Lyophilized Plasma Research Articles

Efficacy and safety of novel freeze-dried plasma products in a porcine combat casualty model.

Hemorrhagic shock is well documented as a leading cause of preventable fatalities among military casualties. During military operations plasma can be transfused while waiting for whole blood. This study was conducted to assess the safety and efficacy of two new freeze-dried plasma formulations in a porcine model of traumatic hemorrhagic shock. In the face of species-specific transfusion, transfusible blood products were derived from porcine sources. The efficacy of three lyophilized plasma (LP) formulations was evaluated: lyophilized plasma (LP), concentrated lyophilized plasma (CLP), and platelet-rich concentrated lyophilized plasma (PCLP). Pigs were subjected to multi-trauma and hemorrhagic shock. Ninety minutes post-shock induction, the animals were treated with one of the three lyophilized products. Monitoring included systolic blood pressure and cardiac output. Point-of-care and laboratory diagnostic tests were used to assess renal function, real-time hemostasis (ROTEM), and coagulation. Histological examinations of kidney, lung, and muscle tissues were conducted 4 h after shock induction. CLP and PCLP significantly improved systolic blood pressure and cardiac output and positively influenced base excess, creatinine, various ROTEM, and coagulation markers compared with standard LP without histologic modification. No adverse effect was associated with the transfusion of any of the plasma products throughout the experimental procedures. Both CLP and PCLP exhibit promising therapeutic potential for managing hemorrhagic shock in scenario where whole blood supplies are limited. However, the distinct physiological and coagulation characteristics of the swine model necessitate further investigation using humanized preclinical models to fully understand their clinical applicability and constraints.

Evaluation of the efficacy and safety of lyophilized plasma

Objective. To assess the hemostatic potential, preclinical safety and in vitro efficacy of lyophilized plasma in patients and blood donors.Materials and methods. The study of acute toxicity was carried out on outbred mice of both sexes (n=120), pyrogenicity – on rabbits of both sexes (n=30), abnormal toxicity – on mice (n=25) and guinea pigs (n=6). Histopathological studies were carried out on preparations of five fragments of the middle part of the mouse tail after intravenous administration of the maximum dose (4500 mg/kg) lyophilized plasma (LP), physiological solution, fresh frozen plasma (FFP) and pathogen-reduced FFP (PRFFP) using the Intercept system. The proand anticoagulant activity was investigated by clotting and chromogenic method to evaluate the hemostatic potential of PL. The effect on hemostasis was studied in vitro by thromboelastometry when FFP and LP were added to blood samples of patients (n = 23) and donors (n = 20). Results. Acute toxicity study showed the absence of death and clinical signs of intoxication in all series. On the 14th day, body weight gain was observed in animals of all series. Regardless of the used LP dosage, no development of effusion and adhesions in the thoracic and abdominal cavities, as well as macroscopically detectable pathologic changes of the main organs were observed in animals. The analysis of the relative weight of internal organs of animals did not reveal statistically significant differences in experimental and control series relative to the values of similar indicators in intact animals. The studied hemostatic potential showed that LP was not inferior to PRFFP in the content of coagulation factors of the blood system. Evaluation of the effect of LP application in vitro in female patients showed statistically significant correction of CT (clotting time) index, which indicates the effectiveness of its application for replenishment of blood coagulation factors (BCF).Conclusion. Preclinical animal studies of LP have proven its safety and efficacy compared to FFP.

Prehospital Freeze-Dried Plasma in Trauma: A Critical Review

Major traumatic hemorrhage is now frequently treated by early hemostatic resuscitation on hospital arrival. Prehospital hemostatic resuscitation could therefore improve outcomes for bleeding trauma patients, but there are logistical challenges. Freeze-dried plasma (FDP) offers indisputable logistical advantages over conventional blood products, such as long shelf life, stability at ambient temperature, and rapid reconstitution without specialized equipment. We sought high level, randomized, controlled evidence of FDP clinical efficacy in trauma. A structured systematic search of MEDLINE/PubMed was carried out and identified 52 relevant English language publications. Three studies involving 607 patients met our criteria: Resuscitation with Blood Products in Patients with Trauma-related Hemorrhagic Shock receiving Prehospital Care (RePHILL, n = 501); Prehospital Lyophilized Plasma Transfusion for Trauma-Induced Coagulopathy in Patients at Risk for Hemorrhagic Shock (PREHO-PLYO, n = 150); and a pilot Australian trial (n = 25). RePHILL found no effect of FDP plus packed red blood cells (PRBC) concentrate transfusion versus saline on mortality. PREHO-PLYO found no effect of FDP versus saline on International Normalized Ratio (INR) at hospital arrival. The pilot trial found that study of PRBC versus PRBC plus FDP was feasible during long air transport times to an Australian trauma centre. Further research is required to determine under what conditions FDP might provide prehospital benefit to trauma patients.

Can volume-reduced plasma products prevent transfusion-associated circulatory overload in a two-hit animal model?

Transfusion-associated circulatory overload (TACO) is a pulmonary transfusion complication and a leading cause of transfusion-related morbidity and mortality. Volume overload and rising hydrostatic pressure as a consequence of transfusion are seen as the central pathway leading to TACO. A possible preventative measure for TACO could be the use of low-volume blood products like volume-reduced lyophilized plasma. We hypothesize that volume-reduced lyophilized plasma decreases circulatory overload leading to a reduced pulmonary capillary pressure and can therefore be an effective strategy to prevent TACO. A validated two-hit animal model in rats with heart failure was used. Animals were randomized to receive 4 units of either solvent-detergent pooled plasma (SDP) as control, standard volume lyophilized plasma (LP-S) or hyperoncotic volume-reduced lyophilized plasma (LP-VR). The primary outcome was the difference between pre-transfusion and post-transfusion left ventricular end-diastolic pressure (ΔLVEDP). Secondary outcomes included markers for acute lung injury. LVEDP increased in all randomization groups following transfusion. The greatest elevation was seen in the group receiving LP-VR (+11.9 mmHg [5.9-15.6]), but there were no significant differences when compared to groups receiving either LP-S (+6.3 mmHg [2.9-13.4], p=0.29) or SDP (+7.7 mmHg [4.5-10.5], p=0.55). There were no significant differences in markers for acute lung injury, such as pulmonary wet/dry weight ratios, lung histopathology scores or PaO2 /FiO2 ratio between the three groups. Transfusion with hyperoncotic volume-reduced plasma did not attenuate circulatory overload compared to standard volume plasma and was therefore not an effective preventative strategy for TACO in this rat model.

Lyophilized plasma resuscitation downregulates inflammatory gene expression in a mouse model of sepsis.

Plasma resuscitation may improve outcomes by targeting endotheliopathy induced by severe sepsis or septic shock. Given the logistical constraints of using fresh frozen plasma in military settings or areas with prolonged prehospital care, dried products such as lyophilized plasma (LP) have been developed. We hypothesized that resuscitation with LP would decrease lung injury, inflammation, and mortality in a mouse sepsis model. Adult male C57BL/6J mice received an intraperitoneal injection of cecal slurry. Twenty-two hours later, the mice were anesthetized, the femoral artery was cannulated, and the mice were randomized to receive resuscitation with LP (10 mL/kg) or lactated Ringer's (LR; 30 mL/kg) for 1 hour. At 48-hours post-cecal slurry injection, bronchoalveolar lavage fluid was collected, the lungs were harvested, and plasma was obtained. Mortality and bronchoalveolar lavage total protein concentration (as an indicator of permeability) were compared between groups. The lungs were analyzed for histopathology and inflammatory gene expression using NanoString, and the plasma was analyzed for biomarkers of inflammation and endothelial function. There was no significant difference in short-term mortality between LR and LP mice, 38% versus 47%, respectively ( p = 0.62). Bronchoalveolar lavage protein levels were similar among mice resuscitated with LR or LP, and there was a lack of significant histopathologic lung injury in all groups. However, LP resuscitation resulted in downregulation of pulmonary inflammatory genes, including signaling pathways such as Janus kinase-signal transducer and activator of transcription and nuclear factor κB, and a circulating inflammatory biomarker profile similar to sham animals. Resuscitation with LP did not improve mortality or reduce permeability or injury in this model compared with LR. However, LP downregulated pulmonary inflammatory gene signaling and may also reduce circulating biomarkers of inflammation. Future studies should evaluate LP resuscitation in combination with antibiotics and other therapeutics to determine whether the anti-inflammatory effects of LP may improve outcomes in sepsis.

Lyophilized plasma attenuates vascular permeability, inflammation and lung injury in hemorrhagic shock.

In severe trauma and hemorrhage the early and empiric use of fresh frozen plasma (FFP) is associated with decreased morbidity and mortality. However, utilization of FFP comes with the significant burden of shipping and storage of frozen blood products. Dried or lyophilized plasma (LP) can be stored at room temperature, transported easily, reconstituted rapidly with ready availability in remote and austere environments. We have previously demonstrated that FFP mitigates the endothelial injury that ensues after hemorrhagic shock (HS). In the current study, we sought to determine whether LP has similar properties to FFP in its ability to modulate endothelial dysfunction in vitro and in vivo. Single donor LP was compared to single donor FFP using the following measures of endothelial cell (EC) function in vitro: permeability and transendothelial monolayer resistance; adherens junction preservation; and leukocyte-EC adhesion. In vivo, using a model of murine HS, LP and FFP were compared in measures of HS- induced pulmonary vascular inflammation and edema. Both in vitro and in vivo in all measures of EC function, LP demonstrated similar effects to FFP. Both FFP and LP similarly reduced EC permeability, increased transendothelial resistance, decreased leukocyte-EC binding and persevered adherens junctions. In vivo, LP and FFP both comparably reduced pulmonary injury, inflammation and vascular leak. Both FFP and LP have similar potent protective effects on the vascular endothelium in vitro and in lung function in vivo following hemorrhagic shock. These data support the further development of LP as an effective plasma product for human use after trauma and hemorrhagic shock.

Resuscitation with Lyophilized Plasma Is Safe and Improves Neurological Recovery in a Long-Term Survival Model of Swine Subjected to Traumatic Brain Injury, Hemorrhagic Shock, and Polytrauma.

We have shown previously that fresh frozen plasma (FFP) and lyophilized plasma (LP) decrease brain lesion size and improve neurological recovery in a swine model of traumatic brain injury (TBI) and hemorrhagic shock (HS). In this study, we examine whether these findings can be validated in a clinically relevant model of severe TBI, HS, and polytrauma. Female Yorkshire swine were subjected to TBI (controlled cortical impact), hemorrhage (40% volume), grade III liver and splenic injuries, rib fracture, and rectus abdominis crush. The animals were maintained in a state of shock (mean arterial pressure 30-35 mm Hg) for 2 h, and then randomized to resuscitation with normal saline (NS), FFP, or LP (n = 5 swine/group). Animals were recovered and monitored for 30 d, during which time neurological recovery was assessed. Brain lesion sizes were measured via magnetic resonance imaging (MRI) on post-injury days (PID) three and 10. Animals were euthanized on PID 30. The severity of shock and response to resuscitation was similar in all groups. When compared with NS-treated animals, plasma-treated animals (FFP and LP) had significantly lower neurologic severity scores (PID 1-7) and a faster return to baseline neurological function. There was no significant difference in brain lesion sizes between groups. LP treatment was well tolerated and similar to FFP. In this clinically relevant large animal model of severe TBI, HS, and polytrauma, we have shown that plasma-based resuscitation strategies are safe and result in neurocognitive recovery that is faster than recovery after NS-based resuscitation.

Early resuscitation with lyophilized plasma provides equal neuroprotection compared with fresh frozen plasma in a large animal survival model of traumatic brain injury and hemorrhagic shock.

Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) is highly lethal. In previous models of combined TBI + HS, we showed that early resuscitation with fresh frozen plasma (FFP) improves neurologic outcomes. Delivering FFP, however, in austere environments is difficult. Lyophilized plasma (LP) is a logistically superior alternative to FFP, but data are limited regarding its efficacy for treatment of TBI. We conducted this study to determine the safety and long-term outcomes of early treatment with LP in a large animal model of TBI + HS. Adult anesthetized swine underwent TBI and volume-controlled hemorrhage (40% blood volume) concurrently. After 2 hours of shock, animals were randomized (n = 5 per /group) to FFP or LP (1× shed blood) treatment. Serial blood gases were drawn, and thromboelastography was performed on citrated, kaolin-activated whole-blood samples. Five hours after treatment, packed red blood cells were administered, and animals recovered. A 32-point Neurologic Severity Score was assessed daily for 30 days (0 = normal, 32 = most severe injury). Cognitive functions were tested by training animals to retrieve food from color-coded boxes. Brain lesion size was measured on serial magnetic resonance imaging, and an autopsy was performed at 30 days. The severity of shock and the degree of resuscitation were similar in both groups. Administration of FFP and LP was well tolerated with no differences in reversal of shock or thromboelastography parameters. Animals in both groups displayed the worst Neurologic Severity Score on postoperative Day 1 with rapid recovery and return to baseline within 7 days of injury. Lesion size on Day 3 in FFP-treated animals was 645 ± 85 versus 219 ± 20 mm in LP-treated animals (p < 0.05). There were no differences in cognitive functions or delayed treatment-related complications. Early treatment with LP in TBI + HS is safe and provides neuroprotection that is comparable to FFP.

Plasma Transfusion: History, Current Realities, and Novel Improvements.

Traumatic hemorrhage is the leading cause of preventable death after trauma. Early transfusion of plasma and balanced transfusion have been shown to optimize survival, mitigate the acute coagulopathy of trauma, and restore the endothelial glycocalyx. There are a myriad of plasma formulations available worldwide, including fresh frozen plasma, thawed plasma, liquid plasma, plasma frozen within 24 h, and lyophilized plasma (LP). Significant equipoise exists in the literature regarding the optimal plasma formulation. LP is a freeze-dried formulation that was originally developed in the 1930s and used by the American and British military in World War II. It was subsequently discontinued due to risk of disease transmission from pooled donors. Recently, there has been a significant amount of research focusing on optimizing reconstitution of LP. Findings show that sterile water buffered with ascorbic acid results in decreased blood loss with suppression of systemic inflammation. We are now beginning to realize the creation of a plasma-derived formulation that rapidly produces the associated benefits without logistical or safety constraints. This review will highlight the history of plasma, detail the various types of plasma formulations currently available, their pathophysiological effects, impacts of storage on coagulation factors in vitro and in vivo, novel concepts, and future directions.

Lyophilized plasma prevents endothelial leak and improves oxygenation following traumatic hemorrhage

Lyophilized plasma (LP) reconstituted to 100% (1×LP) or 50% (2×LP) of its original volume is just as effective as fresh frozen plasma (FFP) in correcting hemodynamic instability and coagulopathy following traumatic hemorrhagic shock. Compared to transfusion with lactated Ringers (LR), FFP also prevents the endotheliopathy of trauma (EOT) following hemorrhagic shock by attenuating vascular endothelial leak, which prevents endothelial damage, edema and end organ injury. Whether LP acts similarly to FFP on the vascular endothelium in preventing EOT is not known. Therefore, we hypothesized that similar to FFP, 1×LP and 2×LP would minimize endothelial permeability and prevent pulmonary dysfunction following traumatic hemorrhage. To test this hypothesis, two experiments were performed. To test for endothelial permeability in vitro, human pulmonary endothelial cells (HPECs) suspended in growth media were treated with FFP, 1×LP or 2×LP. Increased trans‐endothelial membrane resistance (TEER) indicated decreased permeability and barrier function recovery. When HPECs were treated in 10% plasma, the rise in TEER from baseline was similar in cells treated with FFP (22%) and 2×LP (27%), and highest with 1×LP treatment (33%, p&lt;0.05 vs FFP and 2×LP). However, when cells were treated with 2% plasma, the rise in TEER was highest in HPECs treated with 2×LP (29%), followed by FFP (22%), and 1×LP (15%). To test for pulmonary dysfunction in vivo, anesthetized female swine subjected to 30min of uncontrolled hemorrhage from Grade V liver injury were resuscitated with lactated Ringers (LR), FFP, 1×LP or 2×LP. Following conscious recovery, arterial blood gasses were measured 24hrs post‐injury. PaO2 and arterial O2 saturation were low in LR‐treated swine (n=1; PaO2:52mmHg, O2:87%), but improved with FFP (n=2, PaO2:77±1mmHg, O2:97±0%), 1xLP (n=2; PaO2:73±8mmHg, O2:97±1%) and 2×LP (n=2; PaO2:72±4mmHg, O2:96±1%). Only LR‐treated swine exhibited gross pulmonary vascular thrombosis. Take together, these data suggests that LP and FFP act similarly to prevent endothelial permeability and pulmonary dysfunction after injury. Further experiments are required to determine if LP performs as well as FFP to minimize EOT and organ damage following hemorrhagic shock.Support or Funding InformationThis work was funded by DOD US Army Grant W81XWH‐04‐1‐0114 (Schreiber)

Effect of ascorbic acid concentrations on hemodynamics and inflammation following lyophilized plasma transfusion.

Compared with lyophilized plasma (LP) buffered with other acids, LP with ascorbic acid (AA) attenuates systemic inflammation and DNA damage in a combat relevant polytrauma swine model. We hypothesize that increasing concentrations of AA in transfused LP will be safe, will be hemodynamically well tolerated, and will attenuate systemic inflammation following polytraumatic injury and hemorrhage in swine. This prospective, randomized, blinded study involved 52 female swine. Forty animals were subjected to our validated polytrauma model and resuscitated with LP. Baseline control sham (n = 6), operative control sham (n = 6), low-AA (n = 10), medium-AA (n = 10), high-AA (n = 10) groups, and a hydrochloric acid control (HCL, n = 10) were randomized. Hemodynamics, thrombelastography, and blood chemistries were assessed. Inflammatory cytokines (tumor necrosis factor α, interleukin 6 [IL-6], C-reactive protein, and IL-10) and DNA damage were measured at baseline, 2 hours, and 4 hours after liver injury. Significance was set at p < 0.05, with a Bonferroni correction for multiple comparisons. Hemodynamics, shock, and blood loss were similar between groups. All animals had robust procoagulant activity 2 hours following liver injury. Inflammation was similar between groups at baseline, and AA groups remained similar to HCL following liver injury. IL-6 and tumor necrosis factor α were increased at 2 hours and 4 hours compared with baseline within all groups (p < 0.008). DNA damage increased at 2 hours compared with baseline in all groups (p < 0.017) and further increased at 4 hours compared with baseline in HCL, low-, and high-AA groups (p < 0.005). C-reactive protein was similar between and within groups. IL-10 increased at 2 hours compared with baseline in low- and high-AA groups and remained elevated at 4 hours compared with baseline in the low-AA group (all, p < 0.017). Concentrations of AA were well tolerated and did not diminish the procoagulant activity of LP. Within our tested range of concentrations, AA can safely be used to buffer LP.

Reconstitution fluid type does not affect pulmonary inflammation or DNA damage following infusion of lyophilized plasma.

Dysfunctional inflammation following traumatic hemorrhage can lead to multiple-organ failure and death. In our polytrauma swine model, lyophilized plasma (LP) reconstituted with sterile water and ascorbic acid suppressed systemic inflammation and attenuated DNA damage. However, it remains unknown whether the inflammatory response is affected by the type of fluid used to reconstitute LP. We hypothesized that common resuscitation fluids such as normal saline (LP-NS), lactated Ringer's solution (LP-LR), Hextend (LP-HX), or sterile water (LP-SW) would yield similar inflammation profiles and DNA damage following LP reconstitution and transfusion. This was a randomized, prospective, blinded animal study. LP was reconstituted to 50% of original volume with NS, LR, HX, or SW buffered with 15-mM ascorbic acid. Forty swine were subjected to a validated model of polytrauma, hemorrhagic shock, and Grade V liver injury and resuscitated with LP. Serum interleukin 6 (IL-6), IL-10, plasma C-reactive protein, and 8-hydroxy-2-deoxyguanosine concentrations were assessed for systemic inflammation and DNA damage at baseline, 2 hours, and 4 hours following liver injury. Lung inflammation was evaluated by Real Time Polymerize Chain Reaction (RT-PCR). Reconstituted LP pH was similar between groups before resuscitation. IL-6 and IL-10 increased at 2 hours and 4 hours compared with baseline in all groups (p < 0.017). DNA damage increased at 2 hours and 4 hours compared with baseline and from 2 hours to 4 hours in the LP-NS, LP-LR, and LP-SW groups (all p < 0.017). Animals resuscitated with LP-HX not only demonstrated increased DNA damage at 4 hours versus baseline but also had the lowest C-reactive protein level at 2 hours and 4-hours (p < 0.017). Overall, differences between groups were similar for DNA damage and lung inflammation. Reconstitution fluid type does not affect inflammatory cytokine profiles or DNA damage following LP transfusion in this swine polytrauma model. Based on universal availability, these data suggest that sterile water is the most logical choice for LP reconstitution in humans. Prognostic, level II.

Comparison of the hemostatic efficacy of low-volume lyophilized plasma reconstituted using sterile water, lactated Ringer’s, normal saline, and Hextend solutions

Low-volume ascorbic acid-buffered reconstituted lyophilized plasma (LP) provides logistic advantages, reduces the risks for large-volume resuscitation, modulates inflammation, and is equally effective for hemostatic resuscitation as full-volume LP. We compared the physiologic effects of resuscitation using LP reconstituted with sterile water (LP-SW), lactated Ringer's solution (LP-LR), normal saline (LP-NS), and Hextend (LP-Hx). Plasma was collected from swine, lyophilized, and then reconstituted into four test solutions: LP-SW, LP-LR, LP-NS, or LP-Hx. Forty swine were anesthetized and subjected to a validated model of polytrauma and hemorrhagic shock (including a Grade V liver injury), then randomized to receive one of the four test solutions. Physiologic parameters, blood loss, lactate, and hematocrit were followed up. Coagulation status was evaluated using thrombelastography. Inflammatory mediator expression was evaluated by multiplex serum assay. Forty animals were included in the study (10 animals per group). One animal died following LP-Hx resuscitation. There was less blood loss in the LP-SW and LP-LR groups compared with the LP-NS and LP-Hx groups (p < 0.05). The LP-SW group exhibited less early coagulopathic changes by thrombelastography, and the LP-Hx group had persistently elevated international normalized ratios at the end of the study period (p < 0.05). Serum interleukin 6 was lower after 4 hours in the LP-SW group compared with LP-NS (p < 0.05). Resuscitation using low-volume LP-SW and LP-LR buffered with ascorbic acid confers an anti-inflammatory benefit and results in less blood loss. Sterile water is a safe, cost-effective, and universally available fluid for creating a low-volume hemostatic LP resuscitation solution.

Hyperosmolar reconstituted lyophilized plasma is an effective low-volume hemostatic resuscitation fluid for trauma

We performed this study to optimize reconstituted lyophilized plasma (LP) into a minimal volume fluid that provides effective hemostatic resuscitation for trauma while minimizing logistical limitations. We performed a prospective, blinded animal study. Plasma was lyophilized following whole blood collection from anesthetized swine. The minimal volume needed for reconstitution was determined, and this solution was evaluated for safe infusion into the swine. Reconstituted LP was analyzed for electrolyte content, osmolarity, and coagulation factor activity. Twenty swine were anesthetized and subjected to a validated model of polytrauma and hemorrhagic shock (including a Grade V liver injury), then randomized to resuscitation with LP reconstituted to either 100% of the original plasma volume (100%LP) or the minimal volume LP fluid. Physiologic data were monitored, and blood loss and hematocrit were measured. Coagulation status was evaluated using thrombelastography. The minimal volume of reconstituted LP safe for infusion in swine was 50% of the original plasma volume (50%LP). The 50%LP had higher electrolyte concentrations, osmolarity, and increased coagulation factor activity levels by volume compared with 100%LP (p < 0.05). Blood loss, hematocrit, mean arterial pressure, and heart rate did not differ between animals receiving 100%LP (n = 10) or 50%LP (n = 10) at any time point (p > 0.05). International normalized ratio and thrombelastography parameters were not different between groups (R time, α angle, or maximal amplitude, p > 0.05). Resuscitation with 50%LP fluid was well tolerated and equally effective compared with 100%LP, with respect to physiologic and hemostatic properties. The smaller volume of fluid necessary to reconstitute hypertonic LP makes it logistically superior to 100%LP for first responders and may reduce adverse effects of large-volume resuscitation.

The use of lyophilized plasma in a severe multi‐injury pig model

Shock and severe tissue injury lead to an endogenous coagulopathy mediated by activation of Protein C and hyperfibrinolysis known as acute traumatic coagulopathy. Together, hemodilution, acidosis, inflammation, and hypothermia result in a global trauma-induced coagulopathy. Coagulopathy in trauma is associated with mortality. Early and effective hemostatic resuscitation is critical in restoring perfusion, correcting coagulopathy, and saving lives in exsanguinating trauma. Lyophilized plasma (LP) provides a logistically superior alternative to fresh frozen plasma (FFP). Plasma was lyophilized following whole blood collection from anesthetized swine. A series of studies were performed using anesthetized swine subjected to a validated model of polytrauma and hemorrhagic shock including a Grade V liver injury. Animals were randomized to resuscitation using reconstituted LP fluids. Physiologic data and blood loss were measured. Coagulation status and inflammatory mediators were evaluated. Full volume reconstituted LP (100%LP) retains on average 86% coagulation factor activity compared to fresh plasma and when used in 1:1 ratios with red blood cells demonstrated superior hemostatic efficacy compared to FFP. Hypertonic LP reconstituted using 50% of the original plasma volume (50%LP) had higher coagulation factor concentrations, was well tolerated in swine, and equally effective compared to 100%LP with respect to physiologic and hemostatic properties. Buffering with ascorbic acid resulted in significant reductions in serum levels of tumor necrosis factor alpha and interleukin-6. By minimizing the volume of reconstituted LP and optimizing its anti-inflammatory properties, an LP resuscitation fluid may be created to provide effective hemostatic resuscitation with superior logistical properties.

Lyophilized Plasma With Ascorbic Acid Decreases Inflammation in Hemorrhagic Shock

Delivery of a high ratio of plasma to packed red blood cells to patients who require massive transfusion is associated with improved survival. Hemorrhagic shock causes increased production of pro-inflammatory cytokines. These are associated with late morbidity and mortality. The use of fresh frozen plasma makes high ratio resuscitation logistically difficult and does not address dysfunctional inflammation. Lyophilized plasma (LP) is a stable powdered form of plasma that is both safe and easily reconstituted. Previous work demonstrated that LP reconstituted with ascorbic acid (AA) decreased inflammation. Whether the reduction of inflammation was associated with LP or the AA is unknown. Thirty female swine were anesthetized and subjected to a multisystem combat relevant model consisting of femur fracture, controlled hemorrhage, and hypothermia. A standardized grade V liver injury was made and the animals were randomly assigned to receive LP reconstituted with AA, citric acid (CA), or hydrochloric acid (HCl). Blood was drawn at baseline and at 2 hours and 4 hours for interleukin (IL)-6, IL-8, and tumor necrosis factor-α serum concentrations measured by enzyme-linked immunosorbent assay. Lung tissue was harvested and processed for gene expression before euthanizing the animals. No differences were observed in mortality, baseline cytokine serum concentration, or gene expression. Enzyme-linked immunosorbent assay demonstrated that IL-6 concentration increased over time for all groups (p < 0.05), but less so at 2 hours in the AA group compared with CA and HCl. In this animal model of trauma, hemorrhage and resuscitation, AA decreases IL-6 expression relative to CA and HCl. These findings confirm previous work from our laboratory and suggest that AA is responsible for suppression of dysfunctional inflammation in this model.

Lyophilized Plasma Reconstituted With Ascorbic Acid Suppresses Inflammation and Oxidative DNA Damage

Lyophilized plasma (LP) has been shown to be as effective as fresh frozen plasma (FFP) for resuscitation in polytrauma and hemorrhagic shock. LP reconstituted with ascorbic acid is associated with suppression of cytokines when compared with fresh frozen plasma. We aimed to determine the effect of using alternate LP reconstitution acids on physiologic parameters, blood loss, coagulation, oxidative DNA damage, and proinflammatory cytokines in a polytrauma and hemorrhagic shock model. Thirty swine were anesthetized, subjected to polytrauma, hemorrhagic shock, and randomized to resuscitation with LP-ascorbic acid (AA), LP-citric acid (CA), or LP-hydrochloric acid (HCL). Physiologic data were continuously monitored, blood loss measured, and serum collected at baseline, 2 hours, and 4 hours for enzyme-linked immunosorbent assays. Measured 8-OH-2'-deoxyguanosine (8-OHdG) was a biomarker of oxidative DNA damage. No differences were observed in physiologic measures, blood loss, or coagulation parameters. Interleukin-6 increased over time for all groups, but at 2 hours, the concentration in AA (median [minimum, maximum]: 113 ng/mL [0, 244]) was lower compared with CA (181 ng/mL [69, 314], p = 0.01) and HCL (192 ng/mL [41, 310], p = 0.03). Comparing 4 hours to baseline, a significant increase in oxidative DNA damage was observed in CA (22.9 ng/mL [16.3, 34.3] vs. 15.6 ng/mL [13.6, 26.7], p = 0.03) and HCL (19.6 ng/mL [15.7, 56.7] vs. 15.8 ng/mL [11.6, 21.4], p = 0.01) but not in AA (17.9 ng/mL [12.6, 26.9] vs. 17.1 ng/mL [11.8, 18.4], p = 0.24). Resuscitation with AA results in decreased interleukin-6 expression and oxidative DNA damage compared with CA and HCL.

Red Blood Cells Accelerate the Onset of Clot Formation in Polytrauma and Hemorrhagic Shock

Hemorrhage and coagulopathy are major contributors to death after trauma. The contribution of red blood cells (RBCs) in correcting coagulopathy is poorly understood. Current methods of measuring coagulopathy may fail to accurately characterize in vivo clotting. We aimed to determine the effect of RBCs on clotting parameters by comparing resuscitation regimens containing RBCs and plasma with those containing plasma alone. Thirty-two Yorkshire swine were anesthetized, subjected to a complex model of polytrauma and hemorrhagic shock, and resuscitated with either fresh frozen plasma, lyophilized plasma (LP), or 1:1 ratios of fresh frozen plasma:packed RBC (PRBC) or LP:PRBC. Activated clotting time, prothrombin time, partial thromboplastin time, and thrombelastography (TEG) were performed at 1 hour, 2 hours, 3 hours, and 4 hours after resuscitation. Animals treated with 1:1 LP:PRBC had less blood loss than the other groups (p < 0.05). The activated clotting time was shorter in the 1:1 groups when compared with the pure plasma groups at all time points (p < 0.05). The 1:1 groups had shorter TEG R times (time to onset of clotting) at 1 hour, 3 hours, and 4 hours compared with pure plasma groups (p < 0.05). Other TEG parameters did not differ between groups. Partial thromboplastin time was shorter in the pure plasma groups than the 1:1 groups at all time points (p < 0.05). Whole blood assays reveal that RBCs accelerate the onset of clot formation. Coagulation assays using spun plasma underestimate the effect of RBCs on clotting and do not completely characterize clot formation.

Lyophilized Plasma for Resuscitation in a Swine Model of Severe Injury

Lyophilized plasma (LP) is as safe and effective as fresh frozen plasma (FFP) for resuscitation after severe trauma. Multicenter animal study. Animal laboratories, 2 level I trauma centers. Thirty-two Yorkshire crossbred swine. Lyophilized plasma was analyzed for factor levels and clotting activity before lyophilization and after reconstitution. Swine were subjected to complex multiple trauma including extremity fracture, hemorrhage, severe liver injury, acidosis, and hypothermia. They were then resuscitated with FFP, LP, FFP and packed red blood cells (PRBCs) in a ratio of 1:1, or 1:1 LP and PRBCs. Residual clotting activity of LP after reconstitution, swine mortality, hemodynamic measures, total blood loss, coagulation profiles, and inflammatory measures. Lyophilization decreased clotting factor activity by an average of 14%. Survival and heart rate were similar between all groups. Swine resuscitated with LP had equivalent or higher mean arterial pressures. Swine treated with LP had similar coagulation profiles, plasma lactate levels, and postinjury blood loss compared with those treated with FFP. Swine treated with 1:1 FFP-PRBCs were similar to those treated with 1:1 LP-PRBCs. Resuscitation with LP resulted in a reduction in postresuscitation interleukin 6 expression compared with resuscitation with FFP. The process of lyophilization and reconstitution of plasma reduces coagulation factor activity by 14%, without acute differences in blood loss. Lyophilized plasma can be used for resuscitation in a severe multiple trauma and hemorrhagic shock swine model with efficacy equal to that of FFP and with decreased interleukin 6 production.

A comparison of artificially-depleted, lyophilized coumarin and fresh coumarin plasmas in thromboplastin calibration. European Concerted Action on Anticoagulation.

Artificially-depleted lyophilized plasmas and lyophilized coumarin plasmas were prepared and compared with fresh coumarin plasmas to assess their comparative reliability in local thromboplastin calibration using the manual prothrombin time (PT) technique. Their certified PT values were inserted in turn on the vertical axis in place of the PT obtained with fresh coumarin plasmas. PT results were obtained at eight ECAA national laboratories ('test centres') and inserted on the horizontal axis. The resulting thromboplastin calibration slopes were compared with conventional fresh coumarin plasma calibration slopes at the same 'test centres'. When 60 artificially-depleted plasmas were substituted for 60 fresh plasmas, the mean calibration slopes with the human plain International Reference Preparation (IRP) were 4.2% higher. For comparison with 20 lyophilized coumarins, three sets of 20 artificially-depleted plasmas were selected in sequential order from the 60. The lyophilized coumarin plasmas gave a mean deviation of 9.6% from the fresh plasma calibration slopes compared with values of 2.0%, 6.1% and 11.7% for the three sets of 20 depleted plasmas. Although both types of lyophilized plasma calibration slopes give measurable differences from conventional fresh plasmas, these may be regarded as acceptable in clinical terms.