... Key Messages from Individual Speakers and Participants...

... Neuroimaging biomarkers, using CT and MRI, are best established for acute TBI that is moderate and severe in nature, but they face sensitivity limitations in mild and chronic forms of TBI (Wilde)....

... to be most informative, factors such as the effect of time since injury, type of injury, previous brain injuries, clearance rates of proteins, and the origin of biomarkers need to be considered (Gill)....

... Electrophysiological biomarkers provide complementary information. However, significant challenges must be successfully addressed before such biomarkers can enter into routine use in the clinical assessment of TBI (Rapp)....

... Multiple measures of physiological deficits that occur after mild TBI, including eye tracking, auditory testing, sleep monitoring, and postural and gait analyses, may serve as biomarkers for diagnosis, prognosis, and monitoring recovery, and they may play a future role in the design of targeted ...

... need to keep understanding the causes of functional impairments (Master)....

... The development of common analytic pipelines and consistent standards, harmonization of previously collected data, and matching fluid biomarkers with imaging and other types of biomarker information are areas needing further efforts (Gill, Wilde)....

... Expanding analysis methodologies for integrating diverse information from multiple markers is an area of great potential, as are continued applications of ...

... NOTE: This list is the rapporteurs’ summary of points made by the individual speakers identified, and the statements have not been endorsed or verified by the National Academies of Sciences, Engineering, and Medicine. They are not intended to reflect ...

... highlights speaker presentations that focused on the state of the science in four major classes of TBI biomarkers, their current levels of evidence, and where further research is needed....

... Major classes of TBI biomarkers include those identified through neuroimaging, proteins detectable in blood and other biological fluids, electrophysiological signals, and other physiological indicators such as those measured through eye tracking or gait ... . This section describes the landscape of current knowledge on the development and use of these types of biomarkers in TBI care and research....

... Utah, reviewed data on several prominent imaging techniques that can be used as biomarkers of brain injury, particularly computerized tomography (CT) and magnetic resonance imaging (MRI). These are both noninvasive options for imaging the brain. CT remains an important method in clinical guidelines for ...

... sensitivity limitations in cases of acute mild TBI and chronic TBI. CT imaging is less costly and generally faster than MRI and can be a better option in trauma emergencies. However, MRI does not use ionizing radiation as used in CT, and radiation exposures can be a concern ... also provides greater detail in visualizing abnormalities within the brain.2 Neuroimaging markers are best established for acute TBI that is moderate and severe in nature. Progress is being made in developing more advanced imaging biomarkers, including a variety of MRI markers, although many do not yet ... level 1 evidence and require additional validation.3 However, Wilde noted that the literature on imaging for TBI is far ahead of clinical practice, reflecting the ...

... There has been a dramatic increase in TBI imaging publications since 2015, with T1-weighted MRI and diffusion MRI being two modalities growing the fastest. Different imaging modalities are related to the types of pathology they can identify, and use ... different modalities will be necessary to capture the heterogeneous pathology that characterizes TBI. For example, diffusion imaging and susceptibility-weighted MRI imaging are most commonly used to look for vascular changes, metabolic alteration is typically assessed using ... , and positron emission tomography (PET) can be used to detect neuroinflammation....

... Diffusion and volumetric analysis provide insight into structural changes in the brain postinjury and are promising as diagnostic TBI markers. Even in mild TBI, studies have reported abnormalities in diffusion metrics. However, the limited ... over time. Because of the cost of MRI, Wilde explained, imaging is generally taken at several points rather than over multiple, short intervals, and it is difficult to have a precise understanding of the trajectory. Additional questions in using imaging for TBI diagnosis include which modalities ... measure (what), the optimal time points to use (when), and which brain structures to visualize (where), given the diversity of injuries. She also noted the difficulty of validating imaging markers in mild TBI ...

... 2 See https://radiology.ucsf.edu/blog/neuroradiology/exploring-the-brain-is-ct-or-mri-better-for-brain-imaging (accessed December 15, 2022)....

... 3 The strength of evidence in analyses of the literature and development of recommendations can be categorized by different levels, based on how the evidence has been obtained (for example, whether it is drawn ... analysis of one or more well-designed randomized controlled trials [RCTs] versus derived from cohort studies, expert opinion, or other sources). Level 1 evidence is considered the strongest ...

... Neuroimaging markers may also be useful as prognostic and predictive biomarkers. Most studies in adults have found a relationship between diffusion imaging results and symptom presentation, but establishing ... relationship remains tenuous in children. However, Wilde noted a recent pilot study using diffusion imaging and machine learning algorithms to predict symptoms after mild TBI (such as concussion) in children. The method was able to predict recovery, but with ... sensitivity and specificity (Fleck et al., 2021). Another study in children using diffusion imaging and machine learning showed increased diagnostic accuracy (Mayer et al., 2022), but these techniques are not sufficiently advanced to implement in ...

... to monitor injury progression is the area that has seen the least investigation so far, she reported, although some studies have explored brain effects in older adults with a prior history of TBI as well as the alteration of brain development after injury in children....

... for the clinical implementation of imaging biomarkers include the pace of evolution of the field—particularly upgrades in scanner hardware and software that necessitate periodic reanalyses and recalibration of previously collected data so it can be compared across study sites and times. ... using the newest and most specialized imaging sequences versus those that are well validated and widely available across sites is also a challenge. A lack of consensus remains about common analytic pipelines or deployment of new methodologies ... machine learning. One of the most significant challenges in neuroimaging, however, said Wilde, is the lack of normative data and the narrow indications for some existing datasets. Normative data is expensive to collect and has a limited shelf life because of hardware, software, ... may be nearly obsolete by the time researchers are ready to analyze it. Available normative data is also commonly collected from college students and may not reflect sufficient demographic and social variables nor be representative of the full range of TBI patients....

... meeting convened by Cohen Veterans Bioscience (see Box 3-1). Implementing these recommendations involves efforts to develop common analytic pipelines and harmonize previously collected imaging data, use of consistent standards and calibration strategies, and the need for multimodal analysis to ... for inclusion/exclusion criteria in clinical trials, for identifying which patients are most likely to benefit from specific interventions, and for monitoring and assessing the effects of an intervention also remain important directions....

... Gaps and Recommendations for Neuroimaging Biomarker Studies...

... Informed by 2019 Brain Trauma Blueprint State of the Science Summit Convened by Cohen Veterans Bioscience...

... GAP 1: Incomplete large, systematic, observational studies that longitudinally collect and analyze multimodal candidate biomarkers....

... Prospective studies to identify biomarkers to monitor TBI sequelae for pharmacological or therapeutic interventions and study the time course of post-injury trajectories....

... Continued validation of neuroimaging biomarkers for subacute and chronic TBI to understand the underlying injury pathways that correspond to biomarker signatures....

... Establish guidelines to standardize data acquisition and frequency....

... Increase funding both for data collection and also analysis of new or existing data to benefit secondary use of data....

... Harmonize and standardize data across platforms and through data repositories to enable quality control using the same procedures and calibrants....

... Develop reference standards and methods in alignment with other initiatives....

... Foster global team science via an open data sharing and analysis platform to accelerate insights....

... GAP 3: Identify validated, reliable, multimodal biomarker data to improve stratification to guide diagnosis, prognosis, and monitoring....

... Identify and validate underlying injury pathways that correspond to biomarker signatures to determine novel interventional methods....

... Integrate modalities within the same patients and time points....

... Develop an understanding for how biomarker panels and profiles could help guide meaningful indicators of improvement and patient management, which may inform diagnosis, prognosis, and selection of therapy....

... the development of fluid biomarkers for TBI, focusing on proteins detectable in blood while noting that such markers can be studied in saliva, urine, and other bodily fluids. From prior studies it is known that levels of glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH- ... ) are related during the acute period of experiencing a TBI, while neurofilament light chain (NfL) and Tau proteins are associated with recovery over the post-acute period. She highlighted several areas of study that are advancing the identification ...

... Identify novel biomarkers to increase diagnostic and prognostic sensitivity and specificity (moving beyond well-studied markers such as GFAP and UCH-L1), including strategies to measure proteins found in low quantities....

... Map objective fluid and nonfluid (imaging) biomarkers to study the specific pathologies and areas of the brain that have been affected after a TBI....

... Understand the clearance of biomarker proteins from the brain to blood, including patterns associated with different injury mechanisms (blunt force trauma ... blast injury) and in relation to factors that affect the types and concentrations of proteins measured in the blood, including time of day, nutritional status, and hydration....

... Study exosomes to identify the cellular origins of biomarker proteins, and study communication between cells....

... Investigate the roles of individual variability associated with clinical and demographic factors, including underlying, more complex social-environmental factors that shape exposure risk and recovery and that are just starting ...

... Gill shared data from the CARE Consortium,5 which includes baseline data on protein levels and gene activities measured before an individual plays a collegiate sport, facilitating comparison after concussion. She high-...

... 5 The Concussion Assessment, Research and Education (CARE) Consortium was established in 2014 with funding from the Department of Defense and National Collegiate Athletic Association (NCAA) to study mild traumatic brain injury in athletes and military cadets. See https://careconsortium.net (...

... in blood collected from athletes at different time periods after concussion. The results identified the expression of erythrocyte membrane protein band 4.1 (EPB41) as upregulated and alpha-synuclein (SNCA) as downregulated after concussion. Having identified this combination, the investigators have ... working on ways to measure it. Gill emphasized the technical importance of understanding protein isoforms (variants of a protein) and the need for careful testing and validation when developing and optimizing new biomarker assays, processes that can be time consuming. EPB4.1 is an ... protein but has been more challenging to measure and validate, while the measurement of SNCA was being developed by Quanterix and can be measured and validated on their platform....

... Using the CARE cohort, Gill also shared information from ongoing studies measuring blood biomarkers (GFAP, UCH-LI, NfL, and Tau) over 5 years in patients with different severities of TBI (Shahim et al., 2020a,b). Overall, the studied biomarkers map to volume loss changes ... in the brain over the first year, providing an indication that biomarkers can inform TBI risk and can be useful in monitoring....

... Gill highlighted the opportunity to identify how fluid biomarkers relate to brain regions and forms of injury, which involves understanding the clearance of the proteins from the brain to the plasma. A study of 700 acute TBI patients used MRI and CT imaging and gadolinium contrast to ... at brain meninges after injury and 3–5 days later to examine changes (Gill et al., 2018). Within this cohort, they found meningeal enhancement, in which there is a visual difference ... normal in the appearance of the membrane surrounding the brain on imaging, to be the most predictive of long-term symptoms, with longer duration and greater magnitude of enhancement associated with more prolonged recovery. Meningeal enhancement is a difficult part of imaging, and it is not often ... , Gill noted, and it would be useful to have a biomarker that could indicate that a patient would benefit from including this assessment....

... Biomarkers of inflammation have predictive value in TBI and relate to findings from brain imaging, but these inflammatory markers are not specific to neurotrauma. To better distinguish where inflammation is ... by cells throughout the body. A major function of exosomes is in cell–cell communication—in which a nanovesicle secreted from one cell binds to and releases its contents into another cell, thereby affecting that cell’s function. Antibodies can be used to identify the cell type from which the ... came, Gill explained, and exosomes detected in blood can be mapped back to brain cells such as microglia and astrocytes, adding a layer of spatial and functional information. As an example, she shared an early study in people in a particular military role (breachers) that results in signif-...

... markers were significantly increased in the breacher group (Edwards et al., 2022), indicating differences specifically associated with the brain compartment. The findings suggest that exposure to high numbers of even low-level blasts can result in ongoing central inflammation, but that ...

... Putting all of this together, she said, identifying the right fluid biomarkers and matching them with imaging and other types of markers continues to be a challenge, but it is clear that something objective is happening in people with TBI that can be measured and ... . Gill concluded with a call to rethink TBI not as an acute, discrete injury, but as a complex condition with multiple potential physiological and biochemical changes and neurological or psychological effects (see Figure 3-1). This can make it more difficult to recover from a TBI and perhaps ...

... Paul Rapp, Uniformed Services University and University of California, Irvine, described the advantages of using electroencephalograms (EEGs) in the assessment of TBI. EEG is also a noninvasive ... technology. Rather than a static image, it provides information about electrical activity in the brain. EEG technologies are also portable and do not require sophisticated infrastructure, enabling them to be deployed in austere or military far-forward settings. EEG technologies provide ... results and enable repeat EEGs to collect longitudinal data.7...

... More than half a dozen EEG-based biomarkers have been identified for TBI and have assays cleared by the Food and Drug Administration (FDA) for acute structural brain injury, acute functional impairment, and the presence of concussion. EEG is a readily usable ... for TBI,8 although Rapp recognized several challenges associated with measuring EEG and event-related potentials (ERPs). These measurements can be sensi-...

... 6 This section is based on a presentation by Paul Rapp, Uniformed Services University and University of California, Irvine....

... 8 An EEG generally measures brain electrical activity continuously over a period of minutes, while an event-related potential (ERP) is a short segment of an EEG signal, showing the ...

... as circadian rhythm changes, recent exercise, fatigue, or drugs. They can also be sensitive to how they are collected, such as electrode positioning, and they are sensitive to electromagnetic interference. EEG and ERP measures are often used in conjunction with other forms of imaging or clinical ...

... a step back, Rapp reflected on the broader challenges applicable across types of TBI biomarkers, including the potential for a complex presentation and delayed clinical presentation. A single injury event can initiate more than one pathophysiological process, he said, and these can have different ... courses and can interact with each other. A person’s biological response to an event is also dependent on past exposures and history of TBI. Further, people can present in a clinic when their injuries were sustained months or years prior, but their symptoms have reemerged....

... In response to these challenges, Rapp shared examples of actions being taken by the field. Work is being done on expanding the mathematical approaches to analyzing EEG data, he said, including use of interregional synchronization and the development of time-dependent ... entropy. Network analysis—a type of statistical analysis that identifies relationships between items—has also become a major area of study and is being applied to the understanding of information handling in the central nervous system. Lastly, methods are being employed to look at signal ...

... In addition to expanding analysis techniques, efforts to expand data acquisition from EEGs and ERPs are ongoing, as are expanded analyses to incorporate additional metrics for multivariate discrimination. An indicator such as heart rate variability (HRV) can be easily tracked ...

... diverse information is the Integrated Neuropsychiatric Assessment System, which includes an electrode cap, digital amplifiers, tachistoscope, and laptop, all designed for use in rugged environments (Rapp et al., 2015b). The system is designed to handle the simultaneous acquisition of EEGs, ERPs, ... and eye tracking, information that can be combined with patient history, imaging and protein biomarker data, and psychological assessments to produce an integrated analysis. Rapp concluded that collecting multiple types of signals and using mathematical analysis ... will advance the ability to bring information from multiple types of markers and assessments together in a clinically usable fashion. A recent review of psychophysiological biomarkers, such as the integration...

... of data from HRV, EEG, and other psychological and behavioral measures, identifies ongoing challenges and cautionary observations for the field as it continues to advance (Rapp et al., 2022)....

... phenomena after TBI, said Christina Master, Children’s Hospital of Philadelphia. These measures may have potential for improving the understanding of subclinical physiological dysfunction, as well, particularly in areas with functional implications for patients in their everyday lives, such ... visual and vestibular issues. Information from these other types of markers may also identify potential targets for interventions aimed at helping patients ...

... forms of TBI were often written off as insignificant because physiological signs of the symptoms that patients were describing were not observable and deficits were too subtle to measure. As with other forms of TBI, the goal of identifying and using biomarkers for milder injuries is to help diagnose, ... prognose, treat, monitor, and return patients to full recovery and function....

... Master described potential targets for measurement. Autonomic dysfunction is one such area, and applying exercise physiology to TBI has demonstrated exercise intolerance after concussion. A measure such as heart rate threshold on the Buffalo ... Treadmill Test, which is the heart rate at which concussion symptoms are provoked, is prognostic, and patients who could not elevate their heart rate to 135 beats per minute were more likely to have prolonged recovery after TBI (Leddy et al., 2018). ... aerobic exercise has been found to be therapeutic and to aid in preventing persistent postconcussion symptoms (Leddy et al., 2021). There is additional interest in using HRV as a biomarker, as Rapp ... . While more research is needed, HRV is a potentially prognostic biomarker. It is also sensitive to a history of concussion and may reflect residual effects from TBI even after apparent clinical recovery (Harrison et al., 2021)....

... Eye tracking is another active area of research, with some devices demonstrating the ability to distinguish between people with and without concussion (Bin Zahid et al., 2020). After brain injury, a common symptom is convergence insufficiency, in which the eyes do not coordinate ...

... for tracking objects.10 Eye measurement modalities include infrared eye tracking, retinal image-based tracking scanning laser ophthalmoscopes, and measuring smooth pursuit eye movements, with some of these devices achieving FDA clearance as an aid to concussion diagnosis, such as EyeBox (Bin ... et al., 2020) and Eye Sync (Sundaram et al., 2019). Such technologies may potentially be used in the future on the sidelines of a playing field to inform whether ...

... From eye tracking, Master moved to vestibular coordination, where eye tracking also plays an important role, and she said that companies and researchers are looking at both motion sensitivity and motion issues such as balance and gait. Reduced saccade velocity and changes in smooth pursuit ... with the decreased ability of the eyes to track a moving field (optokinetic nystagmus) after concussion (Kelly et al., 2019).11 Postural control and gait are other areas of study within vestibular coordination, and research has found that center-of-mass range of motion and lateral step variability ... in one study did not correlate with objective measures (Smulligan et al., 2021). The experience of mild TBI is still incompletely understood, and current physiological measures may not yet be fully capturing the symptoms experienced by patients....

..., pupillary light reflex (PLR), may be relevant across the spectrum of injury severity, Master said. PLR lends itself to quantitative measurements and to monitoring over time. PLR metrics, in combination with standard demographic factors and CT findings, have been shown to be predictive of poor 6- ... outcome in severe TBI cases (Romagnosi et al., 2022), and can be used to distinguish healthy adolescents from those with concussion (Master et al., 2020). This marker has the sensitivity to detect ... effects and could be used early on to see who might benefit from interventions and how to direct resources (Joseph et al., 2019)....

... 10 https://www.nei.nih.gov/learn-about-eye-health/eye-conditions-and-diseases/convergence-insufficiency (accessed December 20, 2022)....

... 11 Both saccade and smooth pursuit are types of eye movements, with the key point being that traumatic brain injury is associated with changes in the ability of a ...

... to auditory biomarkers, Master explained there is interesting work being done in areas such as the frequency-following response evoked by speech and the implications for TBI diagnosis, prognostication, and clinical care. Even after concussion, a diminished speech-evoked response has been observed, ... symptoms improve, showing its potential use in monitoring recovery. A signature of diminished response remains even after apparent clinical recovery, and so the marker is also sensitive to history of a prior concussion (Kraus et al., 2016, 2017)....

... tends to recover over 4 weeks. However, studies have found poor correlation between self-reported symptoms in those with sport-related concussion and objective measures captured through actigraphy (Considine et al., 2021). This demonstrates that there is still a need to improve understanding of ... physiology after traumatic brain injury and a need to find ways to close the gap between what can be measured and what patients report experiencing. Collecting observable physiological measures through wearables, conducting ecological momentary assessments in ... time and real-life contexts, and incorporating options for remote patient monitoring can all be brought to bear in measuring and monitoring outcomes that are meaningful for patients....

... Initial discussion centered around the interpretation and generalizability of biomarker results, given small sample sizes used in some studies, and the resulting implications for biomarker translation into clinical practice. From a clinician perspective, Master said that new biomarkers need to ... useful information as well as being feasible and user-friendly to be adopted in practice. Rapp indicated that some biomarker results can be overinterpreted and called attention to the importance of ... the confidence intervals when interpreting study results, saying that the intervals need to be tightened to be useful and that failures to replicate biomarker results can discourage clinicians from using them. Wilde added that the numbers of study participants are much ...

... 12 Examples of TBI research consortia mentioned during the workshop and recent report (NASEM, 2022) include the TBI Model Systems Network (https://msktc.org/about-modelsystems/TBI); Transforming Research and Clinical ... in TBI (TRACK-TBI; https://tracktbi.ucsf.edu); CENTER-TBI (https://www.center-tbi.eu); NCAA-DoD Concussion Assessment, Research and Education (CARE) Consortium (https://careconsortium.net); and the Long-Term Impact of Military-Relevant Brain Injury Consortium and the Chronic ...

... last 5 years, she said, investigators have been able to collect large amounts of data that did not previously exist, and this has been of major benefit to the field. Several participants also noted the continued role for increasing understanding on when and how ... can be used to inform TBI diagnosis and classification, prognosis, and recovery monitoring....

... community of interest in TBI. There are currently eight cleared devices, she noted, but DoD is using very few of these yet. While biomarker devices and their cleared uses continue to evolve, she surmised that if a device is not yet usable in a point-of-care battlefield or forward military setting, ... for the biomarker field not to focus only on specialty services for brain injury but also to solicit feedback that informs the development of markers and assays usable in general locations, such as by emergency medical services and in hospital emergency departments. Master noted that in a primary care ... , providers will feel most comfortable using their existing, familiar clinical assessment tools. While new technologies and modalities will inform a more refined characterization of TBI, not all technologies may become a standard part of clinical practice....

... subtypes of TBI. Her lab has used these approaches to collect pilot data, subsequently seeking funding to validate the data in different and expanded cohorts. She called for discovery platforms within TBI consortia that are additive and flexible, and for grant funding to enable findings to be built ...

... pipeline that have level 2 or 3 evidence. In terms of fluid biomarkers, Gill said that NfL protein is performing well across consortia for both acute and chronic TBI patients. Phosphorylated tau has been advancing as a prognostic biomarker, as well, she added. For imaging, Wilde said that diffusion ... does not yet reach level 1 but is the most promising and widely used MRI modality at this point. Master acknowledged that evidence for the categories of physiological markers she discussed are behind ...

... and fluid biomarkers. It will be years before they are ready for clinical use, she noted, but they can be useful in distinguishing subtypes of TBI. ...