MXPA01008092A - Method for diagnosing and distinguishing stroke - Google Patents

Abstract

A method for determining whether a subject has had a stroke and, if so, the type of stroke which includes analyzing the subject's body fluid for at least four selected markers of stroke, namely, myelin basic protein, S100 protein, neuronal specific enolase and a brain endothelial membrane protein such as thrombomodulin or a similar molecule. The data obtained from the analyses provide information as to the type of stroke, the onset of occurrence and the extent of brain damage and allow a physician to determine quickly the type of treatment required by the subject.

Description

METHOD FOR DIAGNOSING AND DISTINGUISHING ATTACK BACKGROUND OF THE INVENTION This application is directed to a method to diagnose if a subject has had an attack and, if so, to distinguish between the various types of attack. More specifically, the method includes analyzing the body fluid of the subject by at least four selected attack markers. Diagnostic devices and equipment are also described for use in the method. The impact of an attack on the health of human beings is very great, when considered in terms of mortality and even more devastating when considering disability. For example, stroke is the third leading cause of death in adults in the United States, after ischemic heart disease and all forms of cancer. For people who survive, the attack is the main cause of the disability. The direct medical costs due to the attack and the cost of lost jobs represent billions of dollars annually. Approximately 85% of all attacks are ischemic (thrombotic and embolic) with the rest that are hemorrhagic. The attack is a market underserved by both therapeutic and diagnostic techniques. In the U.S., only more than 700,000 people have attacks every year. A multiple of this number is suspected to have had attacks with diagnoses only confirmed by expensive technology including computer-assisted tomography scans (CAT = Computer-Assisted Tomography) and magnetic resonance imaging (MRI = Magnetic Resonance Imaging). However, these sophisticated technologies are not available in all hospitals and are not sensitive enough to diagnose ischemic attack at an early stage. The attack is a clinical diagnosis made by a neurologist usually as a consultation. Current methods to diagnose the attack include evaluation of symptoms, medical history, chest X-rays, Electrical Cardiac Activity (ECG), Brain Nerve Cell Activity (EEG = Brain Nerve Cell Activity), CAT scan for estimate the damage to the brain and MRI to obtain internal visuals of the body. A number of blood tests can be performed to explore internal bleeding. These include complete blood count, prothrombin time, partial thromboplastin time, serum electrolytes, and blood glucose. Determining the immediate cause of an attack can be difficult, especially when it occurs when the diagnosis is based primarily on imaging techniques. Approximately 50% of brain infarcts are not visible on a CAT scan. In addition, even when a CAT scan can be very sensitive for the identification of hemorrhagic attack, it is not very sensitive to cerebral ischemia during evaluation of the attack and is usually positive 24 to 36 hours after the onset of an attack. As a result, a window of opportunity for rapid treatment will usually have expired once current diagnostic techniques positively identify an attack. The treatment of the attack includes preventive therapies such as antihypertensive drugs and antiplatelets that control and reduce blood pressure and thus reduce the likelihood of the attack. Also, the development of thrombolytic drugs such as tissue plasminogen activators (t-PA = tissue plasminogen activator) has provided a significant advance in the treatment of ischemic attack victims but to be effective and minimize the damage of the acute attack it is necessary to start treatment very early, for example within approximately three hours after the onset of symptoms. These drugs dissolve blood vessel clots that block blood flow to the brain and are the cause of approximately 80% of attacks. However, these drugs may also have side effects of increased risk of bleeding. Various neuroprotectants such as calcium channel antagonist can stop damage to the brain as a result of ischemic insult. The treatment window for these drugs is typically wider than that for clot solvents and does not increase the risk of bleeding. Techniques for early diagnosis of attack and identification of the type of attack are required to allow the physician to prescribe the appropriate therapeutic drugs at an early stage in the brain event. Different markers for the attack are known and have been described in the specialty analytical techniques for the determination of these markers. As used here, the term "marker" refers to a protein or other molecule that is released from the brain during a cerebral hemorrhagic or ischemic event. These markers include protein isoforms that are unique to the brain. It has been reported in the literature that the concentration of myelin basic protein (MBP = Myelin Basic Protein) in cerebrospinal fluid (CSF Cerebrospinal Fluid) increases after sufficient damage to neuronal tissue, head trauma and AIDS dementia. In addition, it has been reported that ultrastructural immunocytochemistry studies using anti-MBP antibodies have shown that MBP is located exclusively in the myelin lining. Thus, it has been suggested that MBP levels in CSF or serum be used as a marker of brain damage in acute cerebrovascular disease. See Strand, T., et al., Brain and plasma proteins in spinal fluid as markers for brain damage and severity of stroke (Spinal fluid plasma brain proteins as markers for brain damage and attack severity), Stroke (Attack) (1984) 15; 138-144. The increase in concentration of MBP in CSF is more evident in approximately four to five days after the onset of the thrombotic attack while brain hemorrhage and increase was the highest almost immediately after onset. See Garcia-Alex, A, and collaborators, Neuron-specific enolase and myelin basic protein: Relationship of cerebrospinal fluid concentration to the neurologic condition of asphyxiated full-term infants (Myelin basic protein and neuron-specific enolase: ratio of cerebrospinal fluid concentration to the neurological condition of fully developed, asphyxiated infants), Pediatrics (Pediatrics) (1994) 93; 234-240. It has also been found that patients with transient ischemic attack (TIA = Transitory Ischemic Attack) had normal CSF values for MBP whereas those with infarction to the brain and hemorrhages had high values. In infarction to the brain, there was a significant increase in MBP concentration in CSF from the first to the second lumbar puncture while patients with intracerebral hemorrhage reached already markedly elevated levels at the first lumbar puncture. It was reported that the kinetic difference in MBP release may be useful in the differential diagnosis of hemorrhagic and ischemic attack. MBP levels in CSF also correlate with the visibility of brain injury on CT scan and the short-term outcome of patients. In addition, the concentration of MBP increases with the extent of brain injury and high values indicate a poor short-term prognosis for the patient. See Strand, T. and colleagues, previously cited. The S100 protein is another marker that can be taken as a useful marker to assess neurological damage and to determine the extent of brain damage and to determine the extent of brain injury. In this way, its use as an auxiliary in the diagnosis and evaluation of brain injuries and neurological damage due to an attack has been suggested. See Missler, U., eismann, M., Friedrich, C. and Kaps, M., S100 protein and neuron-specific enolase concentrations in blood as indicators of infarction volume and prognosis in acute ischemic stroke (concentrations of neuron-specific enolase and protein SlOO in the blood as indicators of infarct volume and prognosis in acute ischemic attack), Stroke (Attack) (1997) 28; 1956-60. Neuron-specific enolase (NSE = Neuron-Specific Enolase) has also been suggested as a useful marker for neurological damage in the attack study with particular application in the evaluation of treatment. See Teasdale, G. and Jennett, B., Assessment of coma and impaired consciousness, Lancet (1974) 2; 81-84. There continues to be a need for diagnostic techniques that can provide timely information regarding the type of attack suffered by a patient, the onset of the occurrence, the location of the event, the identification of appropriate patients who will benefit from treatment with the appropriate drug and the identification of patients who are at risk of bleeding as a result of treatment. These techniques can provide data that will allow a physician to quickly determine the appropriate treatment required by the patient and allow early intervention. Therefore, it is an object of this invention to provide a method for quickly diagnosing and distinguishing attack.

A further objective of the invention is to provide a method for distinguishing between thrombotic attacks and hemorrhagic attacks. Another object of the invention is to provide such a method that includes analyzing the body fluid of a patient by at least four attack markers. Still another objective is to provide a method that provides information regarding the time of attack initiation. Still another objective is to provide testing devices for diagnosis, for use in the method. COMPENDIUM OF THE INVENTION These and other objects and advantages are achieved in accordance with the invention by providing a method that is capable of determining whether a patient has suffered an attack and if it is thrombotic or hemorrhagic. According to the method, a patient's body fluid is analyzed by four molecules that are cell type specific, three of which are specific ischemic markers, ie SlOO protein, myelin basic protein (MBP) and specific neuronal enolase ( NSE) and an endothelial membrane protein of the brain, for example thrombomodulin (Tm). The method analyzes the isoforms of . . . ^^^ Aea marker proteins that are specific to the brain. The analyzes of these markers can be carried out in the same sample of body fluid or in multiple body fluid samples. In the last modality, the different samples of body fluid can be taken at the same time or in different periods of time. The information that is obtained according to the method of the invention can be provided at critically important early stages of an attack, for example within the first three to six hours after the onset of symptoms, since the analysis of the patient's body fluid It can be carried out in approximately 45 to 50 minutes after the body fluid is collected. The data can be vital for the clinician to assist in determining how to treat a patient who has symptoms of an attack or is suspected of having an attack. The data can rule out the attack or consider it, and differentiate between ischemic and hemorrhagic attack and therefore exclude patients from hemorrhagic attack against the supply of the therapeutic agents that dissolve the clots, due to the risk of increased bleeding. The data can also identify patients who are at risk of bleeding as a result of treatment, ie patients with compromised brain vasculature. In addition, the method can provide at an early stage prognostic information regarding the intervention result that can improve patient selection for appropriate therapeutic agents and intervention. The method of the invention is diagnosed well in advance of imaging technologies. In addition, these data can indicate the location of an attack within the brain and the extent of damage to the brain as well as determine if the extent of the attack increases. Cerebral infarction associated with the attack, consisting of dead brain tissue in the process of death, which is formed due to inadequate oxygenation, typically increases in size during the acute period after ischemia begins. By measuring the markers in body fluid samples taken at different points in time, the progress of the attack can be evaluated. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be further described in detail with respect to various preferred embodiments thereof in conjunction with the accompanying drawings in which: Figure 1 is a graphic illustration of the concentration against time (in minutes) of two '^^^ «« aea marker proteins that are indicative of a brain condition or state; Figure 2 is a flow diagram illustrating how data obtained according to one embodiment of the invention can be used for diagnosis of brain condition or state; and Figures 3-10 are graphic illustrations of the concentration over time (in days) of four marker proteins that are analyzed according to one embodiment of the invention. DESCRIPTION OF PREFERRED MODALITIES The markers, which are analyzed according to the method of the invention, are released into the circulation and are present in the blood and other body fluids. Preferably the blood or any blood product containing them such as for example plasma, serum, cytolyzed blood (for example by treatment with hypotonic buffer or detergents), and their dilutions and preparations are analyzed according to the invention. In another preferred embodiment the concentration of the markers in CSF is measured. The terms "over normal" and "over threshold" as used herein to refer to a level of a marker that is larger than the level of the marker observed in normal individuals, ie individuals who are not subject to or are under brain event, that is, an injury to the brain that can be ischemic, mechanical or infectious. For some markers, infinitesimally low or null levels of the marker may normally be present in the blood of an individual. For others of the markers analyzed according to the invention, detectable levels may be normally present in the blood. In this way, these terms contemplate a level that is significantly above the normal level found in individuals. The term "significantly" refers to statistical significance and generally means a standard deviation of two (SD = Standard Deviation) on the normal or higher concentration of the marker present. The test method by which the analysis for any particular marker protein is carried out, must be sufficiently sensitive to be able to detect the level of the marker that is present over the concentration range of interest and must also be highly specific. The four primary markers that are measured according to the present method are proteins that are released by specific brain cells as the cells are damaged during a brain event. These proteins may already be in their native form or immunologically detectable sub-fragments of the proteins resulting, for example, by enzymatic activity of proteolytic decomposition. The four specific primary markers, when mentioned in the present application, including their claims, are intended to include fragments of the proteins that can be detected immunologically. By "immunologically detectable" is meant that the protein fragments contain an epitope that is specifically recognized by a connate antibody. As previously mentioned, the markers analyzed according to the method of the invention are specific to the cell type. Myelin basic protein (MBP) is a highly basic protein, located in the myelin sheath, and represents approximately 30% of the total protein of myelin in the human brain. The protein exists as a single polypeptide chain of 170 amino acid residues having a rod-like structure with dimensions of 1.5 x 150 nm and a molecular weight of about 18,500 Daltons. It is a flexible protein that exists in a random coil devoid of conformations to β-helices. The increased concentration of MBP in the blood and CSF is most evident approximately four to five days after the onset of an ischemic attack while in the cerebral hemorrhage the increase is higher almost immediately after onset. In addition, patients with TIA have normal values of MBP while those with infarction to the brain and intercerebral hemorrhage have elevated. A normal value for a person who has not had a brain event is from 0.00 to approximately 0.016 ng / mL. MBP has a half-life in serum of about one hour and is a sensitive marker for cerebral hemorrhage. SlOO protein is a cytoplasmic acidic calcium binding protein found predominantly in the gray matter of the brain, primarily in glia and Schwann cells. The protein exists in several homo- and heterodimeric isoforms consisting of two immunologically distinct subunits, alpha (MW = 10,400 Daltons) and beta (MW = 10,500 Daltons) while SlOOas is the aa homodimer that is found primarily in striated muscle, heart and kidney. The SlOOb isoform is the ßß homodimer of 21,000 Daltons. It is present in high concentration in glial and Schwann cells and thus is tissue specific. It is released during acute damage to the central nervous system and is a sensitive marker for infarction to the brain. According to the method of the invention, the assay is specific for the β-subunit of the SlOO protein. The SlOOb isoform is a specific brain marker released during acute damage to the central nervous system. It is eliminated by the kidney and has a half-life of approximately two hours in human serum. Repeated measures of serum levels are useful to follow the course of neurological damage. Additionally, the presence of elevated levels in CSF or serum in association with attack symptoms may be useful in the differential diagnosis of the attack and may be a valuable indicator of infarction to the brain. Enolase enolase (EC 4.2.1.11) catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate into the glycolytic pathway. The enzyme exists in three isoproteins each of the product of a separate gene. The gene sites have been designated ENOl, EN02 and EN03. The ENO1 gene product is non-neuronal enolase (NNE or a), which is widely distributed in various tissues of mammals. The gene product of EN02 is muscle-specific enolase (MSE or ß) which is located primarily in the cardiac and striated muscle, while the product of the EN03 gene is neuronal specific enolase (NSE or?) Which is substantially found in neurons and neuroendocrine cells. The native enzymes are found as homo- and heterodimeric isoforms composed of three immunologically distinct subunits, a, ß and y. Each subunit has an approximate molecular weight of 39,000 Daltons. jafeMaaaaa »The isoforms a? and YY enolase, which have been designated neuronal specific enolase (NSE) each have a molecular weight of approximately 80,000 Daltons. It has been shown that the concentration of NSE in CSF increases after experimental focal ischemia and the release of NSE against damaged brain tissue in CSF reflects the development and size of infarcts. NSE has a serum half-life of approximately 48 hours and its peak concentration has been shown to occur later after cerebral artery occlusion (MCA). NSE levels in CSF have been found elevated in acute and / or extensive disorders including sub-arachnoid hemorrhages and acute cerebral infarction. The fourth marker protein measured according to the invention is an endothelial membrane protein of the brain. The endothelial cells that line the small blood vessels of the brain have unique cell surface expression, receptors, transporters and intracellular enzymes that serve to regulate markedly the exchange of solutes between the blood and the cerebral parenchyma. Brain endothelial membrane proteins include: Thrombomodulin (Tm), a surface glycoprotein of 105,000 Daltons that is involved in the regulation of intravascular coagulation; Glucose transporter (Gluc 1), a transmembrane cell surface protein of 55,000 Daltons that can exist in dimeric or tetrameric form; Neurotelina / HT7, a protein of 43,000 Daltons integrated into the cytoplasmic membrane transport protein; Gamma Glutamyl Transpeptidase, a protein that is found as a heterodimeric isoform composed of subunits of 22,000 and 25,000 Daltons and is involved in the transfer of gamma glutamyl residue from glutathione to amino acids; and P-glycoprotein, a protein that extends into the membrane resistant to multiple drugs. In a preferred embodiment of the Tm method it is the endothelial membrane protein of the brain that is measured. Tm is a sensitive marker for lacunar infarcts. The data obtained according to the method indicate whether an attack has occurred and if so, the type of attack, the location of the damage and the dispersion or extent of the damage. When the levels of all four markers are negative, that is, within a normal range, there is no brain injury. When only the endothelial membrane protein of the brain, for example Tm, is elevated, or positive, ie the level is at least 2SD above normal, the attack is a lacunar infarct present in the basal ganglia and in the deep white matter of the brain. brain. When the level of NSE is positive and the levels of SlOO and / or MBP are negative (the endothelial membrane protein marker of the brain is positive or negative) the patient has suffered a TIA. According to another preferred embodiment, a fifth marker, which is of the cell type specific to one of the three ischemic markers analyzed according to the method of the invention, is measured to provide information regarding the time of onset of the attack. It should be recognized that the onset of attack symptoms is not always known, particularly if the patient is unconscious or older and a reliable medical history is not always available. An indication of the time of onset of the attack can be obtained by resorting to the kinetic differences in the release of brain markers that have different molecular weights. The release in time of markers from the brain to the circulation following the brain injury depends on the size of the marker, with smaller markers that tend to be released earlier in the event while the larger markers tend to be released later. Figure 1 illustrates the release kinetics of two marker proteins that are analyzed according to the method of the invention, ie MBP and SlOO. These data were obtained from fluid collected from the brain tissue of a pig after coronary bypass surgery. Samples were collected at 0, 30, 120, 180 and 240 minutes after the subject was removed from the bypass machine. The concentration values are expressed in multiples of a baseline value that was the concentration at time zero. These data indicate that the release of MBP (MW = 18,500) seems to reach a maximum of approximately 120 minutes after the ischemic event while the release of SlOO (MW = 21,000) does so after approximately 180 minutes. In this way, when measuring an additional protein marker of the cell type specific to one of the three ischemic markers used in the method of the invention, data relating to the start time can be obtained. The start time is defined as the time when the clinical symptoms of the attack appear. In this preferred embodiment, the second marker protein is a marker of higher molecular weight, ie higher, than the primary marker of the same type of cell. The three ischemic markers used according to the invention and various other high molecular weight markers of the same specific cell type are illustrated in Table I. jt _ _,, _, > , __,. . , .. A A In a preferred embodiment of the invention, body fluid samples taken from a patient at different points in time are analyzed. Typically a first sample of body fluid is taken from a patient exhibiting attack symptoms and analyzed according to the invention. Subsequently, some period of time after the presentation, for example, approximately two hours after the presentation, a second sample of body fluid is taken and analyzed according to the invention. Now with reference to Figure 2, there is a flow diagram illustrating how the data obtained from the four marker proteins analyzed according to the invention, in the illustrated embodiment NSE, SlOO, MBP and Tm, can be used for triage of the patient . The data can be used to diagnose attack, rule out attack, distinguish between thrombotic and hemorrhagic attacks, identify the appropriate patients for thrombolytic treatment and determine how the attack evolves. As previously stated, the level of each of the four specific markers in the patient's body fluid can be measured from a single sample or one or more individual markers can be measured in a sample and at least one marker measured in one or more additional samples . By "sample" is meant a volume of body fluid such as blood or CSF that is obtained at a point in time. Furthermore, as will be discussed in detail below, all markers can be measured with a test device or by using a separate assay device for each marker, in which case aliquots of the same fluid sample or different fluid samples can be used. It is apparent that the analyzes should be carried out within a short time frame after the samples are taken, for example in about half an hour, so that the data can be used to determine the treatment as quickly as possible. It is preferred to measure each of the four markers in the same simple sample, regardless of whether the analyzes are carried out in a single analytical device or in separate devices such that the level of each marker simultaneously present in a single sample can be used. to provide insignificant data. Generally speaking, the presence of each marker is determined by using specific antibodies for each of the markers and detecting the immunospecific binding of each antibody to its respective connate marker. Any convenient immunoassay method can be used, including those that are commercially available, to determine the level of each of the specific markers that are measured according to the invention. An extensive discussion of the known immunoassay techniques is not required here since they are known to those skilled in the art. Typical convenient immunoassay techniques include sandwich linked enzyme immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, homogeneous assays, heterogeneous assays, etc. Several of the known immunoassay methods are performed in "Methods in Enzymology" (Methods of Enzymology), 70, pgs. 30-70 and 166-198 (1980). Direct and indirect labels can . » Mtaamtaa is used in immunoassays. A direct label can be defined as an entity, which in its natural state is visible either with the naked eye or with the aid of an optical filter and / or applied stimulus, for example ultraviolet light, to promote fluorescence. Examples of colored labels that may be used include metal sol particles, gold sol particles, coloring sol particles, dyed latex particles, or liposome encapsulated dyes. Other direct labels include radionuclides and fluorescent or luminescent portions. Indirect labels such as enzymes can also be employed according to the invention. Various enzymes are known to be used as labels such as for example, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease. For a detailed discussion of enzymes in immunoassays see Engvall, Enzyme Immunoassay ELISA and EMIT, Methods of Enzymology (ELISA and EMIT Enzyme Immunoassay, and Methods of Enzymology), 70, 419-439 (1980). A preferred immunoassay method for use according to the invention is a double antibody technique for measuring the level of marker proteins in the body fluid of the patient. According to this method, one of the antibodies is a "capture" antibody and the other is a "detector" antibody. The capture antibody is immobilized on a solid support which can be any of several types that are known in the art such as, for example, wells of microtitre plates, beads, tubes and porous materials such as nylon, glass fibers and other materials polymeric In this method, a solid support, for example wells of microtitre plates, coated with a capture antibody, preferably monoclonal, developed against the particular marker protein of interest, constitutes the solid phase. Patient body fluid, diluted, for example serum or plasma, typically approximate 25 μl, standards and controls are added to separate solid supports and incubate. When the marker protein is present in the body fluid it is captured by the immobilized antibody that is specific for the protein. After incubation and washing, an anti-marker protein detecting antibody, for example an anti-polyclonal rabbit marker protein antibody, is added to the solid support. The detector antibody is ligated to the marker protein linked to the capture antibody to form a sandwich structure. After incubation and washing of an anti-IgG antibody, for example a polyclonal goat anti-rabbit IgG antibody, labeled with an enzyme such as horseradish peroxidase (HRP), is added to the solid support. After incubation and washing, a substrate for the enzyme is added to the solid support followed by incubation and the addition of an acid solution to stop the enzymatic reaction. The degree of enzymatic activity of immobilized enzyme is determined by measuring the optical density of the oxidized enzyme product in the solid support at the appropriate wavelength, for example 450 nm for HRP. The absorbance at the wavelength is proportional to the amount of marker protein in the fluid sample. A set of marker protein standards is used to prepare a standard curve of absorbance against the concentration of marker protein. This method is preferred since the test results can be provided in 45 to 50 minutes and the method is both sensitive over the concentration range of interest for each marker and is highly specific. The test methods used to measure marker proteins should exhibit sufficient sensitivity to be able to measure each protein over a range of concentration from normal values found in healthy people at high levels, ie 2SD above normal and beyond. Of course, a range of normal value of marker proteins can be found when analyzing the body fluid of healthy people. For the SlOOb isoform where + 2SD = 0.02 ng / mL, the upper limit of the test range of preference is approximately 5.0 ng / mL. For NSE where + 2SD = 9.9 ng / mL the upper limit of the preference range is approximately 60 ng / mL. For MBP, which has a high level cut-off value of 0.02 ng / mL, the upper limit of the preferred test range is approximately 5.0 ng / mL and for Tm, which has a high level cut-off value of approximately 73 ng / mL, the upper limit of the assay range of preference is approximately 500 ng / mL. The assays can be carried out in various test device formats including those described in U.S. Pat. Nos. 4, 906,439; 5,051,237 and 5,147,609 to PB Diagnostic Systems, Inc. The test devices used in accordance with with the invention can be arranged to provide a semiquantitative or quantitative result. The term "semiquantitative" is understood as the ability to discriminate between a level that is above the high marker protein value and a level that is not above that threshold.

The assays can be carried out in various formats including, as discussed previously, a microtiter plate format that is preferred to carry out the assays in a batch mode. The tests will also be carried out in analyzers of automated immunoassays that are well known in the specialty and they can carry out tests on a number of different samples. These automated analyzers include continuous / random access types. Examples of these systems are described in US Patents. Nos. 5,207,987 and 5,518,688 issued to PB Diagnostic Systems, Inc. Various automated analyzers that are commercially available include the OPUSMR and OPUS MAGNUMMR analyzers. Another test format that can be employed according to the invention is a rapid manual test that can be administered at the point-of-care anywhere. Typically, these point-of-attention testing devices can provide a result that is above or below a threshold value, i.e. a semiquantitative result as previously described. It should also be recognized that the test devices used according to the invention can be provided to carry out a single test for a particular marker protein or to carry out a plurality of tests, from a single volume of body fluid to a corresponding number of tests. different marker proteins. A preferred test device of the latter type is one that can provide a semiquantitative result for the four primary marker proteins that are measured according to the invention, ie SlOOb, NSE, MBP and a brain endothelial marker protein, e.g. . These devices are typically adapted to provide a distinct visually detectable color band wherein the capture antibody for the particular marker protein is localized when the concentration of the marker protein is above the threshold level. For a detailed discussion of test types that may be used in accordance with the invention as well as various test formats and automated analyzer apparatuses see U.S. Pat. No. 5,747,274 awarded to Jackowski. The invention will now be described in more detail with respect to specific preferred embodiments, it being understood that these are intended to be illustrative only and that the invention is not limited to the materials, processes, etc., described therein. EXAMPLE A pilot observational pilot study was conducted in two tertiary care hospitals. The study evaluated thirty-three admitted patients with a clinical diagnosis and computed tomography (CT) of acute ischemic attack. The average age of the patients presenting the attack was approximately 66 years (66.4 + 16.4) with an age range of 27 to 90 years. The average delay between the onset of symptoms and presentation of the hospital was 22 hours with a range of 1 to 72 hours. Registered Rankin scale scores of Admission in National Institutes of Health Attack Scale, Attack and Discharge Scale were recorded. Blood samples were obtained on days 1 (presentation), 3, 5 and 7 in a hospital and days 1, 2 and 3 in the second hospital. All blood samples were centrifuged and aliquots of serum were frozen and stored at -80 ° C until analysis by SlOO, NSE, MBP and Tm. Control subjects included one hundred and three healthy blood donors (age range of 18 to 78 years, average age 54.6 + 15.2 years) whose blood samples were used to determine reference values for levels of SlOO and NSE and twenty-four healthy blood donors who provided samples for reference measurements of MBP and Tm concentrations. All values of reference values are reported as average + 2SD unless otherwise stated. The reference value for SlOO in serum was 0.0067 ng / mL, with a 98th percentile of 0.020 ng / mL. A high SlOO value is taken as any concentration greater than 98th percentile (0.02 ng / mL) normal (normal + 2SD = 0.02 ng / mL). ; t The reference value for serum NSE was 5.03 +2.40 ng / mL. A high NSE value was any concentration greater than 2 SD over normal, 9.85 ng / mL. The reference value for MBP in serum was 0.0162 +0.0019 ng / mL. A high MBP value was any concentration greater than 2SD above normal, 0.02 ng / mL. The reference value for serum Tm was 50.52 +13.62 ng / mL. A high Tm value was any concentration greater than +2SD above normal, 76.14 ng / mL. SlOO and NSE levels were analyzed using the ELISA Exact SlOO and Exact NSE Testing Apparatus, respectively available from Skye Pharma Tech Inc., Mississauga, Canada. Tm levels were analyzed with an ELISA assay available from Diagnostica Stago, 9 rue des Freres Chausson, 92600 Asneres Sur Seine, France. The MBP concentration level was analyzed with an ELISA immunoassay from Diagnostic Systems Laboratories, Webster, Texas, E.U.A. In the tables that show the data obtained "DI" indicates the first day with the first blood sample taken at the time of presentation. Subsequent days of sample collection are indicated by D2, D3, etc. For the values of the concentrations of the markers, + 2SD are above the normal range. "ND" means that no data was obtained. i '^ t-if-rfi ii 10 • & í & . .. * .. í.i i - "* £ * ¿** ... t.1. & A < k X? - LIMA *** á ^^^ i ^ - faAs-jfe. a - * a, éXí &ÁX *. xx i .h. k.

-JJ ¿fil ^ a ¿t üfc ^^ stei £ "». ».. -i * 10 fa 15 Á.ti - »SL * J ¿ The analysis of SlOO, NSE and MBP levels in serum samples of healthy control subjects showed no relationship of levels of these proteins with age or sex. In the case of Tm, the concentrations were higher in serum samples of healthy male subjects than in females (54.62 113.62 ng / mL, 2SI) over normal = 81.86 ng / mL and 43.63 +11.18 ng / mL, 2SD over normal = 68.74 ng / mL, respectively).

Of the thirty-three attack patients, twenty-six were heart attacks (79%) and all five were lacunar (15%) and four had a hemorrhagic attack (12%). Of the hemorrhagic attack patients three had subarachnoid hemorrhages and one had intracerebral bleeding. Three patients (9%) had transient ischemic attacks (TLA). Upon presentation, SlOO levels rose in 44% of patients, NSE levels rose by 59%, MBP levels rose by 40% and Tm levels rose by 57%. The data indicate that when measuring the four marker proteins according to the invention, where any marker was raised, 94% of the patients could be identified upon presentation. Nineteen of the twenty-one non-lacunar infarctions (90%) could be identified upon presentation. The two remaining patients arrived at the hospital at 22 and 72 hours respectively after the onset of symptoms. Each of Figures 3-10 is a graphic illustration of the data obtained from a patient different from the study. The concentration levels are expressed as multiples of a reference value and were obtained by dividing the current measured concentration values by defined reference value for each respective marker protein, ie the 2SD value. All lacunar infarctions, hemorrhagic patients and ATI were identified with presentation with 100% accuracy. All five lacunar infarcts had high levels of Tm upon presentation. In some patients, the only high marker protein was Tm. Now with reference to Figure 3, it can be seen that, for the SM7 patient, the only elevated marker protein was Tm indicating a lacunar infarct. The three AIA patients had high NSE levels and normal SlOO and MBP levels that remained within the normal range. Tm rose in one of the patient AUNT. Now with reference to Figure 4 it can be seen that for the patient SM-24, Tm was slightly elevated and NSE was elevated indicating a TIA. The patient was discharged with a diagnosis of TIA. Now with reference to Figure 5, it can be seen that the patient SM-3 had enormously high levels of MBP and SlOO as well as elevated levels of NSE and Tm indicating a stroke to the brain with damage that is dispersed in the base of the brain. In the four hemorrhagic attack patients, the three subarachnoid hemorrhagic patients had high levels of SlOO and NSE and a normal Tm level. In the patient with an intracerebral hemorrhagic attack, the t ~ t * -. . . i * & lb? M levels of SlOO and NSE were elevated and the MBP level was raised to approximately 250 times. Figure 6 illustrates that patient SJ-16 had an increased 250-fold level of MBP upon presentation as well as elevated levels of SlOO and NSE and suffered from intracerebral hemorrhage. Figure 7 illustrates that the SJ-2 patient had elevated MBPs Tm and SlOO upon presentation and that the levels of MBP and SlOO continue to increase over time indicating a stroke to the brain with the attack increased over time. An initial CAT scan before presentation was negative and became positive only days later. Figure 8 illustrates that patient SJ-18 presented a TIA that evolved into an attack. Tm was in the normal range indicating that the cerebral vasculature was not compromised and thus indicating that the patient was a good candidate for thrombolysis. Figure 9 illustrates that the patient SM-8 presented a cerebral infarction and with Tm in the normal range, was a good candidate for thrombolysis, since the endothelial vasculature was not compromised. Figure 10 illustrates that patient SJ-1 had an infarct to the brain and due to the elevated Tm level he was at risk of bleeding if he was given thrombolytics because the endothelial vasculature was compromised.

For the second serum sample obtained, SlOO levels increased in 73% of attack patients, NSE levels in 54%, MBP levels in 64% and Tm levels in 55%. These data indicate that when measuring the four marker proteins according to the invention, where with any marker that is elevated, 96% of the patients could be identified from the second serum sample obtained. The data indicate that levels of protein markers in subsequent serum samples either increased or decreased depending on whether the severity of the attack increased or decreased.

Of the thirty-three attack patients, eighteen (54%) had a CAT scan performed on presentation. All four haemorrhagic attack patients were CAT positive upon presentation. Nine (50%) of the eighteen patients had a normal CAT on presentation that became positive days later. Eight of the nine patients who had a normal CAT on presentation had high levels of one or more of the four protein markers on presentation.

All nine positive CAT patients on presentation had high levels of one or more protein markers upon presentation. l tt. t SlOO, NSE and MBP peak levels were significantly correlated (Pearson 's) with NIHSS admission ratings (p <0.05) and modified output Rankin classification (p <0.05). The data show that levels of SlOO, NSE, MBP and Tm can be measured easily and reliably in patients with acute ischemic attack and that by measuring these four marker proteins according to the invention, when any marker protein is elevated, sensitivity can be achieved 94% for acute ischemic attack upon presentation. Furthermore, in the hyperacute period of the evolving attack, elevated levels of one or more of these four marker proteins appear to precede irreversible tissue damage and brain edema before detection of this damage by CAT. Although the invention has been described with respect to various preferred embodiments, it is not intended to be limited thereto but rather those skilled in the art will recognize that variations and modifications may be made that are within the spirit of the invention and the scope of the claims. annexes.

Claims (33)

1. CLAIMS 1. A method to diagnose and distinguish attack, characterized because it includes: analyzing the body fluid of a patient to detect the presence and concentration of four attack markers where: i. A first marker is a myelin basic protein, ii. A second marker is the beta isoform of the SlOO protein, iii. A third marker is neuronal specific enolase, iv. A fourth marker is a brain endothelial cell membrane protein and b. From the information obtained from the analyzes, verify if a hemorrhagic or ischemic cerebral event has occurred and differentiate a particular type of brain event.
2. 2. A method according to claim 1, characterized in that the body fluid is selected from the group consisting of blood, blood products and cerebrospinal fluid.
3. 3. A method according to claim 1, characterized in that each of the analyzes is carried out on the same body fluid sample.
4. 4. A method according to claim 1, characterized in that at least one of the analyzes is carried out in a first fluid sample. body and at least one other analysis is carried out in a second body fluid sample.
5. 5. A method according to claim 4, characterized in that the first and second body fluid samples are taken at different periods of time.
6. 6. A method according to claim 1, characterized in that the endothelial cell membrane protein of the brain is selected from the group consisting of thrombomodulin, glucose transporter 1, in the dimeric or tetrameric form, Neurotelin / HT7, Gamma Glutamyl Transpeptidase, P-glycoprotein and its combinations.
7. 7. A method according to claim 1, characterized in that at least one of the analyzes comprises contacting the body fluid with an antibody that is specific for the marker.
8. 8. A method according to claim 7, characterized in that at least one of the analyzes is carried out with an enzyme-labeled immunoassay method.
9. 9. A method according to claim 1, characterized in that it also includes the step of analyzing the body fluid for a fifth marker protein, wherein the fifth marker protein it has the same specific cell type as one of the first, second or third markers and has a higher molecular weight than the first, second or third markers that have the same specific cell type.
10. 10. A method according to claim 9, characterized in that at least one of the analyzes comprises contacting the body fluid with an antibody that is specific for the marker.
11. 11. A method according to claim 10, characterized in that at least one of the analyzes is carried out with an immunoassay method labeled with enzyme.
12. 12. A method according to claim 1, characterized in that it also includes the In a step of analyzing a second sample of body fluid from the patient by the four markers, the second sample of body fluid is taken at a time subsequent to the body fluid analyzed in step a.
13. 13. A method according to claim 1, characterized in that the steps of verifying and differentiating include comparing the level of concentration detected in the analysis for each of the four markers at a predefined threshold level for each marker. t ^ 1 TlffWSWÉIiiii- 1 - "I tff 14. A diagnostic equipment for diagnosing and distinguishing attack, characterized in that it comprises at least four antibodies that are specific for each of four different marker proteins, the antibodies are immobilized on a solid support, characterized in that it comprises: a. a first marker protein is myelin basic protein and a first antibody is specific for it, b. a second marker protein is the beta isoform of SlOO protein and a second antibody is specific for it, c. a third marker protein is neuronal specific enolase and a third antibody is specific for it, and d. a fourth marker protein is a brain endothelial cell membrane protein and a fourth antibody is specific for it and at least four labeled antibodies, each of the labeled antibodies is linked to one of the marker proteins. 15. A diagnostic device according to claim 14, characterized in that each of the four antibodies is immobilized in the same solid support. 16. A diagnostic device according to claim 14, characterized in that at least one of the four antibodies is immobilized in a first solid support and at least one of the four antibodies is immobilized in a second solid support. 17. A diagnostic kit according to claim 14, characterized in that at least one of the labeled antibodies comprises an antibody labeled with enzyme. 18. A diagnostic kit according to claim 14, characterized in that the brain endothelial cell marker protein is selected from the group consisting of thrombomodulin, Glucose 1 transporter in the dimeric or tetrameric form, Neurotelin / H17, Gamma Glutamyl Transpeptidase , P-glycoprotein and its combinations. 19. A diagnostic kit according to claim 14, characterized in that it also includes a fifth antibody that is specific for a fifth marker protein, wherein the fifth marker protein has the same specific cell type as one of the first, second and third. markers and has a higher molecular weight than the first, second and third markers that has the same specific cell type, and a fifth labeled antibody that binds to the fifth marker protein. 20. A diagnostic kit according to claim 19, characterized in that the fifth labeled antibody comprises an antibody labeled with enzyme. 21. A method for the differential diagnosis of cerebral ischemic and hemorrhagic events, characterized in that it comprises: a. analyzing the body fluid of a patient to detect the presence and level of concentration of one or more ischemic marker proteins selected from the group consisting of myelin basic protein, the beta isoform of SlOO protein, neuronal specific enolase and combinations thereof, b. analyzing the patient's body fluid to detect the presence and level of concentration of an endothelial cell membrane protein of the brain, and e. of the information obtained from the analyzes, verify the occurrence of an ischemic or hemorrhagic cerebral event and differentiate a particular type of cerebral event. 22. A method according to claim 21, characterized in that the step of verifying and differentiating includes comparing the levels of concentration detected in the analyzes for one or more ischemic marker proteins and for the endothelial cell membrane protein of the brain, to a predefined threshold level for each ischemic marker protein and for the endothelial cell membrane protein of the brain. ^^^ j ^^ j ^^^^^^^ .Y'iii ^ j-fy ^ '* - £ • ".«. * - - »-' ~ - * & • ** - • ' i & SS & -I- 23. A method according to claim 21, characterized in that the body fluid is selected from the group consisting of blood, blood products and cerebrospinal fluid. claim 21, characterized in that the endothelial cell membrane protein of the brain is selected from the group consisting of Thrombomodulin, Glucose Transporter 1 in dimeric or tetrameric form, Neurotelin / HT7, Gamma Glutamyl Transpeptidase, P-glycoprotein and combinations thereof. A method according to claim 24, characterized in that the brain endothelial cell membrane protein is 1 Thrombomodulin 26. A method according to claim 21, characterized in that it also includes analyzing the body fluid to detect the presence and level of concentration of a secondary marker protein that e has the same specific cell type as one of the myelin basic protein, SlOO protein isoform or neuronal specific enolase, with which the time of onset of a hemorrhagic or ischemic cerebral event can be determined. IAJAM AJJ .- > .. «tri» s i »?? 27. A method according to claim 26, characterized in that the second marker protein has a higher molecular weight than the myelin basic protein, SlOO or neuronal protein isoform having the same specific cell type. 28. A method according to claim 21, characterized in that each of the analyzes is carried out on the same body fluid sample. 29. A method according to claim 21, characterized in that at least one of the analyzes is carried out in a first sample of body fluid and at least another of the analyzes is carried out in a second body fluid sample. 30. A method according to claim 29, characterized in that the first and second body fluid samples are taken at different time periods. A method according to claim 21, characterized in that a plurality of body fluid samples are obtained at predefined time intervals and analyzed and the analysis information is compared as a function of time with what the advance of an event Ischemic or hemorrhagic stroke can be determined. UjAnA-i-rij, *. ****** ". A, i i -i », 32. A method according to claim 21, characterized in that each of the analyzes comprises contacting the body fluid with an antibody that is specific for the protein. 33. A method according to claim 32, characterized in that at least one of the analyzes is carried out with an immunoassay method labeled with enzyme. l *.? *.?. A? - * ~ .. ^. ±? »^ .. t t | .. ^ jjgHfj ^