Nora Philbin

Bovine polymerized hemoglobin (hemoglobin-based oxygen carrier-201) resuscitation in three swine models of hemorrhagic shock with militarily relevant delayed evacuation--effects on histopathology and organ function.

Objective:To test our hypothesis that hemoglobin-based oxygen carrier (HBOC)-201 resuscitation in hemorrhagic shock (HS) will not lead to increased organ injury and dysfunction. Design:Three swine HS models simulating military-relevant delayed evacuation: a) moderate controlled HS, b) severe controlled HS, and c) severe uncontrolled HS. Setting:Military research laboratory. Subjects:Swine. Interventions:Swine were anesthetized/intubated and instrumented. To induce HS, in two controlled hemorrhage experiments, 40% (moderate controlled HS) or 55% (severe controlled HS) of blood volume was withdrawn; in an uncontrolled HS experiment, the liver was crushed/lacerated. During a 4-hr “prehospital phase,” pigs were resuscitated with HBOC-201 (HBOC) or Hextend (HEX) or were nonresuscitated (NON). Upon “hospital arrival,” liver injury was repaired (severe uncontrolled HS), blood or saline was infused, hemodynamics were monitored, and blood was collected. Upon animal death and/or 72 hrs, necropsy was followed by histopathologic evaluation of organ injury (hematoxylin and eosin, electron microscopy) and immunohistochemistry of oxidative potential (3-nitrotyrosine). Significance (p < .05) was assessed by Kruskal-Wallis, analysis of variance/Bonferroni, and mixed procedure tests. Measurements and Main Results:Survival was significantly higher with HBOC than HEX only with severe uncontrolled HS (p = .002). Myocardial necrosis/fibroplasia, fluid requirements, cardiac output, and cardiac enzymes were generally similar or lower in HBOC than HEX pigs, but creatine kinase-MB (but not creatine kinase-MB/creatine kinase ratio) was higher with HBOC in moderate controlled HS. Alveolar/interstitial pulmonary edema was similar with HBOC and HEX, but Po2 was higher with HBOC in severe uncontrolled HS. Jejunal villar epithelial and hepatocellular necrosis were similarly minimal to moderate in all groups. Minimal biliary changes occurred exclusively with HBOC. Aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase were generally higher with HBOC than HEX. Mild renal papillary injury occurred more frequently with HBOC, but consistent patterns for urine output, blood urea nitrogen, and creatinine, were not seen. The 3-nitrotyrosine staining intensity was not different. Conclusions:In comparison with hetastarch, HBOC-201 resuscitation of swine with HS increased survival (with severe HS), did not increase evidence of oxidative potential, and had histopathologic and/or functional effects on organs that were clinically equivocal (myocardium, lungs, hepatic parenchyma, jejunum, and renal cortex/medulla) and potentially adverse (hepatobiliary and renal papilla). The effects of HBOC-201-resuscitation in HS should be corroborated in controlled clinical trials.

Immune effects of resuscitation with HBOC-201, a hemoglobin-based oxygen carrier, in swine with moderately severe hemorrhagic shock from controlled hemorrhage.

HBOC-201, a hemoglobin-based oxygen carrier, improved physiologic parameters and survival in hemorrhagic shock (HS) animal models. However, resuscitation from HS and the properties of different fluids influence immune responses. The aim of this study was to determine if HBOC-201 significantly alters immune function in traumatic HS. Anesthetized pigs underwent soft tissue injury, controlled hemorrhage of 40% of blood volume, and resuscitation with HBOC-201 or Hextend, or no resuscitation. Sequential whole-blood samples were collected for analyses of leukocyte differential (hematology analyzer), T-lymphocyte subsets (CD3+, CD4+, and CD8+) (FACS), lymphocyte adhesion marker CD49d (α4-integrin) expression (FACS), plasma cytokines-tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10-(ELISA), and lymphocyte apoptosis (annexin-V/propidium iodide staining) (FACS). Statistical analyses were performed by the mixed procedure. Total WBC counts decreased posthemorrhage in both resuscitation groups. Lymphocyte percentages decreased and PMN percentages increased around 4 h posthemorrhage in all groups. CD3 cells decreased in all groups, but CD4 and CD8 cells decreased only in the resuscitation groups. TNF-α levels were not detectable in any groups. IL-6 levels were similar across treatment groups (P > 0.05); however, IL-10 levels were higher in the HBOC group, as early as 1 h posthemorrhage (P = 0.04). Increases in lymphocytic CD49d expression levels and apoptosis occurred only in nonresuscitation and Hextend groups, respectively (P ≤ 0.01). In comparison with Hextend, HBOC-201 had no significant adverse or beneficial effects on immune function in this model of moderately severe HS in swine, suggesting that it may be safe as a resuscitation fluid in HS patients.

The Effects of Decreasing Low-molecular Weight Hemoglobin Components of Hemoglobin-based Oxygen Carriers in Swine With Hemorrhagic Shock

BACKGROUND Some hemoglobin-based oxygen carriers (HBOCs) improve outcome in animal models of hemorrhagic shock (HS) in comparison with standard asanguinous resuscitation fluids. Nevertheless, concern about intrinsic vasoactivity, linked in part to low-molecular weight (MW) hemoglobin (Hb), has slowed HBOC development. We assessed the impact of decreasing the low-MW Hb component of bovine HBOC on vasoactivity in severe HS. METHODS Anesthetized invasively monitored swine were hemorrhaged 55% blood volume and resuscitated with bovine HBOC containing 31% (31 TD [HBOC-301]), 2% (2 TD [HBOC-201]), or 0.4% (0.4 TD) low-MW Hb. Pigs received four 10 mL/kg infusions over 60 minutes, hospital arrival was simulated at 75 minutes, organ blood flow (BF) was evaluated by microsphere injection, and monitoring was continued for 4 hours followed by complete necrotic evaluation. RESULTS There were few differences between 2 TD and 0.4 TD. Thirty-one TD pigs had higher systemic and pulmonary blood pressure (BP), systemic vascular resistance index, and pulmonary artery wedge pressure, compared with 2 TD or 0.4 TD (p < 0.01); however, pigs in all groups had at least mildly elevated BP. Transcutaneous tissue oxygenation, base excess, and mixed venous oxygen saturation were similar across groups; lactate and methemoglobin were highest with 0.4 TD (p < 0.03). There were no group differences in BF. Over time, myocardial BF increased and hepatic BF decreased in all groups (for 31 TD, p < 0.05); renal BF was unchanged in all groups. There were no group differences in heart, lung, or liver histopathology, and survival. CONCLUSIONS Although purification from 31% to 2% low-MW Hb content significantly decreased vasoactive responses, further purification to 0.4% had no additional clinically measurable effects in severe HS. If further diminution in HBOC vasoactivity is desired for use in HS, additional technical approaches may be required.

EFFECTS OF BOVINE POLYMERIZED HEMOGLOBIN ON COAGULATION IN CONTROLLED HEMORRHAGIC SHOCK IN SWINE

HBOC-201, a bovine polymerized hemoglobin, has been proposed as a novel oxygen-carrying resuscitative fluid for patients with hemorrhagic shock (HS). Herein, we evaluated the hemostatic effects of HBOC-201 in an animal model of HS. A 40% blood loss-controlled hemorrhage and soft tissue injury were performed in 24 invasively monitored Yucatan mini-pigs. Pigs were resuscitated with HBOC-201 (HBOC) or hydroxyethyl starch (HEX), or were not resuscitated (NON) based on cardiac parameters during a 4-h prehospital phase. Afterward, animals received simulated hospital care for 3 days with blood or saline transfusions. Hemostasis measurements included in vivo bleeding time (BT), thromboelastography (TEG), in vitro bleeding time (platelet function; PFA-CT), prothrombin time (PT), and partial thromboplastin time (PTT). Serum lactate was measured and lung sections were evaluated for microthrombi by electron microscopy. During the prehospital phase, BT remained unchanged in the HBOC group. TEG reaction time increased in HBOC pigs during the late prehospital phase and was greater than in NON or HEX pigs at 24 h (P = 0.03). TEG maximum amplitude was similar for the two fluid-resuscitated groups. PFA-CT increased in both resuscitated groups but less with HBOC (P = 0.02) in the prehospital phase; this effect was reversed by 24 h (P = 0.02). In the hospital phase, PT decreased (P < 0.02), whereas PTT increased above baseline (P < 0.01). Lactic acidosis in HBOC and HEX groups was similar. Aspartate aminotransferase was relatively elevated in the HBOC group at 24 h. Electron microscopy showed no evidence of platelet/fibrin clots or microthrombi in any of the animals. Twenty-four-hour group differences mainly reflected the fact that all HEX animals (8/8) received blood transfusions compared with only one HBOC animal (1/8). In swine with HS, HBOC resuscitation induced less thrombopathy than HEX during the prehospital phase. Mild delayed effects on platelet and clot formation during the hospital phase are transient and likely related to fewer blood transfusions. In swine with HS, HBOC resuscitation induced less thrombopathy than HEX during the prehospital phase but more thrombopathy in the hospital phase. The delayed effects on platelet and clot formation during the hospital phase are transient and may be related to the need for fewer blood transfusions.

Traumatic brain injury and severe uncontrolled haemorrhage with short delay pre-hospital resuscitation in a swine model

INTRODUCTION Unavailability of blood (and oxygen delivery) for pre-hospital resuscitation in haemorrhagic shock patients are major problems, supporting the importance for novel resuscitation strategies. In a combined polytrauma model of uncontrolled haemorrhage and traumatic brain injury (TBI) in swine, we investigated if pre-hospital administration of the haemoglobin based oxygen carrier HBOC-201 will improve tissue oxygenation and physiologic parameters compared to Lactated Ringers (LR) solution. MATERIALS AND METHODS Anaesthetised Yorkshire swine underwent fluid-percussion TBI and Grade III liver laceration. During a 30-min pre-hospital phase, the animals were resuscitated with a single infusion of HBOC-201, LR solution, or nothing (NON). Upon hospital arrival, the animals were given blood or normal saline as needed. Surviving animals were euthanised 6h post-injury. Cerebral blood flow was measured by microsphere injection, and pathology was assessed by gross observation and immunohistochemical analysis. RESULTS Mean TBI force (2.4±0.1atm) (means±standard error of the mean) and blood loss (22.5±1.7mL/kg) were similar between groups. Survival at the 6h endpoint was similar in all groups (∼50%). Cerebral perfusion pressure (CPP) and brain tissue oxygen tension were significantly greater in HBOC-201 as compared with LR animals (p<0.005). Mean arterial pressure (MAP) and mean pulmonary artery pressure (MPAP) were not significantly different amongst groups. Blood transfusion requirements were delayed in HBOC-201 animals. Animals treated with HBOC-201 or LR showed no immunohistopathological differences in glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP-2). Severity of subarachnoid and intraparenchymal haemorrhages were similar for HBOC and LR groups. CONCLUSION In this polytrauma swine model of uncontrolled haemorrhage and TBI with a 30-min delay to hospital arrival, pre-hospital resuscitation with one bolus of HBOC-201 indicated short term benefits in systemic and cerebrovascular physiological parameters. True clinical benefits of this strategy need to be confirmed on TBI and haemorrhagic shock patients.

Shock index and prediction of traumatic hemorrhagic shock 28-day mortality: data from the DCLHb resuscitation clinical trials.

Introduction To assess the ability of the shock index (SI) to predict 28-day mortality in traumatic hemorrhagic shock patients treated in the diaspirin cross-linked hemoglobin (DCLHb) resuscitation clinical trials. Methods We used data from two parallel DCLHb traumatic hemorrhagic shock efficacy trials, one in U.S. emergency departments, and one in the European Union prehospital setting to assess the relationship between SI values and 28-day mortality. Results In the 219 patients, the mean age was 37 years, 64% sustained a blunt injury, 48% received DCLHb, 36% died, and 88% had an SI≥1.0 at study entry. The percentage of patients with an SI≥1.0 dropped by 57% (88 to 38%) from the time of study entry to 120 minutes after study resuscitation (p<0.001). Patients with a SI≥1.0, 1.4, and 1.8 at any time point were 2.3, 2.7, and 3.1 times, respectively, more likely to die by 28 days than were patients with SI values below these cutoffs (p<0.001). Similarly, after 120 minutes of resuscitation, patients with a SI≥1.0 were 3.9× times more likely to die by 28 days (40 vs. 15%, p<0.001). Although the distribution of SI values differed based on treatment group, the receiver operator characeristics data showed no difference in SI predictive ability for 28-day mortality in patients treated with DCLHb. Conclusion In these traumatic hemorrhagic shock patients, the shock index correlates with 28-day mortality, with higher SI values indicating greater mortality risk. Although DCLHb treatment did alter the distribution of SI values, it did not influence the ability of the SI to predict 28-day mortality.

Hematology patterns after hemoglobin-based oxygen carrier resuscitation from severe controlled hemorrhage with prolonged delayed definitive care.

BACKGROUND: The hemoglobin‐based oxygen carrier (HBOC‐201) resuscitation fluid improves outcome in hemorrhagic shock swine models with minimal coagulopathy. Herein, coagulation parameters were evaluated after resuscitation with HBOC‐201 after severe bleeding and prolonged delay to definitive care.

Diaspirin Cross-Linked Hemoglobin Infusion Did Not Influence Base Deficit and Lactic Acid Levels in Two Clinical Trials of Traumatic Hemorrhagic Shock Patient Resuscitation

BACKGROUND Diaspirin cross-linked hemoglobin (DCLHb) has demonstrated a pressor effect that could adversely affect traumatic hemorrhagic shock patients through diminished perfusion to vital organs, causing base deficit (BD) and lactate abnormalities. METHODS Data from two parallel, multicenter traumatic hemorrhagic shock clinical trials from 17 US Emergency Departments and 27 European Union prehospital services using DCLHb, a hemoglobin-based resuscitation fluid. RESULTS In the 219 patients, the mean age was 37.3 years, 64% of the patients sustained a blunt injury, 48% received DCLHb resuscitation, and the overall 28-day mortality rate was 36.5%. BD data did not differ by treatment group (DCLHb vs. normal saline [NS]) at any time point. Study entry BD was higher in patients who died when compared with survivors in both studies (US: -14.7 vs. -9.3 and European Union: -11.1 vs. -4.1 mEq/L, p < 0.003) and at the first three time points after resuscitation. No differences in BD based on treatment group were observed in either those who survived or those who died from the hemorrhagic shock. US lactate data did not differ by treatment group (DCLHb vs. NS) at any time point. Study entry lactates were higher in US patients who ultimately died when compared with survivors (82.4 vs. 56.1 mmol/L, p < 0.003) and at all five postresuscitation time points. No lactate differences were observed between DCLHb and NS survivors or in those who died based on treatment group. CONCLUSIONS Although patients who died had more greatly altered perfusion than those who survived, DCLHb treatment of traumatic hemorrhagic shock patients was not associated with BD or lactate abnormalities that would indicate poor perfusion.

Hematologic effects of recombinant factor Viia combined with hemoglobin-based oxygen carrier-201 for prehospital resuscitation of swine with severe uncontrolled hemorrhage due to liver injury

The combination of traumatic injury, hemorrhage, and fluid resuscitation results in consumption and dilution of coagulation factors, adversely impacting hematology outcome in trauma patients. The hemostatic effects of escalating doses of recombinant factor VIIa added to hemoglobin-based oxygen carrier-201 were assessed as prehospital fluid resuscitation in swine with severe uncontrolled hemorrhage. Swine underwent liver injury causing severe uncontrolled hemorrhage and shock. During a 4-h prehospital phase, either hypotensive or tachycardic, or both, animals were resuscitated with hemoglobin-based oxygen carrier-201 without (0×) or with escalating doses of recombinant factor VIIa [90 μg/kg (1×), 180 μg/kg (2×), or 360 μg/kg (4×)]. The animals received one initial full dose of 10 ml/kg at 15 min and up to four doses of 5 ml/kg thereafter. From 4 to 72 h (hospital phase), animals received either transfusions or isotonic saline or both as needed. Hematology profile (complete blood count), thromboelastography, in-vitro bleeding (platelet function analyzer), and coagulation (prothrombin time) were measured and the results were compared using mixed statistical models. In all groups, dilutional coagulopathy was evidenced by reduced hematocrit, platelets, and thromboelastography-maximum amplitude, and increased platelet function analyzer closure time and thromboelastography-reaction time. During the prehospital phase, hemoglobin-based oxygen carrier-201 restored hemoglobin in all groups. Recombinant factor VIIa decreased prothrombin time in recombinant factor VIIa groups compared with the hemoglobin-based oxygen carrier-201 group (P < 0.01). Unexpectedly, increasing recombinant factor VIIa dosage tended to increase fluid requirement (P > 0.05). Compared with hemoglobin-based oxygen carrier, 1× recombinant factor VIIa tended to decrease blood loss, lactate and thromboelastography-reaction time at 24 h but the 4× group increased these parameters. Platelets and thromboelastography-maximum amplitude decreased (P < 0.01) with the 4× group. In swine with severe uncontrolled hemorrhage, prehospital resuscitation with escalating doses of recombinant factor VIIa in combination with hemoglobin-based oxygen carrier-201 did not change survival or hemostasis. However, there were trends toward possible benefits of low recombinant factor VIIa doses, whereas high recombinant factor VIIa doses adversely affected hemostasis.

The Lack of Consistent Diaspirin Cross-Linked Hemoglobin Infusion Blood Pressure Effects in the US and EU Traumatic Hemorrhagic Shock Clinical Trials

Hemoglobin solutions have demonstrated a pressor effect that could adversely affect hemorrhagic shock patient resuscitation through accelerated hemorrhage, diminished perfusion, or inadequate resuscitation. Data from two parallel, multicenter traumatic hemorrhagic shock clinical trials in 17 US emergency departments and in 27 EU prehospital systems using diaspirin cross-linked hemoglobin (DCLHb), a hemoglobin-based resuscitation fluid. In the 219 patients, patients were 37 years old, 64% sustained blunt injury, 48% received DCLHb, and 36% expired. Although mean systolic blood pressure (SBP) and diastolic blood pressure values differed at 2 of the 10 measured time points, blood pressure (BP) curve analysis showed no SBP, diastolic blood pressure, or MAP differences based on treatment. Although SBP values 160 and 120 mmHg or greater were 2.2× and 2.6× more frequently noted in survivors, they were not more common with DCLHb use or in DCLHb patients who expired in US study nonsurvivors or in any EU study patients. Systolic blood pressure values 160 and 120 mmHg or greater were 2.8× and 1.3× more frequently noted in DCLHb survivors as compared with normal saline survivors. Only 3% of the BP variation noted could be attributed to DCLHb use, and as expected, injury severity and baseline physiologic status were stronger predictors. In the United States alone, treatment group was not correlated by regression with BP at any time point. Neither mean BP readings nor elevated BP readings were correlated with DCLHb treatment of traumatic hemorrhagic shock patients. As such, no clinically demonstrable DCLHb pressor effect could be directly related to the adverse mortality outcome observed in the US study.