Natalie Lott

Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery.

INTRODUCTION Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed from pure mitochondrial DNA (mtDNA) under certain conditions, which is potently proinflammatory. We hypothesized that injury and orthopedic trauma surgery would induce NET production with mtDNA as a structural component. METHODS Neutrophils were isolated 8 trauma patients requiring orthopedic surgery postinjury and up to 5 days postoperatively. Four healthy volunteers provided positive and negative controls. Total hip replacement patients acted as an uninjured surgical control group. Neutrophil extracellular traps were visualized with DNA (Hoechst 33342TM/Sytox Green/MitoSox/MitoTracker) stains using live cell fluorescence microscopy with downstream quantitative polymerase chain reaction analysis of DNA composition. RESULTS Neutrophil extracellular traps were present after injury in all 8 trauma patients. They persisted for 5 days postoperatively. Delayed surgery resulted in NET resolution, but they reformed postoperatively. Total hip replacement patients developed NETs postoperatively, which resolved by day 5. Quantitative polymerase chain reaction analysis of NET-DNA composition revealed that NETs formed after injury and surgery were made of mtDNA with no detectable nuclear DNA component. CONCLUSIONS Neutrophil extracellular traps formed after major trauma and subsequent surgery contain mtDNA and represent a novel marker of heightened innate immune activation. They could be considered when timing surgery after trauma to prevent systemic NET-induced inflammatory complications.

Cell necrosis-independent sustained mitochondrial and nuclear DNA release following trauma surgery.

BACKGROUND Mitochondrial DNA (mtDNA), a potent proinflammatory damage-associated molecular pattern, is released in large titers following trauma. The effect of trauma surgery on mtDNA concentration is unknown. We hypothesized that mtDNA and nuclear DNA (nDNA) levels would increase proportionately with the magnitude of surgery and both would then decrease rapidly. METHODS In this prospective pilot, plasma was sampled from 35 trauma patients requiring orthopedic surgical intervention at six perioperative time points. Healthy control subjects (n = 20) were sampled. DNA was extracted, and the mtDNA and nDNA were assessed using quantitative polymerase chain reaction. Markers of cell necrosis were also assayed (creatine kinase, lactate dehydrogenase, and aspartate aminotransferase). RESULTS The free plasma mtDNA and nDNA levels (ng/mL) were increased in trauma patients compared with healthy controls at all time points (mtDNA: preoperative period, 108 [46–284]; postoperative period, 96 [29–200]; 7 hours postoperatively, 88 [43–178]; 24 hours, 79 [36–172]; 3 days, 136 [65–263]; 5 days, 166 [101–434] [healthy controls, 11 (5–19)]) (nDNA: preoperative period, 52 [25–130]; postoperative period, 100 [35–208]; 7 hours postoperatively, 75 [36–139]; 24 hours postoperatively, 85 [47–133]; 3 days, 79 [48–117]; 5 days, 99 [41–154] [healthy controls, 29 (16–54)]). Elevated DNA levels did not correlate with markers of cellular necrosis. mtDNA was significantly elevated compared with nDNA at preoperative period (p = 0.003), 3 days (p = 0.003), and 5 days (p = 0.0014). Preoperative mtDNA levels were greater with shorter time from injury to surgery (p = 0.0085). Postoperative mtDNA level negatively correlated with intraoperative crystalloid infusion (p = 0.0017). Major pelvic surgery (vs. minor) was associated with greater mtDNA release 5 days postoperatively (p < 0.05). CONCLUSION This pilot of heterogeneous orthopedic trauma patients showed that the release of mtDNA and nDNA is sustained for 5 days following orthopedic trauma surgery. Postoperative, circulating DNA is not associated with markers of tissue necrosis but is associated with surgical invasiveness and is inversely related to intraoperative fluid administration. Sustained elevation of mtDNA levels could be of inflammatory origin and may contribute to postinjury dysfunctional inflammation. LEVEL OF EVIDENCE Prospective study, level III.

Characterization of the hypercoagulable state following severe orthopedic trauma.

BACKGROUND Acute traumatic coagulopathy develops in seriously injured patients, which is followed by a paradoxical hypercoagulable state. The hypercoagulable state contributes to venous thromboembolism, and yet, there are no sensitive tests available to detect it. The aim of this study was to characterize the hypercoagulable state caused by major orthopedic trauma using the overall hemostatic potential (OHP) assay. METHODS Major orthopedic trauma patients admitted during a 7-month period in 2012 were included in the study. Blood samples were drawn 1 hour before surgery, then 1, 7, 24 hours and 3, 5, 10, and 42 days postoperatively. The assay parameters were determined and analyzed according to injury severity (polytrauma or nonpolytrauma), type of surgical intervention, and shock status. Values were compared with 20 healthy controls. RESULTS Forty-one consecutive patients were enrolled (age, 41.5 ± 2.7 years; 70% male; Injury Severity Score [ISS], 21.5 ± 2.1). Hypercoagulability based on OHP was present in the preoperative sample compared with the controls (OHP, 13.8 ± 1.4 U vs. 8.1 ± 0.5 U; p = 0.020) and then further elevated after surgery (1 hour postoperative, 17.8 ± 2.0 U vs. preoperative, 13.8 ± 1.4 U, p = 0.008). Polytrauma patients were more hypercoagulable than nonpolytrauma at the preoperative sample time (17.7 ± 2.6 U vs. 10.7 ± 1.2 U, p = 0.040) and postoperative period (24.3 ± 3.4 U vs. 11.9 ± 1.4 U, p = 0.006). The OHP for patients undergoing open pelvic surgery (28.3 ± 3.0 U) was higher than both intramedullary nailing (16.2 ± 2.0 U) and percutaneous pelvic surgery (17.0 ± 1.7 U) on Day 5 (p < 0.05). Patients demonstrated a higher OHP than controls did at all time points, except at 6 weeks (patients, 10.8 ± 1.7 U vs. controls, 8.1 ± 0.5 U; p = 0.400). CONCLUSION The OHP assay detected the hypercoagulable state following major orthopedic trauma and surgical intervention, which was present for 10 days postoperatively. The extent of hypercoagulability could be associated with polytrauma and the type of surgical intervention; however, further studies are needed to confirm this. LEVEL OF EVIDENCE Epidemiologic study, level III.

Neutrophil oxidative burst capacity for peri-operative immune monitoring in trauma patients

BACKGROUND Post injury immune dysfunction can result in serious complications. Measurement of biomarkers may guide the optimal timing of surgery in clinically borderline patients and therefore prevent complications. AIM peri-operative measurement of neutrophil oxidative burst capacity as an indicator of the immune response to major orthopaedic surgical procedures. METHODS Prospective cohort study of trauma patients aged ≥16 yrs with pelvic, acetabular, femoral shaft or tibial shaft fractures requiring surgical intervention. Blood samples were taken immediately pre-op and at 30 min, 7, 24 and 72-9 6 h post-operatively. Neutrophil oxidative burst capacity was measured both with and without stimulation by formyl-methionyl-leucyl-phenylalanine (fMLP, a chemotactic factor). Clinical outcomes measured were mortality, length of stay, MOF, pneumonia, acute respiratory distress syndrome (ARDS) and sepsis. RESULTS 100 consecutive orthopaedic trauma patients were enrolled over a 16 month period. 78% were male, with a mean age of 42 ± 18 years and an average ISS of 19 ± 13. Neutrophil oxidative burst capacity was significantly elevated at 7 h (p = 0.006) and 24 h (p = 0.022) post operatively. Patients who developed infective complications (pneumonia and sepsis) had higher levels of oxidative burst capacity pre-operatively (pneumonia: 1.52 ± 0.93 v 0.99 ± 0.66 p = 0.032, sepsis: 1.39 ± 0.86 v 0.97 ± 0.56 p = 0.024) and at 24 h post op (pneumonia: 2.72 ± 2.38 v 1.12 ± 0.63 p = < 0.001, sepsis: 2.16 ± 2.09 v 1.10 ± 0.54 p = < 0.001). When analysed by operation type, no statistical difference was seen between major and minor operations. No correlation was found between length of stay, length of ICU stay, ISS or age and neutrophil oxidative burst capacity at any time point. CONCLUSIONS Neutrophil oxidative burst capacity response to orthopaedic trauma surgery is associated with the infective post injury complications. There was no correlation between magnitude of injury or operation and oxidative burst capacity. These results are promising for the development of tools for prediction of post-operative complications and guidance for optimal timing for surgical intervention.

Hospital Smoke-Free Policy: Compliance, Enforcement, and Practices. A Staff Survey in Two Large Public Hospitals in Australia

Background: Smoke-free hospital policies are becoming increasingly common to promote good health and quit attempts among patients who smoke. This study aims to assess: staff perceived enforcement and compliance with smoke-free policy; the current provision of smoking cessation care; and the characteristics of staff most likely to report provision of care to patients. Methods: An online cross-sectional survey of medical, nursing, and allied staff from two Australian public hospitals was conducted. Staff report of: patient and staff compliance with smoke-free policy; perceived policy enforcement; the provision of the 5As for smoking cessation (Ask, Assess, Advise, Assist, and Arrange follow-up); and the provision of stop-smoking medication are described. Logistic regressions were used to determine respondent characteristics related to the provision of the 5As and stop-smoking medication use during hospital admission. Results: A total of 805 respondents participated. Self-reported enforcement of smoke-free policy was low (60.9%), together with compliance for both patients (12.9%) and staff (23.6%). The provision of smoking cessation care was variable, with the delivery of the 5As ranging from 74.7% (ask) to 18.1% (arrange follow-up). Medical staff (odds ratio (OR) = 2.09, CI = 1.13, 3.85, p = 0.018) and full time employees (OR = 2.03, CI = 1.06, 3.89, p = 0.033) were more likely to provide smoking cessation care always/most of the time. Stop-smoking medication provision decreased with increasing age of staff (OR = 0.98, CI = 0.96, 0.99, p = 0.008). Conclusions: Smoke-free policy enforcement and compliance and the provision of smoking cessation care remains low in hospitals. Efforts to improve smoking cessation delivery by clinical staff are warranted.

Clinical implications and pathological associations of circulating mitochondrial DNA.

Mitochondria are membrane-enclosed organelles, the energy-producing centers in almost all eukaryotic cells. The evolutionary emergence of mitochondria is a result of the endocytosis of a-proteobacteria. There are several characteristic features which refer to its prokaryotic ancestors including its independent sets of double-stranded mitochondrial DNA, which is uniquely circular in form and contains a significant amount of unmethylated DNA as CpG islands. Resent research has proven that free mitochondrial DNA found in blood was associated with innate immunomodulation in a broad-range of clinical conditions. Upon release, mitochondrial DNA acts as a danger-associated molecular pattern in the circulation, it is recognized by pattern recognition receptors and it facilitates inflammatory responses. Besides its high receptor activation potential, mitochondrial DNA is likely to perform direct crosstalk with activated leukocytes and to be contributed to other anti-microbial activities. Here we highlight the pathological conditions where cell free mtDNA is involved, describe the potential sources and mechanisms of extracellular mtDNA release and explore evidence for its mechanism of action after being excreted and potential therapeutic strategies.

Smoke-Free Recovery from Trauma Surgery: A Pilot Trial of an Online Smoking Cessation Program for Orthopaedic Trauma Patients

Smoking increases the risk of complications associated with orthopaedic trauma surgery, however delivery of care is low. Online interventions may provide needed smoking cessation care and promote abstinence. This study aims to examine the engagement, acceptability, and retention of an online smoking cessation program (Smoke-Free Recovery; SFR) among a sample of orthopaedic trauma patients, as well as themes around the smoking cessation process. A pilot study of SFR with 31 orthopaedic trauma patients admitted to a public hospital in New South Wales, Australia took place. Semi-structured telephone interviews were conducted following hospital discharge. Thematic analysis and descriptive statistics were used. Engagement was high with 28 participants accessing SFR during admission. Twenty individuals completed follow-up phone calls. Program acceptability was rated favourably. After discharge, changes in smoking habits were noted, with program retention low. Themes on program use included: lack of time or need for additional support; computer illiteracy or technology issues; feeling unready or too stressed to quit; or feeling they had reached the boundary of what could be learnt from the program. This study highlights the difficulties faced by patients following hospital admission, the lack of follow-up support received, and the need for consumer testing prior to roll out. Continuing to develop interventions to promote hospital-initiated cessation attempts that continue post-discharge should be a priority.

Leukodepleted blood components do not remove the potential for long-term transfusion-associated microchimerism in Australian major trauma patients

Despite the introduction of leukodepleted blood components, it has been shown that donor leukocyte engraftment (microchimerism) remains a long-term consequence of red blood cell (RBC) transfusion. The incidence of microchimerism may be affected by international disparities in blood processing methods or variations in transfusion practices. This study was conducted to determine the prevalence of microchimerism in Australian trauma patients. A secondary aim was to examine whether any patient complications correlated to the incidence of microchimerism. Australian trauma patients (n = 86) who had been transfused with red blood cell (RBC) units between 2000 and 2012 with an injury severity score (ISS) of greater than 15 were recruited. The prevalence of microchimerism was determined using genetic screening with a panel of insertion/deletion biallelic polymorphisms. The mean storage age of the transfused RBC units was 20 ± 8 days and the mean length of stay (LOS) in hospital was 40 ± 39 days. There were no significant associations in this study sample to bias for patient age, gender, number of transfused RBC units or ISS. Nine of the 55 (16.3%) patients transfused with non-leukodepleted blood components displayed an incidence of microchimerism. Of the 31 patients transfused with leukodepleted RBC units, 3 (9.6%) displayed an incidence of microchimerism. Therefore, despite the universal introduction of leukodepleted blood components in Australia, the prevalence of transfusion-associated microchimerism was found to be unchanged. Furthermore, half of the patients exhibiting microchimerism were recorded to have had splenic injury or required splenectomy at the time of transfusion.

Smoking, Quitting, and the Provision of Smoking Cessation Support: A Survey of Orthopaedic Trauma Patients

Objective: This study investigates orthopaedic trauma patients smoking cessation history, intentions to quit, receipt of smoking cessation care during hospital admission, and patient-related factors associated with receipt of smoking cessation care. Methods: An online cross-sectional survey of orthopaedic trauma patients was conducted in 2 public hospitals in New South Wales, Australia. Prevalence of smoking and associated variables were described. Logistic regressions were used to examine whether patient characteristics were associated with receipt of smoking cessation care. Results: Eight hundred nineteen patients (response rate 73%) participated. More than 1 in 5 patients (21.8%) were current smokers (n = 175). Of the current smokers, more than half (55.3%) indicated making a quit attempt in the last 12 months and the majority (77.6%) were interested in quitting. More than a third of smokers (37.4%) were not advised to quit; 44.3% did not receive any form of nicotine replacement therapy; and 24.1% reported that they did not receive any of these 3 forms of smoking cessation care during their admission. Provision of care was not related to patient characteristics. Conclusions: The prevalence of smoking among the sample was high. Respondents were interested in quitting; however, the provision of care during admission was low. Smoking cessation interventions need to be developed to increase the provision of care and to promote quit attempts in this Australian population. Level of Evidence: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Development of an online smoking cessation program for use in hospital and following discharge: smoke-free recovery

Background Tobacco smoking can have negative health outcomes on recovery from surgery. Although it is recommended best practice to provide patients with advice to quit and follow-up support, provision of post-discharge support is rare. Developing an online smoking cessation program may help address this gap. Objectives This paper describes the development and pretesting of an online smoking cessation program (smoke-free recovery, SFR) tailored to the orthopaedic trauma population for use while in hospital and post-discharge. Methods Drawing on the DoTTI framework for developing an online program, the following steps were followed for program development: (1) design and development; (2) testing early iteration; (3) testing for effectiveness and (4) integration and implementation. This article describes the first two stages of SFR program development. Results SFR is a 10-module online smoking cessation program tailored for patients with orthopaedic trauma. Of the participants who completed testing early iterations, none reported any difficulties orientating themselves to the program or understanding program content. The main themes were that it was ‘helpful’, provision of ‘help to quit’ was low and SFR increased thoughts of ‘staying quit post-discharge’. Conclusions This study found that a theory and evidence-based approach as the basis for an online smoking cessation program for patients with orthopaedic trauma was acceptable to users. A randomised controlled trial will be conducted to examine whether the online smoking cessation program is effective in increasing smoking cessation and how it can be integrated and implemented into hospital practice (stages three and four of the DoTTI framework).