Trauma is the leading cause of pediatric mortality, potential years of life lost, and accounts for significant medical cost in the developed world (1, 2). While it is understood that coagulopathy is common following major trauma and is associated with poor outcomes in adults (3), the effects of early coagulopathy and physiologic changes in the endothelial microenvironment in the pediatric population are less clear. Coagulopathy associated with trauma has traditionally been thought to be due to consumption of coagulation factors, dilution from intravenous fluids, and/or hypothermia. However, it was recognized in adults, and more recently in children, that at least a quarter of severely traumatized patients have coagulopathy on presentation to the emergency department that is physiologically and mechanistically distinct from classical iatrogenic posttraumatic coagulopathy (4–6). Minimal literature exists on the effect of significant traumatic tissue injury on the endothelial glycocalyx, the release of inflammatory markers, anticoagulation factors, and their subsequent interaction following severe pediatric trauma.
Previous research has shown that tissue injury accompanying massive trauma results in an immediate increase in circulating levels of damage-associated molecular patterns (DAMPs) such as high-mobility group box-1 (HMGB1) and nucleic acids including histone-complexed DNA (hcDNA) (7–11). Although exact mechanisms of coagulation derangements remain elusive, there is emerging consensus that coagulation abnormalities in the adult population are an endogenous response to injury involving the neurohumoral, inflammatory, and hemostatic systems (12–18). Other correlations have been shown to exist between increased extracellular histone levels and injury severity, platelet activation, endothelial damage, and mortality after adult trauma (19, 20). These findings suggest that histone release may be involved in the development of coagulation abnormalities in children after trauma (16). To our knowledge, prospective studies on the proposed mechanisms of posttraumatic coagulopathy in children are limited. Our current understanding of these mechanisms is predicated on data collected from adult samples. However, in addition to the anatomical and physiological differences between adults and children, there is a variance in mechanisms and patterns of injury (5). In the present study, we tested the hypothesis that the release of hcDNA could be detected early after pediatric trauma, and that this would be associated with coagulopathy, platelet dysfunction, endothelial cell damage, and poor outcome in this population.
PATIENTS AND METHODS
The Internal Review Boards of the University of Alabama at Birmingham and of University of Nebraska Medical Center approved this prospective observational cohort study performed between March 2013 and March 2016. Written consent was obtained from the patients or their legally authorized representative once they arrived at the hospital following the traumatic event.
Consecutive pediatric trauma patients admitted to the Children's Hospital of Alabama, the only level 1 pediatric trauma center in the state of Alabama, and University of Nebraska Medical Center were studied. Pediatric trauma patients under the age of 18 who met level 1 trauma criteria (Table 1) were eligible for enrollment. Exclusion criteria included: patients admitted > 6 h after their injury; patients with burns >20% of the total body surface area; patients admitted for primary asphyxiation; patients with known or expected pregnancy, known liver disease, and/or known coagulation disorders. The control cohort was represented by 62 pediatric volunteers (mean age, 6.24 ± 6.2 years). The trauma and control cohort numbers were determined by a set period of time from March 2013 through March 2016.
Sample collection and measurements
Blood samples were collected within 20 min of arrival to the trauma room and at 24 h following admission. The sample was placed in sodium citrated tubes (one part 0.106 mol L−1 sodium citrate and nine parts venous blood) and transported to the laboratory for immediate processing. The plasma from each sample was extracted and stored at −80°C until analysis. Prothrombin time (PT), platelet count, and base deficit (BD) determination were analyzed as part of the standard clinical tests by the hospital laboratory. Control samples were collected from healthy children with no history of coagulation abnormalities who were undergoing elective surgery. Blood was collected at the time of intravenous catheter insertion prior to induction of general anesthesia. The blood was processed in an identical fashion as described above for trauma patients.
Platelet function was assessed at point of care using the Multiplate multiple electrode aggregometer (Verum Diagnostica GmbH; Munich, Germany) immediately after sample collection following admission and at 24 h. Analyses were conducted by diluting 0.175 mL of whole blood in warmed normal saline containing 3 mM CaCl2 and incubating for 3 min at 37°C within a Multiplate mini test cell. Each test cell contains two sets of 3 mm silver-coated copper wires, across which electrical resistance is measured at 0.57 sec intervals. Platelet activation was induced by adenosine diphosphate (ADP, final concentration 6.5 μM; via P2 receptors) and thrombin receptor activating peptide-6 (TRAP, final concentration 32 μM; via PAR receptors). Platelet adhesion to the electrodes was detected as increasing electrical impedance, measured by duplicate sets of sensor wires in each test cell. Agonist responses are reported as area under the aggregation curve (AUC) over a 6-min measurement period. Since there is no normative data in the literature guiding us as to a normal range in pediatric patients, we utilized our control samples to create a normal range. Those patients who fell outside of the range that 95% of our controls fell into were considered abnormal.
Rotational thromboelastometry (ROTEM) is a point of care testing device measuring the viscoelastic properties on multiple aspects of blood coagulation in a sample of citrated whole blood. Unlike conventional coagulation assays, ROTEM assesses the coagulation system as a dynamic process by determining not only the clotting time, but also dynamics of clot formation, mechanical clot stability, and clot lysis over time. Activators are added to whole blood to evaluate the coagulation pathway. The clotting time (CT) is the latency time from addition of the activating reagent until the blood clot starts to form. Mini-cup cells were utilized with 0.150 mL of whole blood according to manufacturer's recommendations. Prolongation of the CT may be a result of coagulation deficiencies or altered levels of coagulation factors. Since there is no normative data in the literature guiding us as to a normal range in pediatric patients, we utilized our control samples to create a normal range. The range of 90 to 232 sec was the range that 95% of our controls fell into allowing this to be our normal range. Patient samples were collected and analyzed immediately after admission and at 24 h.
Enzyme-linked immunosorbent assay (ELISA) measurements
Samples were analyzed at the conclusion of the study by researchers blinded to all patient data. Plasma samples were measured in duplicate for the following: Histone-Complexed DNA fragment (hcDNA, Cell Death Detection ELISA plus, Roche, Conn), and Syndecan-1 (Cell Sciences, Newburyport, Mass). All the measurements were performed in accordance with the manufacturer's instructions.
Data collection, outcome measures
Data were prospectively collected on patient demographics, time of injury, mechanism of injury (blunt or penetrating), Injury Severity Score (ISS), baseline vital signs, transfusion requirements, and prehospital fluid administration. Head Abbreviated Injury Severity (AIS) ≥ 3 was used as a surrogate for the presence of significant traumatic brain injury (TBI). A blood gas was obtained on patient arrival per protocol for the management of high-level pediatric trauma patients. BD was used as a measure of the degree of tissue hypoperfusion. Patients were followed until discharge or death. Secondary outcome measures were recorded for acute kidney injury (defined by the pediatric RIFLE criteria) (21), acute lung injury (defined by the Pediatric Acute Lung Injury Consensus as a PaIO2/FIO2 ratio ≤ 300) (22), and blood transfusion requirements in the first 6 h.
Data analysis was performed by the investigators using Statistical Analysis System (SAS version 9.4, Cary, NC). Data are expressed as median (interquartile range) for continuous variables and count (percent) for categorical variables. Fisher exact test and the Wilcoxon rank sum test were used, as appropriate, to investigate relationships between covariates. Relationships between quartiles of hcDNA and continuous variables were tested with the Kruskal–Wallis test. For any relationships found to be statistically significant, multiple comparison tests were conducted using a non-parametric version of the Tukey–Kramer test. A P value of < 0.05 was chosen to represent statistical significance. Finally, logistic regression was used to assess whether admission levels of hcDNA and syndecan-1 were predictive of patient mortality.
During the study period, 186 patients requiring full trauma team activation were evaluated. A total of 37 patients were deemed ineligible by study exclusion criteria. Patients were excluded for the following reasons: age (n = 2), downgrade to level 2 trauma (less severe) (n = 2), unable to obtain blood (n = 5), dead on arrival (n = 5), missed trauma activation (n = 6), arrival >6 h after injury (n = 8), burns or asphyxiation (n = 9). In all, 149 consecutive pediatric trauma patients were enrolled in the study. Median age was 8.3 years (IQR: 4.6, 12.3) with patients falling into the following age categories: 0 to 2 (n = 25), 3 to 7 (n = 48), 8 to 12 (n = 45), 13 to 17 (n = 31). Median ISS was 20 (IQR: 11, 29), 71% sustained blunt trauma and 36% had severe TBI, defined by a head AIS score ≥ 3. Clinical characteristics of the children included are shown in Table 2. Coagulopathy (defined as PT ratio > 1.2) was present in 29% of patients on arrival to the hospital, and 26% had a base deficit ≥ 6 mmol/L. Overall mortality was 10%. The median time from injury to blood sample collection was 90 min (IQR: 60, 160).
HcDNA plasma level, injury severity, and hypoperfusion
In our injured pediatric population, we observed a median hcDNA plasma level of 3.47 AU (IQR: 1.23, 7.13) compared with 1.67 AU (IQR: 1.26, 3.19) (P = 0.024) in healthy pediatric controls. We performed two analysis evaluating separate cutoffs for “severe injury” with different ISS scores (ISS < or ≥ 15 and ISS < or ≥ 25). Recent literature has demonstrated that an ISS of 25 denotes the most appropriate cutoff for “severe injury” in children and approximates similar mortality to an ISS of 15 in severe adult trauma (23). The release of hcDNA occurred early after trauma and was significantly higher in children with higher ISS scores and higher base deficit versus those with less severe injury, or healthy control patients (P < 0.001, Fig. 1A). All groups of trauma patients have significantly higher hcDNA levels than controls, except the group with ISS < 25 and BD ≥ 6 (P = 0.098). To evaluate the differences between the multiple groups at the time of admission, we performed multiple comparisons with a Bonferroni correction. Group D (ISS ≥ 25 and BD ≥ 6) had a significantly higher hcDNA at admission than Groups A (ISS < 25 and BD < 6; P < 0.001) and C (ISS ≥ 25 and BD < 6; P = 0.002). No other groups significantly differed from each other with multiple comparisons. The difference in hcDNA levels between pediatric trauma patients and controls persisted at 24 h after admission (P = 0.002, Fig. 1B). At 24 h, all groups were different from controls, except the group with ISS < 25 and BD ≥ 6 (P = 0.924). However, the median hcDNA levels of severely injured patients were markedly decreased at 24 h, compared with admission levels, suggesting that the hcDNA levels were beginning to return back to the levels of the control patients. Similar results were seen when utilizing a lower ISS cutoff of 15 with similar BD groups on admission. All groups of trauma patients hcDNA levels were significantly different from control patients (P < 0.001), except the group with ISS <15 and BD <6 (P = 0.469). However at 24 h, fewer trauma groups hcDNA levels were different from controls—those with ISS <15 and BD <6 (P = 0.014) and those with ISS ≥ 15 and BD < 6 (P = 0.001) were significantly different from controls.
Plasma levels of hcDNA and early coagulation derangements in pediatric trauma patients
Trauma patients with clinically significant coagulation abnormalities upon admission (PT ratio >1.2) had significantly higher levels of hcDNA (P < 0.001, Fig. 2A) compared with those patients with a PT ratio of ≤ 1.2. The difference between these groups was not detected 24 h after admission (P = 0.209, Fig. 2B). The median level of hcDNA in coagulopathic patients showed less variability and returned to the level of non-coagulopathic patients. Figure 2C further demonstrates the change in hcDNA level based on different PT ratios after admission. In pairwise comparisons, all of the groups with elevated PT ratio have significantly higher hcDNA levels than the group with a normal PT ratio (P < 0.05, for each pairwise comparison). However, there was no significant difference between severity of PT elevation and absolute hcDNA levels once a threshold had been reached.
In addition to conventional coagulation studies, we utilized ROTEM to evaluate the relationship between coagulopathy and hcDNA levels. The INTEM test evaluates the intrinsic pathway by the addition of an activator to the sample and measuring, among other parameters, the speed of the clotting process with CT and the strength of the clot (amplitude) after 5 min of testing. In our cohort prolonged CT was associated with increase in plasma levels of hcDNA (P = 0.002, Fig. 3A). Furthermore, we demonstrated that those patients with elevated hcDNA levels had a significantly lower clot strength (amplitude, A5) at 5 min (P = 0.049, Fig. 3B). Together these findings demonstrate a correlation between increasing levels of hcDNA, severe traumatic mechanism, and coagulopathy as measured with viscoelastic techniques.
Finally, we evaluated circulating platelet number and function to determine the potential effect of injury and hcDNA release on platelets. In our population, the mean platelet count on admission was normal (288 ± 93.1 × 103/μL). Patients were categorized based on a normal or abnormal AUC for each test, ADP and TRAP based on the values measured in control patients. At admission, patients with abnormal AUC for both ADP and TRAP, compared with controls, had significantly higher median hcDNA levels (Fig. 4A, P = 0.003; Fig. 4B, P < 0.001). However, the difference in hcDNA levels at 24 h among these same groups was not different (Fig. 4C, P = 0.185; Fig. 4D, P = 0.883).
Plasma levels of hcDNA and markers of endothelial cell damage in pediatric trauma patients
To investigate the role of endothelial glycocalyx damage and its association with hcDNA release early after pediatric injury, shedding of the glycocalyx was evaluated by measuring the plasma levels of circulating syndecan-1. We evaluated patients’ syndecan-1 levels in relation to hcDNA levels at admission and 24 h after admission (Fig. 5, A and B). Control patients had low levels of both circulating syndecan-1 levels and hcDNA levels. Pediatric trauma patients were divided into quartiles for their hcDNA levels. Trauma patients in each quartile had significantly higher levels of circulating syndecan-1 when compared with controls (Fig. 5A). Multiple comparisons with a Bonferroni correction were performed for those groups at the time of admission. Group D (hcDNA ≥ 7.12 AU) had significantly higher syndecan-1 levels at admission when compared with Groups A (hcDNA < 1.39 AU; P < 0.001) and B 1.39 ≤ hcDNA < 3.51 AU; P < 0.001). Also, Group C (3.51 ≤ hcDNA < 7.12) had significantly higher syndecan-1 levels at admission than Group A (hcDNA < 1.39 AU; P = 0.001). No other groups showed significant differences in multiple comparisons. These findings persisted at 24 h after admission (Fig. 5B).
Plasma levels of hcDNA and clinical outcome pediatric trauma patients
In evaluating clinical outcomes in regard to coagulopathy and pediatric trauma, consideration of transfusion and head injury in relation to hcDNA levels was evaluated. Patients who required blood transfusion < 6 h after admission had higher plasma levels of hcDNA (7.38 AU (3.33, 11.41)) compared with those patients who did not require transfusion (2.51 AU (1.08, 3.33); P < 0.001). In addition, those patients who required transfusion of blood transfusion < 6 h after admission still had significantly higher hcDNA levels at 24 h following admission (3.38 AU (2.10, 4.85) than patients who did not require transfusion (2.09 AU (1.41, 3.37); P = 0.007). In evaluating hcDNA levels in relation to head injury, we split the population into those with GCS ≤ 8 and > 8. Those patients with GCS ≤ 8 had significantly higher hcDNA levels (4.11 AU, (1.59. 7.39) than those with GCS > 8 (2.51, (0.86, 5.40); P = 0.047) (Fig. 6).
In regard to other clinical outcomes, the median hospital stay for the entire cohort was 7 days (3, 18). Seventy-nine percent of the patients were admitted to the intensive care unit (ICU) and median ICU stay was 4 days (IQR: 2, 8). We found no significant correlation between hcDNA plasma levels, hospital, and ICU length of stay. Of all patients, five children later developed organ dysfunction not due to direct trauma. Only one patient developed acute kidney injury. Those patients (n = 4) who developed acute lung injury (ALI) had a significantly higher admission hcDNA levels than those without ALI (9.35 AU (IQR: 7.18, 10.90) versus 3.47 AU (IQR: 1.26, 7.00) (P = 0.046)).
Finally, to evaluate the direct relationship with mortality, a logistic regression analysis was performed to evaluate if hcDNA and syndecan-1 levels on admission predict mortality. hcDNA is significantly associated with mortality (P = 0.036) (Fig. 7A). The odds ratio (95% CI) associated with a one-unit increase in hcDNA is 1.14 (1.01, 1.30). Syndecan-1 is also significantly associated with mortality by logistic regression (P = 0.026) (Fig. 7B). The odds ratio (95% CI) associated with a 100-unit increase in syndecan-1 is 1.37 (1.04, 1.81).
The main results of this prospective study showed that plasma levels of hcDNA were significantly higher on admission to the hospital in pediatric trauma patients with severe injury (ISS > 25), coagulopathy, and abnormal platelet aggregation. Patients with high hcDNA levels had also significant elevations in plasma levels of syndecan-1, suggesting a significant damage to the endothelial glycocalyx. Finally, significantly higher plasma hcDNA levels were found in children who did not survive their severe trauma. These results demonstrate that hcDNA release may play an important role in the development of coagulopathy, endothelial gylcocalyx damage, and ultimate outcomes in children with severe trauma.
The first result of the present study shows that pediatric trauma patients, like adult trauma patients (19, 20), have an early exaggerated extracellular release of histone-complexed DNA fragments in response to injury. The pediatric patients with higher hcDNA levels also have a higher incidence of early coagulation abnormalities. Previous experimental studies have reported that hcDNA fragments may cause a procoagulant phenotype via a systemic activation of the coagulation cascade by several mechanisms that include binding to platelets and recruiting plasma adhesion proteins, such as fibrinogen, to cause platelet aggregation (24), inhibiting the anticoagulant protein C pathway (25), increasing plasma levels of von Willebrand factor antigen that contributes to platelet activation (26), increasing the extracellular release of HMGB1, a known procoagulant DAMP (27), and binding to negatively charged endothelial heparan sulfate thus disrupting the anticoagulant property of this protein layer (28). The relationship between elevated plasma levels of hcDNA and acute traumatic coagulation abnormalities has been described in adult patients with severe trauma. Interestingly, these studies reported that patients with higher plasma hcDNA levels had significantly higher ISS scores, a higher proportion of coagulopathy, and hyperfibrinolysis that was associated with an enhanced inflammatory response (19, 20). These clinical results indicate that high levels of circulating hcDNA fragments are associated with a coagulopathy phenotype, not a procoagulant one, after severe trauma. The explanation for the difference between the results of experimental and clinical studies could be related to the fact that patients with high plasma levels of hcDNA also have high plasma levels of anticoagulants such as tissue plasminogen activator, tissue factor pathway inhibitor, and activated protein C, suggesting a secondary modulation of the coagulation cascade by these anticoagulant mediators released to prevent diffuse microvascular thrombosis (19).
Although several studies have reported an incidence of acute traumatic coagulopathy in pediatric trauma patients comparable to the one observed in adult trauma patients (5, 16), these studies have not examined whether the extracellular release of hcDNA fragments is associated with similar coagulation abnormalities in traumatized children and whether there are potential differences in the response of children and adults to severe trauma. Our data suggest a relationship between elevated plasma hcDNA levels and worsening coagulopathy on admission to the hospital, as demonstrated by traditional coagulation studies and data generated by thromboelastometry (clotting time and clot strength A5). Indeed, patients with significantly increased hcDNA levels on admission demonstrated significant alterations in time to clotting and strength of the formed clot. In summary, our study demonstrates that pediatric trauma patients, like adult trauma patients, have an early exaggerated extracellular release of histone-complexed DNA fragments in response to severe injury. The pediatric patients with higher hcDNA levels also have a higher incidence of early coagulopathy that correlates with outcome. This study represents the first prospective description of how traumatic injury may affect circulating levels of hcDNA fragments and their relationship with trauma-induced coagulopathy in children with severe trauma.
The second result of this study shows that there is poor aggregation of circulating platelets in response to exposure to ADP or TRAP in severely injured pediatric trauma patients that correlates with high plasma levels of hcDNA. Wohlauer et al. (29) prospectively assessed platelet function in whole-blood samples from 51 adult trauma patients versus controls with thromboelastography-based platelet functional analysis. There were significant differences in platelet response between trauma patients and controls. This study indicated the significant platelet dysfunction manifested after severe trauma and before substantial fluid or blood administration. Furthermore, Kutcher et al. (19) prospectively evaluated platelet function by impedance aggregometry in 101 patients sustaining severe trauma. Likewise, they showed that 45% of these patients had below-normal platelet response at admission and 91% had platelet hypofunction at some point during their ICU stay (19). Although experimental and clinical studies mentioned above showed that hcDNA may activate platelets (indicated increased concentration of soluble CD40 ligand), circulating platelets of adult and pediatric patients with severe trauma show a clear hyporesponsiveness to further stimulation. This circulating platelet dysfunction is likely mediated by the exhausted platelet syndrome that corresponds to an initial platelet activation and depletion of intracellular mediators resulting in platelet hyporesponsiveness to further stimulation (29). Interestingly, pretreatment with valproic acid, an inhibitor of histone deacetylation, attenuated this abnormal responsiveness of circulating platelets in a swine model of traumatic brain injury and hemorrhagic shock (30).
The third result of this study shows a relationship between high levels of circulating hcDNA and endothelial glycocalyx degradation characterized by high plasma levels of syndecan-1 after severe pediatric trauma. This result is of importance because of the critical role of the endothelial glycocalyx layer in controlling the permeability of the vascular endothelium (31). Prior studies of adult trauma patients by Johansson et al. (4) reported that patients with higher circulating markers of endothelial degradation, syndecan-1, had increased evidence of inflammation, coagulation abnormalities, and mortality. Furthermore, they also demonstrated that there was a direct correlation between high circulating syndecan-1 and hcDNA levels in these patients (4). In addition, the same investigators reported in another study that high shedding of syndecan-1 correlated with endogenous heparinization measured by thromboelastography and secondary coagulopathy that is likely secondary to the release of heparan sulfate (31). These results suggest that this coagulopathy may reflect an adaptive response to the trauma-induced release of procoagulant DAMPs to maintain the perfusion of the microcirculation.
The results presented here are subject to the limitations related to the fact that it is a dual center observational study. Although there were a large number of trauma patients and controls in most groups included in the study, care must be taken in interpreting the data as there were limited numbers in some stratified groups utilized in the analysis. The correlations that we observed are not necessarily mechanistic. However, this study represents a severely injured pediatric trauma population in which these observations have not been described to date. This will require further confirmatory studies on larger pediatric trauma populations. A second limitation is the fact that diagnostic tools often used to measure circulating components of the chromatin do not necessarily distinguish free histones and nucleosomes, complexes formed of DNA and DAMPs, such as histones (32). Experimental studies found that free extracellular histones induce endothelial damage, organ failure, and death in sepsis and in animal models confirmed the administration of histones induced endothelial damage, hemorrhage, and thrombosis (33). Free histones cause direct damage to the cell membrane and induce a major calcium flux resulting in cell lysis. In addition, free histones activate Toll-like receptors (TLR) 2 and 4. In contrast, cell-free DNA or nucleosomes follow different routes of immunostimulation that include TLR9 (32). In this study, we measured the level of nucleosomes present in the study plasma, not the levels of free histones. While ELISA developed to quantify histone subtypes are unlikely to solely detect free histones because most of the circulating histones are bound to DNA, we used in the present study an ELISA that combined a monoclonal antihistone antibody with a monoclonal anti-DNA antibody. This assay that is widely used, allows for a reliable measurement of circulating chromatin fragments (32). Interestingly, in a recently published study, Abrams et al. (34) measured both histones by immunoblot and nucleosomes by ELISA in severely traumatized adult patients. The authors found that both plasma levels were elevated during the first hours after trauma. However, plasma histone, but not nucleosome, levels were still elevated 72 h after admission to the hospital (34). These results are in accordance with our own data that showed that although different from controls, the difference between the median levels of severely injured patients at admission and 24 h later is markedly different and suggests that the hcDNA levels are beginning to return back to the levels of the control patients.
In summary, this study demonstrates that severe pediatric trauma induces significant extracellular release of histone-complexed DNA fragments that is associated with early coagulation abnormalities, abnormal circulating platelet aggregation, and endothelial glycocalyx damage. This study also shows that high plasma levels of hcDNA are associated with poor outcome in pediatric patients with severe trauma. As it has previously been reported that extracellular hcDNA causes a systemic activation of the coagulation cascade and may induce a severe inflammatory response via the activation of TLRs 2, 4, and 9, the present clinical results suggest that the release of hcDNA into the blood stream might play an important mechanistic role in the development of coagulation abnormalities and endothelial glycocalyx damage in children with severe trauma.
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