Markers and their use in traumatic brain injury

Abstract

The invention relates to a new biomarker and combination of biomarkers and to their use in screening of traumatic brain injury in paediatric patients and selecting those patients in need of a brain image technique and / or a subsequent treatment intervention. The method allows to rule out the performance of a brain imaging technique if it is determined that the patient does not have any intracranial lesion which would be detectable by means of said brain imaging technique. Particular kits and means for carrying out the methods are also disclosed.

Description

[0001] Markers and their use in traumatic brain injury

[0002] This application claims the benefit of European Patent Application EP24382531.2 filed on May 17, 2024.

[0003] Technical Field

[0004] The invention relates to biomarkers for the diagnostics and prognosis of traumatic brain injury, in particular, mild traumatic brain injury (mTBI), and methods as well as devices (kits) for the detection of the said biomarkers in an individual.

[0005] Background Art

[0006] Brain injuries have a high incidence worldwide. Among them, mild traumatic brain injury (mTBI) has a significant incidence in the world and is responsible for high health cost. In contrast to severe TBI, mTBI related brain damage is not obvious to detect and thus usually a computer tomography (CT) scan is performed before significant brain injury can be ruled in or out. A computed tomography (CT) scan is a by now a standard of care in cases of suspected TBI.

[0007] A mild traumatic brain injury (mTBI), also called concussion, minor head trauma, and minor brain / head injury, is a type of closed head injury, in which the skull and dura mater remain intact, and it is defined as the result of a blunt trauma or acceleration / deceleration forces causing a brief change in mental status (confusion, disorientation or loss of memory) or loss of consciousness for less than 30 minutes. Usually, loss of consciousness is very brief and ranges between a few seconds to minutes. Mild TBI remains the biggest percentage of all closed head, brain injury cases admitted to the hospitals. Mild TBI is the leading cause of death in children under 4 years old and the most common cause of physical disability and cognitive impairment in young people. Currently, the primary criterion for evaluating patients with TBI in clinical setting is the Glasgow Coma Scale (GCS), which assesses the level of consciousness following TBI. A mild traumatic brain injury is most likely to be diagnosed only when there is a change in the mental status at the time of injury or hospital admission (the person is dazed, confused, or loses consciousness, GCS score 13-15). In the USA and Europe around 80-90% of the head injury patients are classified at admission as having a mild TBI.

[0008] Prompt detection and management of ICI are critical, as complications can evolve quickly and can lead to disability or injury-related death. Around 10% of patients with mTBI can suffer long-term disabilities such as headache, fatigue, difficulty thinking, memory problems, attention deficits, mood swings, sleep disorders, frustration, and even epileptic events. In these circumstances, the first objective for clinicians is to detect all patients in need of neurosurgical intervention after TBI but, due to the complicated ethology, it remains challenging to identify which patients with mTBI can be safely sent home without the need for treatment intervention. Currently, intracranial injuries, such as mTBI, may be further diagnosed with tools such as computerized tomography (CT) scans and magnetic resonance imaging (where available). However, in the group of patients with mTBI only around 10% present with an abnormal CT result revealing an acute intracranial lesion in patients. The remaining scans show normal head CT, indicating no complications from injury, exposing patients to unnecessary irradiations and as such are not cost effective and are timeconsuming for both patient and medical staff.

[0009] Moreover, children are particularly at risk of cancer secondary to irradiation and care should be taken to avoid unnecessary exposure to ionizing radiation. Clinicians can follow PECARN (Pediatric Emergency Care Applied Research Network) recommendations to identify children with very low risk of clinically important traumatic brain injury (ciTBI) following head trauma, who would not require imaging. In Europe and the United States, CT are performed in only 10 to 35% of mTBI cases. Most of the children are therefore kept under observation in the emergency department (ED) or hospitalized to monitor their symptoms for 6-24 hours. This observation time is, however, stressful for children and parents and cost consuming for the health care system. The clinical management of pediatric mTBI patients therefore differs compared to the adults.

[0010] The use of biomarkers has been proposed as a means to reduce the amount of unnecessary CT scans and for use in decentralized sites where access to CT equipment is absent. The finding of reliable biomarkers working in the paediatric population is, moreover, of special interest when the target population is the smaller children. Small children (i.e., 0 to 4-5 years old) cannot properly express their pain or health perception state, which greatly complicates management of the patient in the absence of a CT scan.

[0011] Although lately some studies have unravelled biomarkers which can be used for diagnosing mTBI in adults, these findings cannot be directly extrapolated to the paediatric population. It is known that pediatric brain responses after injury differ from adults due to developmental reasons. The stage of brain development at the time of the injury may result not only in a variable age-dependent response but may also have implications for plasticity and repair.

[0012] S100b currently stands as the most extensively studied biomarker. However, the false positive rate is still too high and precludes its use in clinical practice. Clinical studies in pediatric mTBI biomarkers must not only attempt to identify CT-negative patients; it is of outmost importance to identify all patients that need to be kept in observation or intervened, which means that a very high sensitivity is required for use in clinical practice. Besides, Bouvier et al showed that S100b was inversely correlated with age in children and identified at least three age categories. These findings emphasize the difficulties in the future use of S100b and its defined thresholds in routine clinical practice regarding patient's age.

[0013] One of the very few studies related to mTBI biomarkers directed to the pediatric population investigated whether beta-natriuretic peptide in serum of paediatric patients could be used to identify intracranial haemorrhage in case of closed head trauma (Chang and Nager "Pediatric Traumatic Brain Injury: The Utility of Beta-Natriuretic Peptide”, J Trauma- 2010, vol.no. 68, pp.: 1401-1405). However, the authors of this research concluded that BNP levels drawn at the time of the emergency department could not predict intracranial haemorrhage in paediatric trauma patients, such that a head CT was declared to be required for diagnosing intracranial haemorrhage.

[0014] It is clear that an improved management of patients suffering from mTBI is thus needed in ED, specially when the patients are children.

[0015] Summary of Invention

[0016] The inventors have surprisingly found that certain proteins or peptides in isolated biofluid samples are differentially detectable in paediatric patients (subjects) that have suffered TBI and that have intracranial lesions, even though said lesions are not physically or apparently evident at first sight. The inventors have also found that the biomarkers's performance for diagnosing TBI is clinically relevant.

[0017] Moreover, the inventors have detected that the performance of TBI paediatric patients significantly differs from adults. Thus, the present invention is of special interest, since it provides a diagnostic method for TBI customised for the paediatric population.

[0018] The invention thus provides as a first aspect an in vitro method for diagnosing intracranial lesion in a paediatric subject, comprising the steps of:

[0019] (I) determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of interleukin-6 (IL6), neurofilament light protein (NFL), heart fatty acid binding protein (HFABP), brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), S100 calcium binding protein B (S100b), and a fragment of any of the foregoing,

[0020] (ii) comparing the level of the biomarker(s) determined in (I) with a corresponding reference value, and (ill) diagnosing the paediatric patient as having intracranial lesion when the level of the biomarker(s) determined in (I) is(are) higher than the corresponding reference value, or is(are) within a corresponding reference range level of a patient with intracranial lesion detectable by said brain image technique.

[0021] These markers can significantly help in the management of patients in the emergency department, avoiding unnecessary CT scan and helping to reduce length of hospital stay for children and their families.

[0022] As shown in the examples below, the above method provided relevant information for ruling out a CT in mild TBI paediatric patients with a high sensitivity (i.e., even of 100 %) accompanied with a clinically relevant specificity. Specifically, in a paediatric mild TBI cohort, GFAP, HFABP and S100b respectively reached 52%, 41% and 39% specificity at 100% of sensitivity, to rule-out the need of unnecessary CT scans and shorten the length of stay in observation for patients without CT scan. Moreover, their use in combination can even yield more than 60% of specificity. IL6 also achieved 100% sensitivity and 48% specificity in identifying CT- and in- hospital-observation paediatric patients without CT, while excluding all CT+ cases, when sampled within twenty-four hours after the trauma (and 50% specificity when sampled within six hours). Standard analytical technics for the measurement of IL6 in blood samples are already available in routine hospital laboratory medicine, which is an important step, toward its use in clinical diagnosis for mTBI patients. Further, NFL and NTproBNP respectively reached 31% and 26% specificity at 100% sensitivity in identifying CT- and in- hospital-observation paediatric patients without CT, while excluding all CT+ cases.

[0023] This first aspect of the invention can also be drafted as an in vitro method for the classification of a paediatric patient as having a brain damage that is detectable by a brain imaging technique, the method comprising the steps of:

[0024] (I) determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing,

[0025] (ii) comparing the level of the biomarker(s) determined in (I) with a corresponding reference value, and (ill) classifying the paediatric patient as having a brain damage that is detectable by a brain imaging technique when the level of the biomarker(s) determined in (I) is(are) higher than the corresponding reference value, or is(are) within a corresponding reference range level of a patient having brain damage that is detectable by a brain imaging technique.

[0026] All this means that, by practicing the invention disclosed herein, a high number of CT scans (or of any other brain image technique), as well as lengthy hospital stays, can be safely dispensed with, since all the true positive patients (i.e., 100 % of the paediatric patients with intracranial lesion) are detected. Hence, the ruling out of a false negative patient is avoided (paediatric patient with actual intracranial lesion but classified as negative in this kind of lesion according to the method). Additionally, the number of paediatric patients correctly classified as having no intracranial lesion is also increased. Thus, one of the advantages of the method of the invention is the possibility of providing high specificities and, importantly, high sensitivities in a decision to rule out, or to rule in, a CT scan imaging in a paediatric patient to determine the damage in the brain. Once any long-term impairment can be predicted, the patient can be monitored and properly treated to avoid or to mitigate such probable impairments.

[0027] Preferably, the paediatric patient suffering from brain damage is a paediatric patient with a TBI, more in particular mTBI, which means that in the subject has a GCS score of 13-15 (i.e., dazing, confusion, or lost if consciousness, GCS score 13-15).

[0028] Indeed, the first aspect also refers to an in vitro method for selecting a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention, comprising the steps of:

[0029] (I) determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of interleukin-6 (IL6), neurofilament light protein (NFL), heart fatty acid binding protein (HFABP), brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), S100 calcium binding protein B (S100b), and a fragment of any of the foregoing,

[0030] (ii) comparing the level of the biomarker(s) determined in (I) with a corresponding reference value, and (iii) selecting the paediatric patient for a brain image technique and / or for a subsequent treatment intervention when the level of the biomarker(s) determined in (I) is(are) higher than the corresponding reference value, or is(are) within a corresponding reference range level of a patient with intracranial lesion detectable by said brain image technique.

[0031] Related with this capability of correctly predicting an output of a brain imaging technique in paediatric patients with a TBI, a second aspect of the invention refers to an in vitro method for differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique, from a traumatic brain injury paediatric patient with no intracranial lesion, the method comprising the steps of: (I) determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing,

[0032] (ii) comparing the level of the biomarker(s) determined in (I) with a corresponding reference value, and

[0033] (iii) diagnosing traumatic brain injury paediatric patient with intracranial lesion when the level of the biomarker(s) determined in (I) is(are) higher than the corresponding reference value, or is(are) within a corresponding reference range level of a patient with intracranial lesion; or diagnosing traumatic brain injury paediatric patient with no intracranial lesion when the level of the biomarker(s) determined in (I) is(are) equal or lower than the corresponding reference value, or is(are) within a reference range level of a patient with no intracranial lesion.

[0034] The methods of the first and second aspect can be both regarded as methods for screening for TBI.

[0035] The inventors have further found that the proposed biomarkers correlate with the probability of long-term complications in paediatric patients that have experienced TBI. Consequently, the disclosed biomarkers have a prognostic value, such that they are useful to determine the risk of suffering from cognitive and / or emotional and / or behaviour and / or physical impairments in a paediatric subject having experienced TBI.

[0036] A third aspect thus provides an in vitro method for the prognosis of a paediatric patient that has experienced TBI or for determining the risk of suffering from cognitive and / or emotional and / or behaviour and / or physical impairments in a paediatric subject having experienced TBI, comprising the steps of:

[0037] (I) determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing,

[0038] (ii) comparing the level of the biomarker(s) determined in (I) with a corresponding reference value, and

[0039] (iii) determining a bad prognosis or high risk of suffering from cognitive and / or behaviour and / or emotional and / or physical impairments when the level of the biomarker(s) determined in (I) is(are) higher or lower than the corresponding reference value, or is(are) within a reference range level of a patient with intracranial lesion detectable by a brain image technique. Indeed, according to the best of the inventor's knowledge, it is herewith disclosed for the first time an in vitro method for the selection of a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention (i . e. , a method for the correct classification of a paediatric patient as having a brain damage that is detectable by a brain imaging technique), the method comprising the step of determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing.

[0040] A fourth aspect of the invention is the use of a peptide or protein or a combination of peptides or proteins as in vitro biomarker(s) selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing for:

[0041] (a) selecting a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention; or (b) for diagnosing intracranial lesion in case of TBI a in a paediatric subject; or (c) for differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion the diagnosis of intracranial lesion in case of a TBI in a paediatric subject; or (d) for screening for TBI; or (e) for determining the risk of suffering from cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI.

[0042] Inventors have also developed kits and devices for carrying out the method. Thus, a fifth aspect of the invention is a kit or a device comprising means (i.e., reagent means) for determining the level of one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing in a biofluid sample. A sixth aspect is related to the use of said kits or devices for carrying out any of the methods according to the first to third aspects.

[0043] Any analytical method or device available in the art may be used to determine the level of one or more of the above-mentioned biomarkers. In any case, once the level of the marker in the sample is determined, the obtained value can be processed (i.e., computed in a mathematical formula) to give a reliable result.

[0044] Thus, a seventh aspect of the invention provides a computer-implemented method for carrying out any of the methods as defined above in the first to third aspects, in which after the determination of the level of one or more of the biomarkers mentioned above, said level (s) is(are) given a value and / or a score and, optionally, are computed in a mathematical formula to obtain a computed value; wherein, depending of the said level(s), score(s) and or computed value(s), a decision is taken for selecting a paediatric patient suffering TBI (in particular mTBI) for a brain image technique and / or for a subsequent treatment intervention, or a decision is taken about diagnosing a TBI paediatric patient with an intracranial lesion that is detectable by a brain imaging technique or a TBI paediatric patient with no intracranial lesion, or a risk of suffering from cognitive and / or behaviour and / or physical impairments in a paediatric subject having experienced TBI is is determined. The above decisions are taken following the instructions as disclosed in step (iii) of the first to third aspects. In summary, present invention provides methods, kits and assays for screening for TBI and allowing the correct classification of paediatric patients which come to a hospital or ambulatory room with a description of head trauma, and to which the probability of suffering of a non-apparently detectable brain lesion is to be established to proceed further with the best clinical decision.

[0045] Thus, also herewith disclosed, or as an alternative wording of the first and second aspects, are in vitro methods for ruling out of a diagnosis of brain injury by means of (i.e., according to the output of) a brain imaging technique in a paediatric patient TBI, in particular with a suspicion of mTBI, the methods comprising:

[0046] - determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing, comparing the level of the biomarkers(s) with a reference value or reference range of values; and

[0047] - classifying (i.e., selecting) the paediatric patient for a brain image technique and / or for a subsequent treatment intervention when the level of the biomarker(s) determined above is(are) higher than the corresponding reference value, or is(are) within a reference range level of a patient with intracranial lesion detectable by said brain image technique; or ruling out a diagnosis of brain injury (i.e., intracranial lesion) by means of a brain imaging technique if the level of the biomarker(s) determined above is(are) lower than the corresponding reference value, or is(are) within a reference range level of a patient with no intracranial lesion detectable by said brain image technique.

[0048] Also disclosed are, thus, in vitro methods for the diagnosis and prognosis of an intracranial lesion in a paediatric patient suffering of a TBI (in particular mTBI), the methods comprising:

[0049] - determining in an isolated biofluid sample of the paediatric patient the the level of one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing, comparing the level of the biomarkers(s) with a reference value or reference range of values; and

[0050] - classifying (i.e., selecting) the paediatric patient for a brain image technique and / or for a subsequent treatment intervention when the level of the biomarker(s) determined above is(are) higher than the corresponding reference value, or is(are) within a reference range level of a patient with intracranial lesion detectable by said brain image technique; or ruling out a diagnosis of brain injury by means of a brain imaging technique if the level of the biomarker(s) determined above is(are) lower than the corresponding reference value, or is(are) within a reference range level of a patient with no intracranial lesion detectable by said brain image technique.

[0051] Detailed description of the invention All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0052] As used herein, the indefinite articles "a” and "an” are synonymous with "at least one” or "one or more.” Unless indicated otherwise, definite articles used herein, such as "the,” also include the plural of the noun.

[0053] "Brain injury" is any state of a patient or individual which is the cause of sudden impact on the head or the individual. A particular brain injury is TBI or mTBI.

[0054] "TBI" in the sense of the invention is any brain injury caused by a traumatic incident as described above with reference to the prior art. A traumatic brain injury (TBI), also known as an intracranial injury, is an injury to the brain caused by an external force. TBI can be classified based on severity (ranging from mild traumatic brain injury [mTBI / concussion] to severe traumatic brain injury), mechanism (closed or penetrating head injury), or other features (e.g., occurring in a specific location or over a widespread area).

[0055] The term "intracranial lesion” in the context of TBI includes any damage (i.e. degradation from initial healthy state) to the brain tissue due to the traumatic incident. In the particular context of mTBI (most of closed-head injuries) the lesions or injury types that can occur include concussion (temporary dysfunction of normal brain function), intracranial hematomas (i.e., rupture of blood vessel causing pool of blood around the brain, called subdural hematoma, or between the brain and the skull, called epidural hematoma), and cerebral contusions (bruise to the brain tissue as a result of trauma). In the context of mTBI, the concept "intracranial lesion” according to this description refers to an injury of the intracranial tissue with consequences at short or longterm, and which are identified and classified by carrying out a brain image technique (CT, MRI), thus they are lesions identified as a "pathological or positive output” of a brain imaging technique (e.g., pathologycal or positive CT scan). This means that even in a mTBI case, there is often a certain intracraneal lesion or injury of the tissue, but this lesion can easily heal on its own with no long-term complications. On the contrary, the intracraneal lesion can be a health-jeopardizing lesion. Such jealth-jeopardizing intracraneal lesions may often go undetected by the traditional tests, such as the Glasgow Coma Scale and symptoms, used at first instance to classify a patient with a brain trauma.

[0056] In the context of mTBI these possible intracranial brain injuries are not apparent in most of the cases until CT or MRI is performed. Their detection is of vital importance to counter-act possible post-traumatic complications and secondary brain damages in mTBI patients. Said damages include long-term disabilities such as headache, fatigue, difficulty thinking, memory problems, attention deficits, mood swings, sleep disorders, frustration and even epileptic events.

[0057] "Identification" or "identify" or "classify" in the sense of the invention is the analysis of a sample of an individual to assess whether the individual which suffered from a trauma on the head (i.e. , particularly in TBI subjects or mTBI subjects), has an intracranial lesion. This identification of lesions in brain can be verified by use of a CT scan or MRI analysis.

[0058] Indeed, the expression "intracranial lesion detectable by a brain image technique” refers to the fact that the output of the imaging technique is such that a rad lol og i st / pathol og i st would identify or classify it as a healthjeopardizing lesion which needs to be monitored and / or which needs a surgical or pharmacological intervention.

[0059] The term "selecting a paediatric TBI patient or subject for an image technique and / or subsequent treatment", as used herein, relates to the identification of a patient that after the analysis of the biomarker levels in the isolated sample will be further submitted to imaging of the brain to determine the intracranial lesion type and degree. It also relates to the identification of a patient, that as a result of the test sample is determined as having intracranial lesion and requires of the application of therapeutic interventions to avoid complications.

[0060] In this description the term "paediatric patient, subject or population” is used to define a group of people whose age is from birth to the age of majority, which is dependent on national law. In general terms it includes the population being commonly treated by paediatric medicine when required in each country. In a particular embodiment of the invention the paediatric population is that from 0 to 18 years old, more in particular from 0 to 16 years old.

[0061] A biomarker "panel" in the sense of the invention is a combination of at least two biomarkers (the levels of two proteins in the sample), in particular two or three or four markers, optionally used in combination in a suitable setup or device.

[0062] A "sample" or "specimen" in the sense of the invention is any biofluid useful for performing an assay or detection method to identify lesions in a TBI paediatric patient, preferably in an mTBI patient. Preferably the sample is a blood, plasma, serum, saliva, tears, cerebrospinal fluid or urine sample taken from an individual. More preferably the biofluid sample is blood, serum or plasma. If required, the sample may be treated according to generally known procedures in order to determine the level of the biomarkers.

[0063] A method for determining the prognosis in the sense of the invention is or encompasses a method useful in providing information on the risk of the patient suffering from a future negative development after experiencing an impact or a shock or a traumatic incident, e.g. a brain injury, in particular, after a defined time span; said time span may be 1 , 2, 3, 4, 5, or 6 months, or 1 or 2 years. That is, a bad prognosis comprises an elevated risk of the patient suffering from a future negative development after experiencing the impact or a shock or traumatic incident. Siad negative development comprises suffering from cognitive and / or behaviour and / or emotional and / or physical impairments. The term "level of expression of one or more proteins” or "level of one or more proteins in a sample” relates to the amount of the protein expressed as a concentration, usually the weight of the protein in a volume of sample.

[0064] As will be apparent from the experimental part describing the invention, the invention has the advantage that it achieves very reliable test results. Accordingly, in preferred embodiments it provides for methods, kits or devices wherein the sensitivity is more than 90%, in particular more than 95%, preferably 98%, even more preferably 99%, and even more preferably 100%, and the specificity is more than 30%, in particular more than 33 %, more particularly more than 40%, preferably more than 50%, and even more preferably more than 60%. These embodiments allow for safely ruling out an imaging method and / or in-hospital monitoring. In other embodiments, with the aim to rule in all the positive CT scan (i.e., to locate all paediatric patients with healthjeopardizing intracranial lesion determined by imaging techniques), a fixed specificity of more than 95 %, preferably 100% is applied to the method, preferably correlating with sensitivities of more than 60 %, preferably 70 %.

[0065] The term "reference” or "reference value" or "cut off' or "threshold”, as used herein, relates to a predetermined criterion used as a reference for evaluating the values or data obtained from the samples collected from a subject. The reference value or reference level can be an absolute value; a relative value; a value that has an upper or a lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value, such as for example, a value obtained from a sample from the subject being tested, but at an earlier point in time. The reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group or based on a pool of samples including or excluding the sample to be tested. The skilled person in the art, making use of the general knowledge, can choose the subject or group of subjects more adequate for obtaining the reference value for each of the methods of the present invention. Methods for obtaining the reference value from the group of subjects selected are well- known in the state of the art (Burtis C. A. et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values”) In a particular case the "reference value” is a cut-off value or threshold defined, for example, by means of a conventional ROC analysis (Receiver Operating Characteristic analysis). Reference values have been determined for the biomarkers of the invention. The reference value for each of the proteins in this description may be from a lower to an upper value. Range of values of each biomarker (protein levels) and particular combinations of the values of the different biomarkers provide for correct classification of subjects with high sensitivity and specificity.

[0066] In one embodiment the reference value may be the level of the biomarker in a paediatric patient or population of paediatric patients which, even though having suffered a head trauma, have no intracranial lesion or have no health-threatening brain damage. In one example, if the level of the biomarker in the test sample is higher than this reference value, this means that the patient has an intracranial lesion which can be healththreatening and has to be selected for further brain imaging techniques and / or therapeutic approaches to face said intracranial lesion.

[0067] In another embodiment, the reference value may be a range of values for the level of the biomarker in a paediatric patient or population of paediatric patients with intracranial lesion, preferably an intracranial lesion which is detectable or has been detected with a brain imaging technique, such as CT or MRI. In one corresponding example, the tested paediatric patient is diagnosed of intracranial damage and selected for additional brain imaging techniques and / or therapeutic approaches if the level of the biomarker determined for the patient in the isolated sample is within these reference range of values.

[0068] In the sense of the present invention, the level of a biomarker is considered to be higher than its corresponding reference value when it is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% higher than the reference value.

[0069] Likewise, in the context of the present invention, the level of a biomarker is considered to be lower than its corresponding reference value when it is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% lower than the reference value.

[0070] The methods of the invention comprise determining one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, and a fragment of any of the foregoing. In some embodiments, the methods of the invention comprise determining the level of at least one biomarker selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, and a fragment of any of the foregoing. In some embodiments, the methods further comprise determining the level of a biomarker selected from the group consisting of S100b, IL8, IL10, DDi, a fragment of any of the foregoing, and combinations thereof.

[0071] Accuracy of the methods of the invention is generally improved or tuned when determining the level of more than one biomarker. In one embodiment, the methods of the invention comprise determining the level of more than one biomarker selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, and a fragment of any of the foregoing. In another embodiment, the methods of the invention comprise determining the level of more than one biomarker selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, S100b, and a fragment of any of the foregoing. In another embodiment, the methods of the invention comprise determining the level of more than one biomarker selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, IL8, IL10, S100b, DDi, and a fragment of any of the foregoing. In some embodiment, the methods of the invention comprise determining the level of at least two biomarkers selected from the group consisting of IL6 and NFL, S100b and IL6, FABP and BNP, GFAP and IL6, IL6 and FABP, IL6 and DDimer, IL6 and BNP, NFL and FABP, NFL and DDimer, NFL and BNP, S100b and NFL, GFAP and NFL, S100b and FABP, S100b and DDimer, S100b and BNP, GFAP and FABP, GFAP and DDimer, GFAP and BNP, FABP and DDimer, and DDimer and BNP. In particular embodiments, the methods of the invention comprise determining the level of at least three biomarkers selected from the group consisting of IL6, NFL, and S100b; IL6, NFL, and GFAP; IL6, S100b, and GFAP; NFL, S100b, and GFAP; S100b, GFAP, and FABP; NFL, IL6, and BNP; FABP, BNP, and DDimer. Particularly preferred biomarker panels are those comprising IL6 and / or NFL and / or S100b and / or GFAP, e.g. a panel selected from the group consisting of IL6, NFL, and S100b; IL6, NFL, and GFAP; IL6, S100b, and GFAP; NFL, S100b, and GFAP. Also preferred biomarker panels are those comprising IL6, NFL, GFAP, D-Dimer, and / or BNP, e.g. a panel selected from the group consisting of IL6, NFL, and GFAP; GFAP, NFL, and D-Dimer; and D-Dimer, GFAP, and NT-proBNP. In a more particular embodiment, the methods of the invention comprise determining the level of IL6, NFL, GFAP, and a further biomarker selected from DDimer and BNP. In another particular embodiment the biomarker panel comprises IL6, NFL, GFAP, and S100b.

[0072] In preferred embodiments the biomarker proteins to be determined in the methods of the invention are the human proteins. All of them can be determined by methods well known in the art.

[0073] BNP is a hormone secreted by cardiomyocytes in the heart ventricles in response to stretching caused by increased ventricular blood volume. The complete human sequence BNP has the UniProt KB accession number P16860 (version 1 of the sequence). In a particular embodiment of the methods, the fragment of BNP known as N-terminal prohormone of brain natriuretic peptide (NT-proBNP or BNPT) is determined in the isolated sample. The N-terminal fragment of B-type natriuretic peptide (NT-proBNP) is the 76-amino acid N- terminal fragment of the B-type natriuretic peptide prohormone, i.e the fragment consisting of aminoacids in position 27 to 102 of P16860, which has been given UniProt accesion number: PRG_0000451939. Cleaving of pro-BNP yields the NT-proBNP fragment and the active B-type natriuretic peptide (BNP).

[0074] FABP, is fatty acid-binding protein in heart. In a particular embodiment of the methods, the FABP determined in the isolated sample is human FABP (HFABP). The complete sequence for human HFABP has the UniProtKB accession number P05413 (version 4 of the sequence).

[0075] GFAP refers to glial fibrillary acidic protein, an intermediate filament protein that is expressed by numerous cell types of the central nervous system. The complete human sequence for glial fibrillary acidic protein has, in particular, the UniProtKB accession number P14136 (version 1 of the sequence).

[0076] IL6 is interleukine-6. The complete human sequence for IL6 has, in particular, the UniProtKB accession number P05231 (version 1 of the sequence). IL8 is interleukine-8. The complete human sequence for IL8 has, in particular, the UniProtKB accession number P10145 (version 1 of the sequence).

[0077] IL10 is interleukine-10. The complete human sequence for IL10 has, in particular, the UniProtKB accession number P22301 (version 1 of the sequence).

[0078] S100B is S100 calcium binding protein B. The complete human sequence for S100B has, in particular, the UniProtKB accession number P04271 (version 2 of the sequence).

[0079] DDi (d-dimer) is a fibrin degradation product, a small protein fragment present in the blood after a blood clot is degraded by fibrinolysis. It is so named because it contains two D fragments of the fibrin protein joined by a cross-link.

[0080] In embodiments of any of the methods of the first or second aspects, the threshold or cut-off values for the biomarkers when used individually, in particular, in serum, are determined for 95 to 100% sensitivity, in particular for 98%, 99%, or 100% sensitivity.

[0081] In particular embodiments the thresholds or cut-off values for the biomarkers when used individually are around those shown in table 3. In other embodiments, when using a panel of two biomarkers, the thresholds or cut-off values are around those shown in table 4. In other embodiments. The term "around” refers to a value that is ±20%, in particular ±10%, more in particular ±5%, of the given value. The thereshold / cut-off values may vary for each biomarker when used in a panel.

[0082] In particular embodiments of the invention, the brain image technique is computed tomography (CT) scan, and / or magnetic resonance imaging (MRI).

[0083] Also, in particular embodiment of the methods of the invention, the treatment intervention is selected from surgery, in particular for relieving pressure in the skull, and / or for removing clotted blood and / or for repairing skull fractures; medication with neuroprotectants, and / or with anticoagulants and / or with anticonvulsants; and / or physical and / or psycological rehabilitation. Thus, in particular embodiments, the brain image technique is computed tomography (CT) and / or magnetic resonance imaging (MRI); and the treatment intervention is selected from surgery for relieving pressure in the skull, and / or surgery for removing clotted blood and / or surgery for repairing skull fractures, medication with neuroprotectants, and / or medication with anticoagulants and / or medication with anticonvulsants, and / or physical and / or psycological rehabilitation.

[0084] In some embodiments of any of the methods of the inventions, the methods further comprise treating the paediatric patient that has been classified as having intracraneal lesions, or that has been selected for intervention, or thas has been classified as having a high risk of suffering cognitive and / or behaviour and / or emotional and / or physical impairments, to a brain imaging technique, or to an appropriate therapy. In a particular embodiment, such therapy includes surgery for relieving pressure in the skull, and / or surgery for removing clotted blood and / or surgery for repairing skull fractures, medication with neuroprotectants, and / or medication with anticoagulants and / or medication with anticonvulsants, and / or physical and / or psycological rehabilitation.

[0085] This particular embodiment could be drafted as a method of treating a patient suffering TBI, in particular mild TBI, said method comprising carrying out the in vitro method of any of the first to third aspects, and treating the patient with surgery for relieving pressure in the skull, and / or surgery for removing clotted blood and / or surgery for repairing skull fractures, medication with neuroprotectants, and / or medication with anticoagulants and / or medication with anticonvulsants, and / or physical and / or psycological rehabilitation.

[0086] In another embodiment of the in vitro methods of the invention, the biofluid sample is selected from the group consisting of blood, serum, plasma, urine, saliva, lachrymal fluid, cerebrospinal fluid, and combinations thereof. More in particular, the biofluid sample is selected from blood, serum or plasma. Even more in particular it is serum.

[0087] In particular embodiments of the invention, the biofluid sample is obtained from the paediatric patient within 24 post-trauma, more in particular within 12 h, even more in particular within 10 h, even more in particular within 8 h post-trauma. In preferred embodiments of the invention, the biofluid sample is obtained from the paediatric patient within 6 post-trauma.

[0088] In another embodiment of the methods, the traumatic brain injury is mild traumatic brain injury (mTBI). Thus, in particular the patients are paediatric patients with a GCS around 13-15, and the methods of the invention allow the correct sub-stratification in order to see if they have heath-jeopardizing non-apparent lesions, or if they can be safely discharged.

[0089] In another particular embodiment of any of the in vitro methods of the invention, the method further comprises determining one or more clinical parameters, and / or combining the levels of the one or more proteins determined with sex and / or age of the patient. In a more particular embodiment, the above-referred clinical parameters are selected from the group consisting of blood pressure, including systolic blood pressure and / or diastolic blood pressure, scores from systematic assessment tools of TBI (Glasgow Coma Score), and combinations thereof.

[0090] The levels of biomarkers can be determined by measuring the levels of mRNA encoded by the corresponding genes or by measuring the levels of proteins encoded by said genes, and the levels of variants thereof.

[0091] By way of a non-limiting illustration, the expression levels are determined by means of the quantification of the levels of mRNA encoded by said genes. The latter can be quantified by means of using conventional methods, for example, methods comprising the amplification of mRNA and the quantification of the amplification product of said mRNA, such as electrophoresis and staining, or alternatively, by means of Northern blot and the use of suitable probes, Northern blot and use of specific probes of the mRNA of the genes of interest or of their corresponding cDNA / cRNA, mapping with the SI nuclease, RT-PCR, hybridization, microarrays, etc. Similarly, the levels of the cDNA / cRNA corresponding to said mRNA encoded by the marker genes can also be quantified by means of using conventional techniques; in this event, the method of the invention includes a step of synthesis of the corresponding cDNA by means of reverse transcription (RT) of the corresponding mRNA followed by the synthesis (RNA polymerase) and amplification of the cRNA complementary to said cDNA. Conventional methods of quantifying the expression levels can be found in laboratory manuals. In some embodiments, the expression levels, e.g. the mRNA levels, may be determined using fluorescent probes.

[0092] In order to normalize the values of mRNA expression among the different samples, it is possible to compare the expression levels of the mRNA of interest in the test samples with the expression of a control RNA. A "control RNA" as used herein, relates to RNA whose expression levels do not change or change only in limited amounts. Preferably, the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Preferred housekeeping genes for use in the present invention include 18-S ribosomal protein, p-2-microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubulin and p-actin.

[0093] Alternatively, it is also possible to determine the expression levels of the marker genes by determining the levels of the proteins encoded by said genes. Thus, in another particular embodiment of the in vitro methods of the invention, the level of the proteins is determined at the protein level. The protein levels are preferably expressed as concentration in pg / ml, but any other concentration units may be used.

[0094] In particular embodiments, determining the protein level is carried out by an assay or technology selected from the group consisting of an immunoassay, a bioluminescence assay, a fluorescence assay, a chemiluminescence assay, electrochemistry assay, mass spectrometry, and combinations thereof. These assays provide qualitative and / or quantitative information of the proteins in the isolated sample.

[0095] Particular tests that can be implemented in a point of care test format (POCT) are recommended to make easy and fast the determining of protein levels. In a particular embodiment, point of care tests include lateral flow tests, which allow detecting the presence (or absence) of a target analyte in liquid sample (matrix) without the need for specialized and costly equipment, though many lab-based applications exist that are supported by reading equipment.

[0096] In one embodiment, the level of the protein is determined by immunological techniques such as enzyme- linked immunosorbent assay (ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen- antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunocromatography, or other immunoassay formats well-known to the ordinarily skilled artisan, such as radioimmunoassay, as well as protein microarray formats, such as single molecular assay (SIMOA), Western Blot or immunofluorescence.

[0097] Western blot is based on the detection of proteins previously resolved by gel electrophoreses under denaturing conditions and immobilized on a membrane, generally nitrocellulose by the incubation with an antibody specific and a developing system (e.g., chemoluminiscent). The analysis by immunofluorescence requires the use of an antibody specific for the target protein for the analysis of the expression. ELISA is based on the use of antigens or antibodies labelled with enzymes so that the conjugates formed between the target antigen and the labelled antibody results in the formation of enzymatically-active complexes. Since one of the components (the antigen or the labelled antibody) are immobilised on a support, the antibody-antigen complexes are immobilised on the support and thus, it can be detected by the addition of a substrate which is converted by the enzyme to a product which is detectable by, e.g. spectrophotometry, fluorometry, mass spectrometry or tandem mass tags (TMT). SIMOA is a type of assay more sensitive than an ELISA, since it uses arrays of femtoliter-sized reaction chambers, which are termed single-molecule arrays (SimoaTM) that can isolate and detect single enzyme molecules. Because the array volumes are approximately 2 billion times smaller than a conventional ELISA, a rapid build-up of fluorescent product is generated if a labelled protein is present. With diffusion defeated, this high local concentration of product can be readily observed. Only a single molecule is needed to reach the detection limit. Using the same reagents as a conventional ELISA, this method has been used to measure proteins in a variety of different matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.) at femtomolar (fg / mL) concentrations, offering a roughly 1000-fold improvement in sensitivity.

[0098] On the other hand, the determination of the protein expression levels can be carried out by constructing a tissue microarray (TMA) containing the subject samples assembled, and determining the expression levels of the proteins by techniques well known in the state of the art.

[0099] In a preferred embodiment the determination of the levels of the proteins are determined by immunological technique. In a more preferred embodiment, the immunological technique is ELISA.

[0100] When an immunological method is used, any antibody or binding molecule known to bind with high affinity to the target proteins can be used for detecting the amount of target proteins. It is preferred nevertheless the use of antibody, for example polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanised antibodies.

[0101] Thus, in another particular embodiment of the related methods of the invention, the level of the proteins is determined using an antibody or a fragment thereof, both able to bind to the said protein(s).

[0102] In a particular embodiment, the means disclosed above for determining the level of the biomarkers at the mRNA or protein level form part of a kit or a device. Furthermore, devices comprising a biomarker or panel of biomarkers as disclosed above can be used to carry out the methods of the invention. Examples of devices are carrier plates, test stripes, biochip arrays or the like known in the art.

[0103] Thus, herewith disclosed is also the use of a kit, test, assay or device for carrying out the in vitro methods of the invention, said kit, test, assay or device comprising means for determining the level of at least one of the biomarkers disclosed above. All the embodiments described above for the biomarkers, their combinations, and thresholds, also apply to the kits, tests, assays or devices of the invention. For example, in one embodiment the kit or device comprises means for determining the level of two biomarkers selected from the group consisting of IL6 and NFL, S100b and IL6, FABP and BNP, GFAP and IL6, IL6 and FABP, IL6 and DDimer, IL6 and BNP, NFL and FABP, NFL and DDimer, NFL and BNP, S100b and NFL, GFAP and NFL, S100b and FABP, S100b and DDimer, S100b and BNP, GFAP and FABP, GFAP and DDimer, GFAP and BNP, FABP and DDimer, DDimer and BNP, and combinations thereof. In another exemplary embodiment the kit comprises means for determining the level of a panel comprising IL6, NFL, and S100b; IL6, NFL, and GFAP; IL6, S100b, and GFAP; NFL, S100b, and GFAP; S100b, GFAP, and FABP; NFL, IL6, and BNP; FABP, BNP, and DDimer; IL6, NFL, S100b, and GFAP, GFAP, NFL, and D-Dimer; or D-Dimer, GFAP, and NT- proBNP. In particular embodiment the kit comprises means for determining the level of a panel comprising IL6, NFL, and S100b; IL6, NFL, and GFAP; IL6, S100b, and GFAP; NFL, S100b, and GFAP; or IL6, NFL, S100b, and GFAP. In a more particular embodiment, the kit comprises means for determining the level of a panel comprising GFAP, NFL, and D-Dimer; or D-Dimer, GFAP, and NT-proBNP.

[0104] Particular kits, as used herein, refer to products containing the different reagents (or reagent means, or simply means) necessary for carrying out the methods of the invention packed so as to allow their transport and storage. Materials suitable for packing the components of the kit include crystal, plastic (e.g. polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes. Devices according to the invention may also containing the different means necessary for carrying out the methods of the invention. Moreover, in particular embodiments the kits or devices comprise a solid support and / or instructions to carry out the methods of the invention.

[0105] In a more particular embodiment of the kits or devices of the invention, these consist of means for detecting the level of any of the biomarkers or combination of biomarkers disclosed above in a biofluid sample, said means embedded in a single packaging.

[0106] Additionally, the kits of the invention may contain instructions for the simultaneous, sequential or separate use of the different components which are in the kit. Said instructions can be in the form of printed material or in the form of an electronic support capable of storing instructions susceptible of being read or understood, such as, for example, electronic storage media (e.g., magnetic disks, tapes), or optical media (e.g., CD-ROM, DVD), or audio materials. Additionally, or alternatively, the media can contain internet addresses that provide said instructions.

[0107] The reagent means (or simply means) of the kit include compounds that bind specifically to the marker proteins. Preferably, said compounds are antibodies, aptamers or fragments thereof.

[0108] In a preferred embodiment, the reagent is an antibody or fragment thereof as previously indicated. Thus, the reagent means are one or more antibodies that specifically and individually recognize the proteins of interest (i.e., antibodies or fragments that specifically bind to the biomarkers of interest). The antibodies of the kit of this aspect of the invention can be used according to techniques known in art for determining the protein expression levels, such as, for example, flow cytometry, Western blot, ELISA, RIA, competitive EIA, DAS- ELISA, techniques based on the use of biochips, protein microarrays, or assays of colloidal precipitation in reactive strips.

[0109] The antibodies can be fixed to a solid support such as a membrane, a plastic or a glass, optionally treated to facilitate the fixation of said antibodies to the support. Said solid support comprises, at least, a set of antibodies which specifically recognize the marker (i.e., the protein of interest), and which can be used for detecting the levels of expression of said marker. Thus, in another particular embodiment the kits of the invention comprise a solid support for the immobilization of antibodies or of other reagent means capable to bind to the proteins in the sample

[0110] Additionally, the kits or devices of the invention comprise reagents for detecting a protein encoded by a constitutive gene. The availability of said additional reagents allows normalizing the measurements performed in different samples (for example, the sample to be analysed and the control sample) to rule out that the differences in the expression of the biomarkers are due to a different quantity of total protein amount in the sample more than the real differences in the relative levels of expression. The constitutive genes in the present invention are genes that are always active or being transcribed constantly and which encode for proteins that are expressed constitutively and carry out essential cellular functions. Proteins that are expressed constitutively and can be used in the present invention include, without limitation, p-2-microglobulin (B2M), ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH, PSMB4, tubulin and actin.

[0111] In another embodiment, the kits or devices of the invention are conceived as point of care tests. More in particular, they are in form of lateral flow tests.

[0112] In another particular embodiment the kit or device according to the invention comprises a support and two or more sample inlet ports for deposition of a biofluid sample, in particular whole blood or serum; a reaction area comprising the means / reagents that bind specifically to the marker proteins, in particular antibodies; and wherein the sample inlet port is connected with the reaction area. In another more particular embodiment, the kit or device comprises as many sample inlet ports as markers to be detected and corresponding reaction areas connected thereto (multiplex determination). In another embodiment the kit comprises one single inlet import and as capillary tracks connecting to as many reactive areas, said capillary tracks conducting part of the sample to each corresponding connected reaction area. Another aspect of the invention is directed to use of a peptide or protein or a combination of peptides or proteins as in vitro biomarker(s) for the screening of TBI in paediatric patients; or for selecting a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention; or for diagnosing intracranial lesion in case of TBI a in a paediatric subject; or for differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion the diagnosis of intracranial lesion in case of a TBI in a paediatric subject; or for predicting cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI. The peptide or protein may be at least one selected from the group consisting of IL6, NFL, FABP, BNP, GFAP, and a fragment of any of the foregoing. One embodiment provides the use of a combination of peptides or proteins as in vitro biomarker(s) for any of the uses mentioned above, i.e. the use of a panel. Said panel may comprise at least two biomarkers selected from the group of biomarkers consisting of IL6, NFL, FABP, BNP, GFAP, S100b, IL8, IL10, and DDL All the embodiments described above for the biomarkers, their combinations (panels), and thresholds, also apply to this aspect.

[0113] As previously cited, the expression levels of the biomarkers in an isolated sample of a patient, as well as the age, clinical symptoms, and other conditions listed in the methods, kits and systems of the invention, can be determined by measuring both the levels of protein, and the levels of variants thereof, such as fragments (e.g., NT-proBNP), isoforms, analogues and / or derivatives.

[0114] As known in the art the "similarity" between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein to a sequence of a second protein. The variants are defined to include polypeptide sequences different from the original sequence, preferably different from the original sequence in less than 40% of residues per segment concerned, more preferably different from the original sequence in less than 25% of residues per segment concerned, more preferably different from the original sequence in less than 10% of residues per segment concerned, more preferably different from the original sequence in only a few residues per segment concerned and, at the same time, sufficiently homologous to the original sequence to preserve functionality of the original sequence. Variants according to the present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two proteins is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)].

[0115] The proteins can be post-transl ationally modified. For example, post- translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis myristoylation, protein folding and proteolytic processing, etc. Additionally, the proteins may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

[0116] In particular embodiments of the methods of the invention, said methods further comprise the steps of (I) collecting the information provided by carrying out the method, and (ii) saving the information in a data carrier. In particular embodiments, the methods comprise the following steps:

[0117] (I) determining in an isolated biofluid sample of the paediatric patient the level of a biomarker or combination of biomarkers (a panel) of the invention as described in any of the embodiments above,

[0118] (ii) collecting the information provided in (I),

[0119] (ill) saving the information in a data carrier, and

[0120] (iv) processing the information to provide information for selecting a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention; or for diagnosing intracranial lesion in case of TBI a in a paediatric subject; or for differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion the diagnosis of intracranial lesion in case of a TBI in a paediatric subject; or for predicting cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI. The processing of the information may be carried out by al algorithm.

[0121] In the sense of the invention a "data carrier” is to be understood as any means that contain meaningful information data for the selection, presence of intracranial lesion and / or most probable recovery outcome, such as paper. The carrier may also be any entity or device capable of carrying the said information. For example, the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disc or hard disk. Further, the carrier may be a transmissible carrier such as an electrical or optical signal, which may be conveyed via electrical or optical cable or by radio or other means. When the information data are embodied in a signal that may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means. Other carriers relate to USB devices and computer archives. Examples of suitable data carrier are paper, CDs, USB, computer archives in PCs, or sound registration with the same information.

[0122] Another aspect of the invention relates to a computer-implemented method for carrying out any one of the methods of the invention as defined above, in which after determining of level of the biomarker or biomarker panel, and optionally of the one or more clinical parameters, said level(s) are given a value and / or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein in function of the said level (s), score(s) and or computed value(s), a decision is taken for:

[0123] (a) selecting a paediatric patient suffering TBI, in particular suffering mTBI according to GCS, for a brain image technique and / or for a subsequent treatment intervention,

[0124] (b) or for diagnosing a TBI, in particular a mTBI, with intracranial lesion that is detectable by a brain imaging technique or a TBI paediatric patient with no intracranial lesion, or (c) or determining the risk of suffering cognitive and / or behaviour and / or emotional, and / or physical impairments in a paediatric subject having experienced TBI is done.

[0125] In one embodiment, the computer-implemented method comprises the following steps:

[0126] A) Computing in a mathematical formula executed by a computer the level of the biomarker of panel of biomarkers, and optionally of the one or more clinical parameters, to obtain a computed value or score for each of the biomarkers or clinical parameters;

[0127] B) The application of an interpretation algorithm to generate a result from said computed value(s), said result allowing for:

[0128] (a) selecting the paediatric patient suffering TBI, in particular suffering mTBI selecting a paediatric patient suffering TBI, in particular suffering mTBI according to GCS, for a brain image technique and / or for a subsequent treatment intervention,

[0129] (b) or for diagnosing a TBI, in particular a mTBI, with intracranial lesion that is detectable by a brain imaging technique or a TBI paediatric patient with no intracranial lesion, or

[0130] (c) or determining the risk of suffering cognitive and / or behaviour and / or emotional, and / or physical impairments in a paediatric subject having experienced TBI is done.

[0131] In a more particular embodiment of this computer-implemented method, in step (A) the value or score assigned to each of the biomarker and, optionally, to each of the additional clinical parameters, is computed in a mathematical formula to obtain a compiled score or value; and in step (B) the interpretation algorithm generate a result for the selection, diagnosis or prediction of risk of the patient as defined in (a)-(c). Moreover, all the embodiments described above for the biomarkers, their combinations, and thresholds, also apply to the computer-implemented method of the invention.

[0132] The present invention also contemplates a system comprising:

[0133] A) a kit or a device as defined above for determining the level of a biomarker or biomarker panel, and

[0134] B) a processing system configured to compute a value or score for the level of each of the biomarkers determined in (A) and, optionally, for the one or more clinical parameters, and apply an interpretation algorithm to generate a result from said computed value(s).

[0135] The result of the above system provides information for (a) selecting the paediatric patient suffering TBI, in particular suffering mTBI selecting a paediatric patient suffering TBI, in particular suffering mTBI according to GCS, for a brain image technique and / or for a subsequent treatment intervention, or for (b) diagnosing a TBI, in particular a mTBI, with intracranial lesion that is detectable by a brain imaging technique or a TBI paediatric patient with no intracranial lesion, or for (c) determining the risk of suffering cognitive and / or behaviour and / or emotional, and / or physical impairments in a paediatric subject having experienced TBI is done.

[0136] The level of the biomarker or biomarker panel obtained in (A) may be manually introduced in the processing system (B). Alternatively, in particular embodiments, the processing system (B) is configured to directly read the level of the biomarker or panel. For example, when the kit or device in (A) is a lateral flow test, the processing system (B) may be configured to compute a value or score for the biomarker or biomarker panel directly from an image taken from the results of the lateral flow test. Said results may be in the form of coloured bands and the processing system is configured to associate each ban with a biomarker and the intensity of the band with the level of said biomarker. In another example, the results are in the form of fluorescent signals and the processing system is configured to associate each fluorescent signal with a biomarker and its level. In particular embodiments the processing system (B) is a computer-implemented system. In particular embodiments the processing system (B) comprises a computer-implemented method, in particular a computer-implemented method as defined in above.

[0137] In an embodiment the computer-implemented processing system comprises:

[0138] (a) at least a database comprising:

[0139] (i) a first test result collected from a first diagnostic test for a first biomarker as defined above;

[0140] (ii) optionally a second test result collected from a second diagnostic test for a second biomarker as defined above, different than the first diagnostic test;

[0141] (iii) optionally, subsequent test result(s) collected from subsequent diagnostic test(s) different from the previous diagnostic test(s);

[0142] (iv) optionally , secondary subject observations or measurements;

[0143] (v) one or more diagnostic cut-offs or reference values associated with the first diagnostic test, optionally with the second diagnostic test, and optionally with subsequent diagnostic tests, and with the subject observations or measurements, wherein such cut-offs and reference values collectively integrate to assess probability of intracranial lesion in paediatric TBI patient, in particular mTBI;

[0144] (b) one or more processors operatively encoded to automatically:

[0145] (i) apply an interpretation algorithm to generate a subject result coordinate based on the database of test results;

[0146] (ii) optionally apply a second interpretation algorithm to generate a probability of error in the subject result coordinate.

[0147] The algorithm mentioned above may be calculated as a function of a panel defined by the collection of variables considered as input, together with the associated variable thresholds. These thresholds are obtained after a training process. The input variables, (xi, ... . xn), comprise clinical variables (such as patient age, a TBI scale assessment score) and the biomarker concentrations (levels) after the calibration process (bi, b b3,..).

[0148] In one embodiment the algorithm is defined by the following formula: TBI = H(SP- Ts)

[0149] Wherein:

[0150] • H is a Heaviside function, defined as H(x) = 1 when x > 0, 0 otherwise

[0151] • Ts is the total score threshold

[0152] • Sp is the panel score, which is defined by the following formula: n

[0153] SP= si i=1 st = H (sign;(xi - ti)} wherein:

[0154] • H is a Heaviside function, defined as H(x) = 1 when x > 0, 0 otherwise

[0155] • sign; is either a positive sign (+) if xt value is expected to be larger in the TBI cohort, than in the Control group; or a negative sign (-) otherwise

[0156] • xt are the input variables, (xi, ... . xn), comprising clinical variables (such as patient age, a TBI scale assessment score) and the biomarker concentrations (levels) after the calibration process (bi, bi, b3,..)

[0157] • ti. t\, ... . tnare the corresponding variable specific thresholds.

[0158] Herewith disclosed is also a data processing apparatus, device or system comprising means for carrying out the steps of the computer-implemented method of the invention or any of its embodiments.

[0159] Also disclosed is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out the steps of the computer-implemented method of the invention as previously disclosed as an aspect of the invention or in any of its embodiments.

[0160] Herewith disclosed is also a computer-readable (e.g., storage) medium comprising instructions which, when executed by a computer, cause the computer to carry out the steps of the computer-implemented method of the invention as previously disclosed as an aspect of the invention or in any of its embodiments.

[0161] Again, all the embodiments described above for the biomarkers, their combinations, and thresholds, also apply to the systems, data processing apparatuses, computer programs, and computer-readable medium of the invention.

[0162] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise” encompasses the case of "consisting of'. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0163] Examples

[0164] Example 1. IL6, NFL, FABP, BNP, GFAP, and S100b as biomarkers for TBI

[0165] Materials and methods:

[0166] Study population

[0167] Children were recruited in two prospective multicenter cohort studies who took place in 5 pediatric emergency departments in Switzerland between October 2020 and February 2023 and 4 pediatric emergency departments in Spain between 2019 and 2021.

[0168] We included any child of 16 years-old or less with a head trauma within less than 24 hours with, in addition, either 1) Glasgow Coma Scale (GCS) of 14; or 2) GCS of 15 and one of the following symptoms: loss of consciousness of less than thirty minutes within twenty minutes post-trauma, post traumatic amnesia of less than 24 hours within thirty minutes post-trauma, persistent headaches, irritability, three or more episodes of vomiting, confusion, dizziness or transient neurological abnormality such as seizure; or 3) Sign of basal skull fracture (such as hemotympanum, raccoon eyes, CSF otorrhea or rhinorrhea, Battle's sign); or 4) High energy trauma (such as traffic accident or fall from more than 0.9 meter (3 feet) if < 2 years old, more than 1 .5 meter (5 feet) if > 2 years old). Exclusion criteria were patient already included in other clinical study with treatment, proof of alcohol or other substances intoxication, history of TBI of less than one month, convulsion episodes in the previous month, Down syndrome, encephalitis, or meningitis. In one hospital of the Swiss study (Geneva), a control group was also recruited. Inclusion criteria were any child of 16 years-old or less with scheduled blood sample in the ambulatory care unit and without TBI. Exclusion criteria are the same as the TBI group.

[0169] The attending physician obtained written consent from the parents or legal guardians of children meeting the inclusion criteria for participation in the study as well as the child himself if he was over 14 years old. Blood was drawn within 24 hours of the trauma, but as soon as possible. The study did not interfere in any medical decision such as to perform or not a CT scan, to place the patient in observation or to do any other blood test. Study data were collected and managed using REDCap electronic data capture tools hosted at Hopitaux Universitaires de Geneve (HUG).

[0170] CT scan analysis

[0171] All CT scans were reviewed by the same pediatric radiologist (CHM) blinded for the clinical evaluation, biomarkers result and local CT conclusion. CT scan was considered positive in the presence of any of these findings (ICI) : intracranial hemorrhage or contusion, cerebral oedema, traumatic infarction, diffuse axonal injury, shearing injury, sigmoid sinus thrombosis, midline shift of intracranial contents or signs of brain herniation, diastasis of the skull, pneumocephalus or skull fracture depressed by at least the width of the table of the skull (PECARN criteria).

[0172] Blood biomarker analysis

[0173] Serum samples were promptly collected upon arrival at the ED and subsequently centrifuged and stored at - 80°C.

[0174] Blood levels of S100b, GFAP and HFABP were measured by ELISA using respectively R-plex Human S100b (F212E), GFAP (F211 M) and FABP3 / HFABP (F214T) Antibody Sets (Meso Scale Diagnostics, Rockville, MD, USA). LLoD were respectively 1.6 pg / mL with a calibration range of 1,22-5000 pg / mL for S100b, 63 pg / mL and 122-500 000 pg / mL for GFAP, 90 pg / mL and 24,41-100 000 pg / mL for HFABP.

[0175] Blood levels of cytokines IL6, IL8 and IL10, were measured all at once using ELISA V-plex Proinflammatory Panel 1 (Human) antibodies (Meso Scale Diagnostics, Rockville, MD, USA). Lower limits of detection (LLoD) were respectively 0.06 pg / mL with a calibration range of 0.06-488 pg / mL for IL6, 0.07 pg / mL LLoD and 0.07- 375 pg / mL calibration range for IL8, 0.04 pg / mL LLoD and 0.04-233 pg / mL calibration range for IL10. Lower limits of quantification (LLoQ) were 0.633 pg / ml, 0.591 pg / ml, and 0.298 pg / ml for IL6, IL8, and IL10 respectively.

[0176] Blood levels of NFL and NTproBNP, were measured using ELISA R-plex (Human) antibody sets (Meso Scale Diagnostics, Rockville, MD, USA). Lower limits of detection (LLoD) were respectively 5.5 pg / mL with a calibration range of 12.21-50 000 pg / mL for NFL, and 0.30 pg / mL LLoD with 0.12-500 pg / mL calibration range for NTproBNP. Lower limits of quantification (LLoQ) were <12.21 pg / mL, 0.49 pg / mLfor NFL and NTproBNP respectively.

[0177] Lower limit of quantification (LLOQ) was defined as the lowest concentration with a coefficient of variation (CV) below 20% and a recovery between 80 and 120%.

[0178] All kits were used in accordance with the manufacturers' instructions. Duplicate control serum was measured on each plate and, intra- and inter-plate coefficients of variation were below 20%.

[0179] Outcome measures

[0180] For the analyses, patients were classified according to their status: stayed in observation without CT (in- hospital-observation), CT-performed with negative result (CT-), or CT-performed with positive result (CT+). Primary outcome was the presence of ICI on CT. Diagnostic values of the blood biomarkers were evaluated to identify 1) children with CT-negative result (CT-) and 2) children in observation without CT (in-hospital- observation), while all CT-positive (CT+) children were detected. Two separated analyses were performed, with blood sampling done within a) 6 hours and b) 24 hours post-trauma.

[0181] Statistical analysis

[0182] Statistical analysis was performed using R (http: / / www.rproject.org, version 4.3.0) in RStudio (http: / / www.rstudio.com, version 2023.06.0). Biomarkers concentrations were normalized using their medians as correction factors. Patients were dichotomized into the following groups: 1) CT-negative versus CT-positive groups, or 2) in-hospital-observation without CT and CT-negative versus CT-positive groups for statistical analyses. Differences between groups were established using non-parametric Mann Witney U test, given that Kolmogorov-Smirnov test reveals that all proteins were non-normally distributed (p value <0,05). ANOVA was used when comparing more than two groups. Chi-squared test was used for statistical analyses of the clinical data. Statistical significance was inferred at p-value <0.05. The levels of S100b, GFAP and HFABP are presented through box- and dot-plots, with a log 10 Y-scale. Biomarker's ability for classifying patients according to their CT-result group is evaluated using receiver operating characteristic (ROC) curves with the pROC package in R. Iterative combinations of each two-proteins panels were tested with the PanelomiX threshold-based algorithm2425. For each biomarker and their combination, the optimal performance was investigated to identify all patients without ICI, therefore looking for the highest specificity when sensitivity was set at 100%. Presented results were obtained on a selection of patients with blood sampling within 6 hours only and within 24 hours post-trauma.

[0183] Results:

[0184] A total of 376 serum samples were analyzed. This included 74 controls patients without head trauma and 302 mTBI patients. Out of the mTBI patients, 222 patients had blood sampling done within six hours after their head trauma. A total of 179 patients (81%) did not undergo CT scan exam but were kept in observation for symptoms monitoring at the ED for more than six hours (table 1). Within CT-scanned patients (n=43), seven (16%) were positive (table 1 and table 2). Mean age was 8 years old in all groups (SD of 4,93 - 4,43 - 5,08 - 4,93 for respective controls, mTBI without CT, mTBI with CT- and mTBI with CT+ groups). A wide range of ages from newborn to teenagers was observed (from one-month to 16 years old). Most of the patients had a GCS of 15 with associated symptoms. Loss of consciousness, post-traumatic amnesia, persistent headaches and more than three episodes of vomiting were the most frequent symptoms in mTBI patients .

[0185] Out of the seven patients with a CT-positive result, two of them also presented other body fractures and all of them had simple skull fractures seen on CT scan. CT-positive patients often had more than one PECARN criteria that might define a CT-positive result. There were five (71%) cases of intracranial hemorrhage (more specifically a sub-dural hemorrhage), five (71%) pneumocephalus, two (29%) diastasis of the skull, one (14%) midline shift, or intracranial contents or signs of brain herniation and one (14%) skull fracture depressed by at least the width of the table of the skull (table 2).

[0186] Table 2: PECARN criteria for positive CT

[0187] Except for the presence of fractures, no significative differences in clinical parameters were observed comparing patients with or without ICI on CT or kept in observation without CT. Time between head trauma and blood sampling was equivalent within compared groups (median of 4 hours in observed and CT- groups, 3 hours in CT+ group). Blood concentration of the biomarkers were increased in CT-positive patients compared to others and were significantly different when comparing to both CT-negative and in-hospital- observation patients. ROC curve analysis, for diagnostic performances of the biomarkers allowed to select the best specificity when the sensitivity was set at 100% to exclude all children with ICI (table 3). Table 3: Biomarkers performances to rule-out mTBI patients (within 6 hours).

[0188] Two-protein panels showed increased specificity over the best performing single biomarker, GFAP (table 4). Table 4: Biomarker combinations performances to rule-out mTBI patients (within 6 hours).

[0189] The results above were based on the analysis of patients sampled within < 6 hours post-trauma. When the same analysis was performed on samples taken within 24 hours, at 100% sensitivity, S100b performed with 32% SP, IL6 with 48%, NTproBNP with 26%, NFL with 38%, IL10 with 11% and GFAP with 27% SP.

[0190] Age correlation was investigated in the control group without head trauma. Spearman correlation revealed that both S100b and GFAP were age-inversed correlated in the whole control group, while HFABP wasn't (r=- 0,43, p value<0,0001 for S100b, r=-0,71 , p value <0,0001 for GFAP and r=-0,23, p value >0,05 for HFABP). The Spearman correlation matrix also highlighted a significant positive correlation between GFAP and S100b. Furthermore, Spearman correlation revealed that IL6 was significantly, but only slightly age correlated (r=0.24, with p value 0.043), IL10 was significantly age-inversed correlated (r=-0.54, with p value <0.0001), while IL8, NTproBNP and NFL weren't (r=-0.13, with p value >0.1.

[0191] The performances of these biomarkers was deciphered in this prospective pediatric cohort of mTBI patients to discriminate both CT- and in-hospital-observation without CT patients, versus CT+ patients. Blood concentration of GFAP, S100b and HFABP were significantly increased in mTBI children compared to controls. These markers were also significantly increased in CT+ patients compared to both in-hospital- observation and CT- patients. When sampled within six hours after the trauma, GFAP, HFABP and S100b respectively reached 52%, 41% and 39% specificity at 100% of sensitivity, to rule-out the need of unnecessary CT scans and shorten the length of stay in observation for patients without CT scan. The cytokine IL6 is another biomarker for triaging the need for CT scans in pediatric mTBI. IL6 achieved 100% sensitivity and 48% specificity in identifying CT- and in-hospital-observation patients without CT, while excluding all CT+ cases, when sampled within twenty-four hours after the trauma (and 50% specificity when sampled within six hours). This positions IL6 alongside S100b, GFAP, and HFABP as the best blood-based markers for managing pediatric mTBI.

[0192] In addition to IL6, which is primarily pro-inflammatory, we also found that IL10, an anti-inflammatory cytokine, has a role in pediatric mTBI. IL10 might be critical in modulating the inflammatory response after injury, potentially reducing the risk of secondary brain injury. This duality indicates that IL10 not only serves as a marker for inflammation but also represents a prognostic indicator for recovery trajectory. Its observed elevation post-injury might reflect the body's effort to counterbalance the acute inflammatory response.

[0193] Finally, we also investigated IL8's involvement in pediatric mTBI, to explore the chemotactic response that recruits neutrophils to the site of injury, indicating a different aspect of the inflammatory process compared to IL6 and IL10. Its role in the acute phase of inflammation, particularly in the context of blood-brain barrier permeability and the subsequent infiltration of immune cells into the brain, could be crucial for understanding the early pathophysiological changes following mTBI. In our cohort, the observed elevated levels of IL8 in CT- scanned patients might signal a heightened inflammatory response, possibly correlating with more severe clinical presentations.

[0194] Example 2. t-BIOMAP: non-invasive triage solutions for children with mTBI

[0195] The performance of a biomarker panel combining IL6, GFAP and NFLA was also studied in the framework of a larger clinical project where a total of 419 serum samples from mTBI paediatric patients were analysed within less than 24 hours after trauma. From the 419 patients, 401 had a negative CT scan (CT-) or did not undergo CT scan but were kept in observation for symptoms monitoring at the ED, while 18 patients had a positive CT scan (CT+). Mean age was 8. Results showed that in this cohort the panel consisting of the IL6, GFAP, and NFL combination had 100% sensitivity and 67% specificity for discriminating CT- versus CT+ patients (table 5).

[0196] Table 5.

[0197] Variables Sensitivity (%) Specificity(%) Thresholds (pg / ml) Min.nb_rule

[0198] GFAP_pg / ml_NORMmed 100 67.83 75.36 3

[0199] NFL_pg / ml_NORMmed 13.53

[0200] IL6_pg / ml_NORMmed 0.93

[0201] Clauses

[0202] 1 . An in vitro method for screening for traumatic brain injury (TBI) in a paediatric patient comprising the steps of:

[0203] (I) determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of interleukin-6 (IL6), neurofilament light protein (NFL), fatty acid binding protein (FABP), brain natriuretic peptide (BNP), glial fibrillary acidic protein (GFAP), and a fragment of any of the foregoing,

[0204] (ii) comparing the level of the biomarker(s) determined in (I) with a corresponding reference value, and (ill) assessing the paediatric patient for TBI.

[0205] 2. The in vitro method according to claim 1, wherein the assessing the paediatric patient for TBI comprises: (a) selecting the patient for a brain image technique and / or for a subsequent treatment intervention when the level of the biomarker(s) determined in (I) is(are) higher than the corresponding reference value, or is(are) within a corresponding reference range level of a paediatric patient with intracranial lesion detectable by said brain image technique, or (b) differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion, wherein the patient is diagnosed as having intracranial lesion when the level of the biomarker(s) determined in (I) is(are) higher than the corresponding reference value, or is(are) within a corresponding reference range level of a paediatric patient with intracranial lesion; or the patient is diagnosed as having no intracranial lesion when the level of the biomarker(s) determined in (I) is(are) equal or lower than the corresponding reference value, or is(are) within a reference range level of a paediatric patient with no intracranial lesion

[0206] 3.- An in vitro method for determining the risk of suffering cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI, comprising the steps of:

[0207] (I) determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of IL6, NFL, HFABP, BNP, GFAP, and a fragment of any of the foregoing, (ii) comparing the level of the biomarker(s) determined in (I) with a corresponding reference value, and (ill) predicting a high risk of suffering from cognitive and / or behaviour and / or emotional and / or physical impairments when the level of the biomarker(s) determined in (I) is(are) higher or lower than the corresponding reference value, or is(are) within a reference range level of a patient with intracranial lesion detectable by a brain image technique.

[0208] 4. The in vitro method according to according to any one of claims 1 to 3, wherein step (I) further comprises determining the level of a biomarker selected from the group consisting of S100 calcium binding protein B (S100b), interleukin-8 (IL8), interleukin-10 (IL10), DDimer (DDi), a fragment thereof, and combinations thereof.

[0209] 5. The in vitro method according to according to any one of claims 1 to 4, wherein step (I) comprises determining the level of a biomarker panel selected from the group consisting of IL6 and NFL; S100b and IL6; FABP and BNP; GFAP and IL6; IL6 and FABP; IL6 and DDimer; IL6 and BNP; NFL and FABP; NFL and DDimer; NFL and BNP; S100b and NFL; GFAP and NFL; S100b and FABP; S100b and DDimer; S100b and BNP; GFAP and FABP; GFAP and DDimer; GFAP and BNP; FABP and DDimer; DDimer and BNP; IL6, NFL, and S100b; IL6, NFL, and GFAP; IL6, S100b, and GFAP; NFL, S100b, and GFAP; S100b, GFAP, and FABP; NFL, IL6, and BNP; FABP, BNP, and DDimer; and IL6, NFL, S100b, and GFAP.

[0210] 6. The in vitro method according to claim 5, wherein the biomarker panel comprises a biomarker selected from the group consisting of IL6, NFL, S100b, and GFAP, in particular the biomarker panel is selected from the group consisting of IL6, NFL, and S100b; IL6, NFL, and GFAP; IL6, S100b, and GFAP; NFL, S100b, and GFAP; and IL6, NFL, S100b, and GFAP.

[0211] 7. The in vitro method according to any one of claims 1-6, wherein the biofluid sample is selected from the group consisting of blood, serum, plasma, urine, saliva, lachrymal fluid, cerebrospinal fluid, and combinations thereof, in particular, the biofluid sample is selected from the group consisting of blood, serum, and plasma. 8. The in vitro method according to any one of claims 1-7, further comprising determining one or more clinical parameters, and / or combining the levels of the one or more proteins determined with gender and / or age of the patient.

[0212] 9. Use of a peptide or protein or a combination of peptides or proteins as in vitro biomarker(s) for the screening of TBI in paediatric patients; or for selecting a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention; or for diagnosing intracranial lesion in case of TBI a in a paediatric subject; or for differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion the diagnosis of intracranial lesion in case of a TBI in a paediatric subject; or for predicting cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI, wherein:

[0213] (a) the peptide or protein is one or more selected from the group consisting of IL6, NFL, FABP, BNP, GFAP, and a fragment of any of the foregoing; or alternatively,

[0214] (b) the combination of peptides or proteins comprises one selected from the group consisting of IL6 and NFL, S100b and IL6, FABP and BNP, GFAP and IL6, IL6 and FABP, IL6 and DDimer, IL6 and BNP, NFL and FABP, NFL and DDimer, NFL and BNP, S100b and NFL, GFAP and NFL, S100b and FABP, S100b and DDimer, S100b and BNP, GFAP and FABP, GFAP and DDimer, GFAP and BNP, FABP and DDimer, DDimer and BNP, and combinations thereof; or alternatively

[0215] (c) the combination of peptides or proteins comprises one selected from the group consisting of IL6, NFL, and S100b; IL6, NFL, and GFAP; IL6, S100b, and GFAP; NFL, S100b, and GFAP; S100b, GFAP, and FABP; NFL, IL6, and BNP; and FABP, BNP, and DDimer; or alternatively

[0216] (d) the combination of peptides or proteins comprises IL6, NFL, S100b, and GFAP.

[0217] 10. A kit or a device comprising means for determining the level of:

[0218] (a) one or more peptides or proteins selected from the group consisting of IL6, NFL, FABP, BNP, GFAP, and a fragment of any of the foregoing; or alternatively,

[0219] (b) at least two peptides or proteins selected from the group consisting of IL6 and NFL, S100b and IL6, FABP and BNP, GFAP and IL6, IL6 and FABP, IL6 and DDimer, IL6 and BNP, NFL and FABP, NFL and DDimer, NFL and BNP, S100b and NFL, GFAP and NFL, S100b and FABP, S100b and DDimer, S100b and BNP, GFAP and FABP, GFAP and DDimer, GFAP and BNP, FABP and DDimer, DDimer and BNP, and combinations thereof; or alternatively

[0220] (c) at least three peptides or proteins selected from the group consisting of IL6, NFL, and S100b; IL6, NFL, and GFAP; IL6, S100b, and GFAP; NFL, S100b, and GFAP; S100b, GFAP, and FABP; NFL, IL6, and BNP; and FABP, BNP, and DDimer; or alternatively

[0221] (d) at least IL6, NFL, S100b, and GFAP.

[0222] 11 . Use of a kit or device as defined in claim 10 for carrying out the method of any one of claims 1-8. 12. A computer-implemented method for carrying out the method as defined in any of claims 1-8, in which after step (i), the determined level(s) is(are) given a value and / or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein in function of the said level(s), score(s) and or computed value(s), a decision is taken for: selecting a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention; or for diagnosing intracranial lesion in case of TBI a in a paediatric subject; or for differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion the diagnosis of intracranial lesion in case of a TBI in a paediatric subject; or for predicting cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI.

[0223] 13. The method according to any of claims 1-8, the use according to claim 9, the kit or device according to claim 10, the use of the kit or device according to claim 11, or the computer-implemented method according to claim 12, wherein the BNP is the N-terminal prohormone of brain natriuretic peptide (NT-proBNP) and / or the FABP is heart FABP (HFABP).

[0224] 14. The method according to any one of claims 1-8 or 13, the use according to claims 9 or 13, the kit according to any one of claims 10 or 13, the use of the kit or device according to claim 11 or 13, or the computer-implemented method according to any one of claims 12 or 13, wherein traumatic brain injury is mild traumatic brain injury (mTBI).

[0225] 15. The method according to any one of claims 1-8 or 13-14, the use according to claims 9 or 13-14, the kit according to any one of claims 10 or 13-14, the use of the kit or device according to claim 11 or 13-14, or the computer-implemented method according to any one of claims 12 or 13-14, wherein the brain image technique is selected from a tomography computer tomography (CT) and / or magnetic resonance imaging (MRI).

[0226] Citation List

[0227] - Chang and Nager, "Pediatric Traumatic Brain Injury: The Utility of Beta-Natriuretic Peptide”, J Trauma- 2010, vol.no. 68, pp.: 1401-1405

[0228] BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)

[0229] Burtis C. A. et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values”

Claims

Claims1. An in vitro method for screening for traumatic brain injury (TBI) in a paediatric patient comprising the steps of:(I) determining in an isolated biofluid sample of the paediatric patient the level of a panel of biomarkers selected from the group consisting of:- interleukin-6 (IL6), neurofilament light protein (NFL), and glial fibrillary acidic protein (GFAP);- NFL, GFAP, and D-Dimer; and- GFAP, D-Dimer, and N-terminal prohormone of brain natriuretic peptide (NT-proBNP),(ii) comparing the level of the biomarkers determined in (I) with a corresponding reference value, and(ill) assessing the paediatric patient for TBI, wherein the assessing the paediatric patient for TBI comprises:(a) selecting the patient for a brain image technique and / or for a subsequent treatment intervention when the level of the biomarkers determined in (I) are higher than the corresponding reference value, or are within a corresponding reference range level of a paediatric patient with intracranial lesion detectable by said brain image technique, or(b) differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion, wherein the patient is diagnosed as having intracranial lesion when the level of the biomarkers determined in (I) are higher than the corresponding reference value, or are within a corresponding reference range level of a paediatric patient with intracranial lesion; or the patient is diagnosed as having no intracranial lesion when the level of the biomarkers determined in (I) are equal or lower than the corresponding reference value, or are within a reference range level of a paediatric patient with no intracranial lesion2.- An in vitro method for determining the risk of suffering cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI, comprising the steps of:(I) determining in an isolated biofluid sample of the paediatric patient the level of one or more biomarkers selected from the group consisting of:- interleukin-6 (IL6), neurofilament light protein (NFL), and glial fibrillary acidic protein (GFAP);- NFL, GFAP, and D-Dimer; and- GFAP, D-Dimer, and N-terminal prohormone of brain natriuretic peptide (NT-proBNP),(ii) comparing the level of the biomarkers determined in (I) with a corresponding reference value, and (ill) predicting a high risk of suffering from cognitive and / or behaviour and / or emotional and / or physical impairments when the level of the biomarkers determined in (I) are higher or lower than the corresponding reference value, or are within a reference range level of a patient with intracranial lesion detectable by a brain image technique.

3. The in vitro method according to any one of claims 1-2, wherein the biofluid sample is selected from thegroup consisting of blood, serum, and plasma.

4. The in vitro method according to according to any one of claims 1 to 3, wherein step (I) further comprises determining the level of a biomarker selected from the group consisting of interleukin-6 (IL6), neurofilament light protein (NFL), heart fatty acid binding protein (HFABP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP), S100 calcium binding protein B (S100b), interleukin-8 (IL8), interleukin-10 (IL10), DDimer (DDi), a fragment thereof, and combinations thereof.

5. The in vitro method according to according to any one of claims 1 to 4, wherein step (I) comprises determining IL6, NFL, and GFAP.

6. The in vitro method according to claim 5, wherein step (I) further comprises determining NT-proBNP, DDi, or both.

7. The in vitro method according to any one of claims 1-6, further comprising determining one or more clinical parameters, and / or combining the levels of the one or more proteins determined with gender and / or age of the patient.

8. The in vitro method according to any one of claims 1-7, wherein the sample is obtained within twenty-four hours after the trauma.

9. The in vitro method according to claim 8, wherein the sample is obtained within six hours after trauma.

10. Use of a combination of peptides or proteins selected from the group consisting of:- IL6, NFL, and GFAP;- NFL, and GFAP;- NFL, GFAP, and D-Dimer; and- GFAP, D-Dimer, and NT-proBNP as in vitro biomarkers for the screening of TBI in paediatric patients, wherein the screening comprises selecting a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention, or diagnosing intracranial lesion in case of TBI a in a paediatric subject, or differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion; or for predicting cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI.11 . A kit or a device comprising means for determining the level of a combination of peptides or proteins selected from the group consisting of:- IL6, NFL, and GFAP;- NFL, GFAP, and D-Dimer; and- GFAP, D-Dimer, and NT-proBNP.

12. Use of a kit or device as defined in claim 11 for carrying out the method of any one of claims 1-9.

13. A computer-implemented method for carrying out the method as defined in any of claims 1-9, in which after step (I), the determined levels are given a value and / or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein in function of the said levels, scores and or computed value, a decision is taken for: selecting a paediatric patient suffering traumatic brain injury (TBI) for a brain image technique and / or for a subsequent treatment intervention; or for diagnosing intracranial lesion in case of TBI a in a paediatric subject; or for differentiating a traumatic brain injury paediatric patient with intracranial lesion that is detectable by a brain imaging technique from a traumatic brain injury paediatric patient with no intracranial lesion; or for predicting cognitive and / or behaviour and / or emotional and / or physical impairments in a paediatric subject having experienced TBI.

14. The method according to any one of claims 1-9, the use according to claims 10, the kit or device according to claim 11, the use of the kit or device according to claim 12, or the computer-implemented method according to claim 13, wherein traumatic brain injury is mild traumatic brain injury (mTBI).

15. The method according to any one of claims 1-9 or 14, the use according to claims 10 or 14, the kit or device according to any one of claims 11 or 14, the use of the kit or device according to claim 12 or 14, or the computer-implemented method according to any one of claims 13-14, wherein the brain image technique is selected from a tomography computer tomography (CT) and / or magnetic resonance imaging (MRI).

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