Medical Device Link.

ASSAY DEVELOPMENT

Illustration by iStockphoto

There is a ready market for improved laboratory testing in the neurology and neurosurgery fields. New, better, and more complete traumatic brain injury (TBI) diagnosis solutions are of interest to the biotech industry, pharmaceutical companies, the medical community, healthcare providers, health and life insurance companies, and the military.

TBI is a brief episode of neurological dysfunction that can increase the likelihood of stroke or epilepsy. TBI is caused by a blow or jolt to the head or a penetrating head injury that disrupts the normal function of the brain. TBI is complex, presenting with a broad spectrum of symptoms that can result in permanent disabilities. TBI results in both short- and long-term sequelae leading to acute or chronic neurological deficits. Depending on the location of the injury in the brain, TBI can also induce posttraumatic epilepsy and increase the risk for other neurological disorders. Closed head injury is associated with a broad range of physical, cognitive, emotional, and social problems in individuals.

Approximately 1.4 million people in the United States sustain TBI each year, resulting in more than 235,000 hospitalizations and 50,000 deaths. An estimated 5.3 million Americans have current long-term disabilities as a result of TBI.¹ Sports and recreational activities contribute to about 21% of all TBIs among

American children and adolescents. In 2005, nearly 37,000 of 64,500 bicycle-related head injuries treated in U.S. hospital emergency rooms were in children aged 14 years and younger. Mild or subtle TBI is the most prevalent form of injury in military settings. The annual cost to society of TBIs is estimated at $60 billion, which does not include those injured in combat.

In the United States, at least 1% by the age of 20 and 3% by the age of 70 suffer unprovoked convulsions of unknown origin. Stroke and TBI increase seizure likelihood by 50% within the next two years after the incident and can lead to epilepsy. Electroencephalograhs (EEGs) and brain scans are common diagnostic tests for epilepsy with diagnostic probability of 66–70%. Therefore, the search for disease-related biomarkers in the blood that will be able to improve diagnostic certainty of brain-related abnormalities is pertinent. Currently, there is an unmet diagnostic need for a blood test that can assess brain-related seizures or epilepsy, TBI, and both prenatal and natal brain pathology.

Timely and accurate diagnosis is critical: the vast majority of irreversible brain damage is complete within 4-6 hours of a TBI event. With early detection, followed by appropriate treatment, complete recovery is possible. However, there are problems with diagnosing the severity of TBI, namely, the difficulty in diagnosing concussions (mild TBIs), the impossibility of predicting the consequences of moderate brain injury, and the uncertainty about the outcomes associated with severe brain injury.

Pathological changes that occur in the brain soon after head injury can be detected in the blood as biochemical traces, or biomarkers. A blood-based assay that could detect these biomarkers would be a practical instrument that could help provide reliable and rapid identification of patients with brain injury, especially mild TBI, allowing timely, prophylactic, and successful treatment of secondary lesions or posttraumatic seizures.

To date, no commercially available blood test can reliably detect or exclude occurrences of mild TBI. This underscores the diagnostic potential of detecting brain biomarkers. However, the discovery of biomarkers in biological fluids remains an enormous challenge, particularly the discovery of novel biomarkers for brain damage.

Biomarker Assays for TBI Identification

Currently, the medical community faces two principal diagnostic challenges in overall TBI management. First, there are difficulties in diagnosing TBI. Second, there is the challenge of differentiating TBI from other neurological disorders. Therefore, the search for objective biomarkers associated with early neuropathological events following TBI is essential. No perfect biomarker for TBI has yet been discovered.

To be considered perfect, a biomarker or panel of biomarkers must meet the following five requirements:

• Reflect the pathology of brain damage.
• Appear in biological fluids at an early stage of brain damage (within minutes to hours after TBI).
• Correlate with neurological scores and neuroimaging data.
• Predict the outcome of TBI sequelae.
• Allow posttreatment follow-up and adjustment of therapy or medication doses.

Thus far, research to detect TBI has focused on either finding differences in biomarker content in serum or plasma obtained from patients with TBI and appropriate matched controls, or the two-dimensional fractionation of biological fluids.² Both approaches provide a wide range of biomarkers that can be correlated with a pathological profile. Limitations to these approaches include the length of the required procedures, the cross-reactivity in different diseases, and the low specificity of the blood-borne biomarkers for TBI.^3,4

Table I. (click to enlarge) Candidate biomarkers for identification of traumatic brain injury (TBI). GCS = Glasgow coma scale, CNS = central nervous system, CT = computed tomography.

Those researchers who have investigated potential biomarkers of neurological disease—blood-borne cellular proteins, secreted proteins and peptides, and proteolytic fragments—have found this quest to be challenging. There are several biomarkers of brain and nonbrain (blood-borne) origin that are well characterized (see Table I). However, clinical trials of several commercially available markers have demonstrated limited performance characteristics for a diagnosis of TBI. In addition, most of the major requirements of a perfect biomarker were not met.

Feasibility studies also found low assay reliability, with the biomarkers weakly correlating with damage size as defined by radiographic gold standards for computed tomography (CT) scans, diffusion weighted imaging (DWI), and magnetic resonance imaging (MRI) (see Table I).

Table II. (click to enlarge) Pathological features of apoptosis and necrosis.

Predicting long-term outcomes after TBI is an important component of treatment strategy. Despite dramatically improved emergency management of TBI and apparent clinical recovery, most patients with TBI still may have long-term central nervous system (CNS) impairment. Although biomarkers can now be rapidly detected in biological fluids, those currently available cannot predict the consequences of brain injury or define treatment strategy. The pathology of brain injury is complicated and includes several simultaneous processes of necrosis and apoptosis (see Table II) underlying different mechanisms: neurotoxicity, ionic dyshomeostasis, activation of voltage gated ion channels, and failure of high-energy metabolism (see Figure 1). It would seem plausible to search for several brain biomarkers that control key reactions in nervous tissue following brain injury. Combining these biomarkers into a brain panel may improve diagnostic certainty of TBI by providing early identification of brain impairment, aiding in predicting outcomes, and guiding individualized treatment for better patient survival.

Novel TBI Biomarker Assays

Figure 1. (click to enlarge) Candidate biomarkers for identification of traumatic brain injury (TBI). GCS = Glasgow coma scale, CNS = central nervous system, CT = computed tomography.

CNS biomarkers generally represent proteins that are specifically produced in the brain and spinal cord and are relatively sequestered in the CNS, mainly due to the presence of an intact blood-brain barrier (BBB).^5,6 When the BBB is compromised due to brain injury or disorders, brain peptides and proteins are released into circulation and accumulate in the blood. The detection and quantification of CNS-specific proteins released into the blood as the result of brain injury, therefore, provides a potentially attractive means of diagnosing brain injuries using minimally invasive procedures. It is well known that brain injury triggers a cascade of events that results in alteration of cerebral blood flow and metabolism, tissue edema, neuronal degeneration and, finally, cell death.^{7, 8}

When TBI occurs, a cascade of biochemical events is initiated. Mild TBI affects electrical and chemical circuits, subsequently leading to a failure in blood circulation. NMDA and AMPA receptors are key components controlling these processes (see Figure 1).⁹

Neurons and glia regulate brain microvessel functions and the microvessels exchange nutrient (glucose) and oxygen with brain cells through the endothelial cells. It has been demonstrated that NMDA receptors are localized on the surface of epithelia of microvessels that form the BBB.

Compression and microvessel damage after mild TBI lead to edema-activating necrosis. A secondary ischemic area then forms around the edema locus, initiating apoptosis and restricting neuronal oxygen and glucose supply. Excessive glutamate amounts activate NMDA and AMPA receptors and trigger an excessive influx of calcium leading to overexpression of glutamate receptors in extra-synaptic areas.¹⁰ The degradation of receptors by thrombin-activated serine proteases results in peptide fragments entering the bloodstream through the compromised BBB. The immune system recognizes peptides as foreign antigens and generates specific antibodies that capture peptides. Using appropriate technology, peptides and antibodies may be detected directly in the bloodstream.¹¹

Thus, the compression of cerebral microvessels leads to simultaneous necrosis and secondary ischemic damage. Necrosis causes cell swelling and brain spiking activity, while ischemia accompanied with neuronal death causes brain lesion formation.

Research has shown that NMDA and AMPA receptors provide real-time evidence of neurotoxicity following secondary cerebral ischemia and brain-related seizures. On the basis of molecular investigations, NR2 or GluR1 peptides were proposed as novel biomarkers of apoptotic and necrotic events following TBI.

As previously explained, NMDA and AMPA receptor degradation and the resultant apoptosis or necrosis play important roles in brain dysfunction after induced mild TBI and closed head injury. Peptide levels can be detected in the bloodstream immediately after brain injury, and reflect the severity of brain damage.

Animal research has indicated that NMDA and AMPA receptor degradation have different time profiles of expression and bloodstream trafficking depending on the process (necrotic or apoptotic) involved after TBI. Apoptotic neuronal damage was found to initiate NR2 peptide trafficking, while GluR1 peptide was observed when necrotic events were present in nervous tissue following mild TBI. Low-molecular-weight peptides (MWD = 2–2.5 kD) representing fragments of the NR2 subunit of the NMDA receptor and the GluR1 subunit of the AMPA receptor were detected using polyclonal antibodies. Experimental studies using sham controls showed that the peptide profiles were different: GluR1 peptide peaked within minutes after cortical impact, while NR2 peptide was elevated beginning two hours after impact and peaked at 24 hours when compared with the baseline. NR2 peptide levels declined at day three after mild TBI.¹¹

Clinical studies performed in collaboration with the Russian Military Medical Academy (St. Petersburg, Russia) observed 71 patients (20–22 years old, male) with mild closed head injury (CHI). Thirty-five of the patients had a confirmed loss of consciousness following impact, with durations ranging 2–10 minutes. CT head scans were undertaken in observed CHI subjects (n = 71), and the results were normal for all subjects. DWI results depicted edema areas (3–5 cm3) and decreased diffusion in 71% of patients. Apparent diffusion coefficient values were significantly lower in patients with mild CHI than in the age- and gender-matched control group.

The frontal temporal lobe cortex was the region predominantly affected by concussions (43%).

Comparison of mean values of NR2 peptide in control groups demonstrated that values had low distributions, with a cutoff of 0.1 ng/ml. Conversely, significant differences were observed for patients with mild CHI. Ten of the 71 patients (14%) with DWI abnormalities had an increased level of NR2 peptide, while GluR1 peptide (cut off of 0.1 ng/ml) was detected in about 32 patients (45%) within minutes of the brain injury compared with the control level for healthy persons (n = 64). In a separate group of patients with edema defined by DWI, 17 (12%) had above normal levels of both peptides.

TBI patients with increased NR2 peptide developed symptoms of neurological deficit (e.g., temporary visual loss, sensory loss, confusion, or memory problems). Patients presenting with high levels of GluR1 peptide demonstrated spike activity on scalp EEGs; only those with abnormally high levels of both NR2 and GluR1 peptides developed brain-related seizures within a short period after mild CHI.

The time frame of each peptide trafficking into the bloodstream was varied, and the peptide profiles were similar to those observed in animal research. Blood samples from some patients showed abnormal amounts of NR2 peptide within 24 hours of trauma, while elevated GluR1 peptide was detected immediately after the injury (i.e., within 30 minutes of a sport-related injury). Thus, both peptides are detected in the bloodstream within the first 24 hours after brain injury and could help guide treatment procedures.

Antiepileptic drugs (AEDs) are frequently and acutely administered to patients with TBI upon hospital admission to prevent seizures. While the use of long-term AED therapy in absence of seizures remains controversial, a GluR1 peptide assay could navigate the duration of AED therapy due to monitoring actual brain excitation even in absence of seizures. Antiepileptic medication has been found to reduce GluR1 peptide levels, with a favorable patient outcome.

Table III. (click to enlarge) Clinical performance of NR2 peptide assay.

Preliminary feasibility studies showed that cortexin, a natural peptide regulator, significantly decreased edema volume (up to 40%) and improved brain circulation, with a simultaneous reduction of NR2 peptide levels (unpublished data) in TBI patients (n = 12).¹² Polytherapy combining antiepileptic and neuroprotective medications halted the evolution of TBI complications and reduced abundant amounts of both NR2/GluR1 peptides.

NR2 and GluR1 Peptide Assays Available

Table IV. (click to enlarge) Clinical performance of GluR1 peptide assay.

A direct enzyme-linked immunosorbent assay (ELISA) for detecting NR2 or GluR1 peptides by Grace Laboratories (Atlanta) consists of captured antibodies and an indicator reagent comprised of monoclonal IgG conjugated with a signal-generating compound, HRP. The test is intended to assess patients experiencing cerebral ischemia, transient ischemic attack (TIA), or brain-related seizures after TBI. The performance characteristics of the assays for the NR2 and GluR1 peptides are summarized in Tables III and IV.

For the NR2 assay, sensitivity is 92% and specificity 95%; respective performance figures for the GluR1 assay are 90% and 97%. Predictive values are calculated at 90% (GluR1) and 91% (NR2), at a cutoff point of 0.1 ng/ml. Tests are generally

considered conclusive if the positive likelihood ratio is higher than 10 or negative likelihood ratio is less than 0.1. Thus, both tests proved to be conclusive in a population prevalence study that corresponded with an in-hospital clinical study.

Magnetic Particle-Based ELISA (MP-ELISA)

Figure 2. (click to enlarge) Schema of the magnetic particle–based enzyme-linked immunosorbent assay (MP-ELISA).

The magnetic particle–based ELISA (MP-ELISA) employs a custom-modified magnetic particle (MP) with covalently attached hen antibodies (IgY) raised against a peptide (GluR1 or NR2). The peptide (GluR1 or NR2) concentration is deter- mined immunochemically in a serological assay using preformed MP-IgY particles added to each plasma sample and applied to the wells of a microtiter plate (MTP). In the first incubation step, the peptide in the sample reacts with the solid-phase bound antibodies. The MP-IgY-captured peptide complex is then intensively washed using a separation magnet to remove solution from the MTP. The complex reacts in the second incubation step with peroxidase-labeled detection (POD) monoclonal antibodies (see Figure 2).

The immunocomplex that is formed is quantitatively determined and followed by intensive washing in the third incubation step via the POD/ TMB-detection reaction. An acidic stopping solution is then added. The color converts from blue to yellow. The intensity of the yellow color is directly proportional to the concentration of the peptide sample. A dose response curve of the absorbance unit (at dual wavelengths 450 nm and 630 nm) versus the concentration is generated. The amount of the peptide present in the serum samples is determined directly from this calibration curve. The reagent kit contains a set of calibrators (n = 4) and negative and positive controls in ready-to-use solutions for different peptide concentrations. The detection limit is <100 pg/ml and the time of detection is less than 30 minutes.

Svetlana A. Dambinova, DSc, PhD, is visiting professor at Emory University (Atlanta). She is a consultant to CIS Biotech and Grace Laboratories, among others. She can be reached at sdambin@emory.edu.

Conclusion

Diagnosis represents the first line of defense for any medical condition, yet current TBI diagnosis is suboptimal. Clinical use of blood tests that can reliably detect or exclude mild TBI, predict consequences, or forecast recovery and outcome, might improve diagnostic certainty of posttraumatic seizures or cerebrovascular abnormalities (e.g., TIA or stroke).

Early identification of TBI sequelae based on a blood assay that can detect brain-borne biomarkers, namely NR2 and GluR1 peptides, is a key component of successful treatment strategy and outcome monitoring. Advances in analytical assay technologies have made it possible to develop a rapid, cost-effective brain panel that can be used to predict consequences of TBI and clinical outcomes when used in conjunction with neuroimaging.

Timely assessment of TBI with appropriate blood tests would be useful in various clinical settings (e.g., emergency departments, primary physician offices, or in- and out- patient facilities). It has the potential to shorten the often lengthy recovery process after TBI and consequent neurological complications, and it can benefit the healthcare system by reducing direct and indirect costs.