John R. Hess

Intravenous rFVIIa Administered for Hemorrhage Control in Hypothermic Coagulopathic Swine with Grade V Liver Injuries

BACKGROUND Intravenous administration of recombinant activated human clotting factor VII (rFVIIa) has been used successfully to prevent bleeding in hemophilia patients undergoing elective surgery, but not in previously normal trauma patients. This study was conducted to determine whether rFVIIa was a useful adjunct to gauze packing for decreasing blood loss from grade V liver injuries in hypothermic and coagulopathic swine. METHODS All animals (n = 10, 35 +/- 2 kg) underwent a 60% isovolemic exchange transfusion with 6% hydroxyethyl starch and were cooled to 33 degrees C core temperature. The swine then received a grade V liver injury and 30 seconds later, either 180 microg/kg rFVIIa, or saline control. All animals were gauze packed 30 seconds after injury and resuscitated 5.5 minutes after injury with lactated Ringers solution to their preinjury mean arterial pressure. Posttreatment blood loss, mean arterial pressure, resuscitation volume, and clotting studies were monitored for 1 hour. Histology of lung, kidney, and small bowel were obtained to evaluate for the presence of microvascular thrombi. RESULTS At the time of injury, core temperature was 33.3 degrees +/- 0.4 degrees C, hemoglobin was 6 +/- 0.7 g/dL, prothrombin time was 19.1 +/- 1.0 seconds, activated partial thromboplastin time was 29.0 +/- 4.8 seconds, fibrinogen was 91 +/- 20 mg/dL, and platelets were 221 +/- 57 x 105/mL, with no differences between groups (p > 0.05). Clotting factor levels confirmed a coagulopathy at the preinjury point. The posttreatment blood loss was less (p < 0.05) in group 1 (527 +/- 323 mL), than in group 2 (976 +/- 573 mL). The resuscitation volume was not different (p > 0.05). One-hour survival in both groups was 100%. Compared with the control group, rFVIIa increased the circulating levels of VIIa and, despite hypothermia, shortened the prothrombin time 5 minutes after injection (p < 0.05). Laboratory evaluation revealed no systemic activation of the clotting cascade. Postmortem evaluation revealed no evidence of large clots in the hepatic veins or inferior vena cava, or microscopic thrombi in lung, kidney, or small intestine. CONCLUSION rFVIIa reduced blood loss and restored abnormal coagulation function when used in conjunction with liver packing in hypothermic and coagulopathic swine. No adverse effects were identified.

Advanced hemostatic dressing development program: animal model selection criteria and results of a study of nine hemostatic dressings in a model of severe large venous hemorrhage and hepatic injury in Swine.

BACKGROUND An advanced hemostatic dressing is needed to augment current methods for the control of life-threatening hemorrhage. A systematic approach to the study of dressings is described. We studied the effects of nine hemostatic dressings on blood loss using a model of severe venous hemorrhage and hepatic injury in swine. METHODS Swine were treated using one of nine hemostatic dressings. Dressings used the following primary active ingredients: microfibrillar collagen, oxidized cellulose, thrombin, fibrinogen, propyl gallate, aluminum sulfate, and fully acetylated poly-N-acetyl glucosamine. Standardized liver injuries were induced, dressings were applied, and resuscitation was initiated. Blood loss, hemostasis, and 60-minute survival were quantified. RESULTS The American Red Cross hemostatic dressing (fibrinogen and thrombin) reduced (p < 0.01) posttreatment blood loss (366 mL; 95% confidence interval, 175-762 mL) and increased (p < 0.05) the percentage of animals in which hemostasis was attained (73%), compared with gauze controls (2,973 mL; 95% confidence interval, 1,414-6,102 mL and 0%, respectively). No other dressing was effective. The number of vessels lacerated was positively related to pretreatment blood loss and negatively related to hemostasis. CONCLUSION The hemorrhage model allowed differentiation among topical hemostatic agents for severe hemorrhage. The American Red Cross hemostatic dressing was effective and warrants further development.

Dry Fibrin Sealant Dressings Reduce Blood Loss, Resuscitation Volume, and Improve Survival in Hypothermic Coagulopathic Swine with Grade V Liver Injuries

OBJECTIVE The majority of early trauma deaths are caused by uncontrolled hemorrhage, and are frequently complicated by hypothermic and dilutional coagulopathies. Any hemorrhage-control technique that achieves rapid hemostasis despite a coagulopathy should improve the outcome of these patients. We conducted this study to determine whether dry fibrin sealant dressings (DFSD) would stop bleeding from grade V liver injuries in swine that were hypothermic and coagulopathic. METHODS Nineteen swine weighing 39.7 kg (mean and 95% confidence interval, 36.3-43.1), underwent a 60% isovolemic, hypothermic exchange transfusion with 33 degrees C 6% hetastarch to produce a dilutional and hypothermic coagulopathy. The animals then received a grade V liver injury and one of three treatments: DFSD, conventional liver packing with gauze sponges, or immunoglobulin G (IgG) placebo sealant dressing (blinded control). All animals were resuscitated with lactated Ringers solution to their preinjury mean arterial pressure. Blood loss after treatment, mean arterial pressure, resuscitation volume, hematologic variables, and core temperature were monitored for 1 hour. RESULTS At the time of injury, core temperature = 33.3 degrees C (95% confidence interval, 33.2-33.4), hemoglobin concentration = 4.4 g/dL (4.2-4.6), platelet count = 132 x 10(5)/microL, (93-171), prothrombin time = 21.6 seconds (19.6-23.5), activated partial thromboplastin time = 25.2 seconds (range, 22.9-27.5 seconds), and fibrinogen = 83 mg/dL (range, 76-89 mg/dL) across treatments. The posttreatment blood loss in the DFSD group was 669 mL, (range, 353-1,268 mL), which was lower (p < 0.01) than the means of 3,321 mL (range, 1,891-5,831 mL) and 4,399 mL (range, 2,321-8,332 mL) observed in the packing and IgG groups, respectively. The resuscitation volume in DFSD was 2,145 mL (range 1,310-3,514 mL), which was lower (p < 0.05) than the means of 5,222 mL (range 3,381-8,067 mL) and 5,542 mL (range 3,384-9,077 mL) in the packing and IgG groups, respectively. One-hour survival in the DFSD group was 83%, whereas survival in the packing and IgG groups were 0% (p < 0.05). CONCLUSION In swine with a grade V liver injury complicated by a dilutional and hypothermic coagulopathy, DFSD provided simple, rapid hemorrhage control, decreased fluid requirements, and improved survival.

Fibrin sealant foam sprayed directly on liver injuries decreases blood loss in resuscitated rats

OBJECTIVE The majority of early trauma deaths are attributable to uncontrolled hemorrhage from truncal sites. A hemorrhage-control technique that reduced bleeding in the prehospital phase of treatment without requiring manual compression may improve the outcome of these patients. We conducted this preliminary study to determine whether an expanding fibrin sealant foam (FSF) would reduce bleeding from a severe liver injury even during resuscitation. METHODS Rats (n = 31; 291 +/- 5 g; 37.4 +/- 0.3 degrees C; mean +/- SEM), underwent a 60 +/- 5% excision of the median hepatic lobe. The animals received one of three treatments: (1) FSF, (2) immunoglobulin G placebo foam (IgGF), or (3) no treatment. All animals were resuscitated with 40 degrees C lactated Ringers solution at 3.3 mL/ min/kg to a mean arterial pressure of 100 mm Hg. Total blood loss, mean arterial pressure, and resuscitation volume were recorded for 30 minutes. A qualitative measure of foam coverage and adherence to the cut liver edge was recorded. RESULTS The total blood loss was less (p < 0.01) in the FSF group (21.2 +/- 5.0 mL/kg) than in either IgGF (41.4 +/- 4.3 mL/kg) or the no treatment group (44.6 +/- 4.7 mL/kg), which did not differ. The resuscitation volume was not different. The amount of foam used in the treated groups, 9.1 +/- 1.0 g in the FSF group and 10.0 +/- 1.0 g in the IgGF group, did not differ. Survival for 30 minutes was not different among groups. There was no difference in the amount of cut liver covered by either foam, but the clots were more adherent (p < 0.05) in the FSF group than in the IgGF group. CONCLUSION In rats with a severe liver injury, spraying fibrin foam directly on the cut liver surface decreased blood loss when compared with placebo foam and no treatment. This pilot study suggests a future possible treatment for noncompressible truncal hemorrhage.

IMPLICATIONS OF NEW DRY FIBRIN SEALANT TECHNOLOGY FOR TRAUMA SURGERY

Trauma patients have been bleeding to death for thousands of years. The methods used to control hemorrhage (tourniquets, pressure, bandages, and ligatures) have not changed for 2000 years. Technology now exists to amplify the normal clotting system with human proteins, thus providing almost instant hemorrhage control in the face of bleeding. The increasing body of clinical and animal research and safety data regarding new fibrin sealant technologies is compelling. When combined with the evolving concepts of extended trauma resuscitation, acceptance of this technology will finally add a new method of rapid, easy hemostasis to the armamentarium of the surgeon faced with an unstable hemorrhaging patient. Several important issues remain unresolved, such as optimal thrombin and fibrinogen content, amount of material required for hemostasis, long-term effects, distribution of breakdown products, and role of recombinant proteins. These issues are under active investigation. Despite these unanswered questions, the field of absorbable, off-the-shelf, rapidly active hemostatic agents that do not require refrigeration is an exciting area that should yield significant improvements in the care of injured patients.

Pulmonary hypertension and systemic vasoconstriction may offset the benefits of acellular hemoglobin blood substitutes.

OBJECTIVE We tested the hypothesis that the pharmacologic properties of a small volume of alpha alpha-cross-linked hemoglobin (alpha alpha Hb) could effectively resuscitate pigs subjected to hemorrhage. METHODS Fourteen pigs hemorrhaged to a mean arterial pressure (MAP) of 40 mm Hg for 60 minutes were treated with a 4-mL/kg 2-minute infusion of 10 g/dL alpha alpha Hb or 7 g/dL human serum albumin, an oncotically matched control solution. RESULTS The removal of blood (17 +/- 1.5 mL/kg) caused the typical physiologic responses to hemorrhagic hypovolemia. Infusion of alpha alpha Hb restored mean arterial pressure and coronary perfusion pressure, but cardiac output and mixed venous O2 saturation did not improve significantly. Pulmonary arterial pressure and pulmonary vascular resistance increased markedly and were higher than baseline levels after alpha alpha Hb. Infusion of human serum albumin produced only minor hemodynamic changes. Brain blood flow did improve to baseline values after alpha alpha Hb, but was the only tissue to do so. In the human serum albumin group, superior mesenteric artery blood flow recovered to baseline values, whereas brain blood flow did not. Blood flows to other tissues were similar in both groups. CONCLUSION Small-volume infusion of alpha alpha Hb restored mean arterial pressure and brain blood flow, but pulmonary hypertension and low peripheral perfusion may offset benefits for trauma patients.

Liver and Kidney Injury After Administration of Hemoglobin Cross-Linked with Bis(3,5-Dibromosalicyl) Fumarate

Human hemoglobin cross-linked between the alpha chains with bis (3,5-dibromosalicyl) fumarate (DBBF-Hb) was exchange transfused in swine and the histomorphologic changes were evaluated. Following exchange, animals were euthanized and tissues were taken for light and electron microscopy at 7.5 hours and days 1, 4, 7, and 15. Consistent hepatocellular and renal epithelial cell changes were seen. Hepatic injury, evident at 7.5 hours as cellular vacuolization, progressed to necrosis and acute inflammatory cell infiltration by days 1 and 4, was resolving by 7 days and was completely resolved by day 15. Cytochemical stains for iron and hemoglobin revealed positive material in Kupffer cells, endothelial cells, and necrotic hepatocytes. Rabbit anti-human hemoglobin antibody staining revealed immunoreactive material diffusely present at days 1 and 4 and limited to solitary hepatocytes by day 15. Kidney injury began as proximal tubular epithelial vacuolization and intraluminal casts progressing to tubular necrosis by 24 hours, with resolution by day 15. Iron and hemoglobin stains demonstrated these materials in the early lesions. Immunocytochemistries demonstrated human hemoglobin that remained as late as day 15. Electron microscopy revealed degeneration and regeneration of epithelial cells. The renal lesions were consistent with hemoglobinuria. The liver lesion was less well defined but was self limited.

Different hypotensive responses to intravenous bovine and human thrombin preparations in swine.

BACKGROUND Accidental intravenous introduction of commercial bovine thrombin (BT) during use of fibrin glue may result in profound hypotension. Commercial human thrombin (HT) is now available. This study compared the effects of intravenous BT versus HT in swine. METHODS Swine received 30 U/kg BT, 60 U/kg BT, 30 U/kg HT, or 60 U/kg HT intravenously. Mean arterial pressure (MAP) and survival were monitored for 30 minutes. Thrombin purities and in vitro activities were examined. RESULTS MAP nadir was lower (p < 0.05) after BT, 27.7 +/- 3.3% (mean +/- SEM) of pretreatment MAP, compared with 41.1 +/- 3.7% after HT. Five of six animals died after 60 U/kg BT, whereas all others survived (p < 0.05). Histology suggested more severe disseminated intravascular coagulation after BT. HT was purer than BT. In vitro activities were similar. CONCLUSION Both BT and HT produced hypotension. HT appeared safer, because of higher purity. Regardless of source and purity, thrombin must be used with caution.

Blood Substitutes for Surgery and Trauma

Solutions of modified haemoglobin and perfluorocarbon emulsions are currently being tested in advanced human trials. Concerns about renal injury and hypertension with the haemoglobin solutions, and drug retention and pulmonary injury with the perfluorocarbons have been successfully addressed. However, newly recognised inflammatory toxicities of the haemoglobin solutions and the low oxygen-carrying capacity of the perfluorocarbons remain as potential limits on efficacy. As a result, the role of blood substitutes will be less than originally imagined, and none is likely to be licenced for use for several years.

Effects of X-Linked Hemoglobin oph in-Vitro Platelet Function

The in-vitro function of platelets in 1:1 mixtures of fresh whole blood and 10% DBBF alpha-alpha cross-linked hemoglobin in Ringers acetate buffer was assessed by quantitative aggregation after challenge with common agonists and compared to the function of platelets in similar dilutions of whole blood in saline. Whole blood aggregometry was performed after addition of ADP, collagen, and ristocetin. Aggregation was quantified by measuring the change in impedance as drawn on the chart recording. No difference in mean impedance change was noted between the groups of blood samples which were incubated and tested in the hemoglobin solution and those incubated and tested in saline. In addition, incubation and stirring of the above mixtures over a period of six minutes without the addition of agonists did not result in spontaneous platelet aggregation. We conclude that alpha-alpha cross-linked hemoglobin, in the concentration studied, does not affect in-vitro platelet function, as measured by whole blood aggregometry.