Pathogen Reduced Cryoprecipitate and Fibrinogen Concentrate Have Delayed Resuscitation of Simulated Dilutional Coagulopathy in a Microfluidic Model of Bleeding When Compared to Cryoprecipitate

Impaired fibrinogen function, one component of trauma-associated coagulopathy, is highly associated with increased mortality in patients with severe traumatic bleeding. Fibrinogen replacement is crucial for improving outcomes in bleeding patients. The two most common clinically-used hemostatic adjuncts for fibrinogen supplementation are fibrinogen concentrates (FibCon) and cryoprecipitate (Cryo), yet which of these products provides better hemostatic resuscitation remains controversial. Where FibCon is predominantly a source of fibrinogen, Cryo contains additional factors which may enhance hemostatic efficacy, such as FVIII and FXIII (inherent to clot strength and fibrin crosslinking) and von Willebrand factor (VWF, important for platelet adhesion and aggregation). Cryo poses more logistical challenges than FibCon, as Cryo is a frozen product that requires thawing (20 minutes) prior to use, and has a shelf life of 4-6 hours post thaw. FibCon is lyophilized, and can be reconstituted and used within 10 minutes, but with a cost roughly 3 times that of Cryo, use of FibCon can be cost-prohibitive. Ultimately, in the setting of hemostatic resuscitation, every minute matters and each minute delay in blood product transfusion is associated with a 5% increase in mortality. Moreover, Cryo is associated with an increased risk of transfusion transmitted infection (TTI).Thus, there is a need for an immediately available and safe fibrinogen source for use in hemostatic resuscitation. Pathogen reduction (PR) of blood products renders any nucleic acid-containing source replication incompetent via crosslinking using psoralens and ultraviolet light. PR was recently adapted for use with cryoprecipitate, yielding a novel hemostatic adjunct - pathogen reduced cryoprecipitated fibrinogen complex, or PR-Cryo FC. We have previously shown PR-Cryo FC stored out to 10 days performs similarly to Cryo and FibCon in current standard assays used to assess hemostatic function. However, as primary hemostasis is dictated by physiologically relevant flow conditions, we wanted to determine if PR of cryoprecipitate altered its hemostatic function during resuscitation of dilutional coagulopathy using a microfluidic model of hemorrhage. Healthy human whole blood (WB), Cryo, FibCon, and PR-Cryo FC were stained with fluorescent antibodies specific for VWF, CD41, fibrinogen, and FXIII. Stained WB was diluted 3:7 in 0.9% NaCl to induce dilutional coagulopathy (dWB). dWB was resuscitated 1:5 with stained adjuncts (Cryo:dWB, FibCon:dWB, or PR-Cryo FC:dWB) and perfused at three different shear rates (150, 500, 3500 1/s) through a microfluidic model of hemorrhage (a lumen that "bleeds" through an injury site into a collagen/tissue factor-coated extravascular space). Occlusion of the injury site was defined as the point at which clot formation sealed the injury site for > 3 minutes. The time from initial perfusion to occlusion was defined as the bleeding time (BT, seconds). If no seal was formed, the assay was stopped at 20 minutes, and the assay given a BT of 1200 seconds. Real-time phase and fluorescent images of the injury site were acquired. Data was extracted from real-time phase and fluorescent images using MATLAB. Both FibCon:dWB and PR-Cryo FC:dWB had significantly increased BT compared to Cryo:dWB at low shear (150 1/s). PR-Cryo FC:dWB had significantly increased BT compared to Cryo:dWB at medium shear (500 1/s), and at high shear (3500 1/s) there were no significant differences in BT between hemostatic adjuncts. However, kinetic analysis at high shear revealed there was a significant delay in clot formation and accumulation in the injury site, such that by 5 minutes, Cryo:dWB had filled 75% of the injury site and FibCon:dWB and PR-Cryo FC:dWB had only filled 50% of the injury site. Real-time fluorescent image analysis showed that both FibCon:dWB and PR-Cryo FC:dWB had reduced VWF deposition at the injury site compared to Cryo:dWB, and this led to a delays in platelet recruitment. FibCon has less VWF than Cryo, which would explain the delayed VWF deposition and platelet recruitment. In contrast, PR-Cryo FC and Cryo have similar amounts of VWF, suggesting that VWF from PR-Cryo FC has limited binding to the collagen-coated injury site, and as PR-Cryo FC:dWB phenocopies FibCon:dWB during clot formation at high shear, this suggests that pathogen reduction of Cryo may impair early VWF mediated capture of platelets at high shear. Disclosures Spinella: Secure Transfusion Services: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Cerus Corporation: Consultancy, Research Funding.

Combined Administration of Fibrinogen and Factor XIII Concentrate Does Not Improve Dilutional Coagulopathy Superiorly Than Sole Fibrinogen Therapy: Results of an In-Vitro Thrombelastographic Study

The early administration of fibrinogen has gained wide acceptance for the treatment of major hemorrhage, whereas the substitution of coagulation factor XIII (FXIII) is only supported by a low level of evidence. This study aimed to answer the question of whether a combined therapy of fibrinogen/FXIII substitution performs superiorly to sole fibrinogen administration in the treatment of dilutional coagulopathy. An in-vitro model of massive transfusion was used to compare the effect of combined fibrinogen/FXIII administration to that of sole fibrinogen therapy for the treatment of dilutional coagulopathy. For this purpose, the blood of red blood cell concentrates, fresh frozen plasma, and platelet concentrates were reconstituted in a ratio of 4:4:1, and then diluted with gelatin by 20% and 40%, respectively. Clot formation and stability were analyzed by thrombelastography. Both sole fibrinogen therapy (equivalent to 50 mg/kg) and the combined administration of fibrinogen (equivalent to 50 mg/kg) and FXIII (equivalent to 75 International Units (IU)/kg) increased fibrinogen-dependent mean clot firmness independently of the degree of dilution (20% dilution: 7 (6.3–7.8) mm; 20% dilution fibrinogen: 13.5 (13–17.3) mm; 20% dilution fibrinogen/FXIII: 16.5 (15.3–18.8) mm; 40% dilution: 3 (2–3.8) mm; 40% dilution fibrinogen: 8 (7–11.3) mm; 40% dilution fibrinogen/FXIII: 10 (8.3–11.8) mm; all p < 0.01). However, no differences were identified between the two treatment arms. Compared to fibrinogen therapy, no beneficial effect of the combined administration of fibrinogen and FXIII for the treatment of dilutional coagulopathy was detected in this in-vitro massive transfusion model. The result was independent of the degree of dilution.

Plasmatic and cell-based enhancement by microparticles originated from platelets and endothelial cells under simulated in vitro conditions of a dilutional coagulopathy

Background Aggressive fluid management and other external factors may lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to a trauma-induced coagulopathy (TIC) that worsens patients’ outcomes. Procoagulant microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. Methods Blood from eleven volunteers were collected to simulate in vitro conditions of hemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, we measured their concentration-dependent effects (1.000, 10.000 and 15.000 MP/μl blood) on coagulation compared to whole blood (WB). For each condition, coagulation was characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation using Thrombodynamics® technology. Results MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities (10.000 and 15.000 PDMP/μl blood) significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions as well, only high PDMP concentration induced platelet activation, clot growth and size. In contrast, EDMP did not induce platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions. Discussion The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. PDMP affected platelets promoting clot formation likely by providing a surface enlargement. EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous clots. Conclusion Under simulated conditions of a dilutional coagulopathy, MP from different cellular origin indicate a divergent but both procoagulant mechanism within the coagulation process.

Outcome of Major Hemorrhage at a Major Cardiothoracic Center in Patients with Activated Major Hemorrhage Protocol versus Nonactivated Protocol

This study aimed to determine the impact of major hemorrhage (MH) protocol (MHP) activation on blood administration and patient outcome at a UK major cardiothoracic center. MH was defined in patients (> 16 years) as those who received > 5 units of red blood cells (RBCs) in < 4 hours, or > 10 units in 24 hours. Data were collected retrospectively from patient electronic records and hospital transfusion databases recording issue of blood products from January 2016 to December 2018. Of 134 patients with MH, 24 had activated MHP and 110 did not have activated MHP. Groups were similar for age, sex, baseline hemoglobin, platelet count, coagulation screen, and renal function with no difference in the baseline clinical characteristics. The total number of red cell units (median and [IQR]) transfused was no different in the patients with activated (7.5 [5–11.75]) versus nonactivated (9 [6–12]) MHP (p = 0.35). Patients in the nonactivated MHP group received significantly higher number of platelet units (median: 3 vs. 2, p = 0.014), plasma (median: 4.5 vs. 1.5, p = 0.0007), and cryoprecipitate (median: 2 vs. 1, p = 0.008). However, activation of MHP was associated with higher mortality at 24 hours compared with patients with nonactivation of MHP (33.3 vs. 10.9%, p = 0.005) and 30 days (58.3 vs. 30.9%, p = 0.01). The total RBC and platelet (but not fresh frozen plasma [FFP]) units received were higher in deceased patients than in survivors. Increased mortality was associated with a higher RBC:FFP ratio. Only 26% of patients received tranexamic acid and these patients had higher mortality at 30 days but not at 24 hours. Deceased patients at 30 days had higher levels of fibrinogen than those who survived (median: 2.4 vs. 1.8, p = 0.01). Patients with activated MHP had significantly higher mortality at both 24 hours and 30 days despite lack of difference in the baseline characteristics of the patients with activated MHP versus nonactivated MHP groups. The increased mortality associated with a higher RBC:FFP ratio suggests dilutional coagulopathy may contribute to mortality, but higher fibrinogen at baseline was not protective.

Plasmatic and cell-based enhancement by microparticles originated from platelets and endothelial cells under simulated in vitro conditions of a dilutional coagulopathy

Background: Aggressive fluid management and other external factors may lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to a trauma-induced coagulopathy (TIC) that worsens patients’ outcomes. Procoagulant microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. Methods: Blood from eleven volunteers were collected to simulate in vitro conditions of hemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, we measured their concentration-dependent effects (1.000, 10.000 and 15.000 MP/µl blood) on coagulation compared to whole blood (WB). For each condition, coagulation was characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation using Thrombodynamics® technology.Results: MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities (10.000 and 15.000 PDMP/µl blood) significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions as well, only high PDMP concentration induced platelet activation, clot growth and size. In contrast, EDMP did not induce platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions.Discussion: The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. PDMP affected platelets promoting clot formation likely by providing a surface enlargement. EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous clots.Conclusion: Under simulated conditions of a dilutional coagulopathy, MP from different cellular origin indicate a divergent but both procoagulant mechanism within the coagulation process.

Extracellular Mitochondria in Traumatic Brain Injury Induced Coagulopathy

Traumatic brain injury (TBI) induced coagulopathy remains a significant clinical challenge, with unmet needs for standardizing diagnosis and optimizing treatments. TBI-induced coagulopathy is closely associated with poor outcomes in affected patients. Recent studies have demonstrated that TBI induces coagulopathy, which is mechanistically distinct from the deficient and dilutional coagulopathy found in patients with injuries to the body/limbs and hemorrhagic shock. Multiple causal and disseminating factors have been identified to cause TBI-induced coagulopathy. Among these are extracellular mitochondria (exMTs) released from injured cerebral cells, endothelial cells, and platelets. These circulating exMTs not only express potent procoagulant activity but also promote inflammation, and could remain metabolically active to become a major source of oxidative stress. They activate platelets and endothelial cells to propagate TBI-induced coagulopathy and secondary tissue injury after primary traumatic impact. In this review, we discuss recent advances in our understanding of the role of exMTs in the development of TBI-induced coagulopathy.

Perfusion Considerations

The effects of hemodilution during cardiopulmonary bypass (CPB) negatively impact a patient’s hemodynamic and coagulopathic states throughout all operative stages and into the postoperative care environment. Decisions by the surgical team to minimize fluid administration prove beneficial, and combining contemporary perfusion circuit prime reduction techniques, such as body mass index (BMI) correction, venous autologous priming (VAP), retrograde autologous priming (RAP), and antegrade prime displacement, greatly enables efforts of maintaining proper colloidal osmotic pressures (COP) while maximizing delivery of oxygen (DO2) during CPB. Adjuncts such as ultrafiltration (UF), use of vacuum assist venous drainage (VAVD), hyper-osmotic/oncotic solutions administration, and proportional volume replacement better prepare a patient for post-bypass management. Minimizing hemodilution tools facilitates transition onto mechanical circulatory support (MCS) devices such as extracorporeal membrane oxygenation (ECMO) and/or right or left ventricular assist device (RVAD/LVAD). Avoiding heparin rebound and dilutional coagulopathy in the intensive care unit further prevents additional blood product transfusions.