Doug K. Tadaki

Heterotopic Ossification in High-energy Wartime Extremity Injuries: Prevalence and Risk Factors

BACKGROUND Heterotopic ossification in the extremities remains a common complication in the setting of high-energy wartime trauma, particularly in blast-injured amputees and in those in whom the definitive amputation was performed within the zone of injury. The purposes of this cohort study were to report the experience of one major military medical center with high-energy wartime extremity wounds, to define the prevalence of heterotopic ossification in these patients, and to explore the relationship between heterotopic ossification and other potential independent predictors. METHODS We retrospectively reviewed the records and radiographs of all combat-wounded patients admitted to this institution between March 1, 2003, and December 31, 2006. Patients with a minimum of two months of radiographic follow-up who underwent at least one orthopaedic procedure on an extremity constituted our study group; those who underwent at least one orthopaedic procedure but had not had heterotopic ossification develop constituted the control group. Variables recorded for each study subject included age and sex, location and mechanism of injury, method(s) of fracture fixation, number of débridement procedures, duration of negative pressure therapy, location of heterotopic ossification, presence and severity of traumatic brain injury, and Injury Severity Scores. RESULTS During the study period, 1213 war-wounded patients were admitted. Of those patients, 243 (157 in the heterotopic ossification group and eighty-six controls) met the inclusion criteria. The observed rate of heterotopic ossification was 64.6%. A significant relationship was detected between heterotopic ossification and the presence (p = 0.006) and severity (p = 0.003) of a traumatic brain injury. Risk factors for the development of heterotopic ossification were found to be an age of less than thirty years (p = 0.007, odds ratio = 3.0), an amputation (p = 0.048, odds ratio = 2.9), multiple extremity injuries (p = 0.002, odds ratio = 3.9), and an Injury Severity Score of >or=16 (p = 0.02, odds ratio = 2.2). CONCLUSIONS The prevalence of heterotopic ossification in war-wounded patients is higher than that in civilian trauma. Although trends associated with local wound conditions were identified, the risk factors for the development of heterotopic ossification found in this study suggest that systemic causes predominate.

Inflammatory biomarkers in combat wound healing.

Background:Modern war ballistics and blast injuries inflict devastating extremity injuries, violating soft tissue, bone, and neurovascular structures. Despite advances in complex wound management, appropriate timing of war wound closure remains subjective. In addition, the pathophysiology of acute wound failure is poorly defined. Methods:Patients with penetrating extremity wounds sustained during combat were prospectively studied and followed for 30 days after definitive wound closure. The primary outcome was wound healing. Wound dehiscence was defined as spontaneous partial or complete wound disruption after closure. Serum, wound effluent, and wound bed tissue biopsy were collected at each surgical wound debridement. Serum and wound effluent were analyzed with a multiplex array of 22 cytokines and chemokines, and wound tissue for corresponding gene transcript expression. Results:Fifty-two penetrating extremity war wounds in 33 male patients were investigated. Nine (17%) wounds dehisced. Concomitant vascular injury, increased wound size, and higher injury severity score correlated with wound dehiscence. Both serum and wound effluent cytokine and chemokine protein profiles were statistically associated with healing outcome at various time points. Wound biopsy gene transcript expression demonstrated increased tissue inflammation associated with wound failure. Multiple protein and gene transcript biomarkers predictive of wound healing were identified. Conclusions:The cytokine and chemokine protein and gene transcript expression patterns demonstrate a condition of inflammatory dysregulation associated with war wound failure. A molecular biomarker panel may predict combat wound healing outcome and warrants prospective validation.

The majority of US combat casualty soft-tissue wounds are not infected or colonized upon arrival or during treatment at a continental US military medical facility.

BACKGROUND The microbiology of war wounds has changed as medicine and warfare have evolved. This study was designed to determine the microbial flora and bacterial quantification of present-day war wounds in US troops from Iraq and Afghanistan upon arrival at the National Naval Medical Center (NNMC). METHODS Patients with extremity combat wounds treated with a vacuum-assisted wound closure device were enrolled in study. Wounds were biopsied every 48 to 72 hours with quantitative microbiology performed on all biopsies. RESULTS Two hundred forty-two wound biopsies from 34 patients; 167 (69%) showed no growth, and 75 (31%) showed positive growth. The incidence of any bacterial isolation from biopsies weekly from the time of injury was 28% (first), 31% (second), and 37% (≥third). Acinetobacter baumannii was the most prevalent isolate. CONCLUSIONS Most soft-tissue wounds from Iraq and Afghanistan do not have significant bacterial burden upon arrival to and during initial treatment at NNMC. Improved evaluation of combat wound microbiology at all levels of care is warranted to determine shifts in microbiology and to impact care practices.

Evaluation of lyophilized platelets as an infusible hemostatic agent in experimental non‐compressible hemorrhage in swine

Summary.  Introduction: Human lyophilized platelets hold promise as a novel hemostatic infusion agent for the control of traumatic hemorrhage. Rehydrated, lyophilized platelets (Stasix) were investigated as an infusible hemostatic agent in experimental non‐compressible hemorrhage, using a porcine liver injury model. Methods: Yorkshire swine underwent a grade III liver injury and uncontrolled bleeding. After 15 min, animals were infused with Stasix (n = 10) or normal saline vehicle (n = 10). At 2 h, the liver was repaired, and the animals were monitored for another4 h. Resuscitation, including blood transfusion, was administered during the hospital phase. Laboratory data, including arterial blood gas, complete blood count, thromboelastography (TEG), and coagulation parameters, were collected. All animals underwent necropsy with complete histopathologic examination. Results: Overall survival in the Stasix group [8/10 (80%)] was significantly higher than in the control group [2/10 (20%)] (P = 0.023). Mean total blood loss index (g kg−1) was lower in Stasix‐treated animals (22.2 ± 3.5) than in control animals (34.7 ± 3.4) (P = 0.019). Hemodynamic parameters were improved in the Stasix group, and a trend towards higher hemoglobin and lower lactate was observed. Coagulation and TEG parameters were not different between the groups. One surviving animal in the Stasix group had evidence of thrombi on necropsy. Conclusions: This is the first reported study to evaluate rehydrated, lyophilized platelets as an infusible hemostatic agent for non‐compressible hemorrhage. Stasix improved survival and reduced blood loss in a liver injury porcine model. However, evidence of thrombotic complications warrants further investigation prior to human use in the setting of traumatic hemorrhage.

Monitoring the healing of combat wounds using Raman spectroscopic mapping.

Soldiers wounded in modern warfare present with extensive and complicated acute wounds, confounded by an overwhelming inflammatory response. The pathophysiology of acute wounds is unknown and timing of wound closure remains subjective. Collagen gene expression profiles are presented for 24 patients. Impaired healing wounds showed a twofold decrease in the up‐regulation of COL1A1 and COL3A1 genes in the beginning of the wound healing process, compared with normal healing wounds. By the final debridement, however, collagen gene expression profiles for normal and impaired healing wounds were similar for COL1A1 and COL3A1. In addition, Raman spectroscopic maps were collected of biopsy tissue sections, from the first and last debridements of 10 wounds collected from nine patients. Tissue components obtained for the debridement biopsies were compared to elucidate whether or not a wound healed normally. Raman spectroscopy showed a loss of collagen in five patients, indicated by a negative percent difference in the 1,665/1,445 cm−1 band area ratios. Four healed patients showed an increased or unchanged collagen content. Here, we demonstrate the potential of Raman spectroscopic analysis of wound biopsies for classification of wounds as normal or impaired healing. Raman spectroscopy has the potential to noninvasively monitor collagen deposition in the wound bed, during surgical wound debridements, to help determine the optimal time for wound closure.

Non-invasive monitoring of tissue oxygenation during laparoscopic donor nephrectomy.

BackgroundStandard methods for assessment of organ viability during surgery are typically limited to visual cues and tactile feedback in open surgery. However, during laparoscopic surgery, these processes are impaired. This is of particular relevance during laparoscopic renal donation, where the condition of the kidney must be optimized despite considerable manipulation. However, there is no in vivo methodology to monitor renal parenchymal oxygenation during laparoscopic surgery.MethodsWe have developed a method for the real time, in vivo, whole organ assessment of tissue oxygenation during laparoscopic nephrectomy to convey meaningful biological data to the surgeon during laparoscopic surgery. We apply the 3-CCD (charge coupled device) camera to monitor qualitatively renal parenchymal oxygenation with potential real-time video capability.ResultsWe have validated this methodology in a porcine model across a range of hypoxic conditions, and have then applied the method during clinical laparoscopic donor nephrectomies during clinically relevant pneumoperitoneum. 3-CCD image enhancement produces mean region of interest (ROI) intensity values that can be directly correlated with blood oxygen saturation measurements (R2 > 0.96). The calculated mean ROI intensity values obtained at the beginning of the laparoscopic nephrectomy do not differ significantly from mean ROI intensity values calculated immediately before kidney removal (p > 0.05).ConclusionHere, using the 3-CCD camera, we qualitatively monitor tissue oxygenation. This means of assessing intraoperative tissue oxygenation may be a useful method to avoid unintended ischemic injury during laparoscopic surgery. Preliminary results indicate that no significant changes in renal oxygenation occur as a result of pneumoperitoneum.

BAYESIAN MODELING OF PRE-TRANSPLANT VARIABLES ACCURATELY PREDICTS KIDNEY GRAFT SURVIVAL

Introduction: Machine learning can enable the development of predictive models that incorporate multiple variables for a systems approach to organ allocation. We explored the principle of Bayesian Belief Network (BBN) to determine whether a predictive model of graft survival can be derived using pretransplant variables. Our hypothesis was that pretransplant donor and recipient variables, when considered together as a network, add incremental value to the classification of graft survival. Methods: We performed a retrospective analysis of 5,144 randomly selected patients (age ≥18, deceased donor kidney only, first-time recipients) from the United States Renal Data System database between 2000 and 2001. Using this dataset, we developed a machine-learned BBN that functions as a pretransplant organ-matching tool. Results: A network of 48 clinical variables was constructed and externally validated using an additional 2,204 patients of matching demographic characteristics. This model was able to predict graft failure within the first year or within 3 years (sensitivity 40%; specificity 80%; area under the curve, AUC, 0.63). Recipient BMI, gender, race, and donor age were amongst the pretransplant variables with strongest association to outcome. A 10-fold internal cross-validation showed similar results for 1-year (sensitivity 24%; specificity 80%; AUC 0.59) and 3-year (sensitivity 31%; specificity 80%; AUC 0.60) graft failure. Conclusion: We found recipient BMI, gender, race, and donor age to be influential predictors of outcome, while wait time and human leukocyte antigen matching were much less associated with outcome. BBN enabled us to examine variables from a large database to develop a robust predictive model.

Perioperative blood transfusion in combat casualties: a pilot study.

BACKGROUND In recent studies, blood transfusion has been shown to increase the rate of wound healing disturbances in orthopedic patients. Furthermore, our group has determined a correlation between delayed wound healing and elevations in inflammatory mediators in combat casualties. Therefore, we sought to determine the effect of blood transfusion on wound healing and inflammatory mediator release in combat casualties. METHODS Prospective data were collected on 20 severely injured combat casualties sustaining extremity wounds. Patients were admitted to the National Naval Medical Center during a 13-month period from January 2007 to January 2008. Data variables included age, gender, Glasgow coma score (GCS), mechanism of injury, and transfusion history. Injury severity was assessed using the Injury Severity Score (ISS). Serum was collected initially and before each surgical wound debridement and analyzed using a panel of 21 cytokines and chemokines. The association between blood transfusion and wound healing, incidence of perioperative infection, intensive care unit (ICU) admission rate, and ICU and hospital length of stay was assessed. Differences were considered significant when p < 0.05. RESULTS The study cohort had a mean age of 22 +/- 1, a mean ISS of 15.8 +/- 2.6, and a mean GCS 13.9 +/- 0.6; all were men and suffered penetrating injuries (90% improvised explosive device [IED] and 10% gunshot wound [GSW]). The cohort was divided into two groups. Patients receiving <or=4 units of blood initially (group 1, n = 11) were compared with patients who received >4 units of blood initially (group 2, n = 9). There was no significant difference in age, ISS, GCS, or mortality between the two groups. However, group 2 patients had significant impairment in wound healing rate (54% vs. 9%, p < 0.05), higher ICU admission rate (78% vs. 9%, p < 0.01), perioperative infection rate (89% vs. 27%, p < 0.01), and a longer hospital length of stay (49.9 +/- 12.8 vs. 23.8 +/- 2.9, p < 0.05) compared with group 1 patients. In addition, there was a significant correlation between the initial mean serum cytokine/chemokine level of interleukin (IL)-10, IL-8, interferon inducible protein (IP)-10, IL-6, and IL-12p40 and the number of units of blood transfused (p < 0.05). CONCLUSION Allogeneic blood transfusions in combat casualties were associated with impaired wound healing, increased perioperative infection rate, and resource utilization. In addition, the extent of blood transfusion was associated with significant differences in inflammatory chemokine and cytokine release.

Lymphocyte Modulation with FTY720 Improves Hemorrhagic Shock Survival in Swine

The inflammatory response to severe traumatic injury results in significant morbidity and mortality. Lymphocytes have recently been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury. Experimental manipulation of lymphocytes following hemorrhagic shock may prevent secondary immunologic injury in surgical and trauma patients. The objective of this study is to evaluate the lymphocyte sequestration agent FTY720 as an immunomodulator following experimental hemorrhagic shock in a swine liver injury model. Yorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte sequestration agent, FTY720, (n = 9) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period after hemorrhage. Circulating total leukocyte and neutrophil counts were measured. Central lymphocytes were evaluated with mesenteric lymph node and spleen immunohistochemistry (IHC) staining for CD3. Lung tissue infiltrating neutrophils were analyzed with myeloperoxidase (MPO) IHC staining. Relevant immune-related gene expression from liver tissue was quantified using RT-PCR. The overall survival was 22.2% in the vehicle control and 66.7% in the FTY720 groups (p = 0.081), and reperfusion survival (period after hemorrhage) was 25% in the vehicle control and 75% in the FTY720 groups (p = 0.047). CD3+ lymphocytes were significantly increased in mesenteric lymph nodes and spleen in the FTY720 group compared to vehicle control, indicating central lymphocyte sequestration. Lymphocyte disruption significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver immune-related gene expression in the FTY720 treated group. There were no observed infectious or wound healing complications. Lymphocyte sequestration with FTY720 improves survival in experimental hemorrhagic shock using a porcine liver injury model. These results support a novel and clinically relevant lymphocyte immunomodulation strategy to ameliorate secondary immune injury in hemorrhagic shock.

Lymphocyte Depletion in Experimental Hemorrhagic Shock in Swine

BackgroundHemorrhagic shock results in systemic activation of the immune system and leads to ischemia-reperfusion injury. Lymphocytes have been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury, and immunomodulation of lymphocytes may prevent secondary immunologic injury in surgical and trauma patients.MethodsYorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte depletional agent, porcine polyclonal anti-thymocyte globulin (PATG) (n = 8) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period. Circulating lymphocytes were examined with FACS analysis for CD3/CD4/CD8, and central lymphocytes with mesenteric lymph node and spleen staining for CD3. Circulating and lung tissue16 infiltrating neutrophils were measured. Circulating CD3 lymphocytes in the blood and in central lymphoid organs (spleen/lymph node) were stained and evaluated using FACS analysis. Immune-related gene expression from liver tissue was quantified using RT-PCR.ResultsThe overall survival was 22% (2/9) in the control and 75% (6/8) in the PATG groups, p = 0.09; during the reperfusion period (following hemorrhage) survival was 25% (2/8) in the control and 100% (6/6) in the PATG groups, p = 0.008. Mean blood loss and hemodynamic profiles were not significantly different between the experimental and control groups. Circulating CD3+CD4+ lymphocytes were significantly depleted in the PATG group compared to control. Lymphocyte depletion in the setting of hemorrhagic shock also significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver ischemia gene expression.ConclusionsLymphocyte manipulation with a depletional (PATG) strategy improves reperfusion survival in experimental hemorrhagic shock using a porcine liver injury model. This proof of principle study paves the way for further development of immunomodulation approaches to ameliorate secondary immune injury following hemorrhagic shock.