WO2015067915A1

WO2015067915A1 - Biomarkers for traumatic brain injury

Info

Publication number: WO2015067915A1
Application number: PCT/GB2014/000445
Authority: WO
Inventors: Alun CARTER; Gareth David Griffiths; Stuart HARRISSON
Original assignee: UK Secretary of State for Defence
Current assignee: UK Secretary of State for Defence
Priority date: 2013-11-05
Filing date: 2014-11-05
Publication date: 2015-05-14
Anticipated expiration: 2016-05-05
Also published as: GB2525055A; GB201419631D0; GB201319492D0

Abstract

The present invention relates to a method, assay and kit for determining whether a subject is suffering from Traumatic Brain Injury (TBI), utilising biomarkers indicative of TBI.

Description

BIOMARKERS FOR TRAUMATIC BRAIN INJURY

The present invention relates to a subset of brain biomarkers for determining Traumatic Brain Injury (TBI), a method for screening a biological sample from a subject to determine whether the subject is suffering from traumatic brain injury, and an assay and kit for determining whether a subject is suffering from TBI.

The brain is the major organ of the nervous system of all vertebrates and most invertebrates, acting to control a complex network of cell types and organs that coordinate motor output in response to sensory input. The brain is the main sensory processor of the central nervous system (CNS), directing electrochemical signal impulses via the spinal cord to effector cells, such as muscles and glands, to generate a physiological response to a given environmental stimulus. Communication between the CNS and effector cells is achieved via the peripheral nervous system (PNS), which comprises a network of nerves capable of conducting electrochemical signals.

Communication between the brain and the surrounding nervous system is achieved via transmission of electrochemical signals by a specialised cell type termed the neuron. Neurons are one of the primary cell types found in the brain, whilst collections of tightly bundled neurons and associated connective tissue make up the structure of nerves of the nervous system. Neurons comprise an organelle-containing cell body and two types of extending fibres: dendrites, which conduct electrochemical signals towards the cell body; and axons, which conduct electrochemical signals away from the cell body to synaptic terminals. Axon are enclosed in an insulting layer of Schwann cells termed the myelin sheath and typically terminate at synaptic terminals which in turn communicate with other neurons or target cells via neurotransmitters released by the synapse.

Given the vital overarching role of the brain in controlling organs and physiological output, injury to the brain has potentially serious consequences for life. The brain is protected by a layered structure in the form of bone, membrane and fluid which acts to minimise any possible damage. Firstly, the brain is encased in an outer layer of bone termed the skull or cranium, which provides protection against impact. Inside the cranium, three membranous layers termed the dura mater, arachnoid membrane and pia mater, known collectively as the meninges, provide a further lining for the brain. The dura mater is situated as the outermost membranous layer, whilst the pia mater directly encloses the brain. The arachnoid membrane is situated between the dura mater and pia mater. Further cushioning and immunological protection for the brain is provided by cerebrospinal fluid (CSF), a clear liquid found within the subarachnoid space between the arachnoid mater and pia mater. To prevent damage to the brain by substances transmitted via the circulatory blood system, a specialised network of closely-located capillaries termed the blood-brain barrier acts to ensure only certain molecules are able to pass into the brain.

Despite the mechanisms present that attempt to prevent injury to the brain, external forces that include impact from a penetrating or non-penetrating object, rapid

acceleration and/or deceleration, or blast within the vicinity of an individual, are capable of causing intracranial structural damage and/or temporary or permanent damage to brain function. Intracranial injury, also known as TBI, is defined by the Demographics and Clinical Assessment Working Group of the International and Interagency Initiative towards Common Data Elements for Research on TBI and Psychological Health as 'an alteration in brain function, or other evidence of brain pathology, caused by an external force'.

The seriousness of TBI is underlined by statistics associated with this form of injury. There is a prevalence of TBI injury to males of 3:1 , with violence associated with between 7-10% of TBI in civilian practice. Trauma is the leading cause of mortality in the under-35 ages group in England and Wales, with head injury said to account for half of trauma deaths. In Europe, the annual aggregate incidence of hospitalised and fatal TBI has been estimated at approximately 235 per 100,000 people, whilst in the USA there were 403/100,000 Emergency Department visits for TBI per annum and

85/100,000 admissions to hospital for TBI in 2006. The majority of medical interventions for TBI are currently concentrated on those head injuries classified as moderate and severe head injury. Patients who have sustained moderate or severe injury may require care for many years after their head injury. In the USA, the financial burden has been estimated at $60 billion p.a. in 2001. However, the majority of cases of TBI are mild, with it being suggested that only 10-15% require specialist care. There is increasing evidence that this may be an incorrect assumption, as in a proportion of cases of mild TBI there are long lasting anatomical injuries and there is the possibility of being at risk of developing long-term neurological sequelae.

Although the rate of TBI is reducing in the young, the global incidence of TBI is increasing. Possible reasons for this include increased global use of motor vehicles, and the increasingly elderly nature of the general population suffering head injuries as a result of falls. In recent times, there has been renewed focus of the effect of TBI as a result of deployed military personnel sustaining head trauma as a consequence of exposure to explosive blasts. Indeed, it has been suggested that mild TBI is a novel signature injury for British and American troops deployed to Iraq and Afghanistan. In general, four types of blast injury have been described in relation to deployed

serviceman: primary blast injuries (relating to the over-pressure, also termed shock wave); secondary blast injury (relating to primary and secondary fragments); tertiary blast injuries (relating to the blast wind, also termed dynamic over-pressure); and quaternary blast injuries (relating to other injury mechanism).

Due to the variability of brain injury patterns, a problem with TBI is trying to classify this type of injury. One of the most common methods for assessing TBI is based on clinical severity. In particular, the widely used Glasgow Coma Scale (GSC), described initially by Teasdale and Jennett in 1974 (Teasdale, G. & Jennett, B. (1974) Assessment of coma and impaired consciousness. A practical scale. Lancet, 2, 81 -84), is a 15-point scale which looks at motor, vocal and eye-opening responses in a patient after resuscitation, with the summed scores often split into three broad groups of either mild, moderate or severe head injuries. However, several problems have been identified with the GCS, including: assessing intoxicated patients or those who have needed sedation or paralysis; patients with the same score may have very different pathological processes, requiring very different treatments; and localised lesions leading to an inability to assess either the verbal or eye-opening component (for example facial injuries) therefore skewing the score.

An alternative pathology-based classification of TBI bases assessment on an

abnormality needing treatment from an anatomical and physiological perspective. From a patho-anatomical point of view, although this classification is useful for many lesions, it does not allow adequate description of certain diffuse injury patterns such as cerebral ischaemia or oedema, which may be more related to the ongoing process of the injury rather than the initial injury, or for certain mechanisms of injury such as gun-shot wounds, where the track of the injury produces some of the injury but other injury can be caused by energy transmitted from the ballistic round to other surrounding tissues. The most common patho-physiological model used to describe TBI is to look if the injuries are either primary or secondary injuries. It is generally accepted that the primary injury is that which is due to the initial immediate injury and is unavoidable. Secondary injury refers to subsequent injuries which can be avoided, such as hypoxia,

hypertension or hypercapnia. As insight into TBI has increased, the physiological and anatomical information ave been brought together to develop a better understanding of the injuries seen, which can be grouped into four main types: contusion, haematoma, subarachnoid haemorrhage and diffuse axonal injury (Saatman, K.E. et al. (2008) Classification of traumatic brain injury for targeted therapies. J Neurotrauma, 25, 719- 738). However, considering specific secondary injuries such as ischaemia, oedema and vasospasm appears to add to the difficultly of determining TBI in a subject.

In order to overcome the disadvantages associated with assessing TBI based on classification, radiological-based methods such as Computerised Tomography (CT) or Magnetic Resonance Imaging (MRI) can be applied. However, such methods are limited by being single "snapshot" assessments, complex and time consuming. Furthermore, CT has the associated risk of ionising radiation, whilst studies have shown that MRI may provide inconclusive results for white matter injury. In addition, other methods such as Electroencephalogram (EEG) and Intra-Cranial Pressure (ICP) have also been used to look for signs of neurological damages after exposure to non-penetrative blast injury. However, such techniques may require undesired invasive measures. Moreover, studies have also found such methods inconclusive.

The availability of a rapid and relatively simple means of determining whether or not a particular subject has suffered TBI would clearly be desired by clinicians. Such assessment could help inform clinicians in terms of the most appropriate form of treatment as quickly as possible. In particular, the ease and speed of determining TBI is of particular concern with respect to deployed soldiers suffering head injury, as medically-trained staff and state-of-the-art medical facilities may not be immediately and/or readily available. During military operations, it would be desirable to reach a rapid and accurate conclusion as to whether a soldier who has been mildly injured has symptoms of mild TBI and is fit to work or, of greater concern, whether those subjects who are more severely injured will likely require sedation as part of their medical treatment and therefore in whom it is not possible to assess using current clinical methods. An additional complication for military blast casualties is the distinction between symptoms of mild TBI and other traumatic incidents, such as post-traumatic stress disorder, shell shock and post concusional syndrome. Indeed where patients are injured on the battlefield, data collection will often not be complete and patient assessment is performed using retrospective questionnaires. There is a very serious risk of recall bias in such methods which will influence diagnosis of the underlying condition.

Thus, in light of the widespread incidence of TBI, including the recent emerging trends for this type of traumatic injury, as well as the difficultly in terms of assessment and classification, there is a requirement for a simple, cost-effective and accurate method for determining TBI.

A possible solution to determining TBI is to screen a biological sample, such as CSF or blood, for biomarkers indicative of TBI. The term 'biomarker' as used herein is a biological characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes or responses to a therapeutic intervention. Biomarkers can be physical or biochemical measurements which can be used as a validated substitute for a clinical endpoint. Examples of biomarkers include certain cell types, proteins (for example enzymes, hormones, antibodies), gene products, genes or molecules that may indicate normal or abnormal biological processes or states. Clinical screening for biomarkers may offer advantages that include earlier diagnosis of disease severity, allowing better prognostication and prediction of outcomes, and enable monitoring of on-going pathological processes and therapeutic interventions. Examples of biomarkers already used in other pathologies include Troponin-I after myocardial infarction or Prostate Specific Antigen in prostate cancer.

Biomarkers of TBI could potentially offer a high sensitivity and specificity for brain injury, if suitable biomarkers could be identified and assessed. Certain biomarkers may only be released after irreversible destruction of brain tissue, which could provide a good measure of TBI. Furthermore, biomarkers could allow for: better initial assessment of both mild and severe head injury in isolated head and polytrauma patients; monitoring of disease progression; and success of therapy (Neumar, R.W. (2002) Rule out TBI? Serum markers for traumatic brain injury. Ann Emerg Med, 39, 342-343;

Ingebrigtsen, T. & Romner, B. (2002) Biochemical serum markers of traumatic brain injury. J Trauma, 52, 798-808). In mild TBI this may allow identification of those who go on to develop post-concussional syndrome and also those at risk of Secondary Insult Syndrome. In addition to benefiting assessment of TBI in civilians, investigating brain biomarker levels may offer a more standardised method of detecting TBI in military personnel suffering head injuries duririg deployment.

Many attempts have been made to find a biomarker of head injury. For example, potential biomarkers such as Creatine Kinase, Glial Fibrillary Acidic Protein, Lactate Dehydrogenase, Myelin Basic Protein and S100 protein have all been studied to varying degrees of success (Ingebrigtsen, T. & Romner, B. (2002) Biochemical serum markers of traumatic brain injury. J Trauma, 52, 798-808; Kochanek, P.M. et al. (2008)

Biomarkers of primary and evolving damage in traumatic and ischemic brain Injury: diagnosis, prognosis, probing mechanisms, and therapeutic decision making. Curr Opin Crit Care, 14, 135-141 ). Phosphorylated Neurofilament-H (pNF-H) has been identified as a biomarker of TBI (Zurek er a/. (201 1 ) Hyperphosphorylated neurofilament NF-H as a predictor of mortality after brain injury in children. Brain Injury, 25, 221 -226; Anderson er a/. (2008) The Phosphorylated Axonal Form of the Neurofilament Subunit NF-H (pNF- H) as a Blood Biomarker of Traumatic Brain Injury. Journal of Neurotrauma, 25, 1079- 1085), as has Microtubule Associated Protein 2 (MAP2) (PCT/US99/29023 Ά method for detecting neuronal cell damage by quantification of MAP-2 levels in biological fluids'). A single ideal biomarker is, however, unlikely to exist as each head injury has a unique pattern with several different pathological pathways often acting at the same time.

PCT/US2010/048789 'Micro-RNA, Autoantibody and Protein Markers for Diagnosis of Neuronal Injury' teaches that measuring a combination of biomarkers in a subject sample is desirable for determining a neurological condition in a subject. However, this document discloses an exhaustive list of potential biomarkers and does not clearly direct a person skilled in the art to use any specific combination of a small number of biomarkers to determine whether a subject is suffering from TBI. Whilst it is possibly more realistic that a panel of biomarkers is going to be required when screening a biological sample to assist determining TBI, the problem of balancing a practical number of biomarkers for screening with reliability and accuracy remains. To date, a small panel of biomarkers is yet to be identified which is capable of determining TBI especially with a high predictive accuracy, such as at least 85% and preferably higher and,

furthermore, capable of providing a reliable prognosis for instances of TBI.

The present invention thus aims to address the problem of identifying a small subset of brain biomarkers for determining traumatic brain injury, and more specifically a small subset of brain biomarkers capable of providing a reliable prognosis for instances of TBI.

Accordingly, . in a first aspect of the present invention there is provided a method for screening a biological sample from a subject to determine whether the subject is suffering from TBI, the method comprising the steps of measuring the concentration of an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker; and comparing the concentration of each biomarker with a respective control threshold concentration; wherein a concentration higher than its respective control threshold concentration is indicative of TBI.

The term 'biological sample' in relation to the present invention refers to a sample suitable for screening to determine whether the subject is suffering from TBI and includes, but not exclusively, CSF, blood or samples derived thereof (for example whole blood, plasma, serum, cell-free serum, cell-free plasma), tissues, cells, saliva, transpired secretion, urine, faeces, stomach fluid, digestive fluid, nasal fluid, cytosolic fluid or other biological tissue or fluid .sample recognised in the art.

The term 'subject' in relation to the present invention includes, but not exclusively, any person or other animal that; in particular, is in need or thought to be in need of being assessed to determine whether said person or other animal is suffering from TBI.

The Applicant has identified that neuronal damage to axons and dendrites are key elements to TBI, and that in some cases damage to one type of fibre may be more apparent than the other. Potentially one type of fibre may be affected independent of the other. This characteristic has been exemplified by screening a biological sample for the presence and/or elevated concentration of an axonal-associated neuronal biomarker (pNF-H) and a dendritic-associated neuronal biomarker (MAP2), compared with respective control threshold concentrations for each biomarker. By screening for a biomarker related to each fibre, the majority of TBI cases can be captured leading to high predictive accuracies of at least 85%, at least 95% and potentially 100% accuracy.

This aspect of the present invention is also advantageous for a number of further reasons:

Screening a biological sample, which has already been obtained from a subject, potentially circumvents the need for expensive techniques such as radiological-based methods (for example CT, MRI) for screening, wherein the nature of such complex or expensive techniques often allows only a minimum number of investigations to be performed, thus gaining only a limited snapshot of the clinical picture. Screening of a biological sample offers a simpler means to determine whether a subject may be suffering from TBI, and monitoring changes in at least two categories of brain biomarker allows the evolution of the TBI to be followed.

The method provides a simple means for determining whether a subject is suffering from TBI. Given the many varied circumstances in which a subject can succumb to TBI, as well as the many potential biomarkers associated with the nervous system, this present invention has shown that a method comprising a simple combination of a minimum of two biomarkers from different cellular locations (i.e. axons and dendrites) is capable of indicating TBI in a biological sample from a subject. Evaluating biomarkers from at least two different locations within the brain allows a more global assessment of TBI to be conducted.

Thus, a higher concentration of either or both of an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker, relative to the respective control threshold concentration for each biomarker, is indicative of TBI.

Consequently, in one embodiment of the first aspect of the present invention there is provided a method for screening a biological sample from a subject to determine whether the subject is suffering from TBI, the method comprising the steps of measuring the concentration of two biomarkers consisting of an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker; and comparing the

concentration of each biomarker with a respective control threshold concentration;

wherein a concentration higher than its respective control threshold concentration is indicative of TBI.

This embodiment of the present invention is advantageous as it offers a further simplified method for screening a biological sample from a subject to determine whether the subject is suffering from TBI. In particular, evaluation of the concentration of only two biomarkers: a single axonal-associated neuronal biomarker and a single dendritic- associated neuronal biomarker, relative to the respective control threshold

concentration for each biomarker.

In an embodiment of the first aspect of the present invention, the axonal-associated neuronal biomarker is phosphorylated Neurofilament-H (pNF-H) and the dendritic- associated neuronal biomarker is Microtubule Associated Protein 2 (MAP2). pNF-H is the largest of three neurofilament subunits that comprise the neuronal cyctoskeleton which assists the structural integrity of the axon. The molecular weight of mammalian pNF-H is approximately 120 kDa. MAP2 is located in dendrites of neurons and is considered to play a role in the assembly of microtubules, via the cross-linking with intermediate filaments, to lead to the production of the cytoskeleton, which in turn provides the scaffolding network that assists intracellular function and cell division. Mammalian MAP2 exists as different isoforms which can be grouped, for example, according to molecular weight: high molecular weight MAP2 includes MAP2A (280 KDA) and MAP2B (270 kDa); and low molecular weight MAP2 includes MAP2C (70 kDA) and MAP2D (75 kDa). Thus, the term 'MAP2' in relation to the present invention refers to at least one MAP2 isoform from MAP2 isoforms that include MAP2A, MAP2B, MAP2C and MAP2D:

This embodiment of the present invention is advantageous as the combination of pNF-H and MAP2 has been shown to be particularly effective at indicating TBI. Thus, a higher concentration of either or both pNF-H and MAP2, relative to the respective control threshold concentration for each biomarker. is indicative of TBI. The Applicant has shown herein that although there can be a lack of correlation between elevated concentrations of pNF-H and MAP2, relative to respective control thresholds, which the Applicant attributes to different cellular locations of the brain being affected, the combination of both biomarkers can capture the majority of cases of TBI with a high predictive accuracy. The instances of non-elevated concentrations of pNF-H or MAP2, relative to their respective control threshold concentration, highlight that the use of individual biomarkejs alone is not capable of producing high predictive accuracies for TBI but the combination is surprisingly effective. This selection of biomarkers is capable of discriminating between TBI and non-TBI in a subject to a high degree of confidence, for example with at least 85% predictive accuracy, and furthermore potentially up to 100% predictive accuracy.

In a further embodiment of the first aspect the biological sample may be blood (or derived thereof) or CSF.

The Applicant has identified that blood and especially CSF are excellent sources of biomarkers that are indicative of brain activity, or damage to the brain. Blood or samples derived thereof include, but not exclusively, whole blood, plasma, serum, cell-free serum or cell-free plasma.

In a further embodiment of the first aspect for a CSF sample, the pNF-H control threshold concentration may be 327.4 ng/ml and the MAP2 control threshold

concentration may be 0.08 pg/ml.

This embodiment of the present invention is advantageous in that it provides a quantitative control threshold for pNF-H and MAP2 in CSF. Thus, a CSF sample with a pNF-H concentration higher than 327.4 ng ml and/or a MAP2 concentration higher than 0.08 pg/ml would indicate TBI.

In a further embodiment of the first aspect for a blood sample or sample derived thereof, the pNF-H control threshold concentration may be 253.6 ng/ml and the MAP2 control threshold concentration may be 318.1 pg/ml.

This embodiment is advantageous in that it provides a quantitative control threshold for pNF-H and MAP2 in blood or samples derived thereof. Thus, a blood sample or sample derived thereof with a pNF-H concentration higher than 253.6 ng/ml and/or a MAP2 concentration higher than 318.1 pg/ml would indicate TBI.

In a second aspect of the present invention there is provided an assay for determining whether a subject is suffering from TBI, comprising recognition elements specific for an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker.

The term 'assay' in relation to the present invention refers to a qualitative or quantitative investigation, in particular for determining the presence or concentration of an axonal- associated neuronal biomarker and a dendritic-associated neuronal biomarker in a biological sample taken from a subject to determine whether the subject is suffering from TBI. Such assays include, but not exclusively, assays well known to those skilled in the art, for example radio-labelled assays, electrochemical assays, surface plasmon resonance (SPR) assays, mass spectrometry assays and immunoassays. The term 'mass spectrometry' assays includes, but not exclusively, immunoaffinity Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) assays and peptide Multiple Reaction Monitoring (MRM) on protein or immune-enriched proteins or samples that have undergone immune-adsorption-based depletion.

The term 'immunoassay' includes, but not exclusively, Western blotting, Enzyme-Linked Immunosorbant Assays (ELISAs), lateral flow devices, flow cytometry and

immunohistochemisry.

The term 'recognition elements', in relation to the present invention refers to an assay component capable of binding or interacting with a specific target analyte. Recognition elements include, but not exclusively, such elements known to those skilled in the art, for example proteins, polypeptides, peptides, antibodies (such as monoclonal or polyclonal antibodies), enzymes, small molecules, deoxyribose nucleic acid (DNA) or ribonucleic acid (RNA) polynucleotides, synthetic polymers, cells or tissue.

The term 'specific target analyte' in relation to the present invention includes, but not exclusively, an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker.

In an embodiment of the second aspect the axonal-associated neuronal biomarker biomarker is pNF-H and the dendritic-associated neuronal biomarker is MAP2.

In another embodiment of the second aspect the recognition elements comprise a reporter element.

The term 'reporter element' in relation to the present invention refers to an assay component that provides a detectable signal representing a binding or interaction event between a recognition element and a specific target analyte. Reporter elements include, but not exclusively, such elements known to those skilled in the art, for example chromogens, fluorophores, contrast agents, bioluminescent molecules, adsorptive molecules, reflective molecules and radioactive isotopes. In one embodiment of the second aspect the recognition elements comprise aptamers.

The term 'aptamer' in relation to the present invention refers to a nucleic acid or peptide ligand capable of binding or interacting with specific target analyte. The term aptamer includes, but not exclusively, such elements known to those skilled in the art, for example DNA, modified DNA, single strand DNA, modified DNA, RNA, modified RNA and peptide aptamers.

In a third aspect of the present invention there is provided a kit for determining whether a subject is suffering from TBI, comprising one or more reagent solutions comprising recognition elements specific for an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker.

The term 'kit' in relation to the present invention refers to a laboratory-ready or fieldable qualitative or quantitative investigation, in particular for determining the presence or concentration of an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker in a biological sample taken from a subject to determine whether the subject is suffering from TBI.

In an embodiment of the third aspect the axonal-associated neuronal biomarker is pNF- H and the dendritic-associated neuronal biomarker is MAP2.

In another embodiment of the third aspect the recognition elements comprise a reporter element.

In one embodiment of the third aspect the recognition elements comprise aptamers.

The present invention will now be described with reference to the following non-limiting examples and drawings in which:

FIG. 1 is a graph showing the results for pNF-H and MAP2 concentrations in CSF from brain injury and control patients, and FIG. 2 is a graph showing the results for pNF-H and MAP2 concentration in blood plasma from brain injury and control patients.

In both figures, all control patient results fall within the box shown in the left hand corner of the graph.

Example 1

Study groups

The study was designed to focus on a comparison of patients with severe brain injury (study group) compared to those with no head injury (control group).

The study group consisted of patients who had suffered a severe head injury requiring admission to an Intensive Care Unit, assessed on the Glasgow Coma Scale (GCS) as being <8 or those who were felt likely to deteriorate to a GCS <8 with evidence of injury on imaging, and were subsequently managed within the first 72 hours of injury on the Neurolntensive Care Unit. Patients had to need to have insertion of an External

Ventricular Drain (EVD) as part of their normal intensive care management, to allow measurement of intra-cranial pressure and to allow drainage of CSF to reduce their intracranial pressure, to be included in the study.

Patients were excluded from the head injury group if there was evidence of other major traumatic injury i.e. long bone fracture, internal haemorrhage. Those not expected to survive twenty four hours were also excluded. The study was open to both male and female participants with a lower age limit of 18 years and an upper age limit of 90 years.

The control group patients were initially those patients admitted for pituitary surgery and having a lumbar drain (LD) inserted as part of their routine care. Patients with pituitary tumours were chosen as their pathology does not involve neurological tissue and therefore was felt to be less likely to provide a false positive result (as opposed to patients with other neurological conditions requiring lumbar drain or lumbar puncture, for example Normal Pressure Hydrocephalus or Idiopathic Intra-cranial Hypertension, where there is a theoretical risk of ongoing neurological injury which may provide a false negative result). The control group was then further broadened to include those patients admitted for repair of CSF leak, requiring LD insertion, who had no recent (defined as within six months) history of head injury or meningitis. The change was necessary as after opening recruitment to the trial, the number of suitable candidates was only one in the first eight months.

In the control group, patients identified as meeting the inclusion criteria were

approached either at clinic (routine or pre-assessment) or on the day before surgery to consent to take part in the trial. In both groups, the patients were only needed to participate for the time taken to take samples, which was no longer than ten minutes.

Patient recruitment

Between February 201 1 and May 2012, a total of 13 patients were recruited. Ten of these patients were TBI patients, whilst three were control patients (one on the initial trial criteria and two from the amended criteria). All ten trauma patients recruited were male patients. The average time from injury to insertion of an EVD was 46 hr 8 min (minimum: 12 hr 30 min; maximum: 102 hr) and the average time to samples being taken from EVD insertion was 32 hr 31 min (minimum 14 hr; maximum 57 hr 30 min). Of the three control patients, the first was a pituitary tumour in a female patient. The other two were CSF repairs in one male and one female patient. The average age was 48 (range of 42 to 55).

Method and Analysis

As of May 2012, the samples from all the control patients and those from the first seven study group patients had been analysed. At the time of EVD or LD insertion, or as soon as convenient afterwards, after the normal samples required for the patient's

management had been collected, a further CSF sample of 5 ml was taken for analysis. At the same stage, or within 24 hours, 5 ml of blood was withdrawn. It was envisaged that in the trauma group, the sample of CSF and blood would be taken from the patient between 2 and 72 hours after injury had occurred.

Each blood sample was 5 ml drawn into a citrated blood vacutainer tube. In control cases only a single sample was taken, whereas for head injury patients up to four samples could be taken. It was therefore envisaged a maximum blood volume taken in a 72 hour period would be 20 ml, which would have no detectable physiological effect on the head injury patient. Control CSF samples were taken from material which would normally be discarded as part of clinical investigations. The volume of CSF was limited to 2 ml per sample in head injury patients, with a maximum volume of 8 ml over 48 hours. This would be taken from expected EVD drainage.

Sample analysis

The samples were immediately centrifuged at 3000 rpm at 4°C for 10 minutes. The cell- free supernatant was aliquoted and frozen. Western blot analysis was performed to investigate the presence of breakdown products of Alpha II Spectrin. Analysis for the presence of pNF-H and MAP2 was undertaken using commercially available assays (pNF-H Sandwich ELISA kit, Merck Millipore; MAP2 ELISA kit, Cusabio). A direct calculation of the CSF and plasma biomarker concentrations was obtained using a standardisation curve. Any remaining sample was destroyed at completion of the trial. pNF-H and MAP2 results for CSF and plasma

Referring now to FIG. 1 , patients with brain injury have elevated concentrations of either pNF-H or MAP2, or both biomarkers, in CSF which is indicative of either axonal damage (pNF-H) and/or dendritic damage (MAP2). Trie negligible levels of pNF-H and MAP2 concentrations for all three control patients fell below a threshold concentration as represented by the control box shown in FIG. 1.

Referring now to FIG. 2, analysis of pNF-H and MAP2 concentrations in blood plasma produced similar findings to that for CSF analysis of pNF-H and MAP2. Patients with brain injury had elevated concentrations of either one or both of the biomarkers, indicative of either axonal damage (pNF-H) and/or dendritic damage (MAP2), whilst the pNF-H and MAP2 concentrations for all three control patients fell below a threshold concentration as represented by the control box shown in FIG. 2.

FIG. 1 and 2 suggest that different cellular locations of the brain can be affected to different extents by TBI, for example axonal damage may result independent of neuronal damage, as is illustrated by the results for expression of pNF-H (elevated by axonal damage) and MAP2 (elevated by dendritic damage). However by utilising both biomarkers to screen a biological sample for TBI, all cases of TBI were identified. All controls had levels below the threshold concentration indicated in the figures.

Control threshold concentrations in CSF and plasma samples were determined by taking the upper 95% Confidence Index of the negative control values.

This study demonstrated that in each of the TBI patients, a measurable increase in at least one of the candidate biomarkers pNF-H and MAP2 is observed. This finding is in contrast with there being no significant increase in either of the biomarkers in the control patients.

Claims

1. A method for screening a biological sample from a subject to determine whether the subject is suffering from traumatic brain injury (TBI), the method comprising the steps of: measuring the concentration of two biomarkers consisting of an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker; and comparing the concentration of each biomarker with a respective control threshold concentration; wherein a concentration higher than its respective control threshold concentration is indicative of TBI.

2. A method according to Claim 1 , wherein the axonal-associated neuronal biomarker is phosphorylated Neurofilament-H (pNF-H) and the dendritic-associated neuronal biomarker is Microtubule Associated Protein 2 (MAP2).

3. A method according to Claims 1 -2, wherein the biological sample is cerebral spinal fluid (CSF).

4. A method according to Claims 1 -2, wherein the biological sample is blood or derived thereof.

5. A method according to Claim 3, wherein the pNF-H control threshold is 327.4 ng/ml and the MAP2 control threshold is 0.08 pg/ml.

6. A method according to Claim 4, wherein the pNF-H control threshold is 253.6 ng/ml and the MAP2 control threshold is 318.1 pg ml.

7. An assay for determining whether a subject is suffering from TBI, comprising recognition elements specific for an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker.

8. An assay according to Claim 7, wherein the axonal-associated neuronal biomarker biomarker is pNF-H and the dendritic-associated neuronal biomarker is MAP2.

9. An assay according to Claims 7-8, wherein the recognition elements comprise a reporter element.

10. An assay according to Claims 7-9, wherein the recognition elements comprise aptamers.

11. A kit for determining whether a subject is suffering from TBI, comprising one or more reagent solutions comprising recognition elements specific for an axonal-associated neuronal biomarker and a dendritic-associated neuronal biomarker.

12. A kit according to Claim 11 , wherein the axonal-associated neuronal biomarker is pNF-H and the dendritic-associated neuronal biomarker is MAP2!

13. A kit according to Claims 11-12, wherein the recognition elements comprise a reporter element.

14. A kit according to Claims 11-13, wherein the recognition elements comprise aptamers.