The need to objectively measure head injury severity on the battlefield or the football field, at the scene of an auto accident or in the emergency room cannot be overstated. Traumatic brain injury (TBI) remains a major health issue with approximately 1.5 to 2 million incidents occurring annually in just the United States alone. Of these about 50,000 patients die, 500,000 are hospitalized [1-4].
Of the mild TBI patients 40 – 50% suffer persistent neurological problems from one to three months following injury and 25% after one year. This represents over 500,000 new cases of injury-related disability each year . An estimated 90,000 will suffer permanent disabilities . Yet there are no objective, blood based diagnostic tests for TBI, and no effective pharmacological treatments. The absence of clinically validated brain injury diagnostic markers as an internal indicator of tissue damage with the ability to measure changes in the cellular, biochemical and molecular events during the injury response has been identified as a major limiting factor to diagnostic and therapeutic development for brain injury. However, significant strides have been made towards solving these problems.
Proteomics - Major research advances in neurotrauma neuroproteomics have identified several candidate markers that are under evaluation as TBI biomarkers. Animal and human studies are evaluating the clinical relevance of these markers, their correlation with injury severity and potential as diagnostic adjuncts to currently used diagnostic tools such as computer tomography (CT) or magnetic resonance imaging (MRI) scans. These tools have low sensitivity and specificity for diffuse or mild brain injuries, are not universally available [4, 7, 8], are expensive and slow to produce results.
The Glasgow Coma Scale (GCS), a symptoms-based neurological scale used to assess level of consciousness after a TBI, divides patients into broad categories of mild, moderate, and severe injury. While the GCS has proven its utility in the clinical management and prognosis of severe TBI patients, it cannot provide information about the pathophysiological mechanisms responsible for a patient’s neurological deficits. In addition, specific patient populations are difficult to assess with the GCS, particularly those who suffer from mild or moderate TBI, which account for 80 – 90% of all cases.
So there is a need for sensitive and specific biochemical marker(s) of TBI with diagnostic and prognostic capabilities that will allow the clinician to evaluate post-concussive intracranial pathology, to improve patient management and to facilitate therapeutic evaluations . A number of altered neuro-proteins have been identified. They are currently being evaluated for potential to provide insights into injury severity, outcome , and the ability to monitor cellular damage and biochemical and molecular events that occur during the secondary injury phase of TBI as well as cellular repair responses. It is anticipated these biomarkers will be able to assist therapy development by monitoring cellular reactions. The studies identifying biomarkers used a combination of methods. The proteomic approach combines immunoaffinity depletion of abundant CSF proteins, SDS-PAGE gel electrophoresis, and protein identification by mass spectrometry  among other techniques.
More recent proteomic techniques, now being employed, utilize antibody-based methods including high throughput immunoblotting and antibody panel/arrays such as ELISAs. There are several advantages of the antibody-based proteomic approach. For example the antibodies can provide high specificity and selectivity, they are more compatible with complex high-protein content samples (like those found in plasma) and finally, it is possible to rapidly confirm potential hits. On the other hand, the major disadvantages of this method include the potential uneven sensitivity of the antibodies, lack of reliable antibodies to cover all possible proteins and their different isoforms, and the inability to identify potential biomarkers if they are not included on the array.
Biomarkers – Although there are currently no biomarkers with proven clinical utility for diagnosis of brain injury, whether it is caused by TBI, stroke, or other acute brain injuries, research has uncovered several candidates that have shown some preclinical potential. The ones currently generating the most interest include lactate dehydrogenase (LDH), glial fibrillary acid protein (GFAP), neuron specific enolase (NSE), and S-100ß. Although these proteins are currently being assessed, they appear to lack either the necessary sensitivity or brain specificity or both to be used effectively alone [4, 8, 11, 12].
More recently a number of new candidate biomarkers have been discovered. The emerging data suggest UCH-L1, MAP-2, and TAU proteins [13-15], and the ?II-spectrin protein breakdown products (SBDPs) [16-21] have strong possibilities. Currently Banyan Biomarkers, Inc. is performing assay validation of MAP-2 and UCH-L1 sandwich ELISA assays. Clinical validation with human serum samples using these biomarkers is in progress.
Usually, the levels of potential biomarker proteins increase following injury and are found in increasing concentrations in the CSF depending on the injury magnitude. Eventually they find their way into the blood stream via a compromised blood brain barrier . How quickly the biomarkers are cleared from the bloodstream is a major factor in determining its final measurable concentration in the blood. When neuroproteomic studies yield a multitude of potential biomarkers, there are several key factors involved in selection or triage of a particular biomarker. These criteria include preliminary data (literature relevance and proprietary nature of the biomarker), biomarker protein attributes (i.e., molecular weight, proteolytic cleavage, tissue specificity, stability), and cross-species sequence similarity (i.e., human, rat, mouse). Finally, there are two major criteria that need to be critically assessed to determine whether the biomarker is good candidate: 1) whether it is detectable in the blood stream in quantifiable amounts that are indicative of the underlying pathological condition and 2) its specificity to the brain.
Platform - A biomarker’s success depends on the development of a sensitive and reliable platform that is easily readable. Today’s most commonly used assay is one that is ELISA-based. The most critical component of a biomarker platform will be its ability to measure TBI severity as early as possible following injury. That ability, developed and validated in a platform, begins with the capability to develop an assay that can detect the biomarker proteins at extremely low concentrations. To date, the most common validation technology relies on antibodies to ensure accuracy and precision of data. Due to the advances in immunological methods, a wide range of antibody-based diagnostic tools have now been authenticated. To enjoy long term and widespread success the platform needs to be as non-invasive as possible. Moreover, the assays’ clinical usefulness will be governed by multiple clinical determinants such as medical history and medication received and the like.
The ultimate objective, following the successful development of an assay kit, is to translate this into a user friendly, handheld point-of-care (POC) device capable of monitoring a panel of markers in the body fluids such as blood or urine with minimal invasive procedures. Presently, no POCs capable of detecting biomarkers for brain trauma in human biofluids are available, but a number of companies have been drawn to the need and are working on devices. Such a device would be very useful for doctors and EMTs in the civilian population as well as for the military medics in warzones to assess the existence and severity of head trauma. A major challenge however, is that these potential biomarkers exist in blood at extremely low levels, often at or beyond the detection capabilities of conventional ELISA technology. This ultimate challenge may necessitate the use of advanced technology in these devices such as nanotechnology to increase their detection sensitivity as well as their specificity.
Conclusion – The future of biomarker driven assays to diagnose and guide therapeutic development and treatment of TBI is very promising. As studies continually expand our knowledge of the internal workings of the brain under pathological conditions and, as we learn more about the proteins that make up the brain, the closer we come to finding a means to diagnose and treat TBI. The technical, financial, legal and regulatory hurdles that need to be overcome before commercial products will be available are substantial but not insurmountable. And once the platform is available, emergency personnel and clinicians, the ultimate end-users, will need to be trained in the usefulness of these new biomarker tools to foster their widespread application. The future holds great promise for the patients who may sustain a TBI in the future by improving the clinicians’ diagnosis and treatment capabilities.
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Acknowledgements: Supported by DoD Award # DAMD17-03-1-0772; #DAMD17-03-1-0066; NIH Award # R01 NS049175-01; R01-NS052831-01; R01 NS051431-01.
Stephen F. Larner, PhD, CPA; Kevin K.W. Wang, PhD; Monika Oli, PhD; Gillian Robinson, PhD; Andrea Gabrielli, MD; Steven A. Robicsek, MD, PhD; Ronald L. Hayes, PhD
Banyan Biomarkers, Inc., Alachua, FL; University of Florida, College of Medicine, Gainesville, FL