WO2018117913A1 - Predicting the course and outcome of a coma and post-comatose states of unconsciousness (including vegetative states) using haemo-testing - Google Patents

Abstract

The invention relates to the field of diagnostics in emergency medical care, traumatology, intensive care and surgery, and can be used in medical practice. In order to objectively assess the cerebral state of subjects in states of unconsciousness or a coma, to assess damage to nerve tissue and to predict the development of a patient's state, the invention proposes determining the level of antibodies to fragments of specific KAP receptors such as GluR6 and/or GluR7, in body fluids. KAP peptide fragments and antibodies to these indicate structural damage in the brain and are specifically present only in patients in a state of unconsciousness. The level and dynamics of change in the level of these antibodies is used in the present invention to predict both the recovery of a subject's state in a successful outcome, and the likelihood of passing into a persistent vegetative state or fatal outcome.

Description

 Prediction of the course and outcome of coma and post-coma unconscious states (including autonomic ones) using blood tests

Technical field

 The invention relates to emergency medicine, in particular, to emergency and emergency medical care, traumatology, surgery, resuscitation and neurosurgery, in particular, to methods for evaluating unconscious and autonomic conditions, as well as predicting the outcome of coma as a result of damage to the brain structures of traumatic, ischemic or metabolic genesis using blood tests to determine brain-specific protein biomarkers, such as antibodies to kainate receptors.

State of the art

 Unconscious states can be coma and various variants of post-coma disorders of consciousness (autonomic state, akinetic / hyperkinetic mutism) (Alexandrova E.V. et al., "Exit from a protracted unconscious state due to severe diffuse axonal damage to the brain", Neurology, neuropsychiatry, Psychosomatics, Issue N ° 2/2013, pp. 51 - 58). The cause of coma can be structural (traumatic) and non-structural (metabolic) damage. General features of the pathogenesis of all types of unconscious states are in violation of the functions of the cerebral cortex, subcortical formations and the brain stem, as well as in the defeat of the ascending reticular formation of the brain stem, activating the cerebral cortex.

 Severe craniocerebral trauma (BMI) is characterized by the presence of a coma in the acute period, which is most often caused by dysfunction of the brain stem due to its direct traumatic injury or related secondary (non-traumatic) causes, such as edema, dislocation, intracranial hypertension, etc. coma in approximately 35% of cases is accompanied by a transition to another unconscious state (vegetative status, akinetic mutism), the duration of which affects the degree of further the restoration of mental functions, ability to work, and depends on the presence and degree of damage to the deep structures of the brain (trunk, basal ganglia and thalamus) (Alexandrova E.V. et al., "Exit from a protracted unconscious state due to severe diffuse axonal damage to the brain," Neurology, Neuropsychiatry, Psychosomatics, Issue N ° 2/2013, pp. 51-58).

The prognosis of the outcome of coma includes clinical and biochemical diagnosis. Neurological diagnosis of coma using computed tomography (CT) and magnetic resonance imaging (MPT) is to establish the nature, cause and severity of depression of consciousness (using the Glasgow Coma Scale (SCG)). Due to the instability of the patient’s condition, the impossibility of transportation outside the intensive care unit and in the presence of contraindications (the presence of metal objects in the body), carrying out the most informative diagnostic method - brain MRI - is often impossible. In addition, often the usual MRI modes do not allow to evaluate functional disorders (blood flow, biochemical processes) in the brain, which leads to a false-negative diagnosis of brain stem dysfunction. Identification of the causes of coma also includes an assessment of system parameters: blood pressure, gases and acid-base balance (pH) of the blood, biochemical (glucose and ketones, lactate and creatine) and electrolyte parameters. However, these indicators are nonspecific for predicting the outcome of coma, because their values also depend on the speed of peripheral circulation and the severity of the damage to the body.

 It is difficult to predict the way out of a coma in the acute period of PMTCT using only traditional methods. The determination of the prognosis for coma, in addition to purely medical, is of great economic importance because of the need to redistribute material resources and limit the intensive measures in hopeless cases. Until now, the rapid assessment of specific parameters of the state of the brain in dynamics remains an unresolved problem. In this regard, the identification of specific brain biomarkers that reflect the development of the disease and are measured in significant amounts in the peripheral blood is an important task in monitoring and predicting the state of the brain in coma. SUMMARY OF THE INVENTION

 The essence of this invention is that in patients in unconscious states and coma as a result of damage to brain structures, the concentration of antibodies to fragments of specific neuroreceptors, namely kainate receptors, which can be detected in biological fluids of a patient using appropriate biochemical tests, increases. Based on the determination of objective indicators in biological fluids of patients, timely and reasonable appointment of adequate therapy is possible.

It is known that ionotropic glutamate neuroreceptors are divided into three subtypes: NMDA receptors, AMPA, and kainate receptors, which are common in various structures of the central nervous system for the implementation of high-speed signal transmission. NMDA receptors are localized on the surface of the microvascular endothelium mainly in the cerebral cortex. AMPA receptors are localized at synaptic terminals and are found in a number of subcortical brain structures. Finally, kainate receptors (CAR), in particular subtypes such as GluR6 and GluR7, are located mainly in the pathways of the brain stem and spinal cord and are involved in the regulation of cerebral venous circulation, as well as regulate the basic functions of maintaining life.

 CARs are synaptic membrane receptor proteins involved in a cascade of neurotoxicity in brain damage with the formation of secondary vasogenic and / or cytotoxic edema in the deep structures of the brain. The appearance of CAR peptide fragments in biological fluids, including blood, indicates structural damage - pathological changes in the postsynaptic endings of neurons and lesions of the venous circulation of the brain stem. The inventors have found an increased level of CAR fragments formed from GluR6 and / or from GluR7 in patients who are unconscious. In healthy individuals, the formation of CAR fragments in the blood occurs in much smaller amounts, or does not occur at all. Once in the blood, excessive concentrations of CAR fragments can cause an immune response consisting in the production of specific antibodies, the level of which will correlate with the severity and size of damage to brain structures. In this regard, both CAR peptides and antibodies to them can serve as biomarkers of acute and chronic lesions of brain structures.

 The objective of the present invention is an objective assessment of the state of the brain of subjects in unconscious states and coma, as well as predicting the development of the patient's condition, which may consist in restoring the subject's consciousness (favorable outcome), and in the transition to a chronic vegetative state or death (adverse outcome) development of the disease).

 The solution to this problem lies in the fact that in subjects in unconscious states and coma, the level of antibodies to CAR is determined in biological fluids to assess damage to the nervous tissue, on the basis of which a prediction of exit from the unconscious state is carried out. The essence of the proposed method for assessing the state of the patient’s brain is that based on the assessment of brain-specific molecular biomarkers in a patient, it is possible to quickly and dynamically assess the presence and degree of both structural and functional damage to the brain stem and subcortical structures, which cannot be done only using standard neuroimaging methods (changes occur within 5-7 days).

In some embodiments of the invention, a method for predicting an exit from an unconscious state includes: (a) obtaining a biological sample from said subject; (b) at least a single analysis of the specified biological sample to determine the level of antibodies to one or more fragments of kainate receptors GluR6 and / or GluR7; (c) comparing this level with the level of the corresponding antibodies in samples taken from healthy subjects; (d) with a low level of antibodies in a sample of a subject in an unconscious state that does not exceed the control level recorded in samples of healthy subjects, predicting a favorable exit from an unconscious state; (e) with an increased level of antibodies in a sample of a subject in an unconscious state that exceeds the control level recorded in samples of healthy subjects, predicting an unfavorable outcome of the disease.

 In some embodiments of the invention, it is preferable to use a dynamic measurement of the level of CAR receptor fragments released during brain damage and circulating in the blood, which allows to assess the presence, degree of damage and monitor the restoration of the function of deep structures (in particular, the course of a traumatic brain disease), and, therefore, predict the outcome with prolonged chronic conditions. In these cases, at least two, three or more biological samples are collected from a subject in an unconscious state in the range from 1 hour to 90 days after the onset of the unconscious state. After that, a method for predicting an exit from an unconscious state includes: (a) analysis of these biological images to determine the level of antibodies to one or more fragments of the kainate GluR6 and / or GluR7 receptors; (b) comparing the levels of the corresponding antibodies determined in different samples of the same subject, with each other and with the level of the corresponding antibodies in samples taken from healthy subjects; (c) by lowering the level of these antibodies in samples of a subject in an unconscious state, over time, predicting a favorable exit from the unconscious state; (d) with an increase in the level of these antibodies in a sample of a subject in an unconscious state, over time, predicting an unfavorable outcome of the disease, provided that this level exceeds the control level recorded in samples of healthy subjects. To improve prediction, three or more samples are desired. The time intervals between sampling should be determined based on the dynamics of changes in the level of antibodies to fragments of CAR receptors, as well as to fragments of other glutamate receptors to assess the general condition of the glutamatergic system responsible for cortical-subcortical brain functions (for example, in accordance with patents RU2435165 and RU21 12243).

In preferred embodiments of the invention, the subject in an unconscious state is a patient, and the unconscious state is a coma of traumatic, ischemic and / or metabolic origin. In particular embodiments of the invention, whole blood, blood serum, plasma, cerebrospinal fluid, respiratory fumes, sweat and / or saliva obtained from a subject are used as a biological sample.

 Preferably, in the methods of the present invention, the identification and / or quantification of antibodies specific for fragments of kainate receptors is carried out using one or more assay systems, for example, immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RAI) or enzyme immunoassay (EIA); as well as other analytical methods for determining proteins after interaction with specific ligands (for example, mass spectrometry, NMR spectroscopy, liquid chromatography, etc.).

 According to another aspect, the present invention relates to the use of a kit for use in the methods or applications described above, wherein said kit includes a ligand capable of binding to or being specifically recognized by antibodies to fragments of kainate receptors, as well as reporter agents.

 In preferred embodiments of the invention, predicting an unconscious exit from a subject also includes additional clinical, laboratory, and / or instrumental examination methods. For example, additional methods are recommended in cases of cytotoxic edema, diffuse axonal damage and / or hematoma, which are confirmed by neuroimaging methods using special modalities of magnetic resonance imaging (T1, T2, FLAIR, DWI, SWI and SWAN), as well as measuring systemic blood parameters .

The technical result of the present invention lies in the fact that this invention helps to solve the problem of an objective forecast of a favorable or unfavorable outcome after damage to the brain structures, which led to an unconscious state. The invention allows to assess the presence and degree of both structural and functional damage to the trunk and subcortical structures of the brain. Additionally, this invention can allow you to estimate the time of the patient's exit from the unconscious state (hours, days, weeks, months) in accordance with the values of a number of biochemical parameters. The specified forecast allows to increase the accuracy and objectivity of assessing the state of brain structures in individuals in unconscious states and coma. This approach eliminates subjectivity, increases accuracy in predicting a way out of the unconscious state when damage to brain structures of various origins, which will allow for the most optimal therapeutic measures with subsequent improvement in the patient's condition. Definitions and Terms

 In the present description, the terms “includes” and “including” are interpreted as meaning “includes, among other things.” The indicated terms are not

intended to be construed as “consists of only”.

 The term "favorable exit from the unconscious state" means the restoration of consciousness of the subject (patient), while "unfavorable outcome of the development of the disease" means a chronic vegetative state or death.

 By “subject” is meant a human or, less preferably, another mammal endowed with consciousness.

 Kainate receptors (CAR) are one of the subtypes of ionotropic glutamate receptors that are found in the structures of the central nervous system of humans and animals. There are 5 different subunits of kainate receptors (GluR5, GluR6, GluR7, KA1 and KA2) that form homo- or hetero-tetramers to form a functional receptor. In one aspect, the present invention relates to methods for determining the level of antibodies to GluR6 and / or GluR7 fragments that have arisen in an unconscious subject. The GluR6 subunit may alternatively be called GluK2 or GRIK2, and is determined using the sequence SEQ ID NO: 1 (see Figure 1), or variants thereof. The GluR7 subunit may alternatively be called GluK3 or GRIK3, and is determined using the sequence SEQ ID NO: 2 (see Figure 1), or variants thereof.

“Clinical examination methods” should be understood as examination using the Glasgow Coma Scale or the Pittsburgh Scale, while “laboratory examination methods” consist of measuring systemic blood parameters, including measuring the acid-base balance (pCO ₂ , pH) and blood biochemical parameters (ketones , lactate, creatine). By "instrumental examination methods" it is necessary to understand, for example, magnetic resonance imaging, which includes T1 and T2-weighted, FLAIR, DWI, SWI and SWAN modalities.

By “fragments of the kainate receptors GluR6 and / or GluR7” in this description it is necessary to understand both whole protein molecules determined using the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and various variants of these molecules, which may include partial fragments of these molecules, covalent modifications of fragments of these molecules (such as phosphorylation, acetylation, modifications due to oxidative stress, amidation, sulfonation, etc.), crosslinking of partial fragments of these molecules with each other and with other moles molecules are (poliglutamil saturated acid urea, carbamates, aldehydes, esters, nitroanilides, aminomethylcoumarins, polyethylene glycols, etc.), and other variants that can induce the formation of specific antibodies in the body of the subject. For the detection of antibodies specific for fragments of kainate receptors GluR6 and / or GluR7, both whole protein molecules of these receptors and their fragments, as well as various recombinant variants of fragments of kainate receptors GluR6 and / or GluR7, including whole protein molecules, partial fragments, can be used crosslinking of partial fragments, as well as conjugates of these molecules or their fragments with proteins or low molecular weight compounds (biotin, avidin, peroxidase, GABA, AEA, AVA, Ahx).

 Under the "control" or "threshold" level of antibodies to fragments of kainate receptors should be understood as the maximum level of the corresponding antibodies determined in healthy volunteers. The absolute value of this level may vary depending on the specific antibody detection method and should be determined experimentally for each method.

Brief Description of Drawings

Fig. 1. Amino acid sequences of GluR6 (SEQ ID NO: 1) nGluR7 (SEQ ID NO: 2) of kainate receptors.

Fig. 2. A test card for the semi-quantitative determination of CAR antibodies based on the method of immunochromatographic analysis using nanoparticles. 1 - a window for applying a sample of biological fluid, 2 - a double filter, one of which contains nanoparticles with a detecting reagent (3), 4 - a test strip with a recombinant version of GluR6 / 7 coated on a nitrocellulose membrane (5) with an additional control strip (6 ), 7 - a result window, 8 - a filter on which excess reaction solutions are sorbed. The test card with the pressure gauge (9) is assembled in the case of the plastic cassette (10).

Fig. 3. The results of a dynamic measurement of the CAR of antibodies in the blood serum of patients after a head injury and with a different duration of coma (see examples). The red line indicates the threshold CAR values of antibodies (1, 5 ng / ml) determined in the blood serum of healthy volunteers.

Detailed Disclosure of Invention

CAR proteins are specific to the central nervous system; they participate in a cascade of neurotoxicity in brain damage with the formation of secondary vasogenic and / or cytotoxic edema in the subcortical structures of the brain. With structural disturbances in the deep parts of the brain the formation of peptide fragments of CAR in biological fluids, including the formation of new “foreign” autoantigens, which subsequently leads to the development of specific antibodies to peptide fragments of CAR. In PMTCT, the formation of cytotoxic edema and secondary ischemia in hemorrhagic foci is associated with a significant increase in the CAR of antibodies and is a delayed violation of cerebral circulation, which leads to a poor prognosis of the outcome of coma. By analyzing the biological fluids of individuals who do not have a PMI, it is possible to determine threshold values for the level of specific antibodies to specific peptide fragments of CAR. Constantly increased concentrations of CAR antibodies (i.e., above a predetermined threshold value), accompanied by negative values of systemic blood parameters, allows us to predict the risk of an unfavorable outcome of the disease. On the contrary, with a gradual decrease in the level of specific CAR antibodies in the direction of the threshold values, it becomes possible to predict a favorable outcome (recovery of consciousness).

Various biological samples taken from patients in an unconscious state, such as blood, serum or blood plasma, cerebrospinal fluid, saliva, etc., can be used to determine antibodies. Antibodies are preferably determined by enzyme-linked immunosorbent assay (ELISA). The latter consists in the reaction of the analyzed sample with an antigen, a recombinant variant of GluR6 / 7 kainate receptors, immobilized on a solid support, followed by detection of the formed immune complex using a conjugate of antibodies against human immunoglobulins of class G with the corresponding enzyme for detection. Semi-quantitative or quantitative detection of an increase in the level of specific antibodies is carried out by the difference in the values (optical density, fluorescence, luminescence or color change) of the analyzed sample with the same indicator of control samples (with a threshold value) to determine patients with damage to brain structures having high or low recovery potential. This method also differs in that, along with the determination of CAR antibodies, dynamic measurements of systemic blood parameters (lactate, creatine, pC0 ₂ , ketones and pH) are carried out jointly to prescribe timely therapy in patients with traumatic, metabolic or ischemic coma who still have the potential for survival.

The principle of the ELISA-KAP antibody reagent kit consists in the quantitative or semi-quantitative detection of antibodies to peptide fragments of kainate unbound in the immune complex of KAP present in biological fluids human brain receptors. Sorption of a specifically selected recombinant variant of CAP (formed from GluR6 and / or 7 subunits) on the surface of a solid carrier allows selective CAP extraction of antibodies from biological fluids of a patient (20-100 microliters of blood, blood serum, cerebrospinal fluid, respiratory fumes, saliva). The resulting antibody-antigen complex is detected using enzyme-labeled second antibodies against human class G immunoglobulins. An enzyme-linked immunosorbent reaction is measured by a change in optical density (also fluorescence or luminescence) recorded using a portable device for vertical scanning of optical density (450/630 nm) or using a color camera of a mobile device (phone, tablet, etc.) and a software application. Color reaction can also be measured visually (semi-quantitative version) using a color scale. Changes in the concentration of CAR antibodies, including their significant accumulation in the blood of patients, serves as a criterion for the functional state of the brain of these patients. Additional methods for examining patients are based on clinical methods, including determining the severity of the condition on the Glasgow Coma Scale (https://k n rek. Ru / caics / g iasg o. Htm) and the Pittsburgh scale for assessing the state of brain structures (see http: / /refdb.ru/look/2364618-p51 .html). Laboratory examination methods consist in measuring systemic blood parameters: lactate (norm 0.8-4.0 mmol / h * l); pCO ₂ (norm 32.5-47 mm Hg) and pH (<7.3) - to determine the degree of gas acidosis; creatine (normal 76.3-114.5 μmol / L in plasma) - to detect renal failure; and ketones (norm <0.6 mmol / L) - for the assessment of metabolic disorders (metabolic coma); All of the above parameters are evaluated together. Upon admission, magnetic resonance imaging is also performed using a number of modalities, including T1 and T2-weighted, FLAIR to assess lesions of brain structures, DWI to determine cerebral edema, and SWI / SWAN to detect cerebral hemorrhage.

The following examples are provided to illustrate the method according to the present invention and should not be construed as in any way limiting the scope of the invention.

Examples of methods for determining the content of antibodies to a recombinant variant of GluR6 / 7 kainate receptors.

Preparation of blood serum samples for the quantitative ELISA method: blood samples to be analyzed should be diluted 1: 50 before determination; add 20 µl of serum to glass or plastic tubes blood, add 980 μl of phosphate buffer and mix thoroughly with rotary movements. Diluted serum samples can be stored at a temperature of + 2-8 ° C for no more than 7 days.

Conducting quantitative ELISA analysis.

 Quantitative determination of CAR antibodies in biological fluids of patients is carried out by enzyme-linked immunosorbent assay kits of the company LLC “DRD” (Ulan-Ude, RF). Standard solutions, a solution of conjugate labeled with horseradish peroxidase, working buffer and test samples are prepared according to the instructions for the set. All wells of the plate coated with the recombinant variant of GluR6 / 7 kainate receptors are washed once with buffer (200 μl / well) using an ELISA microplate washer. 100 μl of standards or 100 μl of prepared test samples are added to the appropriate wells and incubated for 30 minutes. at + 37 ° C in a microplate incubator shaker. At the end of the incubation, wash the wells of the plate with the working buffer three times using an ELISA of an automatic microplate washer. 100 μl of a solution containing a conjugate of anti-human IgG antibodies with an enzyme (horseradish peroxidase) was added to all wells and the plate was incubated with constant shaking on a shaker for 30 minutes. at + 37 ° C. After incubation, wash the wells of the plate 3 times with a working buffer and, for the last time, with distilled water, as described above; each time the wells are washed, incubate the plate on a shaker for 2 minutes. at + 37 ° C. 100 μl of a solution of tetramethylbenzidine (TMB) are added and incubated for up to 10 minutes. at room temperature in a dark place. Pipette 100 µl of stop solution to stop the reaction, while the blue / blue color should change to yellow. Gently mix until a uniform color in each well and measure the optical density on a vertical scanning photometer at a wavelength of 450 / bZonm. Using the calibration curve of the dependence of the concentration of antibodies on optical density, given in the instructions, determine the concentration of CAR antibodies in the samples in ng / ml.

Conducting a semi-quantitative ELISA.

Semi-quantitative determination of CAR antibodies is carried out on the test card by the method of immunochromatographic analysis (lateral flowtest) (Fig. 2) using an undiluted freshly sampled patient's biological fluid. The reaction is activated by adding one drop of sample (about 20 μl), the antibodies from which react with the GluR6 / 7 peptide. The resulting antigen-antibody complex is detected by visualizing conjugates of antibodies against human IgG with horseradish peroxidase on the test strip in the form of a colored strip. Intensity and rate of manifestation the band corresponds to the concentration of antibodies in the sample, determined by comparison with the color of the bands on the reference map applied to the kit. If only the control band appears, the result is considered negative. Investigation of the correlation of the level of antibodies to fragments of specific CAR receptors (GluR6 / 7) and the dynamics of recovery of patients in an unconscious state.

 Example 1

 Patient K. (female, 19 years old) with a closed severe head injury [4 points on the Glasgow Coma Scale (CCG)] was hospitalized at the Research Institute of Neurosurgery on the 8th day after falling from a height in a coma from a primary hospital.

 Diagnosis: Closed head injury. Diffuse axonal brain damage of the II degree, diffuse cerebral edema. Conclusion on the neurological status upon admission: coma 2. Stem damage to the brain of a predominantly metabolic nature.

 Patient 1 1 day was in a coma and 8 days in a vegetative state, therefore, the duration of the unconscious state as a whole was 19 days. Dynamic measurement of antibodies to kainate receptors in blood serum during the first 7 days (Fig. 3A) showed that their level remained medium-elevated (4.7-4.4 ng / ml at a threshold value of 1.5 ng / ml). Stem symptoms were unexpressed and more consistent with metabolic dysfunction, rather than structural damage. According to MRI performed in T1, T2, FLAIR, and SWAN modes, there was also no damage to the trunk and subcortical structures of the brain.

 Neurological status after 1 month. after PMI: Clear consciousness (dysmnestic syndrome). Mostly left-sided tetraparesis with low muscle tone. Glasgow Outcome Scale (SHIG) after 6 months. amounted to 5 points out of 5 (good recovery).

 By the end of the 1st month, the values of antibodies to CAR (Fig. 1 A) showed a tendency to decrease towards threshold values (1, 5 ng / ml), indicating the restoration of trunk function, which was also noted with a neurological assessment.

Thus, in a patient with no damage to the deep structures of the brain, medium-high values of the CAR of antibodies were revealed during a coma, which quickly returned to normal along with complete regression of stem symptoms and recovery of consciousness. In general, the patient showed a good outcome of PMI after 6 months. Example 2

 Patient Sh. (Male, 44 years old) with a closed severe head injury (MHC = 4) was hospitalized 2 days after a catastrophe in a coma from a primary hospital. Diagnosis: Acute closed PMTCT. Severe contusion of the left frontal lobe with the formation of an intracerebral hematoma, which was removed on the day of admission. Cerebral edema. Conclusion on the neurological status upon admission: coma 2. Stem symptoms from the midbrain, upper and lower parts of the bridge on both sides. According to MRI, significant ischemic and hemorrhagic damage to the brain stem and subcortical structures was revealed.

 Dynamic measurement of anti-CAR antibodies in serum showed that the level of antibodies increased sharply from the threshold on the first day to 8.0 ng / ml by day 7 (Fig. ST). On the 9th day after TBI, negative neurological dynamics was revealed in the form of a decrease in the level of consciousness to coma 3 (GCS 3 points), an increase in stem symptoms from the level of the midbrain, upper and lower parts of the bridge.

 On the 14th day, against the background of a further increase in neurological symptoms, a fatal outcome was recorded. The SHIG score was 1 point.

 Thus, in a patient with severe structural damage to the deep parts of the brain, a significant increase in the titer of antibodies to CAR was revealed, which was accompanied by an aggravation of stem dislocation neurological symptoms. The outcome of the disease is death on the 14th day after the injury.

 Example 3

 Patient L. (male, 39 years old) with a severe head injury was hospitalized on day 3 after an accident (motorcycle injury) in a coma (MHC = 3). Diagnosis: Acute concomitant PMI, severe brain contusion with the formation of contusion foci of type 3 in both frontal and right temporal and parietal lobes, brain stem. Diffuse axonal damage of 4 degrees. Massive subarachnoid hemorrhage was removed on the day of hospitalization, plastic bone defects of the frontal areas from 2 sides. The conclusion about the neurological status upon admission: coma 3, gross trunk symptoms from the level of the midbrain, less pronounced in the upper and lower parts of the bridge, medulla oblongata.

According to CT of the brain, hemorrhages in the frontal, medial parts of the brain and subarachnoid space were identified. Dynamic measurement of antibodies to kainate receptors in the blood serum showed that the antibody level remained at the threshold level in the first three days, at 8, and also from 14-17 days of hospitalization (Fig. ЗВ). However, these values alternated with particularly high levels of antibody CAR at 7, 10-12 days (18-28 ng / ml), indicating a deterioration in the processes in the structures brain stem and patient instability. By the end of the 2nd month of hospitalization, the patient left a coma and remained in a vegetative state for more than 90 days, so the duration of the unconscious state as a whole was more than 120 days. Discharged from the clinic in a vegetative state, SHIG - 2 points.

 Thus, in a patient with severe structural damage to the deep parts of the brain, prolonged coma and vegetative state, a marked increase in the titer of antibodies to CAR was also revealed. The outcome of the disease is a chronic unconscious state.

Example 4. The characteristics of the set of reagents.

The results of testing 143 blood samples in 25 patients in an unconscious state (post-traumatic and metabolic coma), as well as 47 controls showed that the concentration of CAR antibodies determined using the quantitative ELISA test varied over a wide range from <0.6 to 60 ng / ml The values of CAR antibodies were distributed as follows: 89 of 90 samples of blood serum, including controls and a favorable outcome, showed a concentration of CAR antibodies <1, 5ng / ml. Of 53 samples of blood serum in individuals in an unconscious state and with an unfavorable outcome, in 51 samples were detected CAR antibodies ranging from 1, 6 to 60 ng / ml. Determination of the main characteristics of tests for diagnosing exit from coma [disease / test positive (True Positive) = 51, disease / test negative (False Negative) = 2, no disease / test positive (False Positive) = 1, no disease / test negative ( True Negative) = 89] showed a preliminary sensitivity of 96% and a specificity of 98% with a positive Likelihood Ratio of 86.6. Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the gist of the present invention.

Claims

 Claim A method for predicting a way out of an unconscious state in a subject, comprising  a) obtaining a biological sample from the specified subject;  b) at least a single analysis of said biological sample to determine the level of antibodies to one or more fragments of the kainate GluR6 and / or GluR7 receptors;  c) a comparison of this level with the level of the corresponding antibodies in samples taken from healthy subjects;  g) with a low level of antibodies in the sample of a subject in an unconscious state, not exceeding the control level recorded in samples of healthy subjects, predicting a favorable exit from the unconscious state;  e) with an increased level of antibodies in a sample of a subject in an unconscious state that exceeds the control level recorded in samples of healthy subjects, predicting an unfavorable outcome of the disease.  The method according to claim 1, wherein the unconscious state is a coma of traumatic, ischemic and / or metabolic origin.  The method according to claim 1, wherein whole blood, blood serum, plasma, cerebrospinal fluid, respiratory fumes, sweat and / or saliva are used as a biological sample.  The method according to claim 1, in which the level of antibodies is determined using enzyme-linked immunosorbent assay.  The method according to claim 1, including additional clinical, laboratory and / or instrumental examination methods. A method for predicting a way out of an unconscious state in a subject, comprising  a) obtaining at least two biological samples from the specified subject in the range from 1 hour to 90 days; b) analysis of these biological images to determine the level of antibodies to one or more fragments of the kainate receptors GluR6 and / or GluR7; c) comparing the levels of the corresponding antibodies determined in different samples of the same subject, with each other and with the level of the corresponding antibodies in samples taken from healthy subjects; d) by lowering the level of these antibodies in samples of a subject in an unconscious state over time, predicting a favorable exit from the unconscious state;  e) with an increase in the level of these antibodies in samples of a subject in an unconscious state over time, the risk of an unfavorable outcome of the development of the disease is detected, provided that this level exceeds the control level recorded in samples of healthy subjects.  7. The method according to claim 6, in which the unconscious state is a coma of traumatic, ischemic and / or metabolic origin.  8. The method according to claim 6, in which whole blood, blood serum, plasma, cerebrospinal fluid, respiratory fumes, sweat and / or saliva are used as a biological sample.  9. The method according to claim 6, in which the level of antibodies is determined using enzyme-linked immunosorbent assay.  10. The method according to claim 6, including additional clinical, laboratory and / or instrumental examination methods.