,37 potentially explained by the corresponding reduction in hemolysis and maintenance of ATPase cation pump functionality.37 However, the potential impacts of using rejuvenated red cells are unclear, including the contributions of solution components, iron homeostasis, oxidative metabolites (such as hypoxanthine), and long-term circulation post-transfusion. Thus, clinical trials are warranted to validate the safety and efficacy of rejuvenated RBCs, especially for massive transfusion cases. Platelet additive solutions (PAS) are designed to preserve platelet function during storage. PAS also dilute fibrinogen and coagulation factors by replacing 60–70% of plasma, thereby reducing unintended platelet activation and the risk of transfusion reactions.39-43 The components of PAS vary but generally include acetate, citrate, phosphate, potassium, magnesium, and glucose, which serve as metabolic fuels for the citric acid cycle, act as pH buffers, and help maintain normal platelet morphology.40, 44-46 More recently, PAS are also being used for cold-stored platelets (CSP). Traditionally, platelets are stored at room temperature (20–24°C), requiring constant agitation and limiting shelf-life to 5–7 days.47 Murphy and Gardner showed in 1969 that CSP rapidly clears from circulation after transfusion and ultimately may not be the best product for oncology patients.48 These findings led to wide acceptance of room-temperature platelet storage despite reduced aggregation function versus “activated” cold-stored platelets.48, 49 The disadvantages of room-temperature platelets—including increased risk of bacterial growth, storage lesions, and ultimately decreased hemostatic function, which reduce their viability for supporting acute bleeding—brought about efforts to reevaluate CSP in the context of acute hemorrhage, as CSP possess beneficial in vitro characteristics, including increased hemostatic function and procoagulant activity.50-53 In 2023, the FDA granted an exception to practice (variance) allowing platelets to be continuously stored for up to 14 days at 1 to 6°C, intended for use in active bleeding when room temperature platelets are not available or practical.54 The FDA's authorization of this new guidance was enacted to account for the limited bioavailability of platelet supply or when the use of conventional platelets is not practical, including but not limited to prehospital, austere, or rural settings, or in mass casualty/public emergency events.54 This guidance allows platelets to be stored in either plasma or PAS. Although in vitro data from various studies suggest the specific advantages of CSP over room-temperature stored platelets,50-53 clinical data are currently lacking. Efforts to fill this data gap were investigated by Strandenes et al., who compared room-temperature stored platelets versus CSP stored for up to 14 days in randomized adults undergoing semi-urgent complex cardiothoracic surgery.55 Although limited by sample size, the study no significant clinical and platelet by to a the Platelet is a platelet storage study at 20 clinical that as of the transfusion efficacy of room-temperature stored platelets CSP a storage of 21 days in either plasma or in both and patients undergoing complex cardiac The of this study are intended not to platelet but also to that CSP provides hemostatic efficacy versus room-temperature stored for trauma resuscitation and acute hemorrhage RBCs stored for and metabolic due to oxidative which decreases their viability and and to hemolysis and RBC in post-transfusion and storage methods leading to ATP and 2,3-DPG improved RBC and decreased hemolysis oxygen prior to cold storage and this In et al. this process with a in RBC units using AS-3 by of to RBC units were cold-stored in to and compared RBCs with AS-3 had ATP ± ATP at 10 ± for and improved after of with RBCs, to for up to met FDA guidelines overall hemolysis and post-transfusion RBC RBC storage by at least 2 improved autologous post-transfusion RBC mean recovery ± compared to ± Dumont et al. investigated the cause of metabolic by during and that higher 2,3-DPG were associated with while the increased ATP levels were to was granted authorization in the U.S. for a process that the for or a storage The is designed for processing and RBCs for up to 42 days with reduced oxygen and The a and to and with for processing within h of et al. a study with units of cold-stored and the the post-transfusion RBC recovery after 42 days a significant increase of RBCs over ± ± in overall clinical transfusion studies the and safety of RBCs, with the potential to extend preserve and oxygen the and logistical of The logistical of RBCs of blood However, randomized controlled trials in patients be to the of RBCs in of red blood cells is the process of RBC units in which and within RBCs, allowing for long-term was first at U.S. in and successful during the but The of process by per of for the storage of blood products for up to 10 with storage up to 37 However, a to including strategies to reduce processing are warranted to improve operational of the Although not used in the of military blood and it be that the for the of Blood guidance on the of of the major the for in This was limited by a processing and a higher risk of the both and and shelf-life to 14 days when in a military and are limited to 2 and higher due to their shelf-life in the primary for in military is during of increased to improve the process include et that to reduce the of at the of RBC recovery compared to the used a for of platelets at the Blood as a and allowing for storage at for up to 2 also a before of the and reducing to about 10 with a shelf-life of 4 to to platelets and clot by to the of their use is optimal for acute hemorrhage and trauma randomized clinical of patients to conventional platelet transfusion during surgery no and showed decreased blood and decreased overall blood product More recently, a randomized clinical no post-transfusion events in either platelets or has been in military with the Military Blood Bank platelets in and during an of massive transfusion that blood products led to improved rates and a low of transfusion of advancements have in extended storage up to at with an that to and has FDA for the of acute hemorrhage when conventional platelets are not a its or to platelets in patients undergoing The primary was as the blood in 24 h with data the primary efficacy in of the also demonstrated that recovery and demonstrated hemostatic function in unit controlled clinical trials during complex versus platelets and were with results In the context of LSCO or events, a platelet product with a shelf-life will augment the for platelet collection However, it is that the distribution of and will be the military and in to maintain an inventory of or is a that the of and from blood blood products are due to their increased and increased at room storage allows for with an such as on a supply This capability the of blood products in prehospital environments, logistical in blood products in the could reduce or and temperature data collection requirements and allow for and The lyophilization of plasma products to the from canine resuscitation plasma was and by the with 10 units of plasma for In military physician the use of plasma the of the Military Blood which is still for military and civilian plasma has had use in and the with United States Army Operations using an emergency use authorization be with post-transfusion coagulation to of plasma, it as a blood product that transfusion for transport to coagulation levels with an active to of plasma products reduction include such as to the of by for and and by for their advancements in incorporate and improved preservation of factors as In a review of studies, use during major trauma showed no significant in rates or the for transfusion of allogeneic blood products when compared to Although there are currently no approved products, efforts are with FDA approval expected a of FDA-approved plasma with the within the of in The currently U.S. military to and for use in hemorrhage or when plasma is or plasma to through a These are to of up to by and to a This is an process that results in a that be with the plasma logistical to although the products are in vitro study by et al. showed that in decreased and compared to fresh plasma although et al. observed overall higher generation compared to both and studies and in a showed that was as in and improving and products include by which coagulation factors with short times to 4 and storage up to 2 its clinical by the and The limited shelf-life of platelets has a are a hemostatic from blood group O platelets that are to membranes and have a shelf-life at room be in within and with potentially improving bleeding study safety in 24 patients with who of up to are currently undergoing 2 clinical In et al. the of and et al. established that and human RBCs metabolic et al. the by that a solution of human and an increased recovery of studies have demonstrated that trehalose loading in RBCs decreases hemolysis due to the ability to during which cellular membranes and the of in the lyophilization has been to increase survival and reduce hemolysis clinical study of RBC a early clinical study of four low recovery to and However, efforts are using modern and The on blood products and potential supply has in human blood blood products be in the and a viable resuscitation when blood is is a product of human hemoglobin in and serving the of the also be and is an with that its development with demonstrated efficacy in hemorrhage and increase the shelf-life of their blood component is the primary cause of preventable deaths in U.S. civilian trauma and for of potentially survivable prehospital deaths during and Restoring blood is the best to but the on blood product shelf-life often to especially prehospital, in to and supply in This narrative review to methods to prolong the shelf-life of blood products and their resuscitation is at the of studies have of methods that allow for shelf-life extension during the storage of blood products and new to improve blood support current anticoagulant and additive solution has been and in the and or of blood potentially in the and of stored blood products. that a current is that donor not coagulation However, the of the blood product will to with FDA by for its such as and to the FDA allows storage of RBCs for up to 42 days and platelets for 14 days 5–7 day stored platelets are not available or their use is not The of early resuscitation with whole blood are are not the remains the unit was the most product to or support RBCs and platelets have and metabolism, of have been to the storage of the mechanisms the and that occur in cells the of storage will provide new to extend blood In the clinical studies to the specific of are needed to the risk versus of blood product storage to increased blood supply While the blood to to extend blood product during of blood product mass casualty events, blood and transfusion will to from of blood Blood will to increase donor be of the supply approved collection potentially support adequate and or blood to inventory, and inventory will to such as in expired et al. have there was no association in when trauma patients were with expired blood products up to 1 this practice be with and risk new blood products remains due to the of the clinical and the lack of future challenges, blood will to improve on prior and for future the blood has the to blood solutions an which may future blood The have to The have no of not to this as no were or during the current