Traumatic brain injury (TBI) is the leading cause of morbidity and mortality through 45 years of age (1, 2). The severity of patient outcomes is directly related to the magnitude of intracranial hemorrhage (ICH) resulting from TBI (3–5). Occurring in a minority of patients with severe TBI, stable hemorrhage (SH) comprises ICH lesions that do not increase in size. However, up to 60% of patients with TBI experience progressive hemorrhagic injury (PHI) caused by ICH expansion in the initial 4 to 6 h following trauma (3–5). Patients with PHI are at higher risk for complications, chronic morbidity, and mortality than those patients with SH (3–6). Unfortunately, the pathophysiology behind PHI remains poorly understood. A better mechanistic understanding of PHI could provide improved and earlier clinical intervention, which may ameliorate poor outcomes in patients with severe TBI (4–7).
Severe injury and shock initiate maladaptive responses that cause dysregulation of normal hemostatic processes (7–9). It is suspected that failure to maintain adequate intracranial hemostasis may facilitate ICH expansion and result in PHI (4, 5, 7). Published studies to date suggest TBI-associated coagulopathies are driven by thrombocytopenia, platelet dysfunction, and factor deficiency may be the governing forces behind PHI (5–9). However, conflicting reports and a paucity of clinical data have left uncertainties about such mechanisms.
Fibrinolysis has been established as a clinically relevant phenomenon in trauma that is independently associated with mortality (6, 10–13). Additionally, the clinical randomization of an antifibrinolytic treatment in significant hemorrhage (CRASH 2) trial showed improved outcomes with the antifibrinolytic compound tranexamic acid (TXA) in patients with traumatic hemorrhage, if administered soon after injury (14). A nested study of TBI patients in the CRASH 2 trial presents data suggesting that early TXA administration reduces progressive intracranial hemorrhage (15). However, the role of fibrinolysis in TBI has yet to be clearly delineated (6, 7). Recent research by Hijazi et al. (16) is the only causative study to date implicating fibrinolysis as an important mechanism driving PHI. Using genetic knockout mice deficient of various fibrinolytic mediators, Hijazi et al. determined plasminogen activation is the primary process facilitating fibrinolysis and causing PHI. These findings, however, lack clinical generalizability due to potential differences in mouse and human TBI disease processes (17).
An improved understanding of fibrinolysis in patients with TBI may help guide early goal directed clinical management by providing biomarkers for PHI prediction and molecular targets for therapeutic intervention. We hypothesize that PHI is associated with hyperfibrinolysis and that admission levels of profibrinolytic markers can be used to predict PHI. Therefore, the aim of this study was to perform an exploratory analysis to identify such markers of fibrinolysis in need of further investigation to establish their predictive value in PHI following TBI.
This was a single institution, retrospective cohort analysis of prospectively collected data. The data for this study was compiled from highest-level activation adult polytrauma subjects (16–80 years of age) with severe TBI admitted between January 2012 and December 2013. All subjects were treated at Memorial Hermann Hospital in Houston, TX, a Level 1 trauma center in an academic setting, and were appropriately consented into ongoing clinical studies approved by The University of Texas Health Sciences Center at Houston Institutional Review Board (HSC-GEN-11-0174, HSC-GEN-12-0059, HSC-GEN-11-0213). Criteria for inclusion into the study were patients with the highest-level trauma activation, who were estimated to be ≥15 years of age, received directly from the injury scene, transfused with blood products during prehospital transport or within the first hour, and predicted to receive a massive transfusion. These criteria are representative of a majority of patients with polytrauma. To isolate those patients with severe TBI for our study, the patients enrolled into this research study met additional inclusion criteria including a head abbreviated injury scale (AIS) score of ≥3 and evidence of ICH on admission computerized tomography (CT) imaging of the head.
All subjects’ demographic variables and clinical parameters of interest were collected from patient records. Demographic variables included age, gender, mechanism of injury (blunt or penetrating), dates of admission and discharge, mortality, days requiring intensive care unit placement and support with mechanical ventilation. Trauma injury scores indicative of injury magnitude, which include the Glasgow Coma Scale score (GCS), Injury Severity Score (ISS), AIS Head, and the Revised Trauma Score (RTS), were included. Transfusion requirements included units of crystalloid, plasma, red blood cells (RBC), and platelets transfused prehospital, at 6-h, and 24-h time points after admission.
SH and PHI diagnosis
CT imaging of the head was acquired and read as standard of care upon admission for every subject with an AIS Head Score ≥3 if severe TBI was suspected. The radiologist on duty reviewed the admission imaging and documented the presence or absence of ICH. All subjects with an admission ICH received subsequent imaging 6 h after admission, as this interval is the standard protocol used at our institution to assess bleed progression. The radiologist then reviewed both sets of imaging and reported whether the initial ICH was stable or progressed and if any new sites of bleeding were now identified. This study was not designed as a radiologic study and any patient presenting with an AIS Head Score ≥ 3 where TBI is suspected has routine CT imaging read by the radiologist on call. Therefore, since different radiologists often reviewed the CT images in our patient cohort, our clinical authors collectively reviewed each subject's admission and 6 h CT head scans images in parallel to determine bleed progression for research purposes. Trained clinicians who read and utilize these scans in their daily practice performed rereading of the CT scans. There was 100% agreement in our radiological impressions in comparison to the initial radiologist reports. SH was defined as having an ICH on the admission head CT and no new ICH or increase in size of the initial lesion on the subsequent CT, while PHI was defined as the progression of ICH and/or presence of new bleeding foci on repeated imaging.
Upon each subject's admission to the emergency department, nursing staff collected blood samples for coagulation analysis. Rapid thrombelastography (r-TEG) assay was conducted at admission using a TEG 5000 analyzer (Haemonetics, Braintree, Mass) to provide activated clotting time, k-time, maximum amplitude, angle, lysis-30 and lysis-60 values.
Coagulation factor levels
Blood samples were collected by nursing staff for research purposes upon admission and 2, 4, and 6 h later. The samples were centrifuged and separated to yield plasma, which was aliquoted into 1.8 mL tubes and stored at −80°C. Protein measurements were performed on stored plasma for this study alone. Prior to measuring molecular biomarkers, plasma aliquots were thawed to 4°C in an ice bath and centrifuged at 10,000 × g for 10 min. Molecular biomarkers of interest included those proteins involved in fibrinolysis, including plasminogen (PLG), tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), α2-antiplasmin (α2-AP), and D-Dimers (DD). All molecular biomarkers were measured in the plasma samples using respective Human ELISA kits and provided standard protocols (Abcam, San Francisco, Calif).
Descriptive statistics (median, quartile, frequency, proportion, etc.) were provided for demographic and clinical variables. Mann–Whitney test was used to compare continuous variables between SH and PHI groups. Chi square test or Fisher exact test was used to compare categorical variables between the two groups. Multivariable generalized estimating equation (GEE) models were employed to model the probability of developing PHI with regards to changes in levels of fibrinolytic markers across time, with a binomial distribution and logit link. A receiver operating characteristic (ROC) analysis was conducted to evaluate how well admission DD values can differentiate patients with PHI from those with SH. Accuracy of the ROC analysis was then measured by the area under the ROC curve (AUC). Statistical analysis was performed using the STATA Version 12.1 (StataCorp LP, College Station, Tex), R 3.2.3, and SAS software Version 9.3 (SAS Institute, Cary, NC).
After reviewing the charts of 424 patients with polytrauma, 72 severely injured polytrauma patients with TBI and ICH met inclusion criteria (Fig. 1). Of those 72 patients, 25 had repeated blood samples collected and plasma stored at the time of their hospital stay. Therefore, an initial longitudinal analysis was performed on these 25 patients to determine relationships between fibrinolytic proteins and PHI over time. Once completed, the full cohort of 72 TBI patients was used to assess the validity of the fibrinolytic proteins for future use as biomarkers to predict, upon admission, which patients would develop PHI (Fig. 1).
Demographics and clinical parameters
A primary data set of 25 total patients with severe TBI, dichotomized into SH (n = 6) or PHI (n = 19) groups was used to analyze the longitudinal trends of fibrinolysis across time. Patients in the primary data set were predominantly middle aged, male, suffered blunt traumatic injury, and had PHI (Table 1). There were no significant differences between the SH and PHI groups in regards to demographics and indices of injury at admission including GCS, RTS, AIS Head, ISS, and base excess values. Patients with SH had a higher incidence of subarachnoid hemorrhage compared to those with PHI (83.3% vs. 10.5%, respectively, P < 0.01). For all other types of ICH, there were no differences between groups.
Patients with PHI received more transfusions of RBC compared to their SH counterparts in the first 6 h (P = 0.0116) (Table 2). For all other resuscitation variables, both groups received similar types and volumes of prehospital, 4-h, and 24-h transfusions. There were also no group differences in regards to clinical outcomes including ventilator-free days, ICU-free days, hospital-free days, or 24-h mortality (Table 3). Of note, patients with SH had a trend toward lower 30-day mortality rates (16.7% in SH vs. 52.6% in PHI, P = 0.1220); however, this trend did not achieve significance presumably due to limitations in sample size and the severity of injury in both groups.
TEG VALUES AND COAGULATION FACTORS
In the r-TEG data upon admission, we observed that patients with SH demonstrated lower LY-60 values than PHI counterparts (0.5% in SH vs. 2.5% in PHI, P = 0.0845)—a trend which approached, but did not achieve significance.
Also upon admission, levels of tPA and DD were lower in SH patients compared to PHI patients; however, neither trend achieved significance (tPA: 549 pg/mL in SH vs. 998 pg/mL in PHI, P = 0.1117; DD: 11.41 μg/mL in SH vs. 28.10 μg/mL in PHI, P = 0.1360) (Table 4). The SH group had higher levels of PLG and A2-AP (PLG: 74.1% in SH vs. 48.2% in PHI, P = 0.0116; A2AP: 65.0% in SH vs. 49.3% in PHI, P = 0.0302).
Longitudinal box plots for changes in all the fibrinolytic markers and mediators over time for SH and PHI groups are provided (Figs. 2 and 3). The multivariate GEE analysis shows that collectively across time, higher levels of tPA and DD are positively associated with developing PHI (tPA: P = 0.0003 and DD: P = 0.0150) while higher levels of A2AP are negatively associated with developing PHI (P < 0.0010) (Table 5). When time interactions are fitted into the GEE analysis, the interaction between DD and time is additionally significant (P = 0.0027). These data suggest that along the time course of the 6 h after admission, the likelihood of developing PHI increases faster for those patients with higher DD levels compared to those patients with lower DD levels.
To better explore the differences in admission DD levels between SH and PHI patient groups, an expanded cohort of 72 similar patients, dichotomized into SH (n = 29) or PHI (n = 43), was used to conduct a post hoc ROC analysis. Similar to our primary cohort, our expanded cohort was also primarily comprised of middle aged, male patients, with blunt traumatic injuries. Of note, the patients with PHI in our expanded cohort had a greater injury severity as determined by higher AIS Head and ISS scores at admission. However, this expanded cohort was similar to our primary cohort in regards to all other injury scores and ICH sub-types (Table 6). For ROC analysis, we randomly split the data into training and test sets, evenly. Based on the training dataset, the ROC analysis provided an AUC of 0.90, indicating an “excellent” accuracy for differentiating patients with SH from those with PHI based on a Youden-Index cutoff level of 3.04 μg/mL (Fig. 4). This admission DD cutoff level was found, via a validation analysis, to have a sensitivity of 91%, a specificity of 46%, a negative predictive value of 75%, and a positive predictive value of 74%.
PHI has been shown to be a poor prognostic indicator of patient outcomes, including mortality, in TBI patients (3–5). Unfortunately, advances in PHI management are limited because the pathophysiology of ICH expansion remains largely unspecified. Our study aimed to explore the relationship between fibrinolysis and PHI and the potential for fibrinolytic proteins to serve as biomarkers to predict PHI.
A majority of patients with PHI exhibited combined forms of ICH, while most patients with SH exhibited isolated subarachnoid hemorrhage. Interestingly, there was no other admission demographic or injury criteria able to predict or “phenotype” which patients with TBI would develop PHI, emphasizing the need for novel molecular biomarkers. Our data show that severely injured polytrauma patients who develop PHI do, in fact, present with elevated markers of fibrinolysis compared to those with SH. Moreover, admission levels of DD are a readily available clinical biomarker to predict which patients develop PHI. Recent trauma studies have shown that elevations in r-TEG lysis parameters are associated with poor patient outcomes, and that these patients may benefit from individualized hemostatic therapy (11–13, 18, 19). In our initial evaluation of 25 subjects with polytrauma, r-TEG was unable to distinguish patients with and without a subsequent PHI. This is likely due to the reported insensitivity of r-TEG to relatively slight perturbations in coagulation as seen in localized head injuries in the presence of polytrauma and our limited sample size of 25 patients (19).
Increased admission levels of fibrinolytic proteins in PHI patients support the hypothesis that fibrinolytic activation is one of the underlying mechanisms driving PHI. According to our findings, this fibrinolytic process appears to be primarily driven by early release of tPA in addition to lower levels of A2AP in patients with PHI. While we did observe differences in time-dependent regulation of tPA and uPA similar to Hijazi et al., we did not find a relationship between uPA and PHI (16). However, there were key differences in their study design, most importantly that they were able to obtain and make observations in spinal fluid, which we were not. Thus, our observations are limited to systemic effects in the plasma, differences in human versus rodent disease processes, and the polytrauma nature of injury pattern in our patients rather than isolated TBI (16, 17, 20).
Although clinical research implicating fibrinolysis as mediating PHI has been limited, a few studies provide additional context to our findings. Stein et al. and Nakae et al. have reported fibrinolysis as an active process in the early hours following severe isolated TBI and its association with secondary cerebral injury and worsened chronic neurological recovery, respectively (7, 21). Both groups, in addition to findings by Kuo et al., have also suggested that admission DD level may be a strong predictor for differentiating patients with and without TBI- associated complications, including hemorrhagic conversion (7, 21, 22). Moreover, they also suggest DD to have a greater capacity at differentiating patients compared to standard markers of coagulopathy such as platelet counts, PT, and PTT.
Because of the difficulty in predicting PHI, all patients with severe TBI or initial ICH undergo repeat CT imaging of the head, and intensive neurological monitoring. Such management is necessary to ensure those patients who develop PHI receive a high level of care and timely surgical intervention. Unfortunately, this approach also subjects patients with SH to an increased burden of radiation exposure, possible iatrogenic intracranial hemorrhage for intracranial monitor placement, and is associated with increased healthcare costs. Our admission DD cutoff level of 3.04 μg/mL was associated with a high sensitivity and fair negative predictive value. This DD cutoff level, with independent validation, might be used in clinical settings to screen those patients with severe TBI who are unlikely to develop PHI. Consequently, such clinical risk stratification may be used to decrease healthcare costs, reduce unnecessary radiation exposure for patients with SH, and drive early intervention to decrease the incidence of PHI.
Many clinical studies have documented inefficacy of standard treatments including plasma and platelets in reducing the occurrence of PHI (23, 24). These failed efforts in hemostatic resuscitation may suggest that fibrinolysis is the driving mechanism underlying PHI pathophysiology. In this case, the intuitive therapy of choice might be antifibrinolytics. The CRASH 2 trial demonstrated improved survival with TXA administration following traumatic injury compared to placebo; however, there was no difference in bleeding or transfusions between groups, and unfortunately no assessment of coagulation function was performed (15). Currently, however, there are three ongoing large multicenter randomized clinical trials exploring effectiveness of early TXA administration in patients with TBI to improve patient outcomes including chronic neurological outcomes and PHI (25–27). When available, results from these studies will help guide TXA usage in TBI patients.
A major limitation in this study was a small sample size for our primary severe TBI cohort. Given that we had serial samples on a limited number of patients, our sample size was one of convenience. However, our 25 subjects had prospectively collected clinical data across 4 different time points. This repeated sampling increased our power and allowed us to perform longitudinal statistical modeling to analyze the trends of fibrinolytic proteins across time. This analysis guided our selection of potential fibrinolysis biomarkers moving forward and we then used the entire population of 72 patients who met inclusion criteria which all had admission samples available. We feel that this approach of robust longitudinal analytics and validation with a larger 72-subject cohort is sufficient to substantiate our main conclusions. However, a larger study utilizing prospective analysis would be ideal for conclusively defining an ideal DD threshold for risk stratification in TBI patients. Further, we recognize that in the course of conducting a retrospective analysis of prospectively collected data there are limitations. This was a pragmatic clinical study with inherent biases. Much of the data were collected through the course of care of the patient and not specifically for the study. We have attempted to address some of these biases by clearly identifying patients retrospectively with diagnosed TBI, defining the criteria for SH and PHI, and employing independent evaluation of the CT and laboratory data.
There currently exists no admission clinical indicator capable of predicting TBI-associated PHI. Our data support a relationship between markers of fibrinolysis in polytrauma patients with severe TBI and PHI. Upon further investigation, admission DD may provide a new and readily available biomarker to screen TBI patients for PHI in the future.
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