Benefits of Early Plasma Transfusion

The role of plasma in DCR has been reaffirmed time and time again. In 1945, Beecher noted that “Plasma gives more time to get whole blood into the patient” [8]. Multiple US large center retrospective cohorts showed that 24–28% of severely injured trauma patients are found to be coagulopathic on admission. Coagulopathic patients have significantly higher mortality rates, while the degree of coagulopathy correlated with the severity of injury [9–11]. Analysis of the German Trauma Registry yielded similar results with rates of coagulopathy up to 34% as well as a direct correlation between the amount of crystalloid received prehospital and an increasing degree of coagulopathy [12]. Looking back at the first decade of war in Iraq, 33% of combat casualties were coagulopathic on admission, which correlated with a fivefold increase in mortality [13]. These data highlight the importance of recognizing the early onset of the coagulopathy of trauma at the time of injury, as well as endothelial damage, which plasma has been shown to correct [14]. Therefore, correction of coagulopathy should not be delayed until laboratory values are available to guide therapy and goals of care. The approach to the trauma patient should aim at preventing and correcting this coagulopathy as soon as possible to decrease its effects on mortality and progression of shock.

The importance of balanced resuscitation to include a high ratio of plasma delivered early became evident in the Iraq War where a survival advantage was noted in patients receiving close to 1:1 plasma-to-RBC ratio during massive transfusion compared to those who received less plasma [15]. During this conflict, over 335,000 units of blood products were transfused with nearly 110,000 being plasma [16, 17]. Analysis of combat casualties over 10 years revealed improved survival in those receiving a higher ratio of plasma and platelets to blood [13]. By 2010, clinical practice guidelines implemented by the Department of Defense (DoD) led to almost 100% of combat massive transfusions to be at a 1:1:1 ratio [18]. The PROMMTT trial then showed that early plasma administration was associated with reduced mortality in the first 6 h [3]. Following that, the PROPPR trial demonstrated that patients transfused with a higher plasma ratio achieved more hemostasis and had less early death due to exsanguination [19].

A retrospective review of patients receiving thawed plasma available in the ED showed shorter time to plasma transfusion (43 vs. 89 min), a reduction in blood transfused over 24 h, and decreased 30-day mortality [20]. A recent review of US combat casualties in Afghanistan rescued by medical evacuation (MEDEVAC) units showed that early blood product transfusion prehospital or within minutes of injury resulted in greater 24-h and 30-day survival [21]. In a large civilian cohort from a level 1 trauma center, prehospital administration of blood products including RBCs and plasma has been shown to be feasible and beneficial with improved acid-base status on admission and decreased overall blood product use in 24 h as well as a reduction in the risk of death in the sickest patients over the first 6 h [22]. All these findings point toward the need for a method to deliver early plasma in a reliable fashion.

Plasma produces superior volume expansion when compared to crystalloids, allowing less volumes infused to match volume lost, faster hemodynamic recovery, and decreased third-spaced volume [23]. It is important to use plasma as the primary resuscitation fluid for patients who are bleeding [4]. Other benefits of plasma stem from its ability to mitigate the effects of shock on physiology. The role of plasma in the correction of the endotheliopathy of trauma has been reported in multiple studies and is likely more important than correcting coagulopathy. The mechanism of action is thought to be through promoting systemic vascular stability and preventing endothelial permeability, coagulopathy, and inflammation which eventually lead to shock and end-organ failure [24, 25]. Moreover, in a rat model of injury and shock, plasma was able to restore the endothelial glycocalyx, improve syndecan-1 expression , and correct lung injury caused by shock [26].

In a swine model of traumatic brain injury (TBI) and hemorrhagic shock, transfusion of fresh frozen plasma (FFP) decreased neurologic impairment and hastened recovery to baseline cognitive function when compared to saline infusion [27]. Neuroprotective effects of plasma in hemorrhagic shock and TBI are thought to be due to improved cerebral perfusion, decreased glutamate-mediated excitotoxicity, and reduction in mitochondrial dysfunction as demonstrated in animal models [28]. Plasma also reduced the size of brain lesions and swelling in multiple swine models of TBI and hemorrhagic shock [29, 30]. It also incurs neuroprotection by providing higher brain oxygenation and cerebral perfusion profiles [30]. In humans, a large retrospective cohort analysis showed that in the subgroup of patients with multifocal intracranial hemorrhage, early administration of plasma was associated with a survival benefit [31].

Logistical Challenges in Plasma Transfusion

Unfortunately, delivery of early balanced resuscitation is fraught with strategic and logistical challenges. In the USA, half of all trauma patients are cared for outside of levels 1 and 2 trauma centers, where blood products are frequently not readily available [32]. In the military, forward surgical teams (FST) were developed to provide immediate support and treatment to injured soldiers. They used to only carry red blood cells (RBCs) for transfusion, and more recently, they are able to provide plasma but not platelets [33]. Warm fresh whole blood (WFWB) is available in these circumstances and has been shown to improve survival in combat casualties treated by FST [33]. These challenges, along with misconceptions regarding the role of blood products in resuscitation, led to the overuse of crystalloids and its own set of unique complications. Excessive crystalloid resuscitation has been shown to be detrimental, while balanced blood product resuscitation decreases the onset of acute respiratory distress syndrome (ARDS) and abdominal compartment syndrome and improves survival [32, 34].

The recognition of the importance of early, balanced DCR has led many trauma systems in the USA, and around the world, to search for novel approaches to provide blood products as soon as possible to severely injured trauma patients. These include carrying them in the prehospital setting and having them readily available in the ED. The logistical difficulties in providing FFP transfusions early in trauma are based on the need for reliable cold storage facilities, specialized transport equipment and personnel to provide them, and a lengthy thawing process. There is also a significant loss of up to 50% due to bag breakage during transport and thawing [35].

FFP is prepared through separation from whole blood and stored at −18 °C or colder with a shelf life of about 1 year. The thawing process requires a 30–37 °C agitated water bath or a warming device cleared by the US Food and Drug Administration; this takes 15–30 min [36]. Thawed FFP should be immediately used but can be stored between 1 and 6 °C for up to 5 days. All those factors preclude its immediate availability and delay administration in many settings such as austere environments, the battlefield, and smaller hospitals with limited blood banking capabilities. Of great importance and increasing realization is that these same limitations apply in the larger centers as well.

Production and Forms of Dried Plasma

Production of dried plasma is achieved in two ways: freeze-drying, also known as lyophilization , or spray-drying . Lyophilization is achieved by freezing the plasma under a vacuum in a glass container for several days, which decreases the water content to 1–2% [45]. Spray-dried plasma production utilizes atomization of liquid plasma via pressurized drying gas to droplets which are then exposed to hot gas (up to 150 °C) in a drying chamber followed by rapid evaporative cooling. This method can dry a unit of plasma (~250 mL) in approximately 25 min [17, 46]. Dried plasma can then be reconstituted to its original volume or a concentrated form. Multiple pathogen inactivation methods are available to use during the process, and the choice depends on the manufacturer’s preference and experience.

Pathogen Inactivation

Newer and more accurate viral detection and pathogen inactivation methods have led to the improved safety of blood products, reducing the residual risk of transfusion-transmitted HIV-1 and HCV to approximately 1 in 2 million blood units [47]. Pathogen inactivation methods used for plasma include solvent/detergent (S/D) treatment and photochemical inactivation techniques [17]. S/D treatment, which is FDA-approved, binds lipid-enveloped viruses and bacteria to inactivate them followed by a filtration process to remove cells and debris, but it has no effect against non-enveloped viruses and prions [48]. Rigorous screening standards require testing donors for non-enveloped viruses twice at a 6-month interval to decrease the risk of transmission. Moreover, photochemical inactivation utilizes a photosensitizer that binds the DNA and RNA of pathogens, including non-enveloped viruses, and nucleated cells, followed by ultraviolet light exposure to inactivate them [49]. INTERCEPT (Cerus Corp., Concord, CA) is an FDA-approved system, which uses amotosalen (a psoralen molecule) to bind DNA and RNA followed by UV light activation [50]. The Mirasol System (Terumo BCT, Lakewood, CO), which uses riboflavin as the photosensitizer, is currently approved for clinical use in Europe but only approved for investigational use in the USA and Canada [51]. Methylene blue can also be used with visible light exposure [52].

Standard S/D treatment causes a decrease in vWF activity (24%), factor V (37%), protein S (44%), and alpha-2 antiplasmin (79%). Similarly, amotosalen + UV light reduces factor VII (23%) and factor VIII (27%) [17, 48]. A newer S/D treatment product, Octaplas LG (Octapharma, Lachen, Switzerland), received FDA clearance in 2013 and employs a prion reduction step and a modified S/D process that better preserves factor levels [17].

Buffering and Reconstitution

Reconstituted porcine lyophilized plasma is alkalotic with a pH >8.5, making it highly lethal when injected in swine due to their lack of ability to buffer their plasma [53]. Human lyophilized plasma is also alkalotic with a pH near 8; however, it is well tolerated clinically in humans [54]. This increase in pH after lyophilization is attributed to the loss of bicarbonate during the drying process. Multiple acidic buffering solutions were studied to evaluate their effect on the hemostatic properties of lyophilized plasma. For example, when ascorbic acid (vitamin C) is added to lyophilized plasma (LP), 84% of the coagulation factor activity was maintained [55].

In a swine model of polytrauma and hemorrhagic shock, a significant decrease in interleukin-6 (IL-6) levels was observed in all LP-treated animals compared to those receiving FFP, suggesting an anti-inflammatory effect [55]. This was corroborated by another study using concentrated LP (50%) that showed buffering with ascorbic acid resulted in reduced serum levels of IL-6 and TNF [56]. Another study examined the effect of other buffers, such as citric acid and hydrochloric acid, on lyophilized plasma. No difference in physiology, coagulation parameters, or blood loss was noted, but animals receiving ascorbic acid had lower IL-6 levels and less oxidative DNA damage [57]. Using higher concentrations of ascorbic acid didn’t affect the physiologic benefits of LP, but no improvement in the anti-inflammatory effects or further decrease in DNA oxidative damage was detected [53].

The type of fluid used to reconstitute dried plasma has not been shown to affect the degree of inflammation or oxidative DNA damage induced by shock in a swine model of polytrauma and hemorrhagic shock [58]. However, the type of fluid used for reconstitution does affect the hemostatic efficacy and ability of lyophilized plasma (LP) to mitigate the effects of shock. Animals treated with LP reconstituted with sterile water and lactated Ringer’s (LR) had less blood loss compared to those reconstituted in normal saline (NS) and Hextend. The group that received Hextend had persistently elevated INR values and contained the only animal that did not survive the experiment. Serum IL-6 levels were lowest in the sterile water group when compared to NS [59]. The optimal solution for buffering lyophilized plasma in humans is unknown and will require further investigation . For now, sterile water is used to reconstitute human LP.

Lyophilized Versus Fresh Frozen Plasma

The process of freezing and thawing plasma is not benign and has several detrimental effects on coagulation proteins. Never-frozen liquid plasma was found to have a superior coagulation profile and factor activity, as well as thrombin generation potential, when compared to plasma from thawed FFP [60]. Thawed plasma was compared at day 0 and after storage at day 5, and a significant degradation of clotting factors was detected in the older product, with a 40% reduction in thrombin generation potential and significantly decreased hemostatic profile on thrombelastography (TEG) analysis [61]. Thawed FFP decreased vascular permeability in vitro by a factor of 10; however, that effect decreased to only a factor of 2.5 when 5-day-old thawed plasma was used [62]. These findings reinforce some of the advantages of dried plasma over FFP.

Concentrated Dried Plasma

Concentrated, low-volume reconstitution of lyophilized plasma (50%) has been demonstrated to be safe in a swine polytrauma model of hemorrhagic shock with similar physiologic effects, hemostatic properties, and coagulation parameters (INR and TEG) [63]. This could have logistical advantages in packaging and transport on the battlefield as well as in austere environments. The effects of infusing this hypertonic, hyper-oncotic fluid are unknown in humans and will require careful evaluation.

Spray-dried plasma (SDP) at original concentration was compared with triple concentrated SDP in the resuscitation of a swine model of polytrauma and hemorrhagic shock. In vitro evaluation of coagulation parameters of SDP compared with FFP did not reveal any difference between the two products. However, triple-concentration SDP showed an increase in clotting factor activity and prolonged PT/PTT. Treatment with all three formulations corrected INR rapidly and increased clot strength (TEG-maximum amplitude (MA)). This confirmed that concentrated, low-volume SDP is as effective as FFP and regular SDP in reversing trauma-associated coagulopathy [64].

Coagulation Profile of Dried Plasma

The accepted standard for factor loss in frozen then thawed plasma is 25–40% [65]. In vitro analysis of swine FFP vs. lyophilized plasma (LP) coagulation tests (PT, PTT, INR, fibrinogen) did not reveal any statistically significant difference. Reconstituted LP has been shown to maintain an average of 86% of coagulation factor activity when compared to FFP [66]. In comparison, spray-drying causes reduction in several factors including 25% for fibrinogen and protein S, 50% for vWF activity, and 70% for factors V and VIII. However, this has been shown to have no effect on the ability of SDP to generate thrombin [67]. On the contrary, after a year of storage at −25 °C, lyophilized plasma had no significant change in clotting factors activity when compared to fresh plasma [68]. Lyophilized plasma stored as long as 30 years had similar preservation of components [69].

Studies in Animal Models of Shock

Multiple studies demonstrate the safety and efficacy of lyophilized plasma (LP). In a series of studies using a swine model of polytrauma and hemorrhagic shock , LP demonstrated superior hemostatic efficacy to FFP when combined with RBCs in 1:1 ratio. Concentrated LP reconstituted to 50% of its volume was also well tolerated and equally effective in correcting shock physiology when compared to unconcentrated LP [56]. In another study, fresh whole blood (FWB), FFP, and LP all corrected coagulopathy equally in a swine model. There was 85% mortality in the crystalloid only group and no mortality in any of the blood products groups [66]. Another study showed that when compared with colloid alone, 7-day survival was superior in animals that received spray-dried plasma – this effect was equivalent to that seen in animals that received whole blood [70]. Moreover, animals receiving balanced LP-to-RBC had significantly less blood loss than those receiving FFP or LP alone, and LP was as effective as FFP in reversing coagulopathy in this animal model [55].

Lyophilized plasma (LP) demonstrated similar effects as FFP, both in vitro and in vivo , on reducing endothelial cell permeability, increasing trans-endothelial resistance, decreasing leukocyte-endothelial binding, and preserving adherens junctions. In an in vitro mouse model of hemorrhagic shock, LP and FFP both equally reduced pulmonary injury, inflammation, and vascular permeability [71]. Spray-dried plasma (SDP) also reduced vascular permeability and other indicators of endothelial damage as well as FFP [72]. FFP and SDP equally decreased shock-induced pulmonary vascular permeability in vivo. SDP was also equivalent to FFP in the correction of shock in a mouse model. They both reduced alveolar wall thickening, leukocyte infiltration , and the breakdown of EC junctions [73].

Neuroprotective Effects of Lyophilized Plasma

Plasma has been shown to have multiple neuroprotective effects in traumatic brain injury. In a swine model of polytrauma, hemorrhagic shock, and TBI, both lyophilized plasma (LP) and FFP were shown to decrease brain lesion size by 50% after 6 h of injury when compared to saline infusion; swelling was also 54% less in plasma-treated groups [74]. A follow-up study to evaluate the long-term effects of resuscitation with FFP vs. LP on neurological outcomes showed similar recovery of cognitive function in studied animals. The brain lesion size was significantly smaller in LP group on experiment day 3, but this effect dissipated by day 10 [75]. Another large 30-day animal study recently showed similar neuroprotective results with faster return to baseline neurological function in animals treated with LP and FFP vs. NS [76].

French Lyophilized Plasma (FLyP)

Dr. Jean Julliard started producing freeze-dried plasma in 1945 after its inception by the US military in WWII. By 1950, the Centre de Transfusion Sanguine des Armées (CTSA) became the first European center to produce lyophilized plasma. During the Indochina War, almost 40,000 units of lyophilized plasma were delivered to the French military. Production was suspended in 1985 due to concerns for HIV transmission. In 1991, production restarted with the first Gulf War and has continued since that time [54].

Since 1994, French lyophilized plasma (FLyP) is made using a pool of less than 11 donors. Pooling based on blood type selection allows the dilution and neutralization of natural anti-A and anti-B hemagglutinins, making FLyP a universal donor product compatible with any recipient blood type. Since 2003, it is also being leukoreduced. Starting in 2010, plasma from women with a history of pregnancy is tested for HLA antibodies and excluded if positive. That was the same time that FLyP started undergoing amotosalen and UV light processing as a pathogen DNA/RNA inactivation method. This process was chosen over solvent/detergent treatment due to better preservation of clotting factors. The French hemovigilance program has been monitoring FLyP since 1994, and so far, no reactions or infectious complications have been reported out of more than 1100 units transfused [54].

FLyP is packaged in glass bottles, shelf-stable in ambient temperatures between 2 and 25 °C for 2 years, and easily rehydrated with 200 mL of water in less than 3 min, allowing for immediate transfusion with RBCs. FLyP contains all clotting factors and proteins. After more than 2 years of storage at ambient temperature, the fibrinogen and clotting factor levels of FLyP are equivalent to FFP [85]. Despite a certain level of factor reduction (20–25%), lyophilization has not been shown to alter in vitro hemostatic efficacy of plasma. When reconstituted, FLyP has a pH close to 8 [54].

In 2011, FLyP was authorized by the French Agency for the Sanitary Safety of Health Products (AFSSAPS) for use in civilians in austere settings or until thawed plasma became available [54]. Clinical efficacy of FLyP was studied in a prospective trial on 87 ICU patients in Afghanistan and was found to be safe and efficacious in the management of polytrauma and shock [86]. Furthermore, the difference in administration times between FLyP and FFP in a level 1 trauma center was studied. Retrospective analysis showed significantly less time to product administration between patients receiving FLyP vs. FFP (median 15 vs. 95 min). This is consistent with similar reports in the literature of time to FFP transfusion [21]. Subsequently, time to achieve 1:1 resuscitation ratio with RBCs was shorter in FLyP group. There were also significantly less cases of massive transfusion utilization and RBC transfusion in the FLyP group compared to the FFP group (7 vs. 45%). No differences in hospital length of stay, ICU length of stay, or 24-h mortality between the two groups were noted [87].

Recently, a randomized open-label clinical trial of 48 patients who were assigned to receive 4 units of FLyP or FFP within 6 h of injury was completed. Patients in the FLyP group demonstrated less time from randomization to infusion compared to those in the FFP group (median 14 min vs. 77 min). This led to higher levels of fibrinogen achieved within 45 min of randomization, as well as a greater improvement in INR, factor V, and factor II levels. The difference in coagulation parameters between the two groups remained significant at 6 h. However, there was no difference detected in mortality between the two groups [88].

German Lyophilized Plasma (LyoPlas N-w)

In the 1990s, the German Red Cross Blood Service West produced solvent/detergent (S/D) treated lyophilized plasma using pooled plasma. Due to concern for Creutzfeldt-Jakob-type prion disease transmission, which is not inactivated by standard S/D treatment, pooled plasma was replaced with single-donor lyophilized plasma in 2007 [45]. This product is licensed under the name LyoPlas N-w (German Red Cross Blood Service West, Hagen, Germany).

Quarantined plasma from a single donor is stored frozen for at least 4 months until the donor returns for retesting for HIV, hepatitis C virus, hepatitis B virus, hepatitis A virus, and parvovirus B19. After the quarantine, the plasma is thawed and connected by sterile docking to the patented steam-sterilized “bottle-in-bag” system, which consists of a glass bottle and a rubber stopper inside a plastic bag. During transfer into the glass bottle, the plasma passes through a filter with a nominal pore size of 0.2 μm. Once 200 mL of plasma is transferred, the bottle is closed with the stopper and removed from the system. Plasma is then frozen to −30 °C followed by lyophilization in specially designed freeze-dryers. The lyophilization is accomplished by a stepwise increase of the temperature from −45 °C to +15 °C, resulting in water content below 1% [45].

Sterile water for reconstitution (200 mL) is included in the LyoPlas N-w kit and accomplished within 10 min depending on the plasma composition and water temperature. Transfusion can be accomplished via the glass bottle or the plastic bag, which allows for pressure infusion if needed. After storage at 2–8 °C for 24 months, LyoPlas N-w only had a 10% reduction in factor V, VIII, and vWF. All other factors remained stable. Storage at room temperature, however, led to 54% decrease in fibrinogen levels and vWF activity. This is why the shelf life of LyoPlas N-w has been restricted to 15 months only. After reconstitutions, factor degradation increases over time at room temperature. At 48 h, factor VIII and protein S levels decreased by 66 and 50%, respectively. Only 10% degradation of those factors was noted in the first 6 h, however. This is why it is recommended to use LyoPlas N-w within 6 h of reconstitution [45].

From 2007 to 2011, a total of 237,850 units of LyoPlas N-w were provided to hospitals, doctors, and the German military compared to 343,821 units of FFP delivered during the same time period. This reflects the wide use of LyoPlas N-w in Germany (41% of all plasma used) under various clinical settings requiring transfusion and not just trauma resuscitation. The rates and types of transfusion-related complications reported between 2007 and 2011 were similar for FFP and LyoPlas N-w (0.018 vs. 0.023%). No viral transmission has been reported since its inception in 2007 [45].