Title of Invention

" A KITFOR DIFFERENTIAL DIAGNOSIS "

Abstract

The present invention provides a kit for differential diagnosis. The kit provides for the identification and use of diagnostic markers for differential diagnosis of diseases and/or conditions. In a various aspects, the invention relates to methods and compositions able to determine the presence or absence of one, and preferably a plurality, of diseases or conditions that exhibit one or more similar or identical symptoms. Such methods and compositions can be used to provide assays and assay devices for use in determining the disease or condition underlying one or more non-specific symptoms exhibited in a clinical setting.

Full Text

The present invention relates to a kit for the identification and use of diagnostic markers for differential diagnosis of diseases and conditions. In a various aspects, the invention relates to methods and compositions able to determine the presence or absence of one, and preferably a plurality, of diseases and/ or conditions that exhibit one or more similar or identical symptoms. BACKGROUND OF THE INVENTION [0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention. [ 0003 ] The clinical presentation of certain diseases and conditions can often be strikingly similar, even though the underlying diseases, and the appropriate treatments to be given to one suffering from the various diseases, can be completely distinct. For example, subjects may present in an urgent care facility exhibiting a deceptively simple constellation of apparent symptoms (e.g., fever, shortness of breath, dizziness, headache) that may be characteristic of a variety of unrelated conditions. Differential diagnosis methods involve the comparison of symptoms and/or diagnostic test results known to be associated with one or more diseases that exhibit a similar clinical presentation to the symptoms and/or diagnostic results exhibited by the subject, in order to identify the underlying disease or condition present in the subject. [0004] Taking shortness of breath (referred to clinically as "dyspnea") as an example, patients often prqlijit in a clinical setting with this symptom as the initial clinical presentation. This syjcnptom considered in isolation may be indicative of conditions as diverse as asthma, chronic obstructive pulmonary disease ("COPD"), trachea! stenosis, obstructive endobroncheal tumor, pulmonary fibrosis, pneumoconiosis, lymphangitic carcinomatosis, kyphoscoliosis, pleura! effusion, amyotrophic lateral sclerosis, .congestive heart failure, V coronary artery disease, myocardial infarction, cardiomyopafhy, valvular dysfunction, left ventricle hypertrophy, pericarditis, arrhythmia, pulmonary embolism, metabolic acidosis, chronic bronchitis, pneumonia, anxiety, sepsis, acute coronary syndrome, aneurismic dissection, etc. See, e.g..Ketteys Textbook of Internal Medicine, 4th Ed., Lippincott Williams s, Philadelphia, PA, 2000, pp.. 2349-2354, "Approach to the Patient With Dyspnea"; et al., J. Gen. Int. Med. 8: 383-92 (1993). [0005] pjfJCTentijJ^iajmosis in the case of dyspnea involves identifying the particular condition causing shortness of breath in a given subject from amongst numerous possible causes. These methods often require that the climcian integrate information obtained from a battery of tests, leading to a clinical diagnosis that most closely represents the range of symptoms and/or diagnostic test results obtained for the subject. The tests required may include radiography, electrocardiogram, exercise treadmill testing, blood chemistry analysis, echocardiography, bronchoproyocation testing, spirometry, pulse oximetry, esophageal pH i monitoring, laryngoscopy, computed tomography, histology, cytology, magnetic resonance imaging, etc. See, e.g., Morgan rnd Hodge, Am. Fam. Physician 57: 711-16 (1998). Because of the variety of tests that may need to be performed, obtaining sufficient information to arrive at a diagnosis can take hours or even days. [0006] Differential diagnosis .of chest pain requires the clinician to consider many possible causes, including differentiating between respiratory pain and pain associated with angina, or myocardial infarction and pleuritic and chest wall pain. [0007] Differential diagnjQSiSjof diastolic and systolic dysfunction in patients suffering from heart failure is important since the therapies for each dysfunction are different. Further differentiation of atrial fibrillation from heart failure is critical for appropriate therapy. [0008] In the area pfinfection, differential diagnosis of viral versus bacterial is critical to the clinician delivering the appropriate therapy. [0009] The acuteness or jeyerity pf the symptoms often dictates how rapidly a diagnosis must be established and treatment initiated. Immediate diagnosis and care of a patient experiencing a variety of acute conditions associated with dyspnea and chest pain can be critical. See, e.g., Harris, Aust. Fam. Physician 31: 802-06 (2002) (asthma); Goldhaber, Eur. Respir. J. Suppl. 35: 22s-27s (20C2) (pulmonary embolism); Lundergan et al., Am. Heart J. 144:456-62 (2002) (myocardial infarction). However, even in cases where the apparent symptoms appear relatively stable, rapid diagnosis, and the rapid initiation of treatment, can provide both relief from immediate discomfort and advantageous improvement in prognosis. .WQ10] Each reference cited in the preceding section is hereby incorporated by reference in its «*jj#rety, including all tables, figures, and claims. STATEMENT OF THE INVENTION According to the present invention there is provided a kit for differential diagnosis comprising: reagents for analyzing a test sample obtained from a subject for the presence or amount of a plurality of subject-derived markers selected to identify the presence or absence in said subject of a plurality of conditions comprising myocardial infarction, pulmonary embolism, and congestive heart failure, wherein said reagents comprise a plurality of antibodies, said plurality of antibodies comprising individual antibodies that bind to each of said plurality of subject-derived markers. SUMMARY OF THE INVENTION [0011 ] The present invention relates to the identification and use of diagnostic markers for differential diagnosis of diseases and conditions and prediction of clinical outcomes. The methods and compositions described herein can meet the need in the art for rapid, sensitive and specific diagnostic and prognostic assays to be used in the diagnosis and differentiation of various diseases that are related in terms of one or more clinical characteristics. [0012J In various aspects, the invention relates to materials and procedures for identifying the underlying cause of one or more symptoms that, when considered in isolation, may be related to a plurality of possible underlying diseases or conditions; to using such markers in -diagnosing and treating a patient and/or to monitor the course of a treatment regimen; to using such markers to identify subjects at risk for one or more adverse outcomes an underlying disease or condition; and for screening compounds and pharmaceutical compositions that iniglii pio vide a benefit in treating or preventing such diseases or conditions. 10013] In traditional methods to evaluate marker levels in the diagnosis or prognosis of disease, a "threshold" for a marker of interest is typically established, and the concentration of that marker in a sample is compared to that threshold amount; an amount greater than the preestablished threshold is indicative of one state (e.g., disease), and an amount less than the preestablished threshold is indicative of another state (e.g., normal). For example, the American Heart Association has stated that a cardiac troponin I concentration greater that the 99th percentile concentration in the normal population should be used to rule in myocardial infarction. In the methods described herein, such threshold concentrations may be established for one or more markers, and these thresholds used for determining the diagnosis/prognosis of a subject in a similar fashion. As the number of markers in a pane! increase, however, applying individual thresholds to each marker can become unwieldy. Thus, in certain preferred embodiments in which a plurality of markers are evaluated, particulTt thresholds for one or more markers in the marker panel are not relied upon to determine a particular diagnosis and/or prognosis. Rather, the present invention may utilize an evaluation of the plurality of markers as a unitary whole. In a simple example, the ratio of two or more markers, rather than an absolute amount of the markers, may be used to determine a diagnosis/prognosis. Even more preferably, however, a particular "fingerprint" pattern of changes in such a panel of markers may, in effect, act as a specific diagnostic or prognostic indicator. Methods for determininga "panel response value" that integrates a plurality of marker concentrations into a single result are described hereinafter. In. these methods, each marker concentration measured it. a sample contributes to this panel response value, which may be compared to a threshold panel response as if it were simply the concentration of a single marker. This is an exampl; of a diagnostic method wherein the amount of one or more the markers is not compared to a predetermined threshold level. [0014] In a first aspect, the invention discloses methods for determining the presence or absence of a disease or condition (a "diagnosis") in a subject that is exhibiting a perceptible change in one or more physical characteristics (that is, one or more "symptoms") that are indicative of a plurality of possible etiologies underlying the observed symptom(s). These methods comprise analyzirg a te.it sample obtained from the subject for the presence or amount of one or more markers for one or more of the possible etiologies of the observed symptom(s). The presence or amount of such markers) in a sample obtained from the subject can be used to rule in or rule out one or more of the possible etiologies, thereby either providing a diagnosis (rule-in) and/or excluding one or moire diagnoses (rule-out). [0015] In certain embodiments, these markers can be used to rule in or rule out one or more possible etiologies of short-less of breath, or "dyspnea." While the present invention is described hereinafter generally m terms of the differential diagnosis of diseases and conditions related to dyspnea, thy skilled artisan will understand that the concepts of symptom-based differential diagnosis described herein are generally applicable to any physical characteristics that are indicative of a plurality of possible etiologies such as fever, chest pain (or "angina"), abdominal pain, neurologic dysfunction, disturbances in metabolic state, such as aberrant water, electrolyte, mineral, or acid-base metabolism, hypertension, dizziness, headache, etc. [001(|] In preferred embodiments, the present invention relates to methods in which a test X. sample is analyzed for the presence or amount of a plurality of markers related to a plurality of possible etiologies, so that the method is adapted to rule in or out a plurality of possible underlying causes based upon the analysis of a single sample. [0017] In the case of dyspnea, the plurality of markers are preferably selected to rule in or out one or more, and preferably a plurality, of the following diagnoses: asthma, atrial fibrillation, chronic obstructive pulmonary disease ("COPD"), trachea! stenosis, obstructive endobroncheal tumor, pulmonary fibrosis, pneumoconiosis, lymphangitic carcinomatosis, kyphoscoliosis, pleura! effusion, amyotrophic lateral sclerosis, congestive heart failure, coronary artery disease, myocardial infarction, acute coronary syndrome, cardiomyopathy, valvular dysfunction, left ventricle hypertrophy, pericarditis, arrhythmia, pulmonary embolism, metabolic acidosis, chronic bronchitis, pneumonia, anxiety, sepsis, or aneurismic dissection. In a particularly preferred embodiment, the methods relate to defining the cause of dyspnea to rule in or rule out myocardial ischemia and cardiac necrosis, heart failure and pulmonary embolism. In yet another particularly preferred embodiment, the methods relate to defining the cause of dyspnea to rule in or rule out myocard. a! ischemia and cardiac necrosis, hemi fiiiiiuc, pulmonary embolis n and atrial .fibrillation. The plurality of markers may also be used for prediction of risk that a subject may suffer from a future clinical outcome such as death or one or.more nonfatal complications such as might require rehospitalization. The > skilled artisan will understand that the same plurality of markers may provide both diagnostic and prognostic information. The markers used for diagnosis may be the same as those used for prognosis, or may differ in that one or more markers used for one of these purposes may not be used for the other purpose. [0018] Li the case of abdominal pain, the plurality of markers are preferably selected to rule in or out a plurality of the foLowing: aortic aneurysm, mesenteric embolism, pancreatitis, appendicitis, myocardial infarction, one or more infectious diseases described above, influenza, esophageal carcinoma, gastric adenocarcinoma, colorectal adenocarcinoma, pancreatic rumors including ductal adenocarcinoma, cystadenocarcinoma, and insulinoma. [0019]; In the case of disturbances of metabolic state, the plurality of markers are preferably selected to rule in or out a plurality of the following: diabetes mellitus, diabetic ketoacidosis, alcoholic ketoacidosis, respiratory acidosis, respiratory alkalosis, nonketogenic hyperglycemia, hypoglycemia, renal failure, interstitial renal disease, COPD, pneumonia, pulmonary and edema, asthma. [0020] As described in detail herein, a plurality of markers are used as part of panels as described hereinafter to associate diagnosis and/or prognosis to the subject. Such panels may comprise 2,3, 4, 5,6, 7, 8, 9,10,15,20, or more or individual markers, preferred panels for the diagnosis of a cause of dyspnea comprise a. plurality of markers independently selected from the group consisting of specific markers of cardiac injury, specific markers of neural tissue injury, non-specific markers of tissue injury, markers related to blood pressure regulation, markers related to inflammation, markers related to coagulation and hemostasis, markers related to pulmonary injury, and markers related to apoptosis. Exemplary markers in each of these groups are described hereinafter. Preferably, such a panel comprises markers from two, three, four, five, or mere different members of this group. Thus, particularly preferred panels for the diagnosis of a cause of dyspnea comprise one or more specific markers of cardiac injury and one or more markers related to blood pressure regulation; one 01 more specific markers of cardiac injury and one or more markers related to coagulation and hemostasis; one or more markers related to blood pressure regulation and one or more markers related to coagulation ar d hemostasis; or one or more specific markers of cardiac injury, one or more markers related to blood pressure regulation, and one or more markers related to coagulation and hemostasis, where each of these particularly preferred panels may optionally comprise one or more non-specific markers of tissue injury, markers related to inflammation, markers related to pulmonary injury, and/or markers related to apoptosis. One or more markers may lack diagnostic or prognostic value when considered alone, but when used as part of a panel, such marxers may be of great value in determining a particular diagnosis and/or prognosis. [0021 ] Preferred specific markers of cardiac injury for use in the methods described herein comprise, for example, annexin V, (3-enolase, cardiac troponin I (free and/or complexed), cardiac troponin T (free and/or complexed), creatme kinase-MB, glycogen phos^Korylase-BB, heart-type fatty acid binding protein, phosphoglyceric acid mutase-MB, and S-lOOao. Preferred non-specific markers of tissue injury for use in the methods described herein comprise, for example, aspartate aminotransferase, myoglobin, actin, myosin, and lactate dehydrogenase. [0022] Preferred marker(s) related to blood pressure regulation for use in the methods described herein comprise, for example, one or more marker(s) selected from the group consisting of atrial natriuretic peptide ("ANP"), pro-ANP, B-type natriuretic peptide ("BNP"), NT-pro BNP, pro-BNP C-rype ratriuretic peptide, urotensin n, arginine vasopressin, aldpsterone, angiotensin I, angietensin II, angiotensin HI, bradykinin, calcitonin, procalcitonin, calcitonin gene re'ated peptide, adrenomedulUn, calcyphosme, endothelin-2, endothelin-3, renin, and urodilatin, or markers related thereto. [0023] Preferred markers) markers related to inflammation for use in the methods described herein comprise, for example, one or more markers) selected from the group consisting of acute phase reactants, cell adhesion molecules such as vascular cell adhesion molecule ("VCAM"), intercellular adhesion molecule-1 ("ICAM-1"), intercellular adhesion molecule-2 (!wICAM-2il), and intercellular adhesion molecule-3 ("ICAM-3"), myeloperoxidase (MPO), C-reacnve protein, interleukins such as IL-1P, IL-6, and EL-8, interleukin-1 receptor agonist, monocyte chemoattractant protein-1, h'pocalin-type prostaglandin D synthase, mast cell tryptase, eosinophil cationic protein, haptoglobin, tumor necrosis factor a, tumor necrosis factor P, Fas ligand, soluble Fas (Apo-1), TRAIL, TWEAK, fibronectin, macrophage migration inhibitory factor (MIF), and vascular endothelial growth factor ("VEGF"), or markers related thereto. The term "acute phase reactants" as used herein refers to proteins whose concentrntions are elevated in response to stressful or inflammatory states that occur during various msults that include infection, injury, surgery, trauma, tissue necrosis, and the like. Acute phase reactant expression and serum concentration elevations are not specific for the type of insult, but rather as a part of the homeostatic response to the insult. [0024] In addition to those asute phase reactants listed above as "markers related to inflammation," one or more markers related to inflammation may also be selected from the group of acute phase reactants consisting of hepcidin, HSP-60, HSP-65, HSP-70, asymmetric dime&yi'arginme (an endogenous inhibitor of nitric oxide synthase), matrix metalloproteins 11,3, and 9, defensin HBD 1, defensin HBD 2, serum amyloid A, oxidized LDL, insulin like growth factor, transforming growth factor {3, e-selectin, glutathione-S-transferase, hypoxia-inducible factor-la, inducible nitric oxide synthase ("I-NOS"), intracellular adhesion molecule, lactate dehydrogenase, monocyte chemoattractant pep tide-1 ("MCP-1"), n-acetyl aspartate, prostaglandin E2, receptor activator of nuclear factor ("RANK") ligand, TNP receptor superfamily member 1 A, lipopolysaccharide binding protein ("LBP"), and cystatin C, or markers related thereto. [0025] Preferred markers) related to coagulation and hsmostasis for use in the methods described herein comprise, for example, one or more markers) selected from the group consisting of plasmin, fibrinogeii, thrombus precursor protein, D-dimer, p-tbromboglobulin, platelet factor 4, fibrinopeptide A, platelet-derived growth factor, prothrombin fragment 1+2, plasmin-a2-antiplasmin comple::. thrombin-antithrombin TLl complex, P-selectin, thrombin, and von Willebrand factor, tissue factor, or markers related thereto. [0026] Preferred markers related to pulmonary injury for use in the methods described herein comprise, for example, one or more marker(s) selected from the group consisting of neutrophil elastase, 7s collagen fragment, pulmonary surfactant protein(s), dipalmitoylphosphatidyl choline KL-6, and ubiquitin-conjugated lung proteins, or markers related thereto. [0027] Preferred markers) related to apoptosis for use in the methods described herein comprise, for example, one or more marker(s) selected from the group consisting of spectrin, cathepsin D, caspase 3, cytochroJie c, s-acetyl glutathione, and ubiquitin fusion degradation protein 1 homolog. [0028] As described in detail "lereinafter, the methods and compositions of the present invention can be selected to subcivide congestive heart failure by distinguishing between systolic heart failure and diastolic heart failure by analyzing a test sample obtained from the subject for the presence or amc unt of one or more markers, the presence or amount of which can be used to rule in or out systolic heart failure and/or diastolic heart failure, or that can be usedXJp distinguish between these two causes of congestive heart failure. Similarly, the methods and compositions herein may distinguish between atrial fibrillation and heart failure by analyzinga test sample obtained from the subject for the presence or amount of one or more markers, the presence or amount of which can be used to rule in or out heart failure or atrial fibrillation. Likewise, the methods can be used to distinguish between systolic and diastolic dysfunction and atrial fibrillation and/or to distinguish between systolic and diastolic dysfunction, atrial fibrillation,r lyocardial ischemia and cardiac necrosis. [0029] ' For example, the differential diagnosis of various diseases underlying dyspnea may require discrimination between heart failure and atrial fibrillation. A preferred marker panel for performing such discrmination preferably includes a plurality of markers related to blood pressure regulation, prefevably BNP or BNP related peptides, and ANP or ANP related peptides. Additional markers may be added to such a panel to distinguish between systolic and diastolic dysfunction and atrial fibrillation. A preferred marker panel for performing such discrimination preferably includes a plurality of markers related to blood pressure regulation. Most preferably, such a panel comprises BNP, calcitonin gene related peptide, calcitonin, urotensin 1, and ANP, or related peptides. Likewise, markers may be defined to distinguish between systolic and diastolic dysfunction, atrial fibrillation, myocardial ischemia and cardiac necrosis. Preferred marker panels in this case comprise a plurality of markers related to blood pressure regulation, one or more markers of cardiac necrosis, and optionally one or more nonspecific markers of tissue damage. Most preferably, such a panel comprises BNP, calcitonin gene related peptide, calcitonin, iirotensin 1, ANP, and cardiac troponin I or T (free and/or complexed), or related peptides, rind optionally myoglobin, creatine kinase-MB and/or S1 OOao, or related peptides. [0030] Moreover, one or mors markers of coagulation and hemostasis, most preferably D-dimer and/or thrombus precursor protein or related peptides, may be added to assist such panels in ruling in or out pulmonary embolism. Similarly, one or more markers of vascular tissue injury, preferably smooth muscle myosin, and most preferably smooth muscle myosin heavy chain or related peptides, .nay be added to such panels assist such panels in ruling in or out aortic dissection. Finally, one or more markers related to inflammation, preferably XL-Ira, myeloperoxidase, MMP-9, and/or C-reactive protein may also provide additional information to sucn^?n.nels for the further discrimination of disease. [ 0031 ] Particularly preferred markers for distinguishing causes of dyspnea include two or more markers selected from the group consisting of specific markers of cardiac injury, nonspecific markers of tissue injury, markers related to inflammation, markers related to blood pressure regulation, and marker.'; related to coagulation and hemostasis. Most preferred are panels comprising 2, 3,4, 5, 6, 7, 8, or more such markers, which are most preferably selected from the group consisting of cardiac-specific troponin I (free and/or complexed), cardiac-specific troponinT (free and/or complexed), creatine kinase-MB, SlOOao, A-type natnuretic peptide, B-type natnuretic peptide, calcitonin gene related peptide, calcitonin, urotensin 1, myoglobin, smooth muscle myosin light chain, thrombus precursor protein, D-dimer, smooth muscle myosin heavy chain, IL-lra, myeloperoxidase, caspase-3, cytochrome C, C-reactive protein, monocyte chemoattractent peptide-1, and MMP-9, or markers related thereto. One or more markers may be replaced, aided, -or subtracted from this list of particularly preferred markers while still providing clinically useful results. ro0321 These markers may be combined in various combinations. For example, preferred panels may comprise 2,3,4, 5, or more of the following markers: B-type natnuretic peptide or a marker related to B-type natnuretic peptide, creatine kinase-MB, total cardiac troponin I, total cardiac troponin T, C-reactive protein, D-dimer, and myoglobin. [0033] Particularly preferred panels cpmprise creatine kinase-MB, total cardiac troponin I, myoglobin, and B-type natriuret c peptide or a marker related to B-type natnuretic peptide; total cardiac troponin I, C-reactive protein, and B-type natnuretic peptide or a marker related to B-type natnuretic peptide; creatine kinase-MB, total cardiac troponin I, myoglobin, C-reactive protein, and B-type natriuretic peptide or a marker related to B-type natnuretic peptide; myoglobin, C-reactive -protein, and B-type natriuretic peptide or a marker related to B-type natriuretic peptide; creatine kinase-MB, total cardiac troponin I, and myoglobin; or creatine kinase-MB, total cardiar troponin I, C-reactive protein and myoglobin. These particularly preferred panels may further comprise D-dimer and/or myeloperoxidase; and D-dimer and/or myeloperoxidase rray be used to replace one or two of the markers in these particularly preferred panels. [0034f Such panels may diagnose one or more, and preferably distinguish between a plurality of, cardiovascular disorders selected from the group consisting of myocardial infarction, congestive heart failure, acute coronary syndrome, ST elevated ACS, non-ST elevated ACS, unstable angina, and/or pulmonary embolism; and/or predict risk that a subject may suffer from a future clinical outcome such as death, noufatal myocardial infarction, recurrent ischemia requiring urgent revascularization, and/or recurrent ischemia requiring rehospitalization; and/or predict a risk of a future outcome in such diseases. Marker(s) able to differentiate congestive heart failure from diseases or conditions that present similar symptoms, but that are not congestive heart failure ("CHF mimics"), are referred to herein as "CHF differential diagnostic markers;" marker(s) able to differentiate myocardial infarction from diseases or conditions that present similar symptoms, but that are not myocardial infarction ("MI mimics"), are referred to herein as "MI differential diagnostic markers." [0035] In similar fashion, a p.'iciel may comprise a plurality of markers selected to diagnose and/or distinguish amongst a plurality of cerebrovascular disorders. In preferred embodiments, the invention discloses methods for determining a diagnosis or prognosis related to stroke, or for differentiating between types of strokes and/or TIA. These methods comprise analyzing a test sample obtained from a subject for the presence or amount of one or more markers for cerebral injury. These methods can comprise identifying one or more markers, the presence or amount of which is associated with the diagnosis, prognosis, or differentiation of stroke and/or TIA. Once such marker(s) are identified, the level of such marker(s) in a sample obtained from a subject of interest can be measured. In certain embodiments, these markers can be compared to a level that is associated with the diagnosis, prognosis, or differentiation of stroke and/or TIA. By correlating the subject's marker level(s) to the diagnostic marker level(s),: he presence or absence of stroke, the probability of future adverse outcomes, etc., in a patiert may be rapidly and accurately determined. [0036] The invention also discloses methods for determining the presence or absence of a disease or condition in a subject that is exhibiting a perceptible change in one or more symptoms that are indicative of a plurality of possible etiologies underlying the observed symptbin(s), one of which is stroke. These methods comprise analyzing a test sample obtained from the subject for the presence or amount of one or more markers selected to rule in or out stroke, or one or more types of stroke, as a possible etiology of the observed symptom(s). Etiologies other than stroke that are within the differential diagnosis of the symptom(s) observed are referred to herein as "stroke mimics", and marker(s) able to differentiate one or more types of stroke from stroke mimics are referred to herein as "stroke differential diagnostic markers". The preser ce or amount of such marker(s) in a sample obtained from the subject can be used to rule in or rule out one or more of the following: stroke, thrombotic stroke, embolic stroke, lacunar stroke, hypoperfusion, subclinical cerebral ischemia, intracerebral hemorrhage, and s ibarachnoid hemorrhage, thereby either providing a diagnosis (rule-in) and/or excluding a diagnosis (rule-out). [0037] In these aspects related to cerebrovascular disease, preferred marker panels comprise a plurality of markers independently selected from the group consisting of specific markers of neural tissue injury, narkers related to blood pressure regulation, markers related to coagulation and hemostasis, markers related to inflammation, and markers related to *noptosis. Preferably, such a paiel comprises markerd from two, three, four, or five different members of this group. [0038] Exemplary markers related to blood pressure regulation, to inflammation, and to coagulation and hemostasis are described above. One or more markers related to neural tissue injury include those selected from the group consisting of precerebellin 1, cerebillin 1, cerebellin 3, chimerin 1, c'himerin 2, calbrain, calbindin D, brain tubulin, brain fatty acid binding protein ("B-FABP"), brain derived neurotrophic factor ("BDNF"), carbonic anhydrase XI, CACNA1A calcium channel gene, nerve growth factor f), atrophin 1, apolipoprotein E4-1, protein 4.IB, 14-3-3 protein, ciliary neurotrophic factor, creatine kinase-BB, C-tau, glial fibrillary acidic protein ("GFAP"), neural cell adhesion molecule ("NCAM"), neuron specific enolase, S-lOOfi, prostaglandin D synthase. neurokinin A, neurotensin, and secretagogin. Additional exemplary markers related to neural tissue injury are described hereinafter. [0039] Preferred marker panels selected to diagnose and/or distinguish amongst a plurality of cerebrovascular disorders comprise a plurality of markers selected from the group consisting of adenylate kinase, brain-derived neurotrophic factor, calbindin-D, creatme kinase-BB, glial fibrillary acidic protein, lactate dehydrogenase, myelin basic protein, neural cell adhesion molecule (NCAM), c-tau, neuropeptide Y, neuron-specific enolase, neurotrophin-3, proteolipid protein, 8-100(3, thrombomodulin, protein kinase C y, atrial natriuretic peptide (ANP), pro-ANP, B-type natriuretic peptide (BNP), NT-pro BNP, pro-BNP C-type natriuretic peptide, urotensin n, arginine vasopressin, aldosterone, angiotensin I, angiotensin II, angiotensin III, bradykinin, calcitonin, procalcitohin, calcitonin gene related peptide, adrenomedullin, calcyphosine, endothelin-2, endothelin-3, renin, urodilatin, acute phase reactants, MMP-9, cell adhesion molecules, C-reactive protein, interleukdns, iaterleukin-1 receptor agonist, mcnocyte chemotactic protein-1, caspase-3, lipocalin-type prostaglandin D synthase, mast cell tryptase, eosinophil cationic protein, KL-6, haptoglobin, tumor necrosis factor a, tumor necrosis factor |J, Fas tigand, soluble Fas (Apo-1), TRAIL, TWEAK, fibronectin, macropha.'je migration inhibitory factor (MIF), vascular endothelial growth factor (VEGF), caspase-3, cathepsin D, a-spectrin, plasmin, fibrinogen, D-dimer, Pthromboglobulin, platelet factor -4, fibrinopeptide A, platelet-derived growth factor, prothrombin fragment 1+2, plasnjin-a2-antiplasmin complex, thrombin-antithrombm III complex, P-selectin, thrombin, vi n Willebrand factor, tissue factor, and thrombus precursor protein, or markers related thereto. [0040] Most preferred marker panels comprise at least one marker related to neural tissue injury and at least one marker of inflammation, and preferably comprise 2,3,4,5, 6, 7, 8, or more markers selected from the gioup consisting of IL-lra, C-reactive protein, von Willebrand factor (vWF), creatuv; kinase-BB, creatine kinase-MB, c-Tau, D-dimer, thrombus precursor protein, vascular endothelial growth factor (VEGF), matrix metalloprotease-9 (MMP-9), neural cell adhesion molecule (NCAM), BNP, S100J3, cytochrome c, and caspase [0041] Preferred markers oJ'the invention can differentiate between ischemic stroke, hemorrhagic stroke, and TIA. Such markers are referred to herein as "stroke differentiating markers". Particularly preferred are markers that differentiate between thrombotic, embolic, lacunaf, aypoperfusion, intraceiebral hemorrhage, and subarachnoid hemorrhage types of strokes. For purposes of the following discussion, the methods described as applicable to the diagnosis and prognosis of stroke generally may be considered applicable to the diagnosis and prognosis of TIAs. [0042J Still other preferred markers of the invention can identify those subjects suffering from stroke at risk for a subsequent adverse outcome. For example, a subset of subjects presenting with intracerebral hemorrhage or subarachnoid hemorrhage types of strokes may be susceptible to later vascular injury caused by cerebral vasospasm. In another example, a clinically normal subject may be screened in order to identify a risk of an adverse outcome. Preferred markers include caspase-3, NCAM, MCP-1, SlOOb, MMP-9, vWF, BNP, CRP, NT3, VEGF, CKBB, MCP-1 Calbindin, thrombin-antithrombin HI complex, IL-6, IL-8, myelin basic protein, tissue factor, GF/P, and CNP. Each of these terms is defined hereinafter. Particularly preferred markers are those predictive of a subsequent cerebral vasospasm in patients presenting with subarachnoid hemorrhage,, such as von Willebrand factor, vascular endothelial growth factor, matrix metalloprotein-9, or combinations of these markers. Other particularly preferred markers a-e those that distinguish ischemic stroke from hemorrhagic stroke. [0043] Still other particularly preferred markers are those predictive of a subsequent cerebral vasospasm in patients presenting with subarachnoid hemorrhage, such as one or more markers related to blood pressure regulation, markers related to inflammation, markers related to apoptosis, and/or specific markers of neural tissue injury. Again, such panels may comprise 2,3,4, 5, 6, 7, 8, 9,10,15,20, or more or individual markers. Preferred marker(s) for use individually or in panels maybe selected from the group consisting of EL-lra, C-reactive protein, von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), matrix metalloprotease-9 (MMP-9), neural cell adhesion molecule (NCAM), BNP, and caspase-3, or markers related thereto. [0044] Obtaining information on the true time of onset can be critical, as early treatments have been reported to be critical for proper treatment. Obtaining this time-of-onset information can be difficult, and is often based upon interviews with companions of the stroke victim. T^hus, in various embodiments, markers and marker panels are selected to distinguish the approximate time since strode onset. For purposes of the present invention, the term "acute stroke" refers to a stroke that has occurred within the prior 12 hours, more preferably within the prior 6 hours, and most preferably within the prior 3 hours; while the term "nonacute stroke" refers to a stroke that has occurred more than 12 hours ago, preferably between 12 and 48 hours ago, and most preferably between 12 and 24 hours ago. Preferred markers for differentiating between acute and non-acute strokes, referred to herein as stroke "time of onset markers" are described hereinafter. [0045] Preferred panels comprise markers for the following purposes: diagnosis of stroke; diagnosis of stroke and indication if an acute stroke has occurred; diagnosis of stroke and indication if an non-acute stroke has occurred; diagnosis of stroke, indication if an acute stroke has occurred, and indication if an non-acute stroke has occurred; diagnosis of stroke and indication if an ischemic stroke has occurred; diagnosis of stroke and indication if a hemorrhagic stroke has occurred; diagnosis of stroke, indication if an ischemic stroke has occurred, and indication if a hemorrhagic stroke has occurred; diagnosis of stroke and prognosis of a subsequent adverse outcome; diagnosis of stroke and prognosis of a subsequent cerebral vasospasm; and diagnosis of stroke, indication if a hemorrhagic stroke has occurred, and prognosis of a subsequent cerebral vasospasm. [0046] As noted above, panels may also comprise differential diagnosis of stroke; differential diagnosis of stroke and indication if an acute stroke has occurred; differential diagnosis of stroke and indication if an non-acute stroke has occurred; differential diagnosis of stroke, indication if an acute stroke has occurred, and indication if an non-acute stroke has occurred; differential diagnosis of stroke and indication if an ischemic stroke has occurred; differential diagnosis of stroke and. indication if a hemorrhagic stroke has occurred; differential diagnosis of stroke, indication if an ischemic stroke has occurred, and indication if a hemorrhagic stroke has occurred; differential diagnosis of stroke and prognosis of a subsequent adverse outcome; differential diagnosis of stroke and prognosis of a subsequent cerebral vasospasm; differential diagnosis of stroke, indication if a hemorrhagic stroke has occurred, and prognosis of a subsequent cerebral vasospasm. [0047] . The presence or amount of the markers in such panels may be correlated to the presence or absence of a plurality of cerebrovascular disorders. Additional markers are described hereinafter. As described hereinafter, the markers described herein may be indicative of a plurality of diseases, depending on the status of other markers in a panel. For example, certain markers are generally elevated in inflammation resulting from a variety of causes. Thus, alone, a single marker may not be diagnostic per se, but as part of a panel, the marker can provide important diagnostic and/or prognostic information. [0048] In a related aspect, the presence or amount of markers that are selected to diagnose and/or distinguish amongst a plurality of cerebrovascular disorders may also be used prognostically, in order to identity patients at risk for a future onset of a cerebrovascular disorder. Such uses may find particular interest in monitoring patients known to be at increased risk for such onset. For example, patients undergoing carotid endarterectomy are known to be at risk for cerebral ischemia. Outcomes of such ischemia include intraoperative and perioperative stroke, neurologic deficit, and death. Cerebral ischemia is also a risk of procedures such as hypothermic circulatory arrest, aortic valve replacement, mitral valve replacement, coronary artery surgery, endograft repair of aortic aneurism, coronary artery bypass graft surgery, laryngeal mask insertion, and repair of congenital heart defects:. Thus, the present invention also relates to methods and compositions for monitoring the status of patients undergoing such procedures to identify at-risk patients. [0049] In yet another aspect, the present invention describes thrombus precursor protein ("TpP™") and monocyte chemoattractant protein-1 (MCP-1) as representing independent markers for use in risk stratification and diagnosis of patients suffering from vascular diseases. In the case of ACS for example, TpP™ and/or MCP-1 may permit a determination of risk that a subject may suffer from a future clinical outcome such as death, nonfatal myocardial infarction, recurrent ischemia requiring urgent revascularization, and/or recurrent ischemia requiring rehospitalization. The time horizon over which risk stratification may be applied (that is, the period for which prognostic risk may be predicted) may be from 1 day to 5 years, more preferably from 1 week to 2 years, and most preferably from 1 month to 1 year. While described hereinafter with regard to acute coronary syndrom ("ACS") patients, TpP™ and MCP-1 may also he used in various aspects according to the methods described herein to provide diagnostic and prognostic information in a variety of vascular diseases in which coagulation and hemostasis and/or inflammation are implicated. [0050] Preferred diseases to which the various aspects described herein may be applied include one or more diseases selected from the group consisting of sepsis, acute coronary syndrome, atherosclerosis, ischemia stroke, intracerebral hemorrhage, subarachnoid hemorrhage, transient ischemic attack, systolic dysfunction, diastolic dysfunction, aneurysm, aortic dissection, myocardial ischemia, angina pectoris, myocardial infarction, congestive heart failure, dilated congestive cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, cor pulmonale, arrhythmia, valvular heart disease, endocarditis, pulmonary embolism, venous thrombosis, and peripheral vascular disease. [00511 Li a preferred embodiment of this aspect, the invention features methods of predicting a risk of one or more clinical outcomes for a subject suffering from a vascular disease by analyzing a test sampls obtained from the subject for the presence or amount of TpP™ and/or MCP-1, and using die presence or amount of TpP™ and/or MCP-1 measured in the sample to associate a risk of one or more clinical outcomes to the subject. [0052] As described hereinator, TpP™ and/or MCP-1 may be associated with a given risk of one or more clinical outcomes without considering any other markers. Thus, in certain embodiments, such an association may be made simply by providing one or more predetermined threshold concentrations, below which a subject has a first risk level, and above which a subject has a second risk level. A subject may be assigned a a relative prognostic risk based upon a population of vascular disease patients for whom TpP™ and/or MCP-1 concentrations have been measured, and subsequent clinical outcomes followed over a period of days, months, or years. The population TpP™ and/or MCP-1 (as relevant) concentrations may be divided into tertiles, quartiles, quintiles, etc., and an associated risk level determined for each subpopulation by methods known in the art. Patients may then be assigned to one of these prognostic risk subpopulations according to a measured TpP™ and/or MCP-1 concentration. [005.31 , In other embodiments, TpP™ and/or MCP-1 are used as part of panels as described herein to associate a risk of one or more clinical outcomes to the subject. Such panels may comprise 2,3,4,5,6, 7,8,9,10, 15,20, or more or individual markers, at least one of which is TpP™ or MCP -L Preferred panels comprise a plurality of markers independently selected from ths group consisting of TpP™, MCP-1, and one or more additional markers independently selected from the group consisting of specific markers of cardiac injury, specific markers of neural tissue injury, markers related to blood pressure regulation, markers related to inflammation, markers related to coagulation and hemostasis, and markers related to apoptosi;. Exemplary markers in each of these groups are described hereina. One or more markers considered with TpP™ and/or MCP-1 may lack diagnostic or prognostic value when considered alone, but when used as part of a panel, such markers may be of great value in determining a particular diagnosis and/or prognosis. [0054] Suitable additional markers for inclusion in such panels are described in detail hereinafter. Particularly preferred markers for use in such panels in addition to TpP™ include BNP, cardiac troponin I (free aid/or complexed), cardiac troponin T (free and/or complexed), CRP, creatine kinase-MB, MMP-9, caspase-3, myoglobm, myeloperoxidase, sCD40L, or markers related thereto. One or more markers may be replaced, added, or subtracted from this list of particularly preferred markers while still providing clinically useful results. "0055] In another aspect of the present invention, methods of diagnosing a vascular iisease are described. Such methods comprise analyzing a test sample obtained from the iubject for the presence or amouit of TpP™ and/or MCP-1 and one or more additional narkers, and using the presence or amount of TpP™ and/or MCP-1 and the additional narker(s) to determine the presence or absence of the vascular disease in the subject. In this .spect then, TpP™ and/or MCP-1 is used as part of a diagnostic panel. As above, such panels lay comprise2,3,4,5,6,7, 8, 9,10,15,20, or more or individual markers, at least one of fhich is TpP™ or MCP-1. Pref ared panels comprise a TpP™ and/or MCP-1 and one or lore additional markers independently selected from the group consisting of specific markers of cardiac injury, specific markers of neural tissue injury, markers related to blood pressure regulation, markers related to inflammation, markers related to coagulation and hemostasis, and markers related to apoptosis. [0056]" In a related aspect of the present invention, methods of diagnosing atherosclerosis are described. Such methods comprise analyzing a test sample obtained from the subject for the presence or amount of MCP-1 (and optionally one or more additional markers), and using the presence or amount of MCP-1 (and the additional marker(s) if measured) to determine the presence or absence of atherosclerosis in the subject. When MCP-1 is used as part of a diagnostic panel in this aspect, sach panels may comprise 2, 3,4, 5, 6,7, 8,9, 10,15,20, or more or individual markers, at least one of which is MCP-1. Preferred panels comprise MCP1 and one or more additional markers independently selected from the group consisting of specific markers of cardiac injury, specific markers of neural tissue injury, markers related to blood pressure regulation, markers related to inflammation, markers related to coagulation and hemostasis, and markers related to apoptosis. [0057] The marker panels of the present invention may be analyzed in a number of fashions well known to those of skill in the art. For example, each member of a panel may be compared to a "normal" value, or a value indicating a particular disease or outcome. A particular diagnosis/prognosis may depend upon the comparison of each marker to such a value- alternatively, if only a subset of markers are outside of a normal range, this subset may be indicative of a particular diagnosis/prognosis. For example, certain markers in a panel may be used to diagnose (or to rule out) a myocardial infarction, while other members of the panel may diagnose (or rule out) congestive heart failure, while still other members of the panel may diagnose (or rule out) aortic dissection. Markers may also be commonly used for multiple purposes by, for example, applying a different threshold or a different weighting factor to the marker for the different purpose(s). For example, a marker at one concentration or weighting may be used, alone or as part of a larger panel, to to diagnose (or to rule out) a myocardial infarction, and the sai.-e marker at a different concentration or weighting may be used, alone or as part of a larger panel, to diagnose (or rule out) congestive heart failure, etc. [0058] In certain embodimects, one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicators). For example, an assay can be designed so that a positive signal for a marker only occurs above a partic;1.!^ threshold concentration of interest, and below which concentration the assay provides no signal above background. In other embodiments, threshold concentration(s) of diagnostic or prognostic indicators) can be established, and the level of the indicators) in a patient sample can simply be compared to the threshold level(s). [0059] The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test—they also depend on the definition of what constitutes an abnormal result. Tn practice, Receiver Operating Characteristic curves, or "ROC" curves, are typically calculated by plotting the valus of a Variable versus its relative frequency in "normal" and "disease" populations. For any particular marker, a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results dont necessarily give an accurate number. As long as one can rank results, one can create an ROC curve. Fcr example, results of a test on "disease" samples might be ranked according to degree (say l=low, 2=normal, and 3=high). This ranking can be con-elated to results in the "normal" population, and a ROC curve created. These methods are well known in the art. See, e.g., Hanley et al., Radiology 143: 29-36 (1982). Preferably, a threshold is selected to provide ?. ROC curve area of greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. The term "about" in this context refers to +/- 5% of a give: measurement. [0060] As described herinafter, a "panel response value" is preferably determined by plotting ROC curves for the sens tivity of a particular panel of markers versus 1-(specificity) for the panel at various cutoffs. In these methods, a profile of marker measurements from a subject are integrated to provide; a single value that is considered to be the panel "result." Thus, the plurality of markers ic a panel are considered together to provide a global probability (expressed either as a numeric score or as a percentage risk) of a diagnosis or prognosis. In such embodiments, an increase in a certain subset of markers maybe sufficient to indicate a particular diagnosis/prognosis in one patient, while an increase in a different subset of markers may be suffic ient to indicate the same or a different diagnosis/prognosis in another patient. Weighting factors may also be applied to one or more markers in a panel, for example, when a marker is of p irticularly high utility in identifying a particular diagnosis/prognosis, it may be weighted so that at a given level it alone is sufficient to signal a positive result. Likewise, a weighting factor may provide that no given level of a particular marker is sufficient to signal a positive result, but only signals a result when another marker also contributes to the analysis. [0061] In certain embodiments, markers and/or marker panels are selected to exhibit at least about 70% sensitivity, mors preferably at least about 80% sensitivity, even more preferably at least about 85% sensitivity, still more preferably at least about 90% sensitivity, and most preferably at least about 95% sensitivity, combined with at least about 70% specificity, more preferably at least about 80% specificity, even more preferably at least about 85% specificity, still more preferably at least about 90% specificity, and most preferably at least about 95% specificity. In particularly preferred embodiments, both the sensitivity and specificity are at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95%. The term "about" in this co itext refers to +/- 5% of a given measurement. [0062] In other embodiments a positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio is used as a measure of a test's ability to predict risk or diagnose a disease. In the case of a positive likelihood ratio, a value of 1 indicates that a positive result is equally likely among subjects in roth the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group. In the case of a negative likelihood ratio, a value a" 1 indicates that a negative result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a negative result is more likely in die test group; and a value less than 1 indicates that a negative result is more likely in the control group. In certain preferred embodiments, markers and/or markef.jntaels are preferably selected to exhibit a positive or negative likelihood ratio of at least about 1 .5 or more or about 0.67 or less, more preferably at least about 2 or more or about 0.5 or less, still more preferably at least about 5 or more or about 0.2 or less, even more preferably at least about 10 or more or about 0.1 or less, and most preferably at least about 20 or more or about 0.05 or less. The term "about" in this context refers to +/- 5% of a given measurement. [0063] In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group. In certain preferred embodiments, markers and/or marker panels are preferably selected to exhibit an odds ratio of at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less. The term "about" in this context refers to +/- 5% of a given [0064] In the case of a hazard ratio, a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the "diseased" and "control" groups; a value greater than 1 indicates that the risk is greater in the diseased group; and a value less than 1 indicates that the risk is greater in the control group. In certain preferred embodiments, markers and/or marker panels are preferably selected to exhibit a hazard ratio of at least about 1.1 or more or about 0.91 or less, more preferably at least about 1 .25 or more or about 0.8 or less, still more preferably at least about 1.5 or more or about 0.67 or less, even more preferably at least about 2 or more or about 0.5 or less, and most preferably at least about 2.5 or more or about 0.4 or less. The term "about" in this cootext refers to +/- 5% of a given measurement. [0065] The skilled artisan will understand that associating a diagnostic or prognostic indicator, with a diagnosis or with a prognostic risk of a future clinical outcome is a statistical analysis For example,a marker level of greater than X may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to X, as determined by a level of statistical significance. Additionally, a change in marker concentration from baseline leveis may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events. Statistical significance is often detennined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, "• 983. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 9S.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02,0.01, 0.005, 0.001,' and 0.0001. 10066] In yet other embodiments, multiple determinations of one or more diagnostic or prognostic markers described herein can be made, and a temporal change in the marker can be used to determine a diagnosis or prognosis. For example, a marker concentration in a subject sample may be detennined at an initial time, and again at a second time from a second subject sample. In such embodiments, an increase in the marker from the initial time to the second time may be indicative of a particular diagnosis, or a particular prognosis. Likewise, a decrease in the marker from the initial time to the second time may be indicative of a particular diagnosis, or a particular prognosis. This "temporal change" parameter can be included as a marker in the marker panels of the present invention. In a related embodiment, multiple determinations of one or more diagnostic or prognostic markers can be made, and a temporal change in the marker can be used to monitor the efficacy of appropriate therapies. In such an embodiment, one might expect to see a decrease or an increase in the marker(s) over time during the course of e£f active therapy. [0067] The skilled artisan will understand that, while in certain embodiments, comparative measurements are mide of the same diagnostic marker at multiple time points, one could also measurea given marker at one time point, anc a second marker at a second lime point, and a comparison of these markers may provide diagnostic information. Similarly, the skilled artisan will understand that serial measurements and changes in markers or the combined result over timt may also be of diagnostic and/or prognostic value. [0063] In other embodimen s, a threshold degree of change in the level of a prognostic or diagnostic indicator can be established, and the degree of change in the level of the indicator in a patient sample can simply be compared to the threshold degree of change in the level. A preferred threshold change in the level for markers of the invention is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. The term "about" Li this context refers to -*•/- 10%. In yet other embodiments, a "nomogram" can be established, by which a level of a prognostic or diagnostic indicator can be directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages. [0069] In yet another aspect, the invention relates to methods for determining a treatment regimen for use in a subject. Tr « methods preferably comprise determining a diagnosis or prognosis as described herein, and selecting one or more treatment regimens appropriate to the diagnosis. In preferred embodiments, a treatment regimen is selected to improve the subject's prognosis by reducing the disposition for an adverse outcome associated with !h diagnosis. Such methods may also be used to screen pharmacological compounds for agents capable of improving the patient's prognosis as above. [0070] In a further aspect, the invention relates to kits and devices for determining the diagnosis or prognosis of a patimt. .Kits preferably comprise devires and reagents fly performing the assays describee lerein, and instructions for performing the assays. Such devices preferably contain a plurality of discrete, independently addressable locations, or "diagnostic zones," each of whi ;h is related to a particular marker of interest. Following reaction of a sample with the devices, a signal is generated from the diagnostic zone(s), which may then be correlated to the presence or amount of the markers of interest. Optionally, the kits may contain one or more rreans for converting marker level(s) to a diagnosis or prognosis. Such kits preferably contain sufficient reagents to perform one or more such determinations, and/or Food and Drug Administration (FDA)- or other governmentally- approved labeling. BRIEF DESCRIPTION OF THE FIGURES [0071] Fig. 1 shows the relationship of TpP™ concentration to clinical outcome through 12 months following enrollment of ACS subjects in the OPUS-TIMI16 study. [0072] Fig. 2 shows the relationship of MCP-1 concentration to atherosclerosis in subjects not exhibiting clinically apparent atherosclerosis as measured by determining CAC. DETAILED DESCRIPTION OF THE INVENTION [0073] The present invention relates to methods and compositions for symptom-based differential diagnosis of disease* in subjects. [0074] Patients presenting for medical treatment often exhibit one or a few primary observable changes in bodily characteristics or functions that are indicative of disease. Often, these "symptoms" are nonspecific, in that a number of potential diseases can present the same observable symptom or symptoms.A typical list of nonspecific symptoms might include one or more of the following: shortness of breath (or dyspnea), chest pain, fever, dizziness, and headache. These symptoms can be quite common, and the number of diseases that must be considered by the clinician can be astoundingly broad. [0075] Taking shortness of breath (referred to clinically as "dyspnea") as an example, this symptom considered in isolation may be indicative of condi dons as diverse as asthma, chronic obstructive pulmonary disease ("COPD"), tracheal stenosis, pulmonary injury, obstructive endobroncheal tumor, pulmonary fibrosis, pneumoconiosis, lymphangitic carcinomatosis, kyphoscoliosis, pleura! effusion, rnyotrophic lateral sclerosis, congestive heart failure, coronary artery disease, myocard f.l infarction, atrial fibrillation, cardiomyopathy, valvular dysfunction, left ventricle hyperbcphy, pericarditis, arrhythriia, pulmonary embolism, metabolic acidosis, chronic bronchitis, pneumonia, anxiety, nepsis, aneurismic dissection, etc. See, e.g., Kelley's Textbook of Internal Medicine, 4th Ed., Lippincott Williams & Wilkins, Philadelphia, PA, 2000, pp. 2349-2354, "Approach to the Patient With Dyspnea"; Mulrow et al., J. Gen. Int.Med. 8: 383-92 (1993). [0076] Similarly, chest pain, when considered in isolation, may be indicative of stable angina, unstable angina, myoca'dial ischemia, atrial fibrillation, myocardial infarction, musculoskeletal injury, cholecystitis, gastroesophageal reflux, pulmonary embolism, pericarditis, aortic dissection, pneumonia, anxiety,etc. Moreover, the classification of chest pain as stable or unstable angina (or even mild myocardial infarction) in cases other than definitive myocardial infarctior is completely subjective. The diagnosis, and in this case the distinction, is made not by angngraphy, which may quantify the degree of arterial occlusion, but rather by a physician's interrelation of clinical symptoms. [0077] Differential diagnos:s refers to methods for diagnosing the particular disease(s) and/or conditions) underlying the symptoms in a particular subject, based on a comparison of the characteristic features observable from the subject to the characteristic features of those potential diseases. Depending on the breadth of diseases and conditions that must be considered in the differential diagnosis, the types and number of tests that must be ordered by •A clinician can be quite large. In the case of dyspnea for example, the clinician may order tests from a group that includes radiography, electrocardiogram, exercise treadmill testing, blood chemistry analysis, echocardiopaphy, bronchoprovocation testing, spirometry, pulse oximetry, esophageal pH monitoring, laryngoscopy, computed tomography, lii^toiog}, cytology, magnetic resonance imaging, etc. See, e.g., Morgan and Hodge, Am. Fam. Physician 57: 711-16 (1998). The clinician must then integrate information obtained from a battery of tests, leading to a clinical diagnosis that most closely represents the range of symptoms and/or diagnostic test results obtained for the subject. [0078] The present invention describes methods and compositions that can assist in the differential diagnosis of one or iiore nonspecific symptoms by providing diagnostic markers that are designed to rule in or out one, and preferably a plurality, of possible etiologies for the observed symptoms. The concept of symptom-based differential diagnosis described herein can provide panels of diagnostic markers designed to be considered in conceit to distinguish between possible diseases that underlie a nonspecific symptom observed in a patient. [0079] The term "fever" as used herein refers to a body temperature greater than 100 °C orally or 100.8 °C rectally. In the case of fever, a plurality of markers are preferably selected to rule in or out a plurality of the following: sepsis; arteritis; sarcoidosis; and one or more infecfiour. diseases, including infection by Staphyloccus species, Nisseria species, Pneumococcal species, Listeria species, Anthrax, Nocardia species, Salmonella species, Shigella species, Haemophilus species, Brucella species, Vibrio species including V. cholerae, Franciscella tularensis, Yersinia pestis, Pseudomonas species, Clostridia species including C. tetani, C. perfiingens, C. ramosum, C. botulinum, and C. septicum, Actinomyces species, Treponema pallidum, Boirelia species including Brburgdorferi, Leptospira species, Mycobacterium species including M. tuberculosis, M. bovis, M. leprae, and M. africanum, Histoplasma species, EscherichLi coli, Coccidioides species, Blastomyces species, Paracoccidioides species, Sporoihrix species, Cryptococcus species, Candida species, and Aspergillus species; Rickettsial diseases including Rocky Mountain spotted fever, Q fever, typhus, trench fever, and cat-scratch fever; parasitic diseases including Malaria, Babesiosis, African sleeping sickness, Trypanosomiasis, Leishmaniasis, Toxoplasmosis, and Amebiasis; viral infection by influenza virus, parainfluenza virus, mumps virus, adenovirus, respiratory syncytial virus, rhinovirus, polio"irus, coxackievirus, echovirus, rabeola virus, rubella virus, parvovirus, hepatitis A, B, C, D, or E, cytomegalovirus, Epstein-Barr virus, Herpes simplex virus, Varicella-zoster virus, Alpiiavirus, Flaviviruses including yellow fever virus, dengue fever virus, Japanese encephalitis virus, and St. Louis encephalitis virus, West Mile virus, Colorado tick fever virus, Rabies virus, Arenavirus, Marburg agent, and Ebola virus. [OU80] The term "neurologic dysfunction" as used herein refers to a loss of one or more normal physiological or mental functions having a neurogenic etiology. The skilled artisan will understand that neurologic dysfunction is a common symptom in various systemic disorders (e.g., alcoholism, vascu ar disease, stroke, autoimmunity, metabolic disorders, aging, etc.). Specific neurologic dysfunctions include, but are not limited to, pain, headache, aphasia, apraxia, agnosia, amnesii, stupor, coma, delirium, dementia, seizure, migraine insomnia, hypersomnia, sleep apnea, tremor, dyskinesia, paralysis, etc. [0081] The term "hypertension' as used herein refers to a systolic blood pressure of greater than or equal to 140 mm Hg and/or a diastolic blood pressure of greater than or equal to 90 mm Hg. Hypertension can include isolated systolic hypertension (i.e., no elevation in diastolic blood pressure). In the c^se of hypertension, the plurality of markers are preferably selected to rule in or out a plurality of the following: left ventricular failure, atherosclerosis, renal disease including chronic glcmerulonephritis, and polycystic renal disease, coartation of the aorta, renal arterial stenosis, and hyperparathyroidism. [0082] The term "condition v/ithin the differential diagnosis of a symptom" as used herein refers to a pathologic state that is known to be causative of a particular perceptible change in one or more physical characteristics exhibited by a subject suffering from the pathologic state, as compared to a normal subject The concept of differential diagnosis is well established to those of skill in the art. See, e.g., Beck, Tutorials in Differential Diagnosis, Churchill Livingstone, 2002; Zackon, Pulmonary Differential Diagnosis, Elsevier, 2000; Jamison, Differential Diagnosis for Primary Practice, Churchill Livingstone, 1999; Bouchier et al., French's Index of Differential Diagnosis, Oxford University Press, 1997. [0083] The term "marker" at. used herein refers to proteins, polypeptides, glycoproteins, proteoglycans, lipids, lipoproteins, glycolipids, phospholipids, nucleic acids, carbohydrates, etc., small molecules, or other characteristics of one or more subjects to be used as targets for screening test samples obtained Irom subjects. "Proteins or polypeptides" used as markers in the present invention are contemplated to include any fragments thereof, in particular, immunologically detectable fragments. 'Marker" as used herein may also include, derived markers as defined below, and may also include such characteristics as patient's history, age, sex and race, for example. [0084] The term "derived marker" as used herein refers to a value that is a function of one or more measured markers. For example, derived markers may be related to the change over a time interval in one or more measured marker values, may be related to a ratio of measured marker values, may be a marker value at a different measurement time, or may be a complex function such as a panel responf e function. [OOSS] The term "related mirker" as used herein refers to one or more fragments of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent marker'. For example, human BNP is derived by proteolysis of a 108 amino acid precursor molecule, referred to hereinafter as BNPi-ios- Mature BNP, or "the BNP natriuretic peptide," or "BNP-3?" is a 32 amino acid molecule representing amino acids 77108 of t'ois precursor, which may be referred to as BNPyv-ios- The remaining residues 1-76 are referred to hereinafter as BNPi.-,;. Additionally, related markers maybe the result of covalent modification of the parent marker, for example by oxidation of methionine residues, ubiquitination, cysteinylation, nitrosylation, glycosylation, etc. [0086] Further considering BNP as an example, the sequence of the 108 amino acid BNP precursor pro-BNP (BNPi-ios) is is follows, with mature BNP (BNP77-ios) underlined: HPLGSPGSAS DLETSGLQEQ RNHLQGKLSE LQVEQTSLEP LQESPRPTGV 50 MKSREVAT3G IRGHRKMVLY TLRAPRSPKM VQGSGCFGRK MDRISSSSGL IOC GCKVLRRH 108 (SEQIDNO: 1). [0087] BNPi-ios is synthesized as a larger precursor pre-pro-BNP having the following sequence (with the "pre" sequence shown in bold): MDPQTAPSRA LLLLLFLHLA FLGGRSHPLG SPGSASDLET SGLQEQRNHL 50 OGKLSELQVE QTSLEPLQES PRPTGVWKSR EVATEGIRGH RKMVLYTLRA 100 •PRSPKMVQGS GCFQRKMDRI SSSSGLGCKV LRRH 134 (SEQ ID NO: 2). [OOS8] While mature BNP itself may be used as a marker in the present invention, the prepro-BNP, BNPi.ios and BNPi 75 molecules represent BNP-related markers that may be • measured either as surrogates for mature BNP or as markers in and of themselves. In addition, one or more fragments of these rr olecules, including BNP-related polypeptides selected from ihe group consisting of BNP77-106. BNP79-io6, BNP76-i07, BNPes-ios, BNP79-io8,.BNP8o-io8, BNPsi-ios, BNPg3-ioB, BNP3j>-s6, f'NP53-85, BNP66.98, BNP3o-io3, BNPn.,07, BNP9.io6, and BNP3. 113 may also be present in circulaiion. In addition, natriuretic peptide fragments, including BNP fi-agments, may comprise or.e or more oxidizable meth:onines, the oxidation of which to methionine sulfoxide or methionine sulfone produces additional BNP-related markers. See, e.g., U.S. Patent No. 10/419,059: filed April 17, 2003, which is hereby incorporated by reference in its entirety including all tables, figures and claims. i 0089] Because production of marker fragments is an ongoing process that may be a function of, inter alia, the elapsed time between onset of an event triggering marker release into the tissues and the time the sample is obtained or analyzed; the elapsed time between sample acquisition and the time the sample is analyzed; the type of tissue sample at issue; the storage conditions; the quantity of proteolytic enzymes present; etc., it may be necessary to consider tliis degradation when both designing an assay for one or more markers, and when performing such an assay, in order to provide an accurate prognostic or diagnostic result. In addition, individual antibodies that distinguish amongst a plurality of marker fragments may be individually employed to sep arately detect the presence or amount of different fragments. The results of this individual detection may provide a more accurate prognostic or diagnostic result than detecting the plurality of fragments in a single assay. For example, different weighting factors may be applit d to the various fragment measurements to provide a more accurate estimate of the amount of natriuretic peptide originally present in the sample. [0090] In a similar fashion, many of the markers described herein are synthesized as larger precursor molecules, which are then processed to provide mature marker; and/or are present in circulation in the fom?. of fragments of the marker, i hus, 'related markers" u> each of the markers described herein may be identified and used in an analogous fashion to that described above for BMP. f 0091] Removal of polypeptide markers from the circulation often involves degradation pathways. Moreover, inhibitors of such degradation pathways may hold promise in treatment of certain diseases. See, e.g., Trindade and Rouleau, Heart Fail. Monit. 2: 2-7, 2001. However, the measurement of t.ie polypeptide markers has focused generally upon measurement of the intact form without consideration of the degradation state of the molecules. Assays may be designed with an understanding of the degradation pathways of the polypeptide markers and the products formed during this degradation, in order to accurately measure the biologically active forms of a particular polypeptide marker in a sample. The unintended measurement of boHi the biologically active polypeptide markers) of interest and inactive fragments derived from the markers may result in an overestimation of the concentration of biologically active form(s) in a sample. • 0092V The failure to consider the degradation fragments that may be present in a clinical sample may have serious consciences for the accuracy of any diagnostic or prognostic method. Consider for example a simple case, where a sandwich immunoassay is provided for BNP, and a significant amount (e.g., 50%) of the biologically active BNP that had been present has now been degraded into an inactive form. An immunoassay formulated with antibodies that bind a region common to the biologically active BNP and the inactive fragments) will overestimate the amount of biologically active BNP present in the sample by 2-fold, potentially resulting in a 'false positive" result. Overestimation of the biologically active form(s) present in a samp'.e may also have serious consequences for patient management. Considering the BNP example again, the BNP concentration may be used to determine if therapy is effective (e.g., by monitoring BNP to see if an elevated level is returning to normal upon treatment). The same "false positive" BNP result discussed above may lead the physician to continue, increase, or modify treatment because of the false impression that current therapy is ineffective. [0093] Likewise, it may be necessary to consider the complex state of one or more markets described herein. For eximple, troponin exists in muscle ruainly as a "ternary complex' comprising three troponin polypeptides (T, I and C). But troponin I and troponin T circulate :n the blood in forms other than the I/T/C ternery complex. Rather, each of (i) free cardiac-specific troponin I, (ii) binary complexes (e.g., troponin I/C complex), and (iii) ternary complexes all circulate in the blood. Furthermore, the "complex state" of troponin I and T may change over time in a patient, e.g., due to binding of free troponin polypeptides to other circulating troponin polypeptides. Immunoassays that fail to consider the "complex state" of a protein marker may net detect all of the marker present. [00941 Preferably, the methods described hereinafter utilize one or more markers that are derived from the subject. The tena "subject-derived marker" as used herein refers to protein, polypeptide, phospholipid, nucleic acid, prion, glycoprotein, proteoglycan, glycolipid, lipid, lipoprotein, carbohydrate, or sn.all molecule markers that are expressed or produced by one or more cells of the subject. The presence, absence, amount, or change in amount of one or more markers may indicate that a particular disease is present, or may indicate that a particular diseac-« ;.% absent. Additional moikers may be used that are derived not from the subject, such as molecules expressed by patho genie or infectious organisms that are correlated with a particular disease, race, time since onset, sex, etc. Such markers are preferably protein, polypeptide, phospholipid, nucleic acid, prion. or small molecule markers that identify the infectious diseases described above. [0095] The term "test sample" as used herein refers to a sample of bodily fluid obtained tor the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. J n certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred test samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions. In addition, one of skill in the art would realize that some test samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components. [0096] As used herein, a "plurality" refers to at least two. Preferably, a plurality refers to at least 3. more preferably at least 5, even more preferably at least 10, even more preferably at luast 15, and most preterably at 'east 20. In particularly preferred embodiments, apliu is :i large number, i.e., at least 100. 100971 The term "subject" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Further, while a subject is preferably a living organism, the invention described h«rem may be used in post-mortem analysis as well. Preferred subjects are "patients," i.e., living humans that are receiving medical care. This includes persons with no defined illness who are being investigated for signs of pathology. 10098J The term "diagnosis'" as used herein refers to muthods by which the skilled artisan can estimate and/or determine whether or not a patient is suffering from a given disease or condition. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, i.e., a marker, the presence, absence, or amount of which is indicative of the presence, severity, or absence o; the condition. [0(»9j ' Similarly, a "prognosis" is often determined by examining one or more "prognostic indicators." These are markers, the presence or amount of which in a patient (or a sample obtained from the patienO signal a probability that a given course or outcome will occur. For example, when one or more prognostic indicators reach a sufficiently high level in samples obtained from such patients, the level may signal that the patient is at an increased probability for experiencinga fuu:re stroke in comparison to a similar patient exhibiting a ,ower marker level. A level or a change in levei of a prognostic indicator, which in turn is associated with an increased probability of morbidity or deadi,.is referred to as being •';isi=ociated with an increased predisposition to an adverse outcome" in a patient. Preferred prognostic markers can predict the onset of delayed neurologic deficits in a patient after stroke, or the chance of future stroke. 10100] The term "correlating," as used herein in reference to the use of diagnostic and prognostic markers, refers to comparing the presence or amount of the marker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known tc be free of a given condition. As discussed above, a marker level in a patient sample can be compared to a level known to be associated with a specific diagnosis. The sample's marker level is said to have been correlated with a diagnosis; that is, the skilled artisan can use the marker level to determine whether the patient suffers from a specific type diagnosis, and respond accordingly. Alternatively, the sample's marker level can be compared to a marker level known to be associated with a good outcome (e.g., the absence of disease, etc.). In preferred embodiments, a profile of marker levels are correlated to a global probability or a particular outcome. [0101j The phrase "detenninng the diagnosis" as used herein refers to methods by which the skilled artisan can determine tie presence or absence of a particular disease in a patient. The term "diagnosis" does not refer to the ability to determine the presence or absence of a particular disease with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term "diagnosis" refers to an increased probability that a certain disease is present in the subject. In preferred embodiments, a diagnosis indicates about a 5% increased chance that a disease is present, about a ,"•')% chance, about a \5Yo chance, about a 20% chance, about a 25-% chance, about a 30% chance, about a 40% chance, about a 50% chance, abput a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance. The term "about" in this context refers to r/- 2%. [0102] Similarly, the phrase "determining the prognosis" as used herein refers to methods hv which the skilled artisan can determine the likelihood of one or more future clinical outcomes for a patient. The skilled artisan will understand that the term "prognosis" refers to an increased probability that a certain clinical outcome will occur at a future date in the subject, hi preferred embodime its, a prognosis indicates about a 5% increased chance of a certain clinical outcome compared to a "control" population, about a 10% chance, about a 15% chance, about a 20% chanoe, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance., and about a 95% chance. The term "about" in this context refers to +/- 2%. [0103] The term "discrete" as used herein refers to areas of a surface that are non contiguous. That is, two areas are discrete from one another if a border that is not part of either area completely surrounds each of the two areas. [0104] The term "independently addressable" as used herein refers to discrete areas of a surface from which a specific signal may be obtained. f 0105] The term "antibody" as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See. e.g. Fundamental Immunology, 3rd Jidition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. fmmunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody ir.eludes antigen-binding portions, i.e., "antigen binding sites,' (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL. VH, CL and CHI dorrains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:54i-546), which consists cf a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody." f U106] The term "specific marker of myocardial injury" as used herein refers to molecules th;it are typically associated wit!: cardiac tissue, and which can be correlated with a cardiac iujuiy, but are not correlated With other types of injury. Such specific markers of cardiac mjury include annexin V, B-type natriuretic peptide, p-enolase, cardiac troponin I (free and/or complexed), cardiac troponinT (free and/or complexed), creatine kinase-MB, glycogen phosphorylase-BB, heart-type fatty acid binding protein, phosphoglyceric acid mutase-MB, and S-lOOao. f 0107] The term "specific marker of neural tissue injury1' as used herein refers to molecules that are typically associated with neural tissue, and which can be correlated with a neural injury, but are not correlated with other types of injury. Exemplary specific markers of neural tissue injury are describee in detail hereinafter. [010 S] Differential Diagnosis of Dyspnea (Shortness of Breath) f 0 109] The present invention is described hereinafter generally in terms of the differential diagnosis of diseases and conditions related to dyspnea. The skilled artisan will understand, however, that the concepts of symptom-based differential diagnosis described herein are generally applicable to any physical characteristics that are indicative of a plurality of possible etiologies such as fever, neurolo-gic dysfunction, chest pain ("angina"), dizziness, headache, cic. [0110." A first step in the idertification of suitable markers for symptom-bases differential diagnosis requires a consideration of the possible diagnoses vhat may be causative of the nonspecific symptom observed. In the case of dyspnea, the potential causes are myriad. In a preferred embodiment, the following discussion considers three potential diagnoses: congestive heart failure, pulmonary embolism, and myocardial infarction; and three potential markers for inclusion in a differential diagnosis panel for these potential diagnoses: BNP, Dilimer, y^L- cardiac troponin, respectively. In another preferred embodiment, markers for three potential diagnoses, congestive 'leart failure, pulmonary embolism, and myocardial infarction include three potential markers in a differential diagnosis panel, BNP related peptides, Dilimer, and cardiac troponin, respectively. In a preferred embodiment, three potential diagnoses in the case of dyspnea include congestive heart failure, pulmonary embolism, and myocardial infarction, hi a second preferred embodiment, tour potential diagnoses in the case .;!:" dyspnea include congestive heart failure, pulmonary embolism, and myocardial infarction, and atrial fibrillation. Potential -narkers for inclusion in a differential diagnosis panel include one or more of the following: BJNP, BNP related peptides, D-dimer, cardiac troponin, ANP, and ANP related peptides. 101II] BNP 10112] B-type natriuretic peptide (BNP), also called brain-type natriuretic peptide is a 32 amino acid, 4 kDa peptide that ir. involved in the natriuresis system to regulate blood pressure and fluid balance. Bonow, R.CL Circulation 93:1946-1950 (1996). The precursor to BNP is synthesized as a lOS-amino acid molecule, referred to as "pre pro BNP," that is proteolytically processed into a ;d-amino acid N-terminal ptpiide (amino acick l-7u), referred to as "NT pro BNP" and the 32-amino acid mature hormone, referred to as BNP or BNP 32 (amino acids 77-108). It has been suggested that each of these species — NT pro-BNP, BNP-32, and the pre pro BNP - can circulate in human plasma. Tateyama et al., Biochem. Biophys. Res. Commun. 185: 760-7 (1992); Hunt et al., Biochem. Biophys. Res. Commun. 214: 1175-83 (1995). The 2 forms, pre pro BNP and NT pro BNP, and peptides which are derived from BNP, pre pro BNP and NT pro BNP and which are present in the blood as a result of proteolyses of BNP, NT pro BNP and pre pro BNP, are collectively described as markers related to or associated with BNP. f 0113 ] BNP and BNP-relateci peptides are predominantly found in the secretory granules of the cardiac ventricles, and are rsleased from the heart in response to both ventricular volume expansion and pressure' overload. Wilkins, M. et al.} Lancet 349: 1307-10 (1997). Elevations of BNP are associated with raised atrial and pulmonary wedge pressures, reduced ventricular systolic and diastoKc function, left ventricular iiypertrophy, and myocardial infarction. Sagnella, G.A., Cliirc.al Science 95: 519-29 (199S). Furthermore, there are r.umeroas reports of elevated BNP concentration associated with congestive heart failure and renal failure. Thus, BNP levels :n a patient may be indicative of several possible underlying causes of dyspnea. [HI 14] D-dimer j n 11 5] D-dimer is a crosslinked fibrin degradation product with an approximate molecular mass of 200 kDa. The normal plasma concentration of D-dimer is The plasma concentration of D-dimer also will be elevated during any condition associated with coagulation and fibrinolysis activation, including stroke, surgery, atherosclerosis, trauma, and thrcmbotic thrombocytopenic purpura. D-dimer is released into the bloodstream immediately fo> lowing proteolytic clot dissolution by plasmin. The plasma concentration of D-dimer can exceed 2 jig/ml in patients with unstable angina. Gurfinkel, E. i-titi'.. Br Heart J. 71: 151-55(1994). Plasma D-dimer is a specific marker of fibrinolysis and Indicates the presence of a prothrombotic state associated with acute myocardial infarction and unstable angina. The plasma concentration of D-dimer is also nearly always elevated in patients with acute pulmonary embolism; thus, normal levels of D-dimer may allow the exclusion of pulmonary embolitm. Egermayer et al., Thorax 53: 830-34 (1998). [01 17] Cardiac Troponin I Oi l S] Troponin I (Tnl) is a .15 kDa inhibitory element af the troponin complex, found in muscle tissue. Tnl binds to actin m the absence of Ca2+, inhibiting the ATPase activity of aciomyosin. A Tnl isoform that is found in cardiac tissue (cTnl) is 40% divergent from skeletal muscle Tnl, allowing bi th isoforms to be immunologically distinguished. The normal plasma concentration of cTnl is M.J. eta!., Clin. Cardiol. 22: 13-16 (1999); Musso, P. etal.,J. Ml. Cardiol. 26: 1013--23 (1 905); Hclvoet, P. et al., JAMA 281: 1718-21 (1999); Holvoet, P et al., Circulation 98: 14K7-94(1998). [0119] The plasma concentration of cTnl in patients with acute myocardial infarction is sigiiificantly elevated 4-6 hours :ifter onset, peaks between 12-16 hours, and can remain elevated for one week. The release kinetics of cTnl associated with unstable angina may be similar. The measurement of specific forms of cardiac troponin, including free cardiac troponin I and complexes of cardiac troponin I with troponin C and/or T may provide the user with the ability to identify various stages of ACS. Free and complexed cardiac-troponin T may be used in a manner analogous to that described for cardiac troponin I. Cardiac troponin T complex may be useful either;done or when expressed as a ratio with total cardiac troponin I to provide information related to the presence of progressing myocardial damage. Ongoing ischemia may result in the release of the cardiac troponin TIC complex, indicating that higher ratios of cardiac troponin TIC:total cardiac troponin I maybe indicative of continual damage caused by unresolved ischemia. See, U.S. Patent Nos. 6,147,688, 6,156,521, 5,947,124, and 5,795,725, which are hereby incorporated by reference in their entirety, including all tables, figures, and claims. One skilled in the art recognizes that in measuring cardiac troponin, one can measure the different isofonns of troponin I and troponin T. [0120] One skilled in the art recognizes that in measuring cardiac troponin, one can measure the different forms of trooonin I and troponin T. Thus, one may preferably measure free cardiac troponin I, free cardiac troponin T, cardiac troponin I in a complex comprising one or both of troponin T and troponin C, cardiac troponin T in a complex comprising one or both of troponin I and troponin C, total cardiac troponin I (meaning free and complexed cardiac troponin I), and/or total cardiac troponin T, The tern "at least one cardiac troponin form" as used herein refers to any one of these foregoing forms. [0121 j- ANP ."0122] A-type natriuretic peptide (ANP) (also referred to as atrial natriuretic peptide or cjirdiodilatin Forssmann et al Hatochem Cell Biol 110: 335-357 (1998)) is a 28 ammo acid peptide that is synthesized, stored, and released atrial myocytes in response to atrial distension, angiotensin II stimulation, endothelin, and sympathetic stimulation (beta;HIronoceptor mediated). ANP is synthesized as a precursor molecule (pro-ANP) that is converted to an active form, ANP, by proteolytic cleavage and also forming N-terminal ANP (1 -l>8). N-terminal ANP and ANP have been reported to increase in patients exhibiting atrial fibrillation and heart failure (Rossi et al. Journal of the American College of Cardiology 35: 1256-62 (2000). In addition to arial natriuretic peptide (ANP99-126) itself, linear peptide fragments from its N-terminal prohormone segment have also been reported to have biological aclivity. As the skilled artisan will recognize, however, because of its relationship to ANP, the concentration of N-turminal ANP molecule can also provide diagnostic or prognostic information in patients. The phrase "marker related to ANP or ANP related peptide" refers to any polypeptide that originates from the pro-ANP molecule (1-126), other than the 28-amino acid ANP molecule itself. Proteolytic degradation of ANP and of peptides related to ANP have also been described in the literature and these proteolytic fragments are also encompassed it the term "ANP related peptides." 0123] Elevated levels of ANP are found during hypervolemia, atrial fibrillation and :ongestive heart failure. ANP is involved in the long-term regulation of sodium and water Balance, blood volume and arterial pressure. This hormone decreases aldosterone release by he adrenal cortex, increases glomerular filtration rate (GFR), produces natriuresis and liuresis (potassium sparing), and decreases renin release thereby decreasing angiotensin II. hcse actions contribute to reductions in blood volume and therefore central venous pressure '.?VP), cardiac output, and arteria.1. blood pressure. Several isoforms of ANP have been identified, and their relationship Circulation 100:1722-6, 1999; E. trada etal., Am. J. Hypertetis. 7:1085-9,1994. [01241 Chronic elevations of ANP appear to decrease arterial blood pressure primarily by decreasing systemic vascular resistance. The mechanism of systemic vasodilation may involve ANP receptor-mediated elevations in vascular smooth muscle cGMP as well as by attenuating sympathetic vascular tone. This latter mechanism may involve ANP acting upon sites within the central nervous system as well as through inhibition of norepinephrine release i>v sympathetic nerve terminals. ^VNP may be viewed as a counter-regulatory system for the lenin-angiotensin system. A new class of drugs that are neutral endopeptidase (NEP) inhibitors have demonstrated efficacy in heart failure. These drugs inhibit neutral ondopeptidase, the enzyme responsible for the degradation of ANP, and thereby elevate plasma levels of ANP. NEP inhibition is particularly effective in heart failure when the drug lias a combination of both NEP raid ACE inhibitor properties. [0125] Based on the foregoing discussion, the skilled artisan will recognize that, for example, increased BNP is indicative of congestive heart feilure, but may also be indicative of other cardiac-related conditions such as myocardial infarction. Thus, the inclusion of a marker related to myocardial injury such as cardiac troponin I and/or cardiac troponin T can permit further discrimination of the disease underlying the observed dyspnea and the increased BNP level. In tnis case, an increased level of cardiac tropcniu maybe used to rule in myocardial infarction. ! 0 i 26] Similarly, BNP may also be indicative of pulmonary embolism. The inclusion of a marker related to coagulation and hemostasis such as D-dimer can permit further discrimination of the disease underlying the observed dyspnea and the increased BNP level. tii this case, a normal level of D-dimer may be used to rule out pulmonary embolism. j'O L 27] A detailed analysis c f this exemplary marker panel is provided in the examples hereinafter. The skilled artisan will readily acknowledge that other markers may be substituted in or added to such ir arker panels to further discriminate the causes of dyspnea in accordance with the methods fcr identification and use of diagnostic markers described herein. Additional suitable markers are described in the following sections. [0128] As discussed in detail herein, the foregoing principles of marker panel design may bo applied broadly to symptom-based differential diagnosis. For example, in the case of abdominal pain, the pluralityo: markers are preferably selected to rule in or out a plurality of (he following: aortic dissection, mesenteric embolism, pancreatitis, appendicitis, angina, royocanJial infarction, one or more infectious diseases described above, influenza, esophageal carcinoma, gastric adenocarcinoma, colorectal adenocarciiioma, pancreatic tumors including tJiictal adenocarcinoma, cystadcnocarcinoma, and insulinoma. In a preferred embodiment, the potential diagnoses for abdominal pain include aortic aneurysm, mesenteric embolism, pancreatitis, appendicitis, angir.a and myocardial infarction. [0129] The foregoing principles may also be applied to subdivide differential diagnosis to 3 given level of detail required by the clinical artisan. For example, the differential diagnosis of various symptoms may requi-e discrimination between heart failure and atrial fibrillation. An exemplary marker panel for performing such discrimination preferably includes BNP or BMP related peptides, and ANP or ANP related peptides, respectively. Additional markers may be defined to distinguish between systolic and diastolic dysfunction and atrial fibrillation. Preferred markers in this case include BNP, calcitonin gene related peptide, calcitonin and urotensin 1 for differentiation oi"systolic and diastolic dysfunction and ANP or ANP related peptides for the detection of atnal fibrillation. Likewise, markers maybe defined to distinguish between systolic and diastolic dysfunction, atrial fibrillation, myocardial ischemia and cardiac necrosis. Preferred markers in this case include BNP, calcitonin gene related peptide, calcitonin and urotensin 1 for differentiation of systolic and diastolic dysfunction and ANP or ANP related peptides foi the detection of atrial fibrillation and BNP and cardiac troponins for the detection of m>ocardial ischemia and necrosis. [0130] In the case of chest pain, the present invention can provide markers able to distinguish between aortic dissection, myocardial ischemia, and cardiac necrosis; markers able to distinguish between aortic dissection, myocardial ischemia, and myocardial infarction; markers able to distinguish between aortic dissection, myocardial ischemia, cardiac necrosis and heart failure; markers able to distinguish between aortic dissection, myocardial ischemia, cardiac necrosis and myocardial infarction; markers able to distinguish between aortic dissection, myocardial ischemia, cardiac necrosis and atrial fibrillation; and/or markers able to distinguish between aortic dissec'ion, myocardial ischemia and cardiac necrosis, myocardial infarction and atrial fibrillation. In accordance with the foregoing, a particularly preferred marker for aortic dissection is smooth muscle myosin, and most preferably smooth muscle myosi/iJvJ'?vy chain, and a particularly preferred marker for atrial fibrillation is ANP or an 'XNP-reliited marker. I'0131] Preferred marker sets are those comprising smooth muscle myosin (or smooth muscle myosin heavy and/or light chains) and ANP or an ANP-related marker to distinguish aortic dissection and atrial fibrillation; smooth muscle myosin (or smooth muscle myosin heavy and/or light chains), ANP or an ANP-related marker, and BNP or a BNP-related marker to distinguish aortic dissection, atrial fibrillation and myocardial ischemia; smooth muscle myosin (or smooth muscle myosin heavy and/or light chains), BNP or a BNP-related marker, and a cardiac rroponin fc-rm to distinguish aortic dissection, myocardial ischemia, and tnyocardial infarction; and smooih muscle myosin (or smooth muscle myosin heavy and/or light chains), BNP or a BNP-related marker, creatine kinase MB, myoglobin, and a cardiac troponin form to distinguish aort.c dissection, myocardial ischemia, cardiac necrosis, and myocardial infarction. [0132] Congestive heart failure is a heterogenous condition arising from two primary pathologies: left ventricular diastolic dysfunction and systolic dysfunction, which occur either iiion.. or in cc.n'jinatiu;.. Gaasch, JAMA 271: 1276-80 (1994). As many as 40 percent of patients with clinical heart failure have diastolic dysfunction with normal systolic function. Soufer et al., Am. J. Cardiol. 55: 1032-6 (1984). Patient care decisions and prognosis hinge upon determination of the preser.ce of one or both of these pathologies. Shamsham and Mitchell, Am. Fam. Physician 2000; 61:1319-28 (2000). Exemplary marker panels related to differentiating systolic and diastolic function comprise one or more markers selected from the group consisting of BNP, BNP related peptides, aldosterone, ANP, ANP related peptides, nrodilatin, angiotensin 1, angiotansin 2, angiotensin 3, bradykinin, calcitonin, calcitomn gene related peptide, endothelin-2, eniothelin-3, renin, urotensin 1, urotensin 2, antithrombin III, D-dimer, MMP-3, MMP-9, MMP-11, carboxy terminal propeptide of type I collagen (PICP), collagen carboxy terminal telopf-ptide (ICTP), fibrinogen, iibronectin, and vasopressin. Markers related to both systolic and diastolic dysfunction include BNP, ANP and ANP related markers. A preferred list of mar cers for differentiating systolic and diastolic heart failure include one or more markers selected from the group consisting of BNP, BNP related peptides, calcitonin gene related peptide, urotensin 2, endothelin 2, calcitonin and angiotensin 2. i. pnrticuiarly preferred list of markers for differentiating systolic and diastolic dysfunction include; one or more markers selected from the group consisting of BNP, angiotensin 2, urotensin 2, and calcitonin gene related peptide. [0133] Exemplary marker panels related to differentiating aortic dissection, myocardial ischemia, and myocardial infarc'ion comprise one or more markers selected from the group consisting of smooth muscle myosin and/or smooth muscle myosin heavy chain, BNP and/or BNP related peptides, one or more troponin forms, and myoglobin. [0134] Exemplary marker panels related to differentiating atrial fibrillation, myocardial infarction, and/or congestive heart failure comprise markers selected from the group consisting of AMP, ANP related peptides, one or more troponin forms, myoglobin, BNP, and BNP related peptides. [0135] hi the case of disturbzmes of metabolic state, the plurality of markers are preferably selected to rule in or out a plurality of the following: diabetes mellitus, diabetic ketoacidosis, alcoholic ketoacidosis, respiratoiy acidosis, respiratory alkalosis, nonketogenic hyperglycemia, hypoglycemia, riiial failure, interstitial renal disease, COPD, pneumonia, pulmonary edema and asthma. [0136] Differential Diagnosis of Neurologic Dysfunction [0137] In the case of neurologic dysfunction, the plurality of markers are preferably selected to rule in or out a plurality of the following: stroke, brain tumor, cerebral hypoxia, hypoglycemia, migraine, atrial fibrillation, myocardial infarction, cardiac ischemia, peripheral vascular disease and seizure. Preferred markers in this case include specific markers of cerebral injury such as adenylate Jdnase, brain-derived neurotrophic factor, calbindin-D, creatine kinase-BB, glial fibrillary acidic protein, lactate dehydrogenase, myelin basic protein, neural cell adhesion molecule, neuron-specific enolase, neurotrophin-3,proteolipid protein, S1 OOp, thrombomodulin, protein k riase C gamma; and/or one or more non-specific markers of cerebral injury such as p-thromboglobulin, D-dimer, fibrinopeptide A, plasmin-a-2 antiplasmin complex, platelet factor 4, prothrombin fragment 1+2, thrombin-antithrombin III complex, tissue factor, von Willebrand factor, adrenomedullin, cardiac troponin I (for myooardial ischemia and necrosis), head activator, hemoglobin 02 chain, caspase-3, vascular eniiothelial growth factor (VEG?), one or more endothelins (e.g., endothelin-1, endothelin-2, and endothelin-3), interleukin-8, Atrial natriuretic peptide, B-type natriuretic peptide (for myocardial ischemia and necrosis), and C-type natriuretic peptide; and/or one or more acute phase reactants such as C-reacti 'e protein, cemloplasmin, fibrinogen, al-acid glycoprotein, o.l -antitrypsin, haptoglobin, insulin-like growth factor-1, interleukin-lp, interleukin-1 leceotor antagonist, interleukin- metalloproteinase 9 (MMP-9)), :r..onocyte chemotactic prot3in-l, and vascular cell adhesion molecule. [0138] Stroke is a pathological condition with acute onset that is caused by the occlusion or rupture of a vessel supplying blood, and thus oxygen and nutrients, to the brain. The immediate area of injury is refer/ed to as the "core," which contains brain cells that have died as a result of ischemia or physics! damage. The "penumbra" is composed of brain cells that are neurologically or chemically connected to cells in the core. Cells within the penumbra are injured, but still have the ability .o completely recover following removal of the insult caused during stroke. However, as ischemia or bleeding from hemorrhage continues, the core of dead cells can expand from the s te of insult, resulting in a concurrent expansion of cells in the penumbra. The initial volume and rate of core expansion is related to the severity of the stroke and, in most cases, neurological outcome. [0139] The brain contains two major types of cells, neurons and glial cells. Neurons are the most important cells in the brain, and are responsible for maintaining communication within the brain via electrical anc chemical signaling. Glial cells function mainly as structural components of the brain, and they are approximately 10 times more abundant than neurons. Glial cells of the central nervous system (CNS) are astrocytes and oligodendrocytes. Astrocytes are the major interstitial cells of the brain, and they extend cellular processes that are intertwined with and surround neurons, isolating them from other neurons. Astrocytes can nlso form ;end feet" at the end of their processes that surround capillaries. Oligodendrocytes are cells that form myelin sheathes around axons in the CNS. Each oligodendrocyte has the ability to ensheathe up to 50 axois. Schwann cells are glial cells of the peripheral nervous system (PNS). Schwann cells form myelin sheathes around axons in the periphery, and each Schwann cell ensheathes a single axon. i o 140] Cell death during stroke occurs as a result of ischemia or physical damage 10 the fdls of the CNS. During ischerr ic stroke, an infarct occurs, greatly reducing or stopping Mood flow beyond the site of inf nrction. The zone immediately beyond the infarct soon lacks • -iiLiable blood concentrations of .he nutrients essential for cell survival. Cells that lack nutrients essential for the maintenance of important functions like metabolism soon perish. Hernorrhagic stroke can induce cell death by direct trauma, elevation in intracranial pressure, and the release of damaging biochemical substances in blood. When cells die, they release their cytosolic contents into the extracellular milieu. [0141J The barrier action of tight junctions between the capillary endothelial cells of the central nervous system is referred to as the "blood-brain barrier". This barrier is normally impermeable to proteins and other molecules, both large and small. In other tissues such as skeletal, cardiac, and smooth muscle, the junctions between endothelial cells are loose enough to allow passage of most molecules, but not proteins. [0142] Substances that are secreted by the neurons and glial cells (intracellular brain eoiv.partment) of the central nervous system (CNS) can freely pass into the extracellular milieu (extracellular brain compaitment). Likewise, substances from the extracellular brain compartment can pass into the intracellular brain compartment. The passage of substances Between the intracellular and extracellular brain compartments are restricted by the normal cellular mechanisms that regulate substance entry and exit. Substances that are found in the extracellular brain compartment aiso are able to pass freely into the cerebrospinal fluid, and vice versa. This movement is controlled by diffusion. i • j ] 4? 1 The movement of substances between the vasculature and the CNS is restricted by the blood-brain barrier. This restriction can be circumvented oy facilitated transport mechanisms in the endothelial cells that transport, among other substances, nutrients like glucose and ammo acids across tie barrier for consumption by the cells of the CNS. Furthermore, lipid-soluble substances such as molecular oxygen and carbon dioxide, as well as any lipid-soluble drugs or narcotics can freely diffuse across the blood-brain .barrier. [0144] Depending upon their size, specific markers of cerebral injury that are released from injured brain cells during stroke or other neuropathies will only be found in peripheral blood when CNS injury is coupled with or followed by an increase in the permeability of the bli?oii-brain barrier. This is partic nlarly true of larger molecules. Smaller molecules may appear in the peripheral blood as a result of passive diffusion, active transport, or an increase in the permeability of the blood-brain barrier. Increases in blood-brain burner permeability ran arise us a result of physical d sruption in cases such as tumor invasion and extravasation or vascular rupture, or as a result of endothelial cell death due to ischemia. During stroke, the blood-brain barrier is compromised by endothelial cell death, and any cytosolic components o ('dead cells that are present within the local extracellular milieu can enter the bloodstream. 101451 Therefore, specific markers of cerebral injury may also be found in the blood or in blood components such as serum and plasma, as well as the CSF of a patient experiencing stroke or TIAs. Furthermore, clearance of the obstructing object in ischemic stroke can cause injury from oxidative insult during reperfusion, and patients with ischemic stroke can sunk,rime,s experience hemorrhagic transformation as a result of reperfusion or thrombolvtic therapy. Additionally, injury can be caused by vasospasm, which is a focal or diffuse narrowing of the large capacity arteries at the base of the brain following hemorrhage. The increase in blood-brain barrier permeability is related to the insult severity, and its integrity is reestablished following the resolution of insult. Specific markers of cerebral injury will only be present in peripheral blood if * here has been a sufficient increase in the permeability of the Mood-brain barrier that allows these large molecules to diffuse across. In this regard, most specific markers of cerebral injury can be found in cerebrospinal fluid after stroke or any other neuropathy that affects the CNS. Furthermore, many investigations of coagulation or fibrinoiysis markers in stroke are performed using cerebrospinal fluid. 0146] The Coagulation Cast-tide in Stroke [0147] There are essentially two mechanisms that are used to halt or prevent blood loss following vessel injury. The first mechanism involves the activation of platelets to facilitate adherence to the site of vessel ii\iury. The activated platelets then aggregate to form a platelet plug that reduces or temporarily stops blood loss. The processes of platelet aggregation, plug formation and tissue repair are til accelerated and enhanced by numerous factors secreted by activated platelets. Platelet aggregation and plug formation is mediated by the formation of a librinogen bridge between activated platelets. Concurrent activation of the second mechanism, the coagulation cascade, results in the generation of fibrin from fibrinogen and the formation of an insoluble fibrin clot that strengthens the platelet plug. .0 i -IS] The coagulation case jde is an enzymatic pathway that involves numerous serine proteinases normally present in £ n inactive, or zymogen, form. The presence of a foreign surface in tht vasculature or vascular injury results in the activation of the intrinsic and extrinsic coagulation pathways, respectively. A final common pathway is then followed, which results in the generation o.r fibrin by the serine proteiriase thrombin and, ultimately, a cross linked fibrin clot. In the coagulation cascade, one active enzyme is formed initially, which can activate other enzyme.' that active others, and this process, if left unregulated, can continue until all coagulation enzymes are activated. Fortunately, there are mechanisms in place, including fibrinolysis and the action of endogenous proteinase inhibitors that can regulate the activity of the coagulation pathway and clot formation. 10149] Fibrinolysis is the process of proteolytic clot dissolution. In a manner analogous to coagulation, fibrinolysis is mediated by serine proteinases that are activated from their /ymogen form. The serine proteinase plasmin is responsible for the degradation of fibrin into smaller degradation products that are liberated from the clot, resulting in clot dissolution. Fibrinolysis is activated soon after coagulation in order to regulate clot formation. Endogenous serine proteinase inhibitors also function as regulators of fibrinolysis. [01 50] The. presence of a coagulation or fibrinolysis marker in cerebrospinal fluid would indicate that activation of coagulation or fibrinolysis, depending upon the marker used, coupled with increased permeability of the blood-brain barrie.- has occurred. In this regard, more definitive conclusions reg,zjding the presence of coagulation or fibrinolysis markers associated with acute stroke may be obtained using cerebrospinal fluid. [0! 51] Platelets are round or oval disks with an average diameter of 2-4 j.im that are normal^v found in blood at a concentration of 200,000-300,000/^1. They play an essential role in maintaining hemostasis by maintaining vascular integrity, initially stopping bleeding by forming a platelet plug at the site of vascular injury, and by contributing to the process of fibrin formation to stabilize the platelet plug. When vascular injury occurs, platelets adhere to the site of injury and each othei and are stimulated to aggregate by various agents released from adherent platelets and injured endothelial cells. This is followed by the release reaction, in which platelets secrete the contents of their intracellular granules, and formation oi the platelet plug. The formation of fibrin by thrombin in the coagulation cascade allows for consolidation of the plug, followed by clot retraction and stabilization of the plug by crosslinked fibrin. Active thrombin, generated in the concurrent coagulation cascade, also has the ability to induce platelet activation and aggregation. 10152] The coagulation cascade can be activated through either the extrinsic or intrinsic pathways. These enzymatic pathways share one final common pathway. The result of coagulation activation is the formation of a crosslinked fibrin clot. Fibrinolysis is the process of proteolytic clot dissolution tlvt is activated soon after coagulation activation, perhaps in an ^ffou to control the rate ard amount cf clot formation. Urokizase-lype plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) proteoljtically cleave plasminogen, generating the active serine prot&inase plasmin. Plasmin proteolytically digests crosslinked fibrin, resulting in clot dissolution and the production and release of fibrin degradation products. [0 i 53] The first step of the common pathway of the coagulation cascade involves the proteolytic cleavage of prothrombin by the factor Xa/factor Va prpthrombinase complex to yield active thrombin. Thrombir. is a serine proteinase that proteolytically cleaves fibrinogen to form fibrin, which is ultimate'y integrated into a crosslinked network during clot formation. |'0154J Methods and marker sets for differential diagnosis of stroke and other cerebral injuries are described in U.S. PaUvit No. 10/225,082, filed August 20,2002, which is hereby incorporated in its entirety, including all tables figures and claims. As described therein, preferred marker panels diagnose and/or differentiate between stroke, subarachnoid hemorrhage, intracerebral hemorrhage, and/or hemorrhagic stroke; and/or can distinguish hetv.-eeii ischemic and hemorrhr.gic stroke. Particularly preferred are markers that differentiate between thrombotic, embolic, lacunar, hypoperfusion, intracerebral hemorrhage, and subarachnoid hemorrhage types of strokes. Particularly preferred marker sets include BMP, 1L.-6, S-100P, MMP-9, TAT complex, and vWF Al-integrin; BNP, 8-100(3, MMP-9, and vWF-Al-integrin; vWF-Al, VEGF, and MMP-9; caspase-3, MMP-9, and GFAP; caspasc-3, MMP-S, vWF-Al. and BNP; NCAM, BDNF, Caspase-3, MMP-9, vWF-Al, and VEGF; NCAM. BDNF, Caspase-3, MMP-9, vWF-Al, and S-100P; VEGF; NCAM, BDNF. Caspase j. MMP-9. vWF-Al, and MCP-I; VEGF; NCAM, BDNF, Caspase-3, MMP-9, VEGF, and vWF Al-int.egrin; BDNF, MMF-9, S-lOOp, vWF Al-integrin, MCP-1, and GFAP; BDNF, caspase-3, MMP-9, vWF-Al, S-1 OOP, and GFAP; NCAM, BDNF, MMP-9, vWF-Al, S1000, and GFAP; NCAM, BDNF, caspase-3, MMP-9, S-100P, and GFAP; caspase-3, NCAM, MCP-1, S100P, MMP-'), vWF Al-integrin, and BNP; caspase-3, NCAM, MCP-1, S1 OOp, MMP-9, vWF Al, BNP, and GFAP; CRP, NT-3, vWF, MMP-9, VEGF, and CKBB; CRP, MMP-9, VEGF, CKBB, and MCP-1; CRP, NT-3, MMP-9, VEGF, CKBB, and MCP-1; and CRP, MMP-9, VEGF, CKBB, MCP-1. Calbindin, vWF VP1, vWF A3, vWF A1-A3, TAT complex, proteolipid protein, IL-6, IL-8, myelin basic protein, S-100P, tissue factor, GFAP, vWF Al-integrin, CNP, and NCAM. ! 015 5] A panel consisting of the markers referenced herein may be constructed to provide relevant information related to the differential diagnosis of interest. Such a panel may be constructed using 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17,18,19 or 20 individual markers. The analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out by one skilled in the art to optimize clinical sensitivity or specificity in various clinical settings. These include, but are not limited to ambulatory, urgent care, critical care, intensh e care, monitoring unit, inpatient, outpatient, physician office, medical clinic, and health screening settings. Furthemore, one skilled in the art can use a single marker or a subset o." markers comprising a larger panel of markers in combination with an adjustment of the diagnostic threshold in each of the aforementioned settings to optimize clinical sensitivity and specificity. The clinical sensitivity of an assay is defined as the percentage of those with the disease that the assay correctly predicts, and the specificity of an assay is defined as the percentage of those without the disease that the assay correc-tr ^i-edicts (Tietz Textbook of Clinical Chemistry, 2nd edition, Carl Burtis and Edward Ashwood eds., W.B. Saunders and Company, p. 496). The following provides a brief discussion of additional exemplary markers for use in identifying suitable marker panels by the methods described herein. [0156] The Acute Coronary Syndrome [0! 57] Myocardial ischemia is caused by an imbalance of myocardial oxygen supply and demand. Specifically, demand exceeds supply due to inadequate blood supply. The heart accounts for a small percentage of total body weight, but is responsible for 7% of body oxygen consumption. Cardiac tissue metabolism is highly aerobic and has very little reserve to compensate for inadequate bl Dod supply. When the blood supply is reduced to levels that JIG inadequate for myocardial demand, the tissue rapidly becomes hypoxic and toxic cellular metabolites can not be removed Myocardial cells rapidly use oxygen supplies remaining in the local microvasculature, and 'he length of time that aerobic metabolism continues is indirectly proportional to the decree of arterial occlusion. Once the oxygen supply has been exhausted, oxidative phosphory-&.tion can not continue because oxygen is no longer available .is an eisctron acceptor, pyruvate can not be converted to aceiyl cocnzyme A and jmer ihe citric acid cycle. Myocardial metabolism switches to anaerobic metabolism using glycogen and glucose stores, and pyruvate is fermented to lactate. Lactate accumulation is the primary cause of chest pain in individual! with ACS. As ischemia continues, cardiac tissue becomes more acidic as lactate and other jcidic intermediates accumulate, ATP levels decrease, and available energy sources are depleted. Cardiac tissue can recover if it is reperfused 15-20 minutes after an ischemic event. After the cellular glycogen stores have been depleted, the cell gradually displays features of necrosis, including mitochondria! swelling and loss of cell membrane integrity. Upon repe-fusion, these damaged cells die, possibly as a result of the cell's inability to maintain ionic equilibrium. A loss of membrane integrity causes the cell's cytosolic contents to be released into the circulation. fO 158] Stable angina, unstable angina, and myocardial infarction all share one common feature: constricting chest pah> associated with myocardial ischemia. Angina is classified as stable or unstable through a physician's interpretation of clinical symptoms, with or without diagnostic ECG changes. The classification of angina as "stable" or "unstable" does not refer to ihe j.tability of the plaque itself, but rather, the degree of exertion that is required to elicit chest pain. Most notably, the classification of chest pain as stable or unstable angina (or even mild myocardial infarction) in oases other than definitive myocardial infarction is completely .subjective. The diagnosis., and in this case the distinction, is made not by angiography, which may quantify the degree of arte-ial occlusion, but rather by a physician's interpretation of clinical symptoms. [0159] . Stable angina is characterized by constricting chest pain that occurs upon exertion nv stress, and is relieved by rest or sublingual nitroglycerin. Coronary angiography of patients with stable angina usually reveals 50-70% obstruction of at least one coronary artery. Stable angina is usually diagnosed by the evaluation of clinical symptoms and ECG changes. Patients with stable angina may have transient ST segment abnormalities, but the sensitivity and specificity of these change: associated with stable angina are low. [0160] Unstable angina is characterized by constricting chest pain at rest that is relieved by sublingual nitroglycerin. Anginal chest pain is usually relieved by sublingual nitroglycerin, and the pain usually subsides within 30 minutes. There are three classes of unstable angina severity: class I, characterized as new onset, severe, or accelerated angina; class II, subacute angina at rest characterized by increasing severity, duration, or requirement for nitroglycerin; and class III, characterized as acute angina at rest. Unstable angina represents the clinical state between stable angina and AMI and is thought to be primarily due to the progression in the severity and extent of atherosclerosis, coronary artery spasm, or hemorrhage into non-occluding plaques with subsequent thrombotic occlusion. Coronary angiography of patients with unstable angina usually reveals 90% or greater obstruction of at least one coronary artery, resulting in an inability of oxygen supply to meet even baseline myocardial oxygen demand. Slow growth of stable atherosclerotic plaques or rupture of unstable atherosclerotic plaques with subsequent thrombus formation can cause unstable angina. Both of these causes result in critical narrowing of the coronary artery. Unstable angina is usually associated with atherosclerotic plaque nip ure, platelet activation, and thrombus formation. Unstable angina is usually diagnosed by clinical symptoms, EGG changes, and changes in cardiac markers (if any). Treatments for patients with unstable angina include nitrates, aspirin, GPIIb/IIIa inhibitors, heparin, and beta-blockers. ThromboJytic therapy has not beua demonstrated to be beneficial for unstable angina patients, and calcium channel blockers mjy have no effect. Patients may also receive angioplasty and st::nts. Finally, patients with unstable angina are at risk for developing AMI. [0161] Myocardial infarctior- ;s characterized by constricting chest pain lasting longer than 30 minutes that can be accompanied by diagnostic ECG Q waves. Most patients with AMI have coronary artery disease and as many as 25% of AMI cases are "silent" or asymptomatic infarctions, and individuals with diabetes tend to be more susceptible to silent infarctions. Population studies suggest that 20-60% of nonfatal myocardial infarctions are silent infarctions that are not recognized by the patient. Atypical clinical presentations of AMI can include congestive heart failure, angina pectoris without a severe or prolonged attack, atypical location of pain, central nervous system manifestations resembling stroke, apprehension and nervousness, sudden mania or psychosis, syncope, weakness, acute indigestion, and peripheral emboiization. AMI is usually diagnosed by clinical symptoms, ECG changes, and elevations oft ardiac proteins, most notably cardiac troponin, creatine kinase-MB and myoglobin. Treatments of AMI have improved over the past decade, resulting in improved patient outcome and a 30% decrease in the death rate associated witn Aivii. 1'ieatnient of AMI patients is accomplished by administering agents that limit infarct size and improve outcome by removing occlusive material, increasing the oxygen supply to cardiac .•issue, or decreasing the oxygen demand of cardiac tissue. Treatments can include the following: supplemental oxygen, aspirin, GPIIb/IIIa inhibitors, heparin, thrombolytics (tPA), nitrates (nilroglycerin), magnesiu-n, calcium channel antagonists, p-adrenergic receptor blockers, angiotensin-converting enzyme inhibitors, angioplasty (PTCA), and intraluminal coronary artery stents. [0162] The 30 minute time point from chest pain onset is thought to represent the window of reversible myocardial damage caused by ischemia. Stable angina and unstable angina are characterized angiographically as 50-70% and 90% or greater arterial occlusion, respectively, and myocardial infarction is characterized by complete or nearly complete occlusion. A common misconception is that stable angina and unstable angina refer to plaque stability, or thai they, along with myocardial infarction, are separate diseases. Because stable angina often progresses to unstable angina, and unstable angina often progresses to myocardial infarction, stable angina, unstable angina, and myocardial infarction can all be characterized.as coronary anery disease of varying severit i. Recently, the following physiological model of coronary artery disease progression has ba;n proposed: Inflammation -> Plaque Rupture -> Platelet Activation -> Early Thrombosis •••> Early Necrosis. This model is designed to fit the theory that inflammation occurs during stable angina, and that markers of plaque rupture, platelet activation, and early thrombosis can be used to identify and monitor the progressing severity 01"unstable angina. The myocardial damage caused during an anginal attack is, by definition, reversible, while damage caused during a myocardial infarction is irreversible. Therefore, there are two proposed break po;nts in this model for the discrimination of stable angina, unstable angina, and AMI. The tirst occurs between inflammation and plaque rupture, with the theory that plaque rupture does not occur in stable angina. The second occurs between early thrombosis and early necrosis, with the theory that myocardial damage incurred during unstable angina is reversible. It is important to realize that these events, with the exception of early myocardial necrosis, can be associated with all forms of coronary artery disease, and thai progression along this diagnostic pathway does not necessarily indicate disease progression. The progression of coronary artery disease from mild unstable angina to severe unstaole angina and myocardial infarction is related to plaque instability and the degree of arterial occlusion. This progress-on can occur slowly, as stable plaques enlarge and become more occlusive, or it can occur rapidly, as unstable plaques rupture, causing platelet activation and occlusive thrombus formation. Because myocardial infarction most frequently shares the s:une pathophysiology as unstable angina, it is possible that the only distinction between these iwo events is the reversibility of nyocardial damage. By definition, unstable angina causes reversible damage, while myocariial infarction causes irreversible damage. There have been published reports that indicate the presence of myocardial necrosis in patients with unstable angina. By definition, these patients may actually be experiencing early AMI. Nevertheless, even if these patients are diagnosed with unstable angina instead of early AMI, the high degree of severity suggests that they will benefit greatly from early aggressive treatment. Myocardial ischemia is the major determinant in the pathogsnesis of stable angina, unstable angina, and myocardial infarction, and they should not be thought of as individual diseases. Rather, they reflect the increasing severity of myocardial damage from ischemia. f 0163] Inflammatory mecharusms play a pivotal role in the atherosclerotic process. At the base of atherogenesis there are complex interactions between macrophages, T lymphocytes and smooth muscle cells. A growing body of experimental evidence suggests that inflammation is involved in the oathogenesis of ACS and influences its clinical evolution. In patients with ACS, coronary atherosclerotic plaques are characterized by an abundant inflammatory infiltrate. Moreover, in these patients systemic signs of inflammatory reaction can be observed: activated circulating inflammatory cells (neutrophil, monocytes and lymphocytes) and increased concentrations of pro-inflammatory cytokines, such as interleukin (ILV1 and 6, and of acute phase reactants, in particular C-reactive protein (CRP). [0164] Thrombus Precursor Protein f 0165] Thrombin first removes fibrinopeptide A from fibrinogen, yielding desAA fibrin monomer, which can form complexes with all other fibrinogen-derived proteins, including fibrin degradation products, fibrinogen degradation products, desAA fibrin, and fibrinogen. The desAA fibrin monomer is g merically referred to as soluble fibrin, as it is the first product of fibrinogen cleavage, but it is :.iot yet crosslinked via factor Xllla into an insoluble fibrin clot. DesAA fibrin monomer aho can undergo further proteolytic cleavage by thrombin to remove fibrinopeptide B, yielding desAABB fibrin monomer. This monomer can polymerize with other desAABB fibrin monomers to form soluble desAABB fibrin polymer, also referred to as soluble fibrin or thrombus precursor protein (TpP™). [0] 66] TpP™ is the immediate precursor to insoluble fibrin, which forms a "mesh-like" structure to provide structural rigidity to the newly formed thrombus. In this regard, measurement of TpP™ in plasn'a is a direct measurement of active clot formation. The normal plasma concentration of TpP™ was reported to be Carville et al, Clin. Chem. 42:'. 537-1541, 1996). The plasma concentration of TpP™ is also reported to be elevated in patients with unstable angina (Laurino et al., Ann. Clin. Lab. Sci. 2~:338-345,1997), though other workers have found TpP™ levels to be similar in controls, unstable angina, and chronic stable effort angina (Fiotta et al., Blood Coagiil. Fibrinolysis 13: 247-25 -;, 2002). [0167] The concentration olTpP™ in plasma will theoretically be elevated during any condition that causes or is a resi.lt of coagulation activation, including disseminated imravascular coagulation, sepsis1, pulmonary embolism, deep venous thrombosis, congestive heart failure, surgery, cancer, gastroenteritis, and cocaine overdose (Laurino et al., Ann. Clin. Lab. Sci. 27:338-345, 1997; Song et al., Haematologica 87: 1062-1067, 2002; La Capra«?/ al., lilood Coagul. Fibrinolysis 11: 371-377,2000). TpP™ is released into the bloodstream immediately following thrombir activation. TpP™ likely has a short half-life in the bloodstream because it will be n.pidly converted to insoluble fibrin at the site of clot formation. Plasma TpP™ concentrations are reported to peak within 3 hours of AMI onset, returning to normal after 12 hours from onset. The plasma concentration of TpP™ can exceed 30 ng/ml in CVD (Laurino et al.,Ann. Clin. Lab. Sci. 27:338-345, 1997). [0168] MCP-1 [0169] Monocyte chemotactio protein-1 (also called monocyte chemoattractant protein-1) (MCP-1) is a 10 kDa chemotact'c factor that attracts monocytes and basophils, but not ncutrophils or eosiniphils. MCP • I is normally found in equilibrium between a monomeric and homodimeric form, and it is normally produced in and secreted by monocytes and vascular endothelial cells (Yoshi imra, T. et al., FEES Lett. 244:487-493,1989; Li, Y.S. et al., Md. Cell. Biochem. 126:61-68, ] 993). MCP-1 has been implicated in the pathogenesis of a variety of diseases that involve monocyte infiltration, including psoriasis, rheumatoid arthritis, and atherosclerosis. The normal concentration of MCP-1 in plasma is Interestingly, MCP-1 also may 'je involved in the recruitment of monocytes into the arterial wall during atherosclerosis. [0170] Elevations of the serum concentration of MCP-1 are associated with various conditions associated with inflammation, including alcoholic liver disease, interstitial lung disease, sepsis, and systemic lupus erythematosus (Fisher, N.C. et al., Gut 45:416-420, 1999; Saga, M. et al., Enr. Respir. J. 14:376-382,1999; Bossink, A.W. et al., Blood 86:3841-3847, 1995; Kaneko, H. et al. J. Rheiunatol. 26:568-573, 1999). MCP-1 is released into the bloodstream upon activation of -nonocytes and endothelial cells. The concentration of MCP-1 in plasma form patients with AMI has been reported to approach 1 ng/ml (100 pM), and can remain elevated for one month (Soejima, H. el al., J. Am. Coll. Cardiol. 34:983-988, 1999). The kinetics of MCP-1 release iato and clearance from the bloodstream in the context of ACS .ire currently unknown. MCP-1 is a specific marker of the presence of a pro-inflammatory condition that involves monocytt migration. (Table Removed) exemplary specific markers of myocardial injury. This list is not meant to be limiting. 10174] Annexin V, also called lipocortin V, endonexin II, calphobindin I, calcium binding protein 33, placental anticoagulant protein I, thromboplastin inhibitor, vascular anticoagulant(i.. and anchorin CII, is a 33 kDa calcium-binding protein that is an indirect inhibitor and regulator of tissue factor. Giambanco, I. et al., J. Histochem. Cytochem. 39:P1189-1198, 1991; Doubell, A.F. et al., Cardwvasc. Res. 27:1359-1367,1993. The normal plasma concentration of annexin V is 1996). One study has found that the plasma concentration of annexin V is elevated in individuals with AMI, but not significantly elevated in patients with old myocardial infarction, chest pain syndrome, valvular heart disease, lung disease, and kidney disease. Kaneko, N. et al.. Clin. Chim. Acta 251:65-80, 1996. [0175] Enolase is a 78 kDr\ homo- or heterodimeric cytosolic protein produced from a, P, and y subunits. Enolase catal>i:es the interconversion of 2-phosphoglycerate and phosphoenolpyruvate in the gl -colytic pathway. Enolase is present as aa, a(3, pp, ay, and yy isofnngg. The a subunit is found in most tissues, the P subunit is found in cardiac and skeletal muscle, and the y subunit is foiind primarily in neuronal and neuroendocrine tissues, p-enolase is composed of ap and PP enolase, and is specific for muscle, p-enolase is reported to be elevated in the serum of individuals with AMI, but not in individuals with angina (Nomura, VT. eral., Br. Heart J. 58:29-3::, 1987; Herraez-Dominguez, M.V. etal., Clin. Chirn. Ada .')4:307-315. 1975). The plasma concentration of P-enolase is also elevated during heart surgery, muscular dystrophy, a id skeletal muscle injury (Usui, A. et aL, Cardiovasc. Res. 1-5:737-740. 1989; Kato, K. et a,'., Clin. Chim. Acta 131:75-85, 1983; Matsuda, H. et al.. Forensic Sci. Int. 99:197-208, ' 999). [0176] Creatine kinase (CK) is an 85 kDa cytosolic eireyme that catalyzes the reversible formation ADP and phosphocreatine from ATP and creatine. CK is a homo- or heterodimer composed of M and B chains. CK is composed of 2 subunits, each with a molecular weight of 43 kDa. Three isoenzymes result from various pairings of two different subunits: B (for brain) and M (for muscle). CK-MM piedominates in skeletal muscle (approximately 99 percent of total CK) and heart muscle (approximately 55 percent of total CK); CK-BB predominates in brain tissue (over 90 percent of total CK); and CK-MB is most prevalent in heart muscle (up to about 45 percent of total CK). After myocardial infarction, CK-MB levels become elevated wiUiin 3 to S hours, peak within 9 to 30 hours, and return tc normal after 48 to 72 hours.Thygesen, K. et al., Eur. J. Clin. Invest. 16:1-4,1986; Koukkunen, H. et al., Ann. Med. .Vi.488-496, 1998; Bertinchant, J.P.etal., Clin. Biochem. 29:587-594, 1996; Benamer, H. et al.. Am. J. Cardiol. 82:845-850, 1998; Norregaard-Hansen, K. et al.,Eur. Heart J. 13:188193, 1992. CK-MB may be usetul in determining the severity of unstable angina because the extent of myocardial ischemia is directly proportional to unsrtable angina severity. [0 i 77] Glycogen phosphoryl ise (GP) is a 188 kDa intraoellular allosteric enzyme that catalyzes the removal of glucose (liberated as glucose-1-phosphate) from the nonreducing ends of glycogen in the presence cf inorganic phosphate during glycogenolysis. GP is present ;:.s ;i homodimer, which associates with another homodimer to form a tetrameric enzymatically active phosphorylase A. There jre three isoforms of GP that can be immunologically distinguished. The BB isoform is found in brain and cardiac tissue, the MM isoform is found in skeletal muscle and cardiac tissue, and the LL isoform is predominantly found in liver (Ma.'r. J.j:t al., Br. Heart .7. 72:125-127,1994). The plasma GP-BB concentration is significantly elevated in patients with AMI and unstable angina with transient ST-T elevations, but not stable angina (Mair, J. et al., Br. Heart J. 72:125-127, 1994; Mair, J., dm. C/iim. Ada 272:79-86, 1998; Rabitzsch, G. etal., Clin. Chem. 41:966-978, 1995; Rabitzsch, G. at al.. Lancet 341:1032-1033, 1993). GP-BB also can be used to detect perioperative AMI and myocardial ischemia in patients undergoing coronary artery bypass surgery (Rabitzscll, G. i-:' <.il. biomed. biochim. acta s584-s588 mair p. et al. eitr. j. clin. chem. lliochem. because it is also found in the brain plasma gp-bb concentration may be elevatsd during ischemic cerebral injury.> (0178] Heart-type fatty acid binding protein (H-FABP) is a cytosolic 15 kDa lipid-binding protein involved in lipid metabolism. Heart-type FABP antigen is found not only in heart tissue, but also in kidney, skeletal muscle, aorta, adrenals, placenta, and brain (Veerkamp, J.H. and Maatman, R.G., Prog.L-pidRes. 34:17-52, 1995; Yoshimoto, K. et al.,Heart Vessels 10:304-309, 1995). The plasma H-FABP concentration is elevated in patients with AMI and unstable angina (Ishii, J. et al., Clin. Chem. 43:1372-1378,1997; Tsuji, R. et al., Int. J. Cardiol. 41:209-217,1993). Myocardial tissue as a source of H-FABP can be confirmed by detemiimng tne ratio of myoglobin/FABP (grams/grams). Van Nieuwenhoven, F.A. et al., Circulation 92:2848-2854, 1995. The plasma H-FABP concentration can be significantly elevated 1-2 hours after the onset of chest pain, earlier than CK-MB and myoglobin (Tsuji, R. & al.. Int. J. Cardiol. 41:209-217. 1993; Van Nieuwenhoven, F.A. etal, Circulation 92:28482S54, 1995; Tanaka, T. etal., Clin. Biochem. 24:195-201,1991). [0179J Phosphoglyceric acid nutase (PGAM) is a 57 kDa homo- or heterodimeric intracellular glycolytic enzyme co uposed of 29 kDa M or B aubunits that catalyzes the interconversion of 3-phosphoglycorate to 2-phosphoglycerate in the presence of magnesium. Cardiac tissue contains isozymes MM, MB, and BB, while skeletal muscle contains primarily PGAM-MM, and most other tissues contain PGAM-BB (Durany, N. and Carreras, J., Comp. Biochem. Physiol. B. Biochem.Mc.l. Biol. 114:217-223, 1996). [0180] S-100 is a 21 kDa bomo- or heterodimeric cytosolic Ca2+-binding protein produced from a and ft subunits. It is tho jght to participate in the activation of cellular processes along the Ca2+-dependent signal transduction pathway (Bonfrer, J.M. et al., Br. J. Cancer 77:2210 22 i 4, 199S). S-lOOao (aa isofonn) is found in striated muscles, heart and kidney, S-lOOa (ap isofonrO is found in glial cells, but not in Schwann cells, and S-100J3 (PP isoform) is found in high concentrations in glial cells and Schwann cells, where it is a major cytosolic component UCato, K. and Kimura, S.. Biochim. Biophys. Acta 842:146-150,1985; Hasegawa, S. et aL Eur. Urol. 24:393-396, 1993). The serum concentration of S-lOOao was reported to be elevated in patients with AMI, but not in patients with angina pectoris with suspected AMI riisui, A. at al., Clin. Chem. 36:539-641, 1990). The serum concentration of S-lOOao is significantly elevated on admission in patients with AMI, increases to peak levels 8 hours after admission, decreases and n-.turns to baseline one week later (Usui, A. et al., Clin. Chem. 3o:639-641, 1990). Furthermore, S-lOOao appears to be significantly elevated earlier after AMI onset than CK-MB (Usui, A. et al., Clin. Chem. 36:639-641,1990). f 01S1] (ii) Exemplar) • Markers Related To Blood Pressure Regulation [0182] In addition to ANP, BMP, and markers related thereto, discussed in detail above, the following represent exemplaiy markers that are known in the art to be related to blood pressure regulation. This list is not meant to be limiting. [0183] C-type natriuretic pepiide (CNP) is a 22-amino acid peptide that is the primary active nairiuretic peptide in the human brain; CNP is also considered to be an endothelium-derived relaxant factor, which acts in the same way as nitric oxide (NO) (Davidson et al., Circulation 93:1155-9, 1996). CNP is structurally related to Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (Bi JP); however, while ANP and BNP are synthesized predominantly in the myocardium, CNP is synthesized in the vascular endothelium as a precursor (pro-CNP) (Prickett etcl,Biochem. Biophys. Res. Commun. 286:513-7, 2001). [9184] Urotensin II is a peptide having the sequence Ala-Gly-Thr-Ala-Asp-Cys-Phe-Trp-Lys-T yr-Cys-Val, with a disulfidc bridge between Cys6 and Cys 11. Human urotensin 2 (1JTN) is synthesized in a prep:o form. Processed urotensin 2 has potent vasoactive and cardiostimulatory effects, acting on the G protein-linked receptor GPR14. [ 0185] Vasopressin (arginire vasopressin, AVP; antidiuretic hormone, ADH) is a peptide hormor.e released from the posterior pituitary. Its primary function in the body is to regulate extracellular fluid volume by affecting renal handling of water. There are several mechanisms regulating release of AVP. Hyp^volemia, as occurs during hemorrhage, results in a decrease in atrial pressure. Specialized stretch receptors within the atrial walls and large veins (cardiopulmonary baroreceptors) entering the atria decrease their firing rate when there is a rail in alrial pressure. Afferent iiom these receptors synapse within the hypothalamus; atrial receptor firing normally inhibits the release of AVP by the posterior pituitary. With hypovolemia or decreased cential venous pressure, the decreased firing of atrial stretch receptors leads to an increase in AVP release. Hypothalamic osmoreceptors sense extracellular osmolarity and stimulate AVP release when osmolarity rises, as occurs with dehydration. Finally, angiotensin II receptors located in a region of the hypothalamus regulate AVP release — an increase in angiotensin II simulates AVP release. [0186] Calcilonin gene related peptide (CGRP) is a polypeptide of 37 amino acids that is a product of the calcitonin gene derived by alternative splicing of the precursor mRNA. The calcitonin gene (CALC-I) primaiy RNA transcript is processed into different mRNA segments by inclusion 01 exclusion of different exons as part of the primaiy transcript. Calcitonin-encoding mRNA is the main product of CALC-I transcription in C-cells of the thyroid, whereas CGRP-I mRNA (CGRP = calcitonin-gene-related peptide) is produced in nervous tissue of the central anri peripheral nervous systems. In the third mRNA sequence, the calcitonin sequence is lost and alternatively the sequence of CGRP is encoded in the mRNA. CGRP is a markedly vasoactive peptide with vasodilatative properties. CGRP has no effect on calcium and phosphate metabolism and is synthesised predominantly in nerve cells related to smooth muscle cells of the blooil vessels. ProCGRP, the precursor of CGRP, and PCT have partly identical N-terminal amino acid sequences. [0187] Procalcitonin is a 1 lo amino acid (14.5 kDa) protein encoded by the Calc-1 gene located on chromosome 1 Ipl5/. The Calc-1 gene produces two transcripts that are the result of alternative splicing events. Pie-procalcitonin contains a 25 amino acid signal peptide which is processed by C-cells in the thyroid to a 57 amino acid N-terminal fragment, a 32 amino acid calcitonin fragment, and a I'.l amino acid katacalcin fragment. Procalcitonin is secreted intact us a^gjycosylated product by other body cells. Whicher et al., Ann. Clin. Biochem. 38: 483-93 (.JG01). Plasma procalcitonin has been identified as a marker of sepsis and its severity. Vukioka ei al., Ann. Acad. Med. Singapore 30: 528-31 (2001); Pettila et al., Intensive Care Med. 28: 1220-25 (2002). f oi SS] Angiotensin II is an cr.tapeptide hormone formed by renin action upon a circulating substrate, angiotensirogen, that undergoes proteolytic cleavage to from the ilccapeptidc angiotensin I. Vasci'lar endothclium, particularly in the lungs, has an enzyme, angiotensin converting enzyme (.ACE), that cleaves off two amino acids to form the . octapeptide, angiotensin II (All). [i)189] Adrenomedullin (AM) is a 52-amino acid peptide which is produced in many (issues, including adrenal medulla, lung, kidney and heart (Yoshitomi et al., Clin. Sci. (Colch) c)4:135-9, 1998). Intravenous adiunistration of AM causes a long-lasting hypotensive effect, accompanied with an increase in the cardiac output in experimental animals. AM is synthesized as a precursor molec jle (pro-AM). The N-terminal peptide processed from the AM precursor has also been reported to act as a hypotensive peptide (Kuwasako et al., Ann. Clin. Biochem. 36:622-8, 1999). (0190] The endothelins are tbree related peptides (endothelin-1, endothelin-2, and endothelin-3) encoded by separate genes that are produced by vascular endothelium, each of which exhibit potent vasoconstricting activity. Endothelin-1 (ET-1) is a 21 amino acid residue peptide, synthesized as a 212 residue precursor (preproET-1), which contains a 17 residue signal sequence that is removed to provide a peptide known as big ET-1. This molecule is further processed by hydrolysis between trp21 and va!22 by endothelin converting enzyme. .Both big ET-1 and ET-1 exhibit biological activity; however the mature ET-1 form exhibits .greater vasoconstricting activity (Brooks and Ergul, J. Mol. Endocrinol. 21:307-15, 199S). Similarly, endothelin-2 and endoihelin-3 are also 21 amino acid residues in length, and are produced by hydrolysis of big eidothelin-2 and big endothelin-3, respectively (Yap et al., Br. ./. Phannacol. 129:170-6, 2000; Lee et al., Blood 94:1440-50, 1999). '0191] (iii)Exemplary Marltars Related to Coagulation and Hemostasis [0192] Elevations in the sernn concentration of markers related to coagulation and hemostasis may be associated wilh clot presence, or any condition that causes or is a result of llbrinolysis activation, including atherosclerosis, disseminated intravascular coagulation. :icute myocardial infarction, surgery, trauma, unstable angina, stroke, pulmonary embolsim. venous thrombosis, and thrombctic thrombocytopenic purpura. In addition to D-dimer and TpP, described in detail above, the following are exemplary markers related to coagulation •and hemostasis. This list is not meant to be limiting. [0193] Plasmin is a 78 kDa serine proteinase that proteolytically digests crosslinked fibrin, resulting in clot dissolution. The 70 kDa serine proteinase inhibitor a2-antiplasmin (cx2AP) regulates plasmin activity by forming a covalent 1:1 stoichiometric complex with piasmin. The resulting ~150 kD.i plasmin-oc2AP complex (PAP), also called plasmin inhibitory complex (PIC) is fonr ed immediately after ct2AP comes in contact with plasmin that is activated during fibrinolys is. [0194] p-thromboglobulin (fTG) is a 36 kDa platelet a granule component that is released upon platelet activation. Plasma levels of P-TG appear to be elevated in patients wiui unstable angina and acute myocardial infarction, but not stable angina (De Caterina, R. et al.. Ew. Heart J. 9:913-922, 1988; Bazzan, M. et al., Cardiologia 34, 217-220, 1989). Plasma BTG elevations also seem to be correlated with episodes of ischemia in patients with unstable angina (Sobel, M. et al., Circulation 63:300-306, 19S1). Plasma concentrations of [3TG associated with ACS can approach 70 ng/ml (2 nM), but this value may be influenced by platelet activation during the sampling procedure. [0195J Platelet factor 4 (PF4) is a 40 kDa platelet a granule component that is released upon platelet activation. PF4 is a marker of platelet activation and has the ability to bind and .leutralize heparin. The plasma concentration of PF4 appears to be elevated in patients with acute myocardial infarction and i nstable angina, but not stable angina (Gallino, A. et al., Am. Heart J. 112:285-290,1986; Szkata, K. etal.,Jpn. Circ. J. 60:277-284,1996; Bazzan, M. et a!., Cai-diologia 34:217-220,1989). Plasma PF4 elevations also seem to be correlated with episodes of ischemia in patients1, with unstable angina (Sobel, M. et al., Circulation 63:300 306, 19SP. [0196] Fibrinopeptide A (F?A) is a 16 amino acid, 1.5 kDa peptide that is liberated from amino terminus of fibrinogen by the action of thrombin. The plasma FPA concentration is elevated in patients with acute myocardial infarction, unstable angina, and variant angina, but not stable angina (Gensini, G.F. et al., Thromb. Res. 50:517-525, 1988; Gallmo, A. et al., Am. Hi-art J. 112:285-290,19S6; Sakata, K. et al.,Jpn. Circ. J. 60:277-284, 1996; Theroux, P. et i;/.. Circulation 75:156-162, 19E7; Merlini, P.A. et al., Circulation 90:61-68, 1994; Manten, A. ct al., Cardiovasc. Res. 40:389-395,1998). Furthermore, plasma FPA may indicate the severity of angina (Gensini, G.F. et al., Thromb. Res. 50:517-525, 1988). f0197] Platelet-derived growth factor (PDGF) is a 28 kDa secreted homo- or heterodimeric protein composed of the homologous subunits A and/or B (Mahadevan, D. et al.,J. Biol. Chem. 270:27595-27600, 1995). PDGF is released by aggregating platelets and monocytes near sites of vascular injur, and has been implicated in the pathogenesis of atherosclerosis. Plasma PDGF CDncentrations are higher in individuals with acute myocardial infarction and unstable angina than in healthy controls or individuals with stable angina (Ogawa, H. ct al., Am. J. Cardiol 69:453-456, 1992; Wallace, J.M. et al.,Ann. Clw. Biochem. 35:236-241, 1998; Ogawa, H. ei al., Coron. Artery Dis. 4:437-442, 1993). [0198] Prothrombin fragment 1+2 is a 32 kDa polypeptide that is liberated from the amino terminus of thrombin during thrombin activation. The plasma concentration of Fl+2 is reportedly elevated in patients with acute myocardial infarction and unstable angina, but not stable angina (Merlini, P.A. et ai., Circulation 90:61-68,1994). Other reports have indicated that there is no significant chang:; in the plasma Fl+2 concentration in cardiovascular disease (Biasucci, L.M. et al., Circulation 93:2121-2127, 1996; Maiiten, A. etal, Cardiovasc. Res. 40:389-395, 1998). [01991 P-selectin, also called granule membrane protein-140, GMP-140, PADGEM, and C.D-62P, is a ~140 kDa adhesion molecule expressed in platelets and endothelial cells. P-selectin is stored in the alpha tianules of platelets and in the Weibel-Palade bodies of endothelial cells. Membrane-bo md and soluble forms of P-selectin have been identified. Soluble P-selectin may play an important role in regulating inflammation and thrombosis by blocking interactions between le ikocytes and activated platelets and endothelial cells (Gamble, J.R. et al., Science 249:414-417,1990). The plasma soluble P-selectin concentration was significantly elevated in patients with acute myocardial infarction and unstable angina, but not stable angina, even following an exercise stress test (Ikeda, H. et al.. Circulation 92:1693-1696, 1995; Tomoda, H. and Aoki,N.,Angiology 49:807-813, 199S; Hollander, J.E. at til., J. Am. Coif. Cardiol. 34:95-105, 1999; Kaikita, K. et al., Circulation 32: H26-1730, 1995; Ikeda, H. et al., Coron. ArteiyDis. 5:515-518,1994). The sensitivity :ind specificity of membrane-bouud P-selectin versus solubla P-selectin for acute myocardial infarction is 71% versus 76% ana 32% versus 45% (Hollander, J.E. et al., J. Am. Coll. Canhol. 34:95-105,1999). The sensitivity and specificity of membrane-bound P-selectin versus soluble P-selectin for unstable angina + acute myocardial infarction is 71% versus 79% and 30% versus 35% (Hollander J.E. etal., J. Am. Coll. Cardiol. 34:95-105,1999). [ 0200] Thrombin is a 37 kDa sierine proteinase that proteolytically cleaves fibrinogen to form fibrin, which is ultimately integrated into a crosslinked network during clot formation. .-\jitithrombin HI (ATIII) is a 65 VDa serine proteinase inhibitor that is a physiological regulator of thrombin, factor XIa. factor Xlla, and factor IXa proteolytic activity. The normal plasma concentration of the approximately 100 kDa thrombin-ATID. complex (TAT) is [0201] von Willebrand factor (vWF) is a plasma protein produced by platelets, megakaryocytes, and endothelial cells composed of 220 kDa monomers that associate to form a series of high molecular weight multimers. These multimers normally range in molecular weight from 600-20,000 kDa. The Al domain of vWF binds to the platelet glycoprotein Ib-IX-V complex and non-fibrillar collagen type VI, and the A3 domain binds fibrillar collagen types I^rind III (Emsley, J. et al, J. Biol. Chem. 273:10396-10401,1998). Other domains present in the vWF molecule include the integrin binding domain, which mediates platelet-platelet interactions, the the protease cleavage domain, which appears to be relevant to the pathogenesis of type 11A von Willebrand disease. Measurement of the total amount of vWF would allow one who is skilled in the art to identify changes in total vWF concentration. This measurement could be performed through the measurement of various forms of the v\VF molecule. Measurement of the Al domain would allow the measurement of active vWF in tiie circulation, indicating that a pro-coagulant state exists because the Al domain is accessible for platelet binding. Ii this regard, an assay that specifically measures vWF molecules with both the exposed Al domain and either the integrin binding domain or the A3 domain would also allow for the identification of active vWF that would be available for mediating platelet-platelet interactions or mediate crosslinking of platelets to vascular subendothelium, respectively. f 0202] Tissue factor (TF) is a 45 kDa cell surface protein expressed in brain, kidney, and heart, and in a transcriptionally regulated manner on perivascular cells and monocytes. Tissue factor can be detected in the bloodstream in a soluble form, bound to factor Vila, or in a cumplux witn factor Vila, and tissue factor pathway inhibitor that can also include factor Xa. TF also is expressed on the surface of macrophages, which are commonly found in atherosclerotic plaques. TF is elevated in patients with unstable angina and acute myocardial infarction, but not in patients with stable angina (Falciani, M. et al.. Thromb. Haemosi. 79:495-499,199S; Suefuji, H. et al, Am. Heart J. 134:253-259,1997; Misumi, K. et al., Am. .1. Cardiol. 81:22-26, 1998). Furthermore, TF expression on macrophages and TF activity in atherosclerotic plaques is more common in unstable angina than stable angina (Soejima, H. et al.. Circulation 99:2908-2913, 19£»9; Kaikita, K. et al, Arterioscler. Thromb. Vase. Biol 17:2232-2237, 1997; Ardissino, D. etal. Lancet 349:769-771,1997). [01103] (iv) Exemplary Markers Related to the Acute Phase Response [0204] Acute phase proteins are a group of proteins, such as C-reactive protein and mannose-binding protein, produced by cells in the liver and that promote inflammation, acti vate the complement cascade, and stimulate chemotaxis of phagocytes. In addition to \TCP-l7 described in detail above, the following are exemplary markers related to the acute phase response. This list is not meant to be limiting. "0205] Human neutrophil elastase (HNE) is a 30 kDa serine proteinase that is normally contained within the azurophilic granules of neutrophils. HNE is released upon neutrophil activation, and its activity is regulated by circulating aj-proteinase inhibitor. The plasma HNE concentration is usually measured by detecting HNE-cti-PI complexes. The normal concentration of these complexes is 50 ng/ml, which indicates a normal concentration of approximately 25 ng/ml (0.8 nM) for HNE. HNE release also can be measured through the specific detection of fibrinopeptide Bfao-43, a specific HNE-derived fibrinopeptide, in plasma. Plasma HNE is elevated in patients with coronary stenosis, and its elevation is greater in patients with complex plaques fian those with simple plaques (Kosar, F. et al., Angiology 49:193-201, 1998; Amaro, A. et al, Eur. Heart J. 16:615-622, 1995). Plasma HNE is not significantly elevated in patients with stable angina, but is elevated inpatients with unstable angina and acute myocardial infarction, as determined by measuring fibrinopeptide Bfao^j, with concentrations in unstable angina being 2.5-fold higher than those associated with acute myocardial infarction (Dinerman, J.L. etal., J. Am. Coll. Cordial. 15:1559-1563, 1990; Mehta, J. et al., Circulation 79:549-556, 19S9). [0206] Inducible nitric oxide synthase (iNOS) is a 130 kDa cytosolic protein in epithelial cells macrophages whose expression is regulated by cytokines, including interferon-y, interleukin-1 p, interleukin-6, and tumor necrosis factor a, and lipopolysaccharide. iNOS catalyzes the synthesis of nitric oxide (NO) from L-arginine, and its induction results in a sustained high-output production of NO, which has antimicrobial activity and is a mediator of a variety of physiological and irilammatory events. NO production by iNOS is approximately 100 fold more than the amount produced by constitutively-expressed NOS (Depre, C. et al.. Cardiovasc. Res. 41:465-472, 1999). iNOS expression during myocardial ischemia may not be elevated, suggesting that iNOS may be useful in the differentiation of angina from acute myocardial infarction (Hammerman, S.I. et al, Am. J. Physiol. 277:H1579H 1 592, 1999; Kaye, D.M. et al , Life Sci 62:883-887, .1998). f 0?,07] Lysophosphatidic acid (LPA) is a lysophospholipid intermediate formed in the synthesis of phosphoglycerides and triacylglycerols. In the context of unstable angina. LPA is most likely released as a direct result of plaque rupture. . [0208] Malondialdehyde-mr dified low-density iipoprotein (MDA-modified LDL) is formed during the oxidation of the apoB-1 00 moiety of LDL as a result of phospholipase activity, prostaglandin synthesis, or platelet activation. Plasma concentrations of oxidized LDL are elevated in stable angina, unstable angina, and acute myocardial infarction, indicating that it may be a marker of atherosclerosis (Holvoet, P., Acta Cardiol. 53:253-260, 1 998; Holvoet, P. et al.. Circulation 98:1 487-1494, 1998). Plasma MDA-modified LDL is not elevated in stable angina, bu: is significantly elevated in unstable angina and acute myocardial infarction (Holvoet, P., Acta Cardiol. 53:253-2 f0209] Matrix metalloproteiuase-1 (MMP-1), also called collagenase-1, is a 41/44 kDa /.inc- and calcium-binding proteuase that cleaves primarily type I collagen, but can also ..-.leave collagen types II, III, VII and X. The active 41/44 kDa enzyme can undergo autolysis ;:o the still active 22/27 kDa forrr.. MMP-1 can be found in the bloodstream either in a free form or in complex with TIMP-1, its natural inhibitor. MMP-1 is found in the shoulder region of atherosclerotic plaques, which is the region most prone to rupture, and may be involved in atherosclerotic plaque destabilization (Johnson, J.L. et al., Arteriosder. Thromb. I'asc. Bio!. 18:1707-1715, 1998). Furthermore, MMP-1 has been implicated in the pathogenesis of myocardial reperfusion injury (Shibata, M. et al., Angiology 50:573-582, (021 0] Lipopolysaccharide V nding protein (LBP) is a ~ 60 kDa acute phase protein produced by the liver. LBP binds to lipopolysaccharide and is involved in LPS handling in humans. LBP has been reported :o mediate transfer of LPS to the LPS receptor (CD 14) on mononuclear cells, and into HDL. LBP has also been reported to protect mice from septic shock caused by LPS. [0211] Matrix metalloproteiriase-2 (MMP-2), also called gelatinase A, is a 66 kDa zinc-and calcium-binding proteinase that is synthesized as an inactive 72 kDa precursor. Mature MMP-3 cleaves type I gelatin and collagen of types IV, V, VII, and X. MMP-2 is usually found in plasma in complex with IIMP-2, its physiological regulator (Murawaki, Y. et al.. J. Hepatol. 30:1090-1098, 1999). MMP-2 expression is elevated in vascular smooth muscle cells within atherosclerotic lesions, and it may be released into the bloodstream in cases of plaque instability (Kai, H. ei al.. J. Am. Coll. Cardiol. 32:368-372, 1998). Serum MMP-^ concentrations were elevated in patients with, stable angina, unstable angina, and acute myocardial infarction, with elevations being significantly greater in unstable angina and acute myocardial infarction than in stable angina (Kai, H. et al., J. Am. Coll. Cardiol. 32:368-372, J.99S). MMP-2 was elevated on admission in the serum of individuals with unstable angina and acute myocardial infarction, with maximum levels approaching 1.5 ug/ml (25 nM) (Kai, H. et al., J. Am. Coll. Cardiol. 31:368-372, 1998). [0212] Matrix metalloproteir ase-3 (MMP-3), also called stromelysin-1, is a 45 kDa zinc-and calcium-binding proteinase that is synthesized as an inactive 60 kDa precursor. The bcrnm. MMP 2 concentration in males is approximately 2 times higher than in females (Manicourt., D.H. et al., Arthritis Rheum. 37:1774-1783,1994). MMP-3 is found in the shoulder region of atherosclerotic plaques, which is the region most prone to rupture, and may be involved in atherosclerotic plaque destabilization (Johnson, J.L. et al., Arterioscler. Tliromb. Vase. Biol. 18:1707-1715, 1998 [0213] Matrix metalloproteinase-9 (MMP-9) also called gelatinase B, is an 84 kDa zinc-and calcium-binding proteinase tnat is synthesized as an inactive 92 kDa precursor. MMP-9 exists as a monomer,a homodimer, and a heterodimer with a 25 kDa aj-microglobulin-related protein (Triebel, S. etal.,FEBSlett. 314:386-388, 1992). Plasma MMP-9 concentrations are significantly elevated in patients v/ith unstable angina and acute myocardial infarction, but not stable angina (Kai, H. et al., J. Am. Coll. Cardiol. 32:368-372, 1998). [0214] The balance between matrix metalloproteinases and their inhibitors is a critical factor that affects tumor invasion and metastasis. The TIMP family represents a class of small (21 -28 kDa) related proteins tha; inhibit the metalloproteinases. Tissue inhibitor of metaiteptpfcinase 1 (TIMP1) is reportedly involved in the regulation of bone modeling and remodeling in normal developing human bone, involved in the invasive phenotype of acute myelogenous leukemia, demonstrating polymorphic X-chromosome inactivation. TIMP1 is known to act on MMP-1, MMP-2, MMP-3, MMP-7, MMP-S, MMP-9, MMP-10, MMP-l I, MMP-12, MM.P-13 and MMP-bi. Tissue inllibitor of metalloproteinase 2 (TMP2) complexes with metalloproteinases (such as oollagenases1) and irreversibly inactivates them. TIMP 2 is known to aci on MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13. MMP-14, MMP-15, MMP-16 and MMP-19. Two alternatively spliced forms may be associated with SYN4, and involved in the invasive phenotype of acute myelogenous leukemia. Unlike the inducible expression of some other TIMP gene family members, the expression of this gene is largely constitutive. Tissue inhibitor of metalloproteinase 3 (TIJVIP3) antagonizes matrix metalloproteinase activity and can suppress tumor growth, angiogenesis, invasion, and metastasis. Loss of T1MP-3 has been related to the acquisition of tumorigenesis. [0215] (v) Exemplary Markers Related to Inflammation [02! 6] Acute phas? proteins are part of a larger group of proteins that are related to local or systemic inflammation. The following exemplary list of additional markers related to inflammation is not meant to be limiting. [0217] Interleukins (ILs) are part of a larger class of polypeptides known as cytokines. These are messenger molecules that transmit signals between various cells of the immune system. They are mostly secretec by macrophages and lymphocytes and their production is induced in response to injury or infection. Their actions influence other cells of the immune system as well as other tissues arid organs including the liver and brain. There are at least 18 I La described. IL-lp, IL-2, IL-4, TL-6, IL-S and IL-10 are preferred for use as markers in the present invention. The following table shows selected functions of representative interleukins. Table 1: Selected Functions of Representative Interleukins* (Table Removed) [02IS] Interleukin-lp (IL-1 P) is a 17 kDa secreted proinflamrnatory cytokine that is involved in the acute phase response and is a pathogenic mediator of many diseases. IL-lp is normally produced by macrophages and epithelial cells. IL-lp is also released from cells undergoing apoptosis. Elevations of the plasma IL-lp concentration are associated with activation of the acute phase response in proinflamrnatory conditions such as trauma and iniection. [0219] Interleukin-1 receptor antagonist (IL-lra) is a 17 kDa member of the IL-1 family predominantly expressed in hepatocytes, epithelial cells, monocytes, macrophages, and neutrophils. 1L- Ira has both intrasellular and extracellular forms produced through alternative splicing. IL-lra is thought to participate in the regulation of physiological IL-1 activity. The plasma concentration of IL-lra is elevated in patients with acute myocardial infarction and unstable angina that proceeded to acute myocardial infarction, death, or refractory angina (Biasucci, L.M et al. Circulation 99:2079-2084,1999; Latmi, R. et al.. J. Cardiovasc. Pharmacol. 23:1-6, 1994). Furthermore, IL-lra was significantly elevated in severe acute myocardial infarction as compared to uncomplicated acute myocardial infarction (Latini, R. et al., J. Cardiovasc. Pharmacol. 23:1-6, 1994). [02201 Interleukin-6 (IL-6) is a20 kDa secreted protein that is ahematopoietin family proinflammatory cytokine. Its major function is to mediate the acute phase production of hepatic proteins, and its synthesis is induced by the cytokine IL-1. IL-6 is normally produced by macrophages and T lymphocytes. The plasma concentration of IL-6 is elevated in patients with aciue rnyocardial infarction md unstable angina, to a greater degree in acute myocardial infarction.(.Biasucci, L.M. et ai, Circulation 94:874-877,1996; Manten, A. et al, Cardiovtsc. Res. 40:389-395,1938; Biasucci, L.M. etal, Circulation 99:2079-2084, 1999). IL-6 is not significantly elevated in the plasma of patients with stable angina (Biasucci, L.M. t/ al.. Ci>-ciilcition 94:874-877,1996; Manten, A. etal.. Cardiovasc. Res. 40:389-395, 1998). The plasma concentration of IL-6 is elevated within 8-12 hours of acute myocardial infarction onset, and can approach 100 pg/ml. The plasma concentration of IL-6 in patients with i msUible angina was elevated at peak levels 72 hours after onset, possibly due to the severity oi" insult (Biasucci, L.M. et al.. Circulation 94:874-877, 1996). [0221] Inlerleukin-S (IL-8) is a 6.5 kDa chemokine produced by monocytes, endothelial cells, alveolar macrophages and fibroblasts. IL-8 induces chemotaxis and activation of neutrophils and T cells. [0222] Tumor necrosis factor a (TNFa) is a 17 kDa secreted proinflammatory cytokine that is involved in the acute phase response and is a pathogenic mediator of many diseases. TNF-alpha is a protein of 185 amino acids glycosylated at positions 73 and 172. It is synthesized as a precursor proteii. of 212 amino acids. Monocytes express at least five different molecular forms of TNI'-alpha with molecular masses of 21.5-28 kDa. They mainly differ by post-translational alterations such as glycosylation and phosphorylation. The normal serum concentration of TNFa is [022?] Soluble intercellular adhesion molecule (sICAM-1), also called CD54, is a S5-110 kDa cell surface-bound immunoglobulin-like integrin ligand that facilitates binding of leukocytes to antigen-presenting cells and endothelial cells during leukocyte recruitment and migration. The plasma concentration of sICAM-1 is significantly elevated in patients with acute myocardial infarction and unstable angina, but not stable angina (Pellegatta, F. et at., J. Cardiovi'ic. Pharmacol. 30:455-460,1997; Miwa, K. etai. Cardiovasc. Res. 36:37-44, 1997; Ghaisas, N.K. et at., Am. J. Cardiol. 80:617-619,1997; Ogawa, H. et al, Am. J. Cardiol. S.":3S-42,1999). Furthermore, ICAM-1 is expressed in atherosclerotic lesions and in areas predisposed to lesion formation, so it may be released into the bloodstream upon plaque rupture (Liyama, K. et al., Circ. Res. 85:199-207, 1999; Tenaglia, A.N. et al., Am. J. Cardiol. 79:742-747, 1997). Additional ICAM molecules are well known in the art, including ICAM2 (also called CD 102) and ICAM.-3 (also called CD50), which may also be present in the Mood. [0224] Vascular cell adhesion molecule (VCAM), also called CD106, is a 100-110 kDa cell surface-bound immunoglobulin-like integrin ligand tha: facilitates binding of B lymphocytes and developing T lymphocytes to antigen-presenting cells during lymphocyte recruitment. The plasma concentration of sVCAM-1 is marginally elevated in patients with acute myocardial infarction, unstable angina, and stable angina (Mulvihill, N. et al., Am. J. Cardiol. 83:1265-7, A9,1999; Ghaisas, N.K. etal., Am. J. Cardiol. 80:617-619, 1997). However, sVCAM-1 is expressed in atherosclerotic lesions and its plasma concentration may correlate with the extent of atherDSclerosis (liyama, K. et al, Circ. Res. 85:199-207, 1999; Peter, K. etal., Arterioscler. Thromb. Vase. Biol. 17:505-512, 1997). [0225] Macrophage migraticr. inhibitory factor (MIF) is a lymphokuie involved in cell-mediated immunity, immunoregulation, and inflammation. Like TNFa and IL-lfJ, MIF plays a central role in the host response to endotoxemia. Coinjection of recombinant MIF and LPS exacerbates LPS lethality, whereas neutralizing anti-MIF antibodies fully protect mice from endotoxic shock. [0226] Hemoglobin (Hb) is an oxygen-carrying iron-containing globular protein found in erythrocytes. It is a heterodimer of two globin subunits. 0:272 is referred to as fetal Hb, aifa is called adult HbA, and cc262 is called adult HbA2. 90-95% of hemoglobin is HbA, and the cc2 giobin chain is found in all Hb types, even sickle cell hemoglobin. Hb is responsible for carrying oxygen to cells throughout the body. Hbcci is not normally detected in serum. [0227] ^ Oxysterols (oxidized derivatives of cholesterol) and oxidized lipoproteins have been ideiuilied in atherosclerotic lesions, and are suggested to play a role in the pathogenesis of coronary heart disease. See, e.%., Staprans et al.,Arterioscler. Thromb. Vase. Biol. 20: 70814, 2000. Recently, an aldol condensation product believed to be formed by ozonolysis of cholesterol in atherosclerotic plaques was reported to be detectable in plasma from subjects with advanced atherosclerotic disease. It was suggested that this molecule may be a marker of arterial inflammation in atherosc'erosis. Wentworth et al., Science 302: 1053-6, 2003. This publication is hereby incorporate'! by reference in its entirety. [022S] Human lipocalin-type prostaglandin D synthase (hPDGS), also called p-trace, is a .'50 kDa glycoprotein that catalyzes the formation of prostaglandin D2 from prostaglandin H. Elevations of hPDGS have been identified in blood from patients with unstable angina and cerebral infarction (Patent No. EP0999447A1). Furthermore, hPDGS appears to be a useful marker of ischemic episodes (Patent No. EP0999447A1). [0229] Mast cell tryptase, also known as alpha tryptase, is a 275 amino acid (30.7 kDa) protein that is the major neutral protease present in mast cells. Mast cell tryptase is a specific marker for mast cell activation, aid is a marker of allergic airway inflammation in asthma and in allergic reactions to a diverse set of allergens. See, e.g., Taira et al., J. Asthma 39: 315-22 (2002); Schwartz et al., N. Engl../. Med. 316: 1622-26 (1987). Elevated serum tryptase levels f > i ng/mL) between 1 and 6 hours after an event provides a specific indication of mast cell lie^ranulaiion. 10230J Eosinophil cationic protein (ECP) is a heterogeneous protein with molecular weight variants from 16-24 kDa and a pi of pH 10.S. Assessment of serum ECP may be assumed to reflect pulmonary inflammation in bronchial asthma. Koller et al.,Arch. Dis. Childhood 73: 413-7 (1995); see also, Sorkness et al., Clin. Exp. Allergy 32: 1355-59 (2002); Badr-elDin et al., East Mediterr. Health J. 5: 664-75 (1999). [0231] Merleukin 10 ("IL-10") is a 160 ammo acid (18.5 kDa predicted mass) cytokine that is a member of the four a-he!ix bundle family of cytokines. In solution, EL-10 forms a homodimer having an apparent molecular weight of 39 kDa. The human IL-10 gene is located on chronv^nme 1. Viera et al.,Proc. Natl. AcadSci. USA 88: 1172-76 (1991); Kim et al., J. [mmunol. 148: 3618-23 (1992). Dverproduction of IL-10 has been identified as a marker in sepsis, and is predictive of severty and mortality. Gogos et al., J. Infect. Dis. 181: 176-80 i;!000). [0232] (vi) Exemplary Markers of Pulmonary Injury i 02L'3] KJL-6 (also referred to as MUCI) is a high molecular weight (> 300 kDa) mucirous glycoprqtein expressed on pneumonocytes. Serum levels of KL-6 are reportedly elevated in interstitial lung diseases, which are characterized by exertional dyspnea. KL-6 has been shown to be a marker of various interstitial lung diseases, including pulmonary fibrosis, interstitial pneumonia, sarcoidosis, and interstitial pneumonitis. See, e.g., Kobayashi and Kitamura, Chest 108: 311-15 (1995); Kohno, J. Med. Invest. 46:151-58 (1999); Bandoh et a/., Ann. Rheum. Dis. 59: 257-62 (2000); and Yamane etal, J. Rheumatol. 27: 930-4 (2000). [0234] Surfactant proteins are a family of apoproteins, which are associated in a complex with phospholipids. There are four main surfactant proteins, known as SP-A, B, C, and D. Various of the surfactant proteins have been associated with pulmonary disease. See. e.g., Doyle et al.. Am. J. Respir. Crit. CareMed. 156: 1217-29,1997; Bersten et al.. Am. J. Respir. Chi. Care Med.\64: 648-52, 2001; Suwabe, RibnshoByori 50: 1061-66, 2002; Cheng et al., C>-it. Care Med. 31:311-13, 2003; Hastings, J. Clin. Monit. Comput. 16: 385-92, 2000. [0235] Neutrophil elastase, a proteolytic enzyme, has long been measured as a marker of pulmonary injury, both in the systemic circulation and in bronchoalveolar lavage fluid. See, •?.?., Moraes et al., Crit. CareMs.d. 31(4 Suppl): SI89-94, 2003 [0236] The products associa'ed with the breakdown of :ype IV collagen (a main constituent of basement membrane), such as the 7S protein fragment of collagen, have been used to mark lung injury. Increased 7S protein levels have been shown to be associated with high matrix metalloproteinase (MMP; proteolytic enzyme) and neutrophil concentrations in the bronchoalveolar lavage fluid of patients after they have undergone cardiopulmonary bypass. See, e.g., Owen et al. Am. J. Respir. CellMol. Bio,'. 29: 283-94,2003. [02? 7] (vii) Exemplary Specific Markers of Neural Tissue Injury |'023SJ In the case where a \ascular disease affects tissues other than myocardium (e.g., in stroke), specific markers of tissi e damage other than markers of myocardial tissue damage may be particularly useful. Conj.idering stroke as an example, the following list of exemplary specific markers of neural tissue injury is provided. This list is not meant to be limiting. f 0239] Adenylate kinase (AK) is a ubiquitous 22 kDa cytosolic enzyme that catalyzes the interconversion of ATP and AM^ to ADP. Four isoforms of adenylate kinase have been identified in mammalian tissues Yoneda, T. et al.. Brain Res Mol Brain Res 62:187-195, 1V!>S). The AK1 isoform is fourxl in brain, skeletal muscle, heart, and aorta. Serum AKl appears to have the greatest specificity of the AK isoforms as a marker of neural tissue injury. AK. may be best suited as a cerebrospinal fluid marker of cerebral ischemia, where its dominant source would be neural tissue. [0240] Neurotrophins are a family of growth factors expressed in the mammalian nervous system. Some examples include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). Neurotrophins exert ilu-ij effects primarily as target-dsrived paracrine or autocrine neiurotrophic factors. The role of the neurotrophins in survival, differentiation and maintenance of neurons is well known. They exhibit partially overlapping but distinct patterns of expression and cellular targets. In addition to the effects in the central nervous system, neurotrophins also affect peripheral aiTcrent and efferent neurons. [0241] BDNF is a potent neu.otrophic factor which supports the growth and survivability of nerve and/or glial cells. BDNF1 is expressed as a 32 kDa precursor "pro-BDNF" molecule mat is cleaved to a mature BDNF form. Mowla et al., J. Biol. Chem. 276: 12660-6 (2001). The most abundant active form ot human BDNF is a 27 kDa homodimer, formed by two identical 119 amino acid subunits, which is held together by strong hydrophobic interactions; however, pro-BDNF is also released extracellularly and is biologically active. [0242] NT-3 is also a 27 kDa 'lomodimer consisting of two 119-amino acid subunits. The addition of NT-3 to primary cortical cell cultures has been s/iown to exacerbate neuronal death caused by oxygen-glucose deprivation, possible via oxygen free radical mechanisms (Bates et\ir.. Neurobiol. Dis. 9:24-37,2002). NT-3 is expressed as an inactive pro-NT-3 molecule, which is cleaved to the mature biologically active form. [0243] Gilbindin-D is a 2S lOa cytosolic vitamin D-dependent Ca2+-binding protein that may serve a cellular protective fvnction by stabilizing intracellular calcium levels. Calbindin-P is found in the central nervous system, mainly in glial cells, and in cells of the distal renal tubule (Hasegawa, S. etai, J. Urol. 149:1414-1418, 1993). The normal serum concentration of c;i!bindin-D is [0244] Creatine kinase (CK) is a cytosolic enzyme that catalyzes the reversible formation of ADP and phosphocreatine from ATP and creatine. The brain-specific CK isoform (CKBB) is an 85 kDa cytosolic protein that accounts for approximately 95% of the total brain CK activity. It is also present in significant quantities in cardiac tissue, intestine, prostate, rectum, stomach, smooth muscle, thyroic. uterus, urinary bladdei, [0245] Glial fibrillary acidic protein (GFAP) is a 55 kDa cytosolic protein that is a major structural component of astroglisJ filaments and is the major intermediate filament protein in astrocytes. GFAP is specific to sistrocytes, which are interstitial cells located in the CNS and can be found near the blood-brain barrier. Serum GFAP is elevated following ischemic stroke (Miebroj-Dobosz, I., et al., Folia Neuropathol. 32:129-137, 1994). Serum concentrations G.FAP appear to be elevated soon after the onset of stroke, continuously increase and persist for an amount of time (weeks) thit may correlate with the severity of damage. [0246] Lactate dehydrogenise (LDH) is a ubiquitous 135 kDa cytosolic enzyme. It is a •w tetramer of A and B chains that catalyzes the reduction of pyruvate by NADH to lactate. Five isoforms of LDH have been identified in mammalian tissues, and the tissue-specific isoforms are made of different combinations of A and B chains. Elevations in serum LDH activity are reported following both ischemi-: and hemorrhagic stroke, but further studies are needed in serum to confirm this observation and to determine a correlation with the severity of injury :md neurological outcome (Agguwal, S.P. et al., J. Indian Med. Assoc. 93:331-332, 1995; Maiuri, V. er al., Neural. Res. 11:6-8,1989). [0248] Myelin basic protein «MBP) is actually a 14-21 kDa family of cytosolic proteins generated by alternative splicing of a single MBP gene that is likely involved in myelin compaction around axons during the myelination process. MBP is specific to oligodendrocytes in the CNS and in Schwann cells of the peripheral nervous system (PNS). Serum MBP is elevated after all cypes of severe stroke, specifically thrombotic stroke, embolic stroke, intracerebral hemorrhage, and subarachnoid hemorrhage, while elevations in MBP concentration are not reported in the serum of individuals with strokes of minor to moderate severity, which would include lacunar infarcts or transient ischemic attacks (P:ilfreynian, J.W. et al., Clin. Chvn. Acta 92:403-409,1979). The serum concentration of MBP has been reported to correlate with the extent of damage (infarct volume), and it may also correlate with neurological outcome. [024SJ Neural cell adhesion molecule (NCAM), also called CD56, is a 170 kDa cell surface-bound immunoglobulin-ljjce integrin ligand that is involved in the maintenance of neuronal and glial cell interactiors in the nervous system, where it is expressed on the surface of astrocytes, oligodendrocytes, Sihwann cells, neurons, and axons. NCAM is also localized to developing skeletal muscle myotubes, and its expression is upregulated in skeletal muscle during development, denervation i':id renervation. [0249] Proteolipid protein (PLP) is a 30 kDa integral membrane protein that is a major structural component of CNS myelin. PLP is specific to ohgodendrocytes in the CNS and accounts for approximately 50% of the total CNS myelin protein in the central sheath, although: wuremely low levels of PLP have been found ( (PNS) myelin. Scrum PLP is elevated after cerebral infarction, but not after transient ischcmic attack (Trotter, J.L. et al.,Ann. Neural 14:554-558,1983). Elevations of PLP in SL-nim can be attributed to neural tissue injury due to physical damage or ischemia caused by infarction or cerebral hemorrhage, coupled with increased permeability of the blood brain barrier. [0250] S-tOOp is elevated in serum after 4 hours from stroke onset, with concentrations reaching a maximum 2-3 days a7:er onset. After the serum concentration reaches its maximum, which can approach 20 ng/ml (1-9 mM), it gradually decreases to normal over approximately one week. Because the severity of damage has a direct effect on the release of S-100P, it will affect the release kinetics by influencing the length of time that S-100p is elevated in the serum. S-lOOp will be present in the serum for a longer period of time as the seventy of injury increases. Furthermore, elevated serum concentrations of S-100P can indicate complications related to neural tissue injury after AMI and cardiac surgery. [0251] Thrombomodulin (TM) is a 70 kDa single chain integral membrane glycoprotein found on the surface of vascular eudothelial cells. Current reports describing serum TM concentration alterations follow .ng ischemic stroke are mixed, reporting no changes or significant increases (Seki, Y. e\ al., Blood Coagul. Fibrinolysis 8:391-396, 1997). Serum elevations of'TM concentration reflect endothelial cell injury and would not indicate coagulation or fibrinolysis activation. [0252] The gamma isoform of protein kinase C (PKCg) is specific for CNS tissue and is not normally found in the circul ttion. PKCg is activated during cerebral ischemia and is present in the ischemic penumb.~n at levels 2-24-fold higher than in conrralateral tissue, but is not elevated in infarcted tissue (Krupinski, J. et al., Acta Neurobiol. Exp. (Warz) 58:13-21, 998). Additional isoforms of PK.C, beta I and beta II were found in increased levels in the infarcted core of brain tissue from patients with cerebral ischemia (Krupinski, J. et al.,Acta Neurobioi Exp. (Wan.) 58:13-21,1998). Furthermore, the alpha and delta isoforms of PKC (PKCa and PKCd, respectively) have been implicated in the development of vasospasm following subarachnoid hemorrhage using a canine model of hemorrhage. Therefore, it may be of k*saeiit to measure various isoforms of PKC, either individually or in various combinations thereof, for the identification of cerebral damage, the presence of the ischemic penumbra, as well as the develcpment and progression of cerebral vasospasm following subarachnoid hemorrhage. Rat os of PKC isoforms such as PKCg and either PKCbl, PKCbll, CM- both also may be of benefit ui identifying a progressing stroke, where the ischemic penumbra is converted to irreversibly damaged infarcted tissue. "Yi2x>J (viii) Non-Specific Markers for Cellular Injury [0254] Myoglobin is a smaK (17.8 kDa) heme protein transports oxygen within muscle cells, and constitutes about 2 percent of muscle protein in both skeletal and cardiac muscle. Because of its low molecular we,ght, myoglobin is rapidly released into the circulation and is the first marker to exhibit rising levels after an AMI: elevated levels appear in the circulation after 0.5 to 2 hours. However, elevated levels may also be ielated to various skeletal muscle traumas and renal failure, and are therefore not specific for cardiac muscle injury. [0255] Human vascular endothelial growth factor (VEGF) is a dimeric protein, the reported activities of which incl ide stimulation of endothelial cell growth, angiogenesis, and capillary permeability. VEGF is: secreted by a variety of vascularized tissues. In an oxygendeticient environment, vascular endothelial cells may be damaged and may not ultimately survive. However, such endothdial damage stimulates VEGF production by vascular smooth muscle cells. Vascular endothelial cells may exhibit increased survival in the presence of VEGF, an effect that is believed to be mediated by expression of Bcl-2. VEGF can exist as a variety of splice variants known as VEGF(1S9), VEGF(165), VEGF(164), VEGFB(155), VEGF(148), VEGF(145), and V1T.GF(121). [0256] Insulin-like growth ft.ctor-1 (IGF-1) is a ubiquitous 7.5 kDa secreted protein that mediates the anabolic and somatogenic effects of growth hormone during development (1,2). In the circulation, IGF-1 is nornfcilly bound to an IGF-binding protein that regulates IGF activity. Serum IGF-1 concentrations are reported to be significantly decreased in individuals with ischemic stroke, and the magnitude of reduction appears to correlate with the severity of injury (Schwab, S. et al, Stroke 28:1744-1748, 1997). Serom IGF-1 may be a sensitive indicator -pf neural tissue injury. However, the ubiquitous expression pattern of IGF-1 indicates that all tissues can pott ntially affect serum concentrations of IGF-1. [0257] Adhesion molecules are involved in the inflammatory response can also be considered as acute phase reactarts, as their expression levels are altered as a result of insult. L samples of such adhesion molecules include E-selectin, intercellular adhesion molecule-1, vascular cei] adhesion molecule, and the like. [0258] E-selectin, also called ELAM-1 and CD62E, is a 140 kDa cell surface C-type lectin expressed on endothelial cells in response to IL-1 and TNFa that mediates the "rolling" interaction of neutrophils with endothelial cells during neutxophil recruitment. Some investigations report increases itt serum E-selectin concentration following ischemic stroke, while others find it unchanged (Bitsch, A. et al., Stroke 29:2129-2135, 1998; Kim, J.S., J. Xeurol. Sci. 137:69-78, 1996; Shyu, K.G. et al.,J. Neurol. 244:90-93,1997). E-selectin concentrations are elevated in the CSF of individuals with subarachnoid hemorrhage and may predict vasospasm (Polin, R.S. et al, J. Neurosurg. 89:559-567, 1998). Serum E-selectin concentrations are elevated in individuals with atherosclerosis, various forms of cancer, preeclampsia, diabetes, cystic fihrosis, AMI, and other nonspecific inilammatoi> scaies fHwang, SJ. et al, Circulation 96:4219-4225, 1997; Banks, R.E. et al, Br. J. Cancer 68:122114, 1993; Austgulen, R. et al.,jlur. J. Obstet. Gynecol Reprod. Biol. 71:53-58, 1997; Steiner, M. et al, Thromb. Haeniost. 72:979-984,1994; De Rose, V. et al, Am. J. Respir. Crit. CareMed. 157:1234-1239, 1998). [0259] Head activator (HA) is an 11 amino acid, 1.1 kDa neuropeptide that is found in the hypothalamus and intestine. It w as originally found in the freshwater coelenterate hydra, where it acts as a head-specific growth and differentiation factor. [0260] Glycated hemoglobin HbAlc measurement provides an assessment of the degree to which blood glucose has been elevated over an extended time period, and so has been related to the extent diabetes is controlled in a patient. Glucose binds slowly to hemoglobin A, forming the Ale subtype. The reverse reaction, or decomposition, proceeds relatively slowly, so any buildup persists for roughly4 weeks. With normal blood glucose levels, glycated hemogHmi is expected to be 4.5% to 6.7%. As blood glucose concentration rise, however, more binding occurs. Poor blood sugar control over time is suggested when the glycated hemoglobin measure exceeds S.0%. [0261] (ix) Markers related to apoptosis [0262] Apoptosis refers to th; eukaryotic '•programmed cell death" pathway. The pathway is dependent upon intracellular proteases and nucleases, leading ultimately to fragmentation ofgenomic DNA and cell death. The following exemplary list of markers related to apoptosis is not meant to be limiting. [0263] Caspases are a familj of proteases that relay a "doomsday" signal in a step-wise manner reminiscent of signaling by kinases. Caspases are present in all cells as latent enzymes. They are recruited to receptor-associated cytosolic complexes whose formation is initiated by receptor oligomerization (e.g., TNF receptors, FAS, and TRAIL receptors) and to other cytoplasmic adaptor proteins, such as APAF-1. Recruitment of caspases to oligomerized receptors leads to activation via d.merization or dimerizatioi accompanied by autoproteolytic cleavage. Active caspases can prDteolyze additional caspases generating a caspase cascade that cleaves proteins critical for cell survival. The final outcome of this signaling pathway is a form of controlled cell death termed apoptosis. [0264] The subgroup of casp.ises involved in apoptosis has been referred to as either initiators or effectors. Caspases-tt, -9, and -10 (possibly, -2 and -5) can initiate the caspase activation cascade and are therefore called initiators. Based on the prototypes, caspases-8 and -9, initiators can be activated eittur by dimerization alone (caspase-9) or by dimerization with concomitant autoproteolysis (casp ase-8). The effector caspases-3, -6, and -7 propagate the cascade and are activated by prottjolytic cleavage by other caspases. Although an initiator cuspase may not be responsible for starting the caspase cascade, it can become activated and involved in later steps of the cascade. Thus, in the latter scenario, the caspase would be more appropriately termed an effector [0265] Caspase-3, also called CPP-32, YAMA, and apopain, is an interleukin-lp convertir.g; enzyme (ICE)-like intracellular cysteine proteinase that is activated during cellular apoptosis. Caspase-3 is present us an inactive 32 kDa precursor that is proteolytically activated during apoptosis induc'ion into a heterodimer of 20 kDa and 11 kDa subunits fFernandesrAlnemri, T. el al, J. BioL Chem. 269:30761-30764, 1994). Its cellular substrates include poly(ADP-ribose) polyrr.erase (PARP) and sterol regulatory element binding proteins rSREBPs) (Liu, X. et al.. J. Biol Chem. 271:13371-13376, 1996). The normal plasma concentration of caspase-3 is unknown. There are no published investigations into changes in the plasma concentration of casp.ise-3 associated with ACS. There are increasing amounts of evidence supporting the hypothesis of apoptosis induction in cardiac myocytes associated with ischemia and hypoxia (Sannte, A., Herz 24:189-195,1999; Ohtsuka, T. etal., Coron. Artery Dis. 10:221-225, 1999; Jttnes, T.N.. Coron. Artery Dis. 9:291-307, 199S; Bialik, S. et al.../. Clin. Invest. 100:1363-1372, 1997; Long, X. et al., J. Clin. Invest. 99:2635-2643. 1997). Elevations in the plasma caspase-3 concentration may be associated with any physiological event mat involves apoptosis. There is evidence that suggests apoptosis is induced in skeletal muscle during and following exercise and in cerebral ischemia (Carraro, U. and Franceschi, C., Aging (kFlano) 9:19-34, 1997; MacManus, J.P. et al., J. Cereb. Blood FlowMetab. .19:502-510,1999). [0266] CathepsinD (E.C.3.4.23.5.) is a soluble lysosomal aspartic proteinase. It is synthesized in the endoplasmic reticulum as a preprocathepsin D. Having a mannose-6phosphate tag, procathepsin D in recognized by a mannose-6-phosphate receptor. Upon entering into an acidic lysosome.. the single-chain procathepsin D (52 KDa) is activated to cathepsin D and subsequently to a mature two-chain cathepsin D (31 and 14 KDa, respectively). The two mannose-Sphosphate receptors involved in the lysosomal targeting of procathepsin D are expressed both intracellularly and on the outer cell membrane. The glycosylation is believed to be cnicial for normal intracellular trafficking. The fundamental role of cathepsin D is to degrade intracellular and internalized proteins. Cathepsin D has been suggested to take part in antigen processing and in enzymatic generation of peptide hormones. The -.issue-specific function of -';athepsin D seems to be connected to the processing of prolactin. Rat mammary glands use this enzyme for the formation of biologically active fragments of prolactin. Cathepsin D is functional in a wide variety of tissues during their ir-rtg or regression, and in apoptosis. j i J267] Brain a spectrin (also referred to as a fodrin) is a cytoskeletal protein of about 284 kDa thai interacts with calmodulin in a calcium-dependent manner. Like erythroid spectrin, iiriiin a spectrin forms oligomers (in particular dimers and tetrarners). Brain a spectrin contains two EF-hand domains and 23 spectrin repeats. The caspase 3-mediated cleavage of a spectrin during apoptotic cell death may play an important role in altering membrane stability ;ind thelbrmation of apoptotic bodies. [0268] The following table provides a list of various preferred markers, associated with a classification of the marker to a group of related markers. A.s understood by the skilled artisan and described herein, markers may indicate different conditions when considered with additional markers in a panel; alternatively, markers may indicate different conditions when considered in the entire clinical context of the patient. (Table Removed) 3arboxytermin?J.propeptide of type I procollagen (PICP) ollagen carboxyterminal telopeptide (ICTP) Glutathionf, S Transferase HIF 1 ALPHA IL-10 IL- .-Beta n-lra I .-6 Lysophosuhatidic acid JVIDA-mcclified LDL Human neuirophil elastase C-reactive protein Insulin-like growth factor Inducible nitric oxide synthase Intracellular ac'hesion molecule Lactate dehydrogenase MCP-1 MDA-LDL MMP-1 MMP-2 MMP-3 MMP-9 TIMP-1 TIMP-2 TIMP-3 n-acetyl Espartate TNF Receptor Superfamily Member 1A Transforming growth factor beta Tumor necrosis factor alpha Vascular cell adhesion molecule Vascular endothelial growth factor cystelin C substance P Myeloperoxidase (MPO) macrophage inhibitory factor Fibronectin cardiotrjphin 1 Haptotjlobin PAI'PA S-CD40 ligand* HMG Collagen synthesis Collagen degradation Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory . Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory Inflammatory .'L-l Inflammatory IL-2 Inflammatory If ,-4 Inflammatory 1L-6 Inflammatory IL-8 Inflammatory IL-10 Inflammatory IL-11 Inflammatory IL-13 Inflammatory IL-1S Inflammatory Eosinophil :ationic protein Inflammatory Mast Cijil tryptase Inflammatory VCAM Inflammatory sIC:AM-l Inflammatory TNFa Inflammatory Osteoprotegerin Inflammatory Prostaglanoin D-synth£se Inflammatory Prostaglandin B2 Inflammatory RANECligand Inflammatory HSP-60 Inflammatory Serum Amyloid A Inflammatory s-iL 18 receptor Inflammatory S-iL-1 receptor Inflammatory s-TNF P55 Inflammatory s-TNF P75 Inflammatory TGF-beta Inflammatory MMP-11 Inflammatory BetaNGF Inflammatory CD44 Inflammatory E(3F Inflammatory E-sciectin Inflammatory Fibronectin Inflammatory Neutrop'iil elastase Pulmonary injury KL-6 Pulmonary injury LAMPS Pulmonary injury LAMP3 Pulmonary injury Lung Surfactant protein A Pulmonary injury Lung Surfactant protein B . Pulmonary injury Lung Surfactant protein C Pulmonary injury Lung Surfactant protein D . Pulmonary injury phospholipase D Pulmonary injury PLA2G5 Pulmonary injury SFfPC Pulmonary MAPK10 Neural tissue injury KCNK4 Neural tissue injury (Table Removed) an important role in the control of numerous processes, such as the way in which extracellular materials are incorporated into a cell, the movement of biochemical signals from the cell msmbrane, and the regulation of cellular functions such as transcrfptional on-off switches. The ubiquitin system has been implicated in the immune response and development. Ubiquitin is a 76-amino acid polypeptide that is conjugated to proteins targeted for degradation. The ubiquitin-protein conjugate is recognized by a 263 proteolytic complex that splits ubiquitin from the protein, which is subsequently degraded. Levels of ubiquitinated proteins generally, or of specific ubiquitin-protein conjugates or /rigments thereof, can be measured as additional markers of the iirvS.itian. Moreover, circuiting levels of ubiquitin itself or its fragments can be a useful marker in the methods describee herein. See. e.g., Hu et al., J. Cereb. Blood Flow Metab. 21: $05-75,2001. [0271] The skilled artisan w 11 recognize that an assay f :>r ubiquitin may be designed that recognizes uhiquitin itself, ubiq'ii tin-protein conjugates, or 3Oth ubiquitin and ubiquitinprotein conjugates. For example, antibodies used in a sandvich immunoassay may be selected so that both the solid phase antibody and the labeled antibody recognize a portion of ubiquitin that is available for binding in be th unconjugated ubiquitin and ubiquitin conjugates. Alternatively, an assay specific far ubiquitin conjugates of:: marker of interest could use one antibody (on a solid phase or labsl) that recognizes ubiquitin, and a second antibody (the other of the solid phase or label) that recognizes the marker protein. [0272] The present invention contemplates measuring ubiquitin conjugates of any marker described herein. [0273] Use of marker panels and the "panel response value" [0274] As discussed above, traditional methods to evaluate marker levels in the diagnosis or prognosis of disease typically oamprise establishing a "threshold" for a marker of interest. The concentration of that marker in a sample is then compaied to that threshold amount, and 101 amount greater than the pre-e? tablished threshold is indie a-ive of one state (e.g., disease), while an amount less than the pp.- established threshold is indicative of another state (e.g.. normal). One skilled in the art w 11 recognize that univariate analysis of markers can be performed and the data from the \ livariate analyses of mult pie markers can be combined to form panels of markers to differentiate different disease conditions. [0275] As the number of mar cars in a panel increase, ho wever, applying individual thresholds to each marker can be ;c me unwieldy. While the use of individual thresholds for one or more markers in a panel if within the scope of the present invention, the following section describes exemplary methods by which a plurality of markers are evaluated, in which particular thresholds for one or more markers in the marker' panel are not relied upon in correlating a marker level to a particular diagnosis and/or prognosis. Rather, the plurality of markers considered as a unitary whole. A simple example of integrating markers to form a unitary result can be calculating the ratio of two or more markers. In the following exemplary methods, each marker concentre tion measured in a sample contributes to a "panel response value," which may be compared 10 a "threshold" panel response as if it were simply the concentration of a single markei. This is an example of a diagnostic method wherein the amount of one or more the markers is not compared to a predetermined threshold level. [0276] Suitable methods for identifying markers useful for the diagnosis of disease states are described in detail in U.S. Piovisional Patent Application No. 60/436,392, entitled METHOD AND SYSTEM FOP. DISEASE DETECTION USING MARKER COMBINATIONS (attorney docket no. 071949-6801), filed December 24, 2002; U.S. Patent Application No. 10/331,127, entitled METHOD AND SYSTEM FOR DISEASE DETECTION USING MARKER COMBINATIONS (attorney docket no. 071949-6802), filed December 27, 2002; and PCT application no. , filed December 23, 2003 (Arty Docket No. 071949-6805), each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims. One skilled in the art will also recognize that univariate analysis of markers can be performed and the data from the univariate analyses of multiple markers can be combinsd to form panels of markers to differentiate different disease conditions. [0177] In developing a panel of markers useful in diagnosis, data for a number of potential markers may be obtained from a group of subjects by testing for the presence or level of certain markers. The group of subjects is divided into two sets, and preferably the first set and the second set each have an approximately equal number of subjects. The first set includes subjects who have been confirmed as having a disease or, more generally, being in a first condition state. For example, this first set of patients may be those ACS patients who have recently had a subsequent adverse outcome. Hereinafter, subjects in this first set will be referred to as "diseased". [0278] The second set of subjects is simply those who do not fall within the first set. Subjects in this second set may is "non-diseased;" that is, normal subjects. Alternatively, subjects in this second set may he selected to exhibit one symptom or a constellation of symplsras tiiat mimic those symptoms exhibited by the "diseased" subjects. In the case of the ACS ex;r.nple described hereinafter, the "non-diseased" group may be those ACS patients ••vho, over the same time period, did not suffer a subsequent adverse outcome. [0279] The data obtained frcrn subjects in these sets includes levels of a plurality of markers. Preferably, data for the same set of markers is available for each patient. This set of markers may include all candidate markers thai may be suspected as being relevant to the detection of a particular disease or condition. Actual known relevance is not required. •Embodiments of the methods and systems described herein maybe used to determine which of the candidate markers are mowl relevant to the diagnosis of the disease or condition. The levels of each marker in the two .sets of subjects may be distributed across a broad range, e.g., as a Gaussian distribution. However, no distribution fit is required. [0280] As noted above, a marker often is incapable of definitively identifying a patient as either diseased or non-diseased. For example, if a patient is measured as having a marker level that falls within the overlapping region, the results of the test will be useless in diagnosing the patient. An artificial cutoff may be used to distinguish between a positive and a negative test result for the detection of the disease or condition. Regardless of where the cutoff is selected, the effectiveness of the single marker as a diagnosis tool is unaffected. Changing the cutoff merely trades off between the number cf false positives and the number of false negatives resulting from the use of the single marker. The effectiveness of a test having such an overlap is often expressed using a ROC (Receiver Operating Characteristic) curve. ROC curves are well kno vn to those skilled in the art. [0281] The horizontal axis of The ROC curve represents [1- specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cutoff selected, the value of (1spacincity) may be determined, a^d a corresponding sensitivity may be obtained. The area under the ROC curve is a measurs of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveress of the test. [0252] While the measurement of the level of a single marker may have limited usefulness, the measurement of additional markers provides additional information. But the difficulty lies in properly combining the levels of two potentially unrelated measurements. In The methods and systems according to embodiments of the present invention, data relating to levels of various markers for the sets of diseased and non-diseased patients may be used to cioveiop a panel of markers to provide a useful panel response. The data may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file. The database or data file may contain, for example, a patient identifier such as a name or number, tlie levels of the various markers present, and whether the patient is diseased or non-diseased. [0283] Next, a "window" region may be initially selected for each marker. The location of the window region may initially be selected to include any concentrations of the marker, but the selection may affect the optimization process described below. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In a preferred method, the center of the window region is initially centered about the center of the overlap region of the two seis of patients. In one embodiment, the "window" region may simply be a cutoff point or threshold, as is known in the art. In other embodiments, the window region may span a defined concentration range for the marker. In this regard, the window region may be defined by a center value and a width. In practice, the initial selection of the limits of the window region may be determined according to a pre-selected percentile of each set of subjects. For example, a point above which a pre-selected percentile of diseased patients are measured may be used as the right (upper) end of the window range. [0284] Each marker value for each patient may then be mapped to an indicator. The indicator is assigned one value below the window region and another value above the window region. For example, if a marker .generally has a lower value for non-diseased patients and a higher value for diseased patients, a zero indicator will be assigned to a low value for a particular marker, indicating a potentially low likelihood of'a positive diagnosis. In other embodiments, the indicator may be calculated based on a polynomial. The coefficients of the polynomial may be determined based on the distributions of the marker values among the diseased and non-diseased subjscts. In the exemplary embodiments described in detail below, marker concentrations less thar. the window region are assigned a value of 0, and marker conc greater than the window region are assigned a value of 1. Within the window region, concentrations are linearly interpolated to a value between 0 and 1. While the choice of a linear function within the window is used in the examples below, in principle any nonlinear function may also be ipplied to the marker concentrations within the window, as described in the next paragraphs. [0285] In many disease states, nonspecific markers associated with that state are elevated. But above a certain threshold, higher values of the marker may not relate to a higher probability of disease state. Below a certain threshold, lower marker values may not relate to a lower probability of disease sta e. In this situation the indicator function may not increase linearly with the marker value. A preferred embodiment is an indicator function that is a function that has a high and monotonic rate of change betwsen the thresholds, and a small rate of change elsewhere. Examples of this type of function are the ramp, step, or sigmoid functions. One may associate th-i lower threshold with the start of an overlap region (or window region"), and the upper threshold with the end of the: overlap region. Below the lower threshold the probability of diseise is substantially 0, while above the upper threshold the probability of disease is 1. Note that in the case where the indicator function is a. step function and the weighting value is 1 for each marker, then the panel response is simply the number of markers above the window. This case is identical to the example used above where one is searching for the best panel with n of m. markers above their window. Allowing the indicator to vary continuously near the threshold enables the panel response to be sensitive to a marker just under the window. This information is not lost as it is ir. the n of m marker example or the step function example, where the indicator value is not continuous. Another common approach of summing over M*W forces the linear relation with M. But as discussed above the most appropriate indicator function may not increase linearly with the marker value. In a further preferred embodiment the ramp function is used as an elevation indicator function. The indicator values within the window regions may vary linearly from a value of zero at one aid to a value of one at the other ;nd. In other embodiments, non-linear variations of the indicator value may be used. The ramp function has the advantage of simplicity, and may be good approximation to other function in this class. With proper choices of parameters, the ramp function can be equivalent to the step function or can increase linearly with the marker value. [00100] In some disease state.1;, for example unstable angina, a specific marker such as the cardiac troponins (including isoforms of cardiac troponin, comprising troponin I and T and complexes of troponin I, T and C) may be elevated above the normal population, but further elevation indicates an acute condition, in this case a myocardial infarction. Unstable angina is an ischemic condition that leads to minor necrosis of cardiac tissue. During a myocardial infarction, there is major necrosis of cardiac tissue. Cardiac troponin, which is specific to cardiac necrosis, is elevated in both conditions, but the amount of elevation is related to the amount of necrosis. The best indicator function of cardiac troponin in diagnosing unstable angina may not be an elevation indicator function. In a preferred embodiment the indicator function may be a function that is peaked near the expected values of unstable angina, and decreases when the marker value is above or below the expected value. Examples of this type of function include a Gaussian, triangle, trapezoid, or square function. These functions tend to localize the marker value of interest around a specific value. Another example of use for such an indicator function is in cases where a pattern of markers values indicates a disease state. For example, a disease conditior. may be indicated when one or more markers are within a range of values. When desired, the use of this type of indicator may allow for recognition of patterns of marker values. [0286] The relative importance of the various markers may be indicated by a weighting factor. The weighting factor may initially be assigned as a coefficient for each marker. As with the cutoff region, the initial selection of the weighting factor may be selected at any acceptable value, but the selection may affect the optimization process. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In a preferred method, acceptable weighting coefficients may range between zero and one, and an initial weighting coefficient for each marker may be assigned as 0.5. In a preferred embodiment, the initial weighting coefficient for each marker may be associated with the effectiveness of that marker by itself. For example, a ROC curve may be generated for the single marker, and the area under the ROC curve may be used as the initial weighting coefficient for that marker. [0287] Next, a panel response may be calculated for each subject in each of the two sets. The panel response is a function of the indicators to which each marker level is mapped and the weighting coefficients for each marker. In a preferred embodiment, the panel response (R ) for a each subject (j) is expressed as: where i is the marker index, j is the subject index, w, is the weighting coefficient for marker i, I is the indicator value to which the marker level for marker i is mapped for subject j, and Z is the summation over all candidate markers i. [0288] One advantage of using an indicator value rather than the marker value is that an extraordinarily high or low marker levels do not change the probability of a diagnosis of diseased or non-diseased for that particular marker. Typically, a marker value above a certain level generally indicates a certair. condition state. Marker values above that level indicate the condition state with the same certainty. Thus, an extraordinarily high marker value may not indicate an extraordinarily high probability of that condition state. The use of an indicator which is constant on one side of the cutoff region eliminates this concern. [0280] The panel response may also be a general function of several parameters including the murker levels and other factors including, for example, race and gender of the patient. Oilier factors contributing to the yanel response may include the slope of the value of a particular marker over time. For example, a patient may be measured when first arriving at the hospital for a particular marker. The same marker may be measured again an hour later, and the level of change may be reflected in the panel response. Further, additional markers may be derived from other markers and may contribute to the value of the panel response. For example, the ratio of values of two markers may be a. factor in calculating the panel response. [0290] Having obtained pan Jl responses for each subject in each set of subjects, the distribution of the panel responses for each set may now be analyzed. An objective function may be defined to facilitate the sslection of an effective panel. The objective function should generaly be indicative of the effectiveness of the panel, as may be expressed by, for example, overlap of the panel responses of the diseased set of subjects and the panel responses of the non-diseased set of subjects. In this manner, the objective function may be optimized to maximize the effectiveness of the panel by, for example, minimizing the overlap. [0291] In a preferred embodiment, the ROC curve representing the panel responses of the two sets of subjects may be used to define the objective function. For example, the objective furiction may reflect the area unotir the ROC curve. By maximizing the area under the curve, one may maximize the effectiveness of the panel of markers. In other embodiments, other features of the ROC curve may be used to define the objective function. For example, the point at which the slope of the ROC curve is equal to one may be a useful feature. In other embodiments, the point at which the product of sensitivity and specificity is a maximum, sometimes referred to as the "knee," may be used. In an embodiment, the sensitivity at the knee may be maximized. In further embodiments, the sensitivity at a predetermined specificity level may be used to define the objective function. Other embodiments may use the specificity at a predetermined sensitivity level may be used. In still other embodiments, combinations of two or more of *.hese ROC-curve features may be used. [0292] It is possible that one of the markers in the panel is specific to the disease or condition being diagnosed. When such markers are present at above or below a certain threshold, the panel response may be set to return a "positive" test result. When the threshold is not satisfied, however, the levels of the marker may nevertheless be used as possible contributors to the objective function. [02931 An optimization algorithm may be used to maximize or minimize the objective function. Optimization algorithms are well-known to those skilled in the art and include several commonly available minimizing or maximizing functions including the Simplex method and other constrained optimization techniques. It is understood by those skilled in the an that some minimization functions are better than others at searching for global minimums. rather than local minimums. ID the optimization process, the location and size of the cutoff region for each marker may be allowed to vary to provide at least two degrees of freedom per marker. Such variable parameters are referred to herein as independent variables. In a embodiment, the weighting coefficient for each marker is also allowed to vary across iterations of the optimization algorithm. In various embodiments, any permutation of these parameters may be used as independent variables. [0294] In addition to the above-described parameters, the sense of each marker may also be used as an independent variable. For example, in many cases, it may not be known whether a higher level for a certain marker is generally indicative of a diseased state or a non-diseased state. In such a case, it may be useful to allow the optimization process to search on both sides. In practice, this may ae implemented in several ways. For example, in one embodiment, the sense may be a truly separate independent variable which may be flipped between positive and negative by the optimization process. Alternatively, the sense may be implemented by allowing the weighting coefficient to be negative. [0295] Within the teachings of this document it is often assumed for simplicity that markers that are elevated in patients with the disease or "positive sense" markers. However this is not always the case, and o^en, particularly with poor univariate markers, it is not clear from univariate analysis whether the marker when used in conjunction with the other markers in the panel, is best utilized in a positive or negative sense, .{f the sense of a marker is inverted, then it is straightforward to invert the indicator function for that marker. If the sense is not known, then the search engine may include this as a degree of freedom. For example, in one embodiment, the sense may be a truly separate independent variable, which may be flipped between positive and negalive by the optimization process. For optimal performance, the sense should map smoothly from improper to proper, and there should be pressure that allows the search engine to move toward the proper sense. In a preferred embodiment the sense is switched by allowing the weighting coefficient of th«; analyte to go negative. If the wrong sense is selected, the weighting coefficient will be driven towards zero since inclusion of the marker in the panel response negatively impacts the objective function. The search engine will be able to drive the weighting coefficient across zero to the proper sense. [0296] The optimization algorithm may be provided with certain constraints as well. For example, the resulting ROC curve may be constrained to provide an area-under-curve of greater than a particular value. rtOC curves having an area under the curve of 0.5 indicate andomness, while an area under the curve of 1.0 reflects perfect separation of the two sets. Thus, a minimum acceptable value, such as 0.75, may be used as a constraint, particularly if the objective function does not incoiporate the area under the curve. Other constraints may include limitations on the weighting coefficients of particular markers. Additional constraints may limi'. the sum of all the weighting coefficients to a particular value, such as 1.0. [0297] The iterations of the optimization algorithm generally vary the independent parameters to satisfy the constraints while minimizing or maximizing the objective function. The number of iterations may be limited in the optimization process. Further, the optimization process may be terminated when the difference in the objective function between two consecutive iterations is below a predetermined threshold, thereby indicating that the optimization algorithm has reached a region of a local minimum or a maximum. [ 00101] hi practice diagnosis of a disease statejrom multiple markers can be confusing. Often the individual marker values may seem to contradict one another. In panels where the individual markers are not very effective, it is extremely difficult to understand their meaning. in a. preferred embodiment, a function that combines the marker values into a scalar value that increases with increasing likelihood of disease is defined. In this manner, the information from multiple markers may be presented in a useable form. This defined function is referred to herein as the panel response (PR), and is a function of the marker values (M0_n), written as PR =f(M0_H). The panel response may be scaled such that all values are between 0 and 1. Because the effectiveness of the test may not depend on a scaling of the panel response, scaling may not influence the result of the method. However forcing the panel response to be a given scale may remove an unneeded redundancy, as panel response functions that differ only by a scaling factor may in fact represent the same solution. The panel response may also he a general function of several parameters including the marker levels and other factors including, for example, a patient's history, age, race and gender of the patient. [00102] In a preferred embodiment, the panel response (PR) for each subject is expressed where i is the marker index, Ws is the weighting coefficient for the marker i, M; is the marker value for arker i, I is an indicator function for marker i, and Z is the summation over all candidate markers. The weighting factors scale the indicator functions and may allow for more important or specific markers to have a greater impact on the final panel response. The indicator function maps the marker value into a functional form appropriate to the marker's pathology. The indicator functions can be complex and should be chosen to match the marker. This will be illustrated in the embodiments described below. The indicator function may be a diff'erent functional form for each marker. In one example, the indicator function can map the marker value into a probability of the disease state. This mapping may not be a simple function of the marker value. In this example the said indicator from each marker can be summed to determine a relative index which is related to the probability of the patient being diseased. In a preferred embodiment the sum of all the weighting coefficients is constrained to a particular value, such as 1.0. In a preferred embodiment the indicator function is constrained to values between 0 and 1. In a further preferred embodiment, both of the above constraints ire satisfied, thus, the panel response is also constrained to a value between 0 and 1. [0298] Thus, the optimization process may provide a panel of markers including \veighting coefficients for each marker and. cutoff regions for the mapping of marker values to indicators. In order to develop lower-cost panels that require the measurement of fewer marker levels, certain markers may be eliminated from the panel. In this regard, the effective contribution of each marker in the panel may be determined to identify the relative importance of the markers. In onu embodiment, the weighting coefficients resulting from the optimization process may be used to determine the relative importance of each marker. The markers with the lowest coefficients may be eliminated. [0299] In order to determine a suitable panel, which for practical reasons may often mean 10 or less markers, one must find a way to systematically remove markers that do not significantly contribute to the overall result. This is accomplished by calculating the contribution from each marker. A method to accomplish this is to remove an analyte from the panel, and recalculate the objective function. The change in the objective function is related to the contribution of the marker. The markers may then be arranged in order of decreasing contribution. In embodiments where a weighting coefficient is applied to each analyte, the weight for the analyte can be set to zero to remove the analyte from the panel. In embodiments where a weighting coefficient is applied to each analyte, one cannot simply use the weights as the contribution. An example of why this does not give the proper result is the case where a marker has zero impact on the test. In this case, the weight it is given by the search program can be any value, so it is possible that its weight will be the highest. [0300] In certain cases, the lower weighting coefficients may not be indicative of a low importance. Similarly, a higher weighting coefficient may not be indicative of a high importance. For example, the optimization process may result in a high coefficient if the associated marker is irrelevant to the diagnosis. In this instance, there may not be any advantage that will drive the coefficient lower. Varying this coefficient may not affect the value of the objective function. [0301] Panel response values themselves may also be used as markers in the methods described herein. For example, a panel may be constructed from a plurality of markers, and . -ach marker oflhe panel may be described by a function and a weighting factor to h^ applied to that marker (as determined by the methods described above). Each individual marker level is determined for a sample to be tested, and that level is applied to the predetermined function and weighting factor for that paracular marker to arrive at a sample value for that marker. The sample values for each marker are added together to arrive at the panel response for that particular sample to be tested. Foe a "diseased" and "non-diseased" group of patients, the resulting panel responses may be treated as if they were just levels of. another disease marker. [0302] One cound use such £ method to define new "markers" based on panel responses, and even to determine a "panel response of panel responses." For example, one may divide ACS and non-ACS subjects as follows: (1) ACS + adverse outcome; (2) ACS - adverse outcome; (3) normals. One would define a first panel constricted from a plurality of markers as described above, and obtain the panel responses from this first panel for all the subjects. Then, the members of any one of these 3 groups may be compared to the panel responses of the members of any other of these groups to define a function and weighting factor that best differsntvites these two groups b:ised on the panel responses. This can be repeated as all 3 groups are compared pairwise. Tie "markers" used to define a second panel might include anjr or all of the following as a ne.w "marker": (1) versus (2) as marker 1; (1) versus (3) as marker 2; (2) versus (3) as marker 3. [0303] Measures of test accuracy may be obtained as described in Fischer et al.. Intensive Care Med. 29: 1043-51, 2003; Zl'ou et al., Statistical Methods in Diagnostic Medicine, John Wik-y & Sons, 2002; and Motulsky, Intuitive Biostatistics, Oxford University Press, i995; anl other publications well known to those of skill in the art. and used to determine the effectiveness of a given marker or panel of markers. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, hazard ratios, and ROC curve areas. As discussed above, suitable tests may exhibit one or more of the following results on these various measures: [0304] A ROC curve area of greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about O.S5, and most preferably greater than itbout 0.9; [0305] a positive or negative likelihood ratio of at least about 1.1 or more or about 0.91 or Lss, more preferably at least about 1.25 or more or about 0.8 or less, still more preferably at least about 1.5 or more or about 0.67 or less, even more preferably at least about 2 or more or about 0.5 or less, and most preferably at least about 2.5 or more or about 0.4 or less; an odds ratio of at leas'- about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.3 J or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or mors or about 0.1 or less; and/or [0307] a hazard ratio of at least about 1.1 or more or about 0.91 or less, more preferably at least about 1.25 or more or about 0.8 or less, still more preferably at least about 1.5 or more or about 0.67 or less, even more preferably at least about 2 or more or about 0.5 or less, and mos preferably at least about 2.5 or more or about 0.4 or less. [0305] Measures of diagnostic accuracy such as those discussed above are often reported together with confidence intervals or p values. These may be calculated by methods well known in the art. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, Now York, 19S3. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, L"0309] Assay Measurement Strategies [0310] Numerous methods and devices are well known to the skilled artisan for the detection and analysis of the markers of the instant invention. With regard to polypeptides or proteins in patient test samples, immunoassay devices and methods are often used. See, e.g., U.S. Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615: 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. These devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors nnH optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Patents 5,631,171; and 5,955,377. each of which is hereby incorporated by reference in its entiiety, including all tables, figures and claims. One skilled in the art also recognizes that robotic instrumentation including but not limited to Beckman Access, Abbott AxSym, Roche ElecSys, Bade Behring Stratus systems are among the immunoassay analyzers that are capable of performing the immunoassays taught herein. [0311 ] Preferably the markers are analyzed using an immunoassay, although other methods are well known to those skilled in the art (for example, the measurement of marker RNA levels). The presence or amount of a marker is generally determined using antibodies specific for each marker and detecting specific binding. Any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and.(he like. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Direct labels include fluorescent or lummw,cj!Qt tags, metals, dyes, radionuclides, and the like, attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the Ike. [0312] The use of immobilized antibodies specific for the markers is also contemplated by the present invention. The antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatogri>ohic matrix particles, the surface of an assay place (such as rnicrotiter wells), pieces of a solM substrate material or membrane (such as plastic, nylon, paper), and the like. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. [0313 j The analysis of a plurality of markers may be carried out separately or simultaneously with one test sample. For separate or sequential assay of markers, suitable apparatuses include clinical laboratory analyzers such as the ElecSys (Roche), the AxSym (Abbott), the Access (Beckman), the ADVIA® CENTAUR® (Bayer) immunoassay systems, the NICHOLS ADVANTAGE® (Nichols Institute) immunoassay system, etc. Preferred, apparatuses or protein chips perform simultaneous assays of a plurality of markers on a single surface. Particularly useful physical formats comprise surfaces having a plurality of discrete, aclressable locations for the detection of a plurality of different analytes. Such formats include protein microarrays, or "protein chips" (see, e.g., Ng and Hag, J. CellMol. Med. 6: 329-340 (2002)) and certain capillary devices (see, e.g., U.S. Patent No. 6,019,944). In these embodiments, each discrete surface location may comprise antibodies to immobilize one or :nore analyte(s) (e.g., a marker) fir detection at each location. Surfaces may alternatively comprise one or more discrete panicles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, whine the microparticles comprise antibodies to immobilize one analyte (e.g., a marker) for detection. [0314] Several markers may be combined into one test for efficient processing of a multiple of samples. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, a1, successive time points) from the same individual. Such testing oi'?erial samples will allow the identification of changes in-marker levels over time. Increases or decreases in marker levels, as well as the absence of change in marker levels, would provide useful information about the disease status that includes, but is not limited to identifying the approximate time from onset of the event, the presence and amount of salvagable tissue, the appropriateness of drug therapies, the effectiveness of various therapies as indicated by reperfusion or rerolution of symptoms, differentiation of the various types of NT'S, identification of the severity of the event, identification of the disease severity ajici identification of the patient's outcome, including risk of future events. [0315] A panel consisting of the markers referenced above may be constructed to provide relevant information related to differential diagnosis and/or prognosis. Such a panel may be constucted using U 2,3,4, 5,6,7, 8, 9, 10, 15,20, or more or individual markers. The analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out by one skilled in die art to optimize clinical sensitivity or specificity in various clinical settings. These include, but are not U mi ted to ambulatory, urgent care, critical care, intensive care, monitoring unit, i ipatient, outpatient, physician office, medical clinic, and health screening settings. Furthermore, one skilled in the art can use a single marker or a subset of markers comprising a larger panel of markers in combination with an adjustment of the diagnostic threshold in each of the aforementioned settings to optimize clinical sensitivity and specificity. The clinical sensitivity of-an assay is defined as the percentage of those with the disease that the assay correctly predicts, and the specificity of an assay is defined as the percentage of those without the disease that the assay correctly predicts (Tietz Textbook of Clinical Chemistry, 2nd edition, Carl Burtis and Edward Ashwood eds., W.B. Saunders and Company, p. 496). [0316] The analysis of markers could be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings. [03 1 7] In another embodiro ent, the present invention provides a kit for the analysis of Such a kit preferably comprises devises and reagents for the analysis of at least one test sample and instructions for performing the assay. Optionally the kits may contain one or more means for using information obtained from immunoassays performed for a marker panel to rule in or out certain diagnoses. [ 0318] Selection of Antibodies [0319] The generation and selection of antibodies may be accomplished several ways. For example, one way is to purify polypeptides of interest or to synthesize the polypeptides of interest using, e.g... solid phase peptide synthesis methods well known in the art. See, e.g.. Guide to Protein Purification, Murray?. Deutcher, ed., Meth. Enzymol. Vol 182 (1990); Solid Phase Peptide Synthesis, Greg B. Fields ed., Meth. Enzymol. Vol 289 (1997); Kiso et al., Chem. Pharm. Bull. (Tokyo) 38: U92-99, 1990; Mostafkvi etal., Biomed. Pept. Proteins Nucleic Acids 1: 255-60, 1995; Fujiwara et al, Chem. Pharm. Bull. (Tokyo) 44: 1326-31, 1996. The selected polypeptides may then be injected, for example, into mice or rabbits, to generate polyclonal or monoclon il antibodies. One skilled in the art will recognize that many procedures arc available for the production of antibodies, for example, as described in Antibodies, A Laboratory Manua1, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Karbor, N.Y. One skilled in the art will also appreciate that binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (Antibody Engineering: A Practical Approach (Borrebaeck, C., ed.), 1995, Oxfoid University Press, Oxford; J. Immunol. 149, 3914-3920 (1992)). [ 03 20] In addition, numerous |. association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physic,'Association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See. e.g., U 3. Patent No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims. [0321 ] The antibodies that arc generated by these methods may then-be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mous-j antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized poh peptide(s) are present. ['0322] The antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody. [0323] Those skilled in the art will recognize that man}' approaches can be taken in producing antibodies or binding fragments and screening and selecting for affinity and specificity for the various polypeptides, but these approaches do not change the scope of the invent > [0324] Selecting a Treatment Regimen [0325] The appropriate treat nents for various types of vascular disease may be large and diverse. However, once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with he diagnosis. Accordingly, the present invention provides methods of early differential diagnosis to allow for appropriate intervention in acute time windows. The skilled artisan is aware of appropriate treatnuzits for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, NJ, 1999. [0326] The following provides a brief discussion of additional exemplary markers for use in identifying suitable marker panels by the methods described herein. [0327] Examples [0328] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention. [0329] Example 1. Blc od Sampling [0330] Blood specimens were collected by trained study personnel using EDTA as the anticoagulant and centrifuged for greater than or equal to 10 minutes. The plasma component was transferred into a sterile crycvial and frozen at -20° C or colder. Specimens from the following population of patients and normal healthy donors were collected (Table 1). Clinical histories were available for each c r the patients to aid in the statistical analysis of the assay data. [0331 ] Example 2. Biochemical Analyses [0332] Markers were measured using standard immunoassay techniques. These techniques involved the use of antibodies to specifically bind the protein targets. A monoclonal antibody directed against a selected marker was biorinylated using N-hydrav"! J.'iccinimide biotin (NHS-biotin) at a ratio of about 5 NHS-biotin moieties per antibody. The antibody-biotin conjugate was then added to wells of a standard avidin 384 well microtiter plate, and antibody conjugate not bound to the plate was removed. This formed the "anti-marker" in the microtiter plate. Another monoclonal antibody directed against the same marker was conjugated to alkaline phosphatase using succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC) and //-succinimidyl 3-[2-pyridyldithiojpropionate (SPDP) (Pierce, Rockford, TL). [0333] Immunoassays were .jsrformed on a TECAN Genesis RSP 200/8 Workstation. Biotinylated antibodies were pipetted into microtiter plate wells previously coated'with avidin and incubated for 60 min. The solution containing unbound antibody was removed, and the wells were washed with a wash rmffer, consisting of 20 mM borate (pH 7.42) containing 150 mM NaCl, 0.1% sodium azide, a^d 0.02% Tween-20. The plasma samples (10 uL) were pipeted into the microtiter plate wells, and incubated for 60 min. The sample was then removed and the wells were .wash3d with a wash buffer. The antibody- alkaline phosphatase conjugate was then added to the wells and incubated for an additional 60 min, after which time, the antibody conjugate was removed and the wells were washed with a wash buffer. A substrate, (AttoPhos®, Promega, Madison, WI) was added to the wells, and the rate of formation of the fluorescent product was related to the concentration of the marker in the patient samples. [0334] . Example 3. Dyspnea Analysis [0335] The following table compares levels of pulmonary surfactant protein D ("SP-D"), D-dimer, BNP, total cardiac trope nin I ("Tnl"), and the ratio of BNP:D-dimer ("Ratio") in individual patients presenting with clinical dyspnea and in normal subjects. Dyspnea patients were subdivided into patients receiving a clinical diagnosis of congestive heart failure ("CHF"), and those receiving a conical diagnosis of pulmonary embolism ("PE"). All units are ng/ml except BNP (pg/ml) and ratios. (Table Removed) [03 36] These data indicate that the median D-dimer levels in the patients diagnosed with pulmonary embolism is higher than for the CHF patients, which is itself higher than normal subjects. Pulmonary surfactant protein D levels appears to be elevated over normals to nearly the same extent in both disease groups compared to normals. Using 3.4 as the rule-out cutoff would again result in one false negative diagnosis, but would correctly rule out 25 of the 35 CHF patients. The low cardiac troponin I level in all disease and normal subjects correctly rules out the occurrence of myocardial infarction in the entire test population. This example demonstrates that the differential diagnosis of causes of dyspnea can be accomplished through the measurement of d-dimer, BNP and cardiac troponin. Additionally, pulmonary embolism can be ruled in when BNP, d-dimer and pulmonary surfactant protein D levels are elevated above normal levels and tropomr. levels are normal. Pulmonary embolism can be ruled out when d dimer levels are in the normal range. When BNP levels are above normal, one can rule in congestive heart failure. When cardiac troponin levels are above normal, either cardiac ischemia or necrosis can be ruled in. ['0337] Example 4. Identification of diastolic dysfunction [0338] The following table compares levels of BNP, vasopressin, endothelin-2, calcitonin gene rel-ated peptide, urotensin 2, ANP, angiotensin II, the ratios of BNP : CGRP, BNP : ANP, BNP : urotensin 2, and calcitonin in heart disease patients and normal subjects. The heart disease patients are subdivided according to the New York Heart Association classification of functional capacity and objective assessment. See, Nomenclature and Criteria for Diagnosis of Diseases of the Heart and Great Vessels. 9th ed. Boston, Mass: Little, Brown & Co; 1994, pp. 253-256. The classification is made as follows: (Table Removed) is of diastolic dysfunction, and exhibit an ejection fraction of > 50%. Low ejection fraction (EF) patients are those exhibiting an ejection fraction of • CT&bi systolic, rather than diastolic, dysfunction. All units are ng/ml except BNP (pg/ml) and ratios, and N is the number of subjects in each group(Table Removed) [0340] These data indicate that Urotensin-2 and ANP can distinguish diastolic dysfunction from systolic dysfltnction. In both cases, the levels are higher in systolic dysfunction than in diastolic dysfunction. Moreover, with the addition of BNP, the the ability to discriminate diastolic dysfunction from systolic dysfunction is enhanced, as elevation of both BNP and AN? appears to be indicative of systolic dysfunction while elevation of BNP with ANP at or below normal levels appears to be indicative of diastolic dysfunction. Urotensin 2 shows a similar pattern. CGRP contributes to the ability to distinguish diastolic from systolic dysfunction when expressed as a ratio with BNP where the ratio is greater in cases of systolic dysfunction relative to diastolic dysfunction. [0341 ] Hammer-Lercher discusses the significance, or lack thereof, of NT-proBNP levels in controls and in patients with diastolic dysfunction (Hammer-Lercher et al., Clin. Chim. Ada 310(2): 193-7 (2001). In a preferred embodiment, a panel consisting of BNP and NTproBNP can distinguish heart failure patients with diastolic dysfunction. When both NTproBNP and BNP are elevated above the cutoff, the patient has systolic dysfunction. When NTproBNP is not elevated, but BNP is elevated above the cutoff, this would signify that the patient suffers from diistolic dysfunction. [03421 Example 5. Marker Panels for Cardiac Differential Diagnosis [0343] Exemplary marker panels were selected initially comprising a marker related to blood pressure regulation and a plurality of markers related to myocardial injury in order to develop a panel for diagnosing and/or distinguishing congestive heart failure, acute coronary syndromes, and myocardial infarction, and for guiding therapy in response to the results of the assay. For this purpose, BNP, cardiac troponin I (free and complexed), creatine kinase-MB, and myoglobin were selected. Threshold levels for comparison of measured marker concentrations were established in this example using the upper end of normal values for CKMB (4.3 ng/mL), myoglobm (107 ng/mL) and troponin I (0.4 ng/mL). Elevation and/or Temporal changes in these three markers, coupled with chest pain for a period of at least 20 minutes is highly indicative of myocardial infarction. In addition, BNP concentrations in excess of 80 pg/mL BNP can provide additional risk stratification in these patients, as this level of BNP is related to increased rates of death, myocardial infarction, and congestive heart failure in comprarison to patients having a BNP level below this threshold. Moreover, even in subjects experiencing no clinical symptoms of disease, a BNP level in excess of 100 pg/mL is associated with a substantially higher incidence of congestive heart failure. Thus, this multimarker strategy can provide substantially more clinically relevant information than can individual markers. [0344] The addition of other markers to the multimarker panel can provide additional clinical information for both risk stratification and differential diagnosis. For example, D- dimer may be added to the par el as a marker of coagulation and hemostasis. As discussed above, the addition of D-dimer can permit the differentiation of pulmonary embolism and/or deep venous thrombosis from myocardial infarction and congestive heart failure, despite the fact that the subjects may present to the clinician with substantially similar symptoms. In this case, a threshold level of about about 1 ug/mL may be established. In addition, or in the alternative, to D-dimer, C-reactive protein, a relatively nonspecific indicator of inflammation, can provide additional risk stratification to the panel. While the data is not presented here, CK-MB and royoglobin can also provide for distinguishing ST-elevation and non-ST-elevation ACS. [0345] As the number of markers in a panel increases, the determination of a single panel response and its correlation to various disease states by the methods described herein can be advantageous. An example of such a panel may include specific markers of cardiac injury (e.g., cardiac troponin I ,md/or T (free and complexed), creatine kinase-MB, etc.), and non-specific markers of tissue injury (e.g., myoglobin), where none of the markers are compared to a predetermined threshold. Starting with a number of potential markers, an iterative procedure was applied, m this procedure, individual threshold concentrations for the markers were not used as cutoffs perse. Rather, a "window" of assay values between a minimum and maximum marker concentration was determined. Measured marker concentrations above the maximum are assigned a value of 1 and measured marker concentrations below the minimum are assigned a value of 0; measured marker concentrations within the window are linearly interpolated to a value of between 0 and 1. The value obtained for a given marker concentration was then multiplied by a weighting factor. The absolute values of the weights for all of the individual markers used in a panel add up to 1. A negative weight for a marker implies that the assay values for the control group are higher than those for the diseased group. A "panel response" is calculated by summing the weighted values tor each marker in the panel. The panel responses for the entire population of "disease group" and "controls" are subjected to ROC analysis, and a panel response threshold was selected to yield the desired sensitivity and specificity for the panel. After each set of iterations, the weakest contributors to the equation may be eliminated and the iterative process started again with the reduced number of markers. [0346] The following panels represent such marker panels identified for the ability to discriminate subjects suffering from acute myocardial infarction (labeled "Disease group") from "control" subjects. Panel 1 represents the results obtained from a "first draw" at clinical presentation, while Panels 2-4 represent the results obtained using 60,90, and ISO minute draws, respectively. Using the "gold standard" of cardiac troponin I alone, first, 60, 90, *nd 180 minute draws provide 25.9%, 28.7%, 55.6% and 100% specificity, respectively, atl>2.5% sensitivity. (Table Removed) [0347] In contrast to the results obtained from cardiac troponin I alone, the panels described above provide improved specificity at early time points. In these time points, myoglobin and CK-MB are included at an increased weight. Not surprisingly, at the final time point (where cardiac troponin I alone achieves 100% specificity, cardiac troponin I dominates the panel response. Additional panels may include additional markers as described herein, particularly ir.eluding markers related to blood pressure regulation (e.g., BMP), markers related to coagulation and hemostasis (e.g., D-dimer, TpP), markers related to apoptosis (e.g., caspase-3, c>tochrome c), and/or markers related to inflammation (e.g., MMP-9, CRP, myeloperoxidasrt, IL-lra, MCP-1). In addition, the change in one or more of the foregoing markers over tim« is preferably included as an additional marker in such panels. [0348] Using this same methodology, similar marker panels can be defined in order to distinguish acute myocardial infarction and mimic conditions such as non-cardiac chest pain and unstable angina. The following tables compare samples obtained from subjects suffering from these mimic conditions and samples obtained within 10 hours of presentation from subjects suffering from an acute myocardial infarction. (Table Removed) [0349] The following pane Is represent prognostic marker panels used to analyze test samples obtained from ACS pi .tients having an adverse event (death, acute myocardial infarction, congestive heart failure, labeled "Disease group11) within 30 days to a "control" group representing ACS patierts not having such an event. An odds ratio was calculated based on these results for the ability of each panel to predict such an adverse event. (Table Removed) [0350] The following panels consider death within 180 days alone as the adverse event. (Table Removed) [0351 ] And the following panels consider death within 90,180,365, and 740 days, respectively, as the adverse evrat. (Table Removed) [0352] The skilled artisan will understand that additional markers may be included or substituted into the foregoing panels. Additional markers may include single concentrations of markers, or may include a rr£rker "slope" (i.e., relative changes in markers over time, ratios of two markers, etc. The skilled artisan will also understand that the same panel may provide both diagnostic and prognostic information. The markers used for diagnosis may be the same as those used for prognosis, or may differ in that one or more markers used for one of these purposes may not be used for the other purpose. [03 S'jj Example 6. Diagnosis of Subclinical Atherosclerosis Usint [0354] MCP-1 has been identified as an independent risk predictor in ACS. See, e.g., de Lemos et al., Circulation 107: 690-95 (2003), which is hereby incorporated by reference in its entirety. The following data demonstrates the use of MCP-1 in the diagnosis of subclinical atherosclerosis. Baseline MCP-1 levels were measured in 3499 patients not exhibiting symptoms of atherosclerosis (based on clinical presentation). A subset of 2733 patients was given electron beam computerized tomography (EBCT) scans. EBCT is an imaging procedure that uses a CT scanner to measure the amount of calcium found in the arteries of the heart. Subclinical coronary artery disease can be detected without the need of surgery or the injection of tracking fluids by measuring coronary artery calcium ("cac"). See. e.g., Khaleeli et al, Am. Heart J. 141: 637-44, 2001. [0355] Distribution of MCP-1 among the 3499 patients from whom it was measured: (Table Removed) MCP-1 Levels and Cardiovascular Risk Factors MCP-1 Quartiles (Table Removed) These associations are among nil 3499 patients. LDL: Each quartile contains about 875 patients; HTN: 1060 patients had hypertension; smoking: 1013 patients were current smokers; family hx: 1139 patients had a family h/o cad; DM: 402 patients had DM. Associations (not shown) between baseline variables and MCP-1 levels were also performed among the subset of patients that had EBCT scans (n=2733). [0357] Figure 2 shows the association of MCP-1 to subclinical atherosclerosis in 2733 patients who had an EBCT scan. Of these, 581 patients had evidence of subclinical atherosclerosis defined as a coronary calcification score ^10. Additional evidence suggests a significant association between the degree of cac (categorical) and MCP-1 levels (continuous). • [0358] Relative Risk for Subclinical Atherosclerosis (CAC>10) in Multivariate Analysis (Excluding Age) (Table Removed) [0359] Multivariate Model for Subclinical Atherosclerosis stratified by intermediate or highest age fertile (>= 40yeais; n=1831) (Table Removed) [0360] In the case of acute myocardial infarction, panels, window values, .and weighting factors are selected that, using a panel response value, preferably provide a sensitivity of at least 80% at greater than 90% specificity. [0361] Example 7. sis for Cerebrovi . Diagnosis [0362] In the case of cerebrovascular differential diagnosis, marker panels were selected comprising a marker related to blood pressure regulation and a plurality of markers related to neural tissue injury in order to develop a panel for diagnosing and/or distinguishing stroke from patients referred tc herein as "stroke mimics." Additional classes of markers tested to increase marker panel response include markers of apoptosis, markers of inflammation, and/or acute phase reactants. A final exemplary panel was identified that provided a sensitivity of at leas' 80% at greater than 90% specificity. Starting with a number of potential markers, an iterative procedure was applied. In this procedure, individual threshold concentrations for the markers were not used as cutoffs per se. Rather, a 'window" of assay values between a minimum and maximum marker concentration was determined. Measured marker concentrations above the maximum are assigned a value of 1 and measured marker concentrations below the minimum are assigned a value, of 0; measured marker concentrations within the window are linearly interpolated to a value of between 0 and 1. The value obtained for a given marker concentration was then multiplied by a weighting factor. The absolute values of the weights for all of the individual markers used in a panel add up to 1. A negative weight for a marker implies that the assay values for the control group are higher than those for the diseased group. Again, none of the markers are compared to a predetermineri threshold. Instead, a "panel response" is calculated by summing the weighted values for each marker in the panel. The panel responses for the entire population of "disease group" and "controls" are subjected to ROC analysis, and a panel response threshold was selected to yield the desired sensitivity and specificity for the pane*. After each set of iterations, the weakest contributors to the equation may be eliminated and the iterative process started again with the reduced number of markers. (Table Removed) [0363] In addition, panels were assessed for the ability to identify severity of neurologic deficit in stroke patients. In these panels, controls were subjects exhibiting an NIH stroke scale ("NIHSS") score of 5. As shown in the following table, simply changing the panel parameters (e.g., the width and position of the window and/or weighting) while using the same eight markers described above for stroke diagnosis, can provide important information about the severity of neurologic deficit. (Table Removed) [0364] Interestingly, MMP--9 shows a negative correlation with neurologic deficit, indicating that, while MMP-9 i.; increased in stroke patients relative to mimics, MMP-9 is actually decreased in the case of stroke patients exhibiting an increased neurologic deficit, relative to subjects with less severe neurologic deficit. High MMP-9 may be indicative of increased revascularization, and therefore may be a marker of positive prognosis in stroke patients. In addition, thrombolytic treatment may be less advantageous in stroke patients with high MMP-9, as revascularization is providing additional perfusion of the lesion. Such panels may provide prognostic information in diseases and procedures that are associated with a risk of neurologic deficit Such procedures include carotid endarterectomy, hypothermia circulatory arrest, aortic valve replacement, mitral valve replacement, coronary artery surgery, endograft repair of aortic aneurism, coronary artery bypass graft surgery, laryngeal mask insertion, and repair of congenital heart defects. [0365] Additional panels may be provided that utilize fewer markers, with no to moderate loss of sensitivity and specificity, as shown in the following tables: (Table Removed) [0366] Panels defined in accordance with the foregoing principles may be selected to differentiate subjects suffering from stroke (ichemic and/or hemorrhagic) from age-matched normal subjects. Such panels r;an be used to identify those subjects in a stroke mimic population that suffer from aciite ischemia that does not rise to the level of a diagnosis of stroke. For example, ausing sush panels to screen a mimic population (e.g., subjects suffering from TIA, syncope, peripheral vascular disease, etc.), can identify a subpopulation exhibiting a panel response thet could be considered a "false positive" stroke diagnosis. This subpopulation may be suffering from a significant stroke-like episode, but because of the location of the lesion, may not be exhibiting a sufficient neurologic deficit to fall within the clinical diagnosis of stroke. Such subjects may benefit from more aggressive treatment than mimic subjects who appeal "normal" according to the panel response. This mimic population is referred to herein as suffering from "subclirtcal stroke" or "subclinical ischemia," and the methods def-Bribed herein can be used for the diagnosis and/or prognosis of such subclinical conditions. [0367] Examule 8. Diagnosis of Stroke [0368] A panel that includes any combination of the above-referenced markers may be constructed to provide relevant information regarding the diagnosis of stroke and management of patients with stoke and TIAs. In addition, a subset of markers from this larger panel may be used to optimize sensitivity and specificity for stroke and various aspects of the disease. The example presented here describes the statistical analysis of data generated from immunoassays specific for BNP, IL-6, S-100p\ MMP-9, TAT complex, and the Al and integrin domains of vWF (vWF Al-integrin) used as a 6-maiker panel The thresnolds used for these assays are 55 pg/ml for BNP, 27 pg/ml for IL-6,12 pg/ml for S-lOOp. 200 ng/ml for MMP-9. 63 ng/ml for TAT complex, and 1200 ng/ml for vWF Al-integrin. A statistical analysis of clinical sensitivity and specificity was performed using these thresholds in order to determine efficacy of the marker panel in identifying patients with ischemic stroke, subaraconoid hemorrhage, intracerebral hemorrhage, all hemorrhagic strokes (intracranial hemorrhage), all stroke types, and TIAs. Furthermore, the effectiveness of the marker panel was compared to a current diagnostic method, computed tomography (CT) scan, through an analysis of clinical sensitivity and specificity. [0369] The computed tomography (CT) scan is often used in the diagnosis of stroke. Because imaging is performed on the brain, CT scan is highly specific for stroke. The sensitivity of CT scan is very high in patients with hemorrhagic stroke early after onset. In contrast, the sensitivity of CT scan in the early hours following ischemic stroke is low, with approximately one-third of patients having negative CT scans on admission. Furthermore, 50% patients may have negative CT scans within the first 24 hours after onset. The data presented here indicates that the sensitivity of CT scan at admission for 24 patients was consistent with the expectation that only one-third of patients with ischemic stroke have positive CT scans. Use of the -5 -marker panel, where a patient is positively identified as having a stroke if at least two markers are elevated, yielded a sensitivity of 79%, nearly 2.5 times higher than CT scan, will high specificity (92%). The specificity of CT scan in the study population is assumed to be close to 100%. One limitation of this assumption is that CT scans were not obtained from individuals comprising the normal population. Therefore, the specificity of CT scan in th allow the early identification of patients experiencing ischemic stroke with high specificity and higher sensitivity than CT scan. (Table Removed) [03 70] The sensitivity and specificity of the 6-marker panel was evaluated in the context of ischemic stroke, subarachnoid hemorrhage, intracerebral hemorrhage, all hemorrhagic stroke (intracranial hemorrhage), and all stroke types combined at various times from onset. The specificity of the 6-markei; panel was set to 92%, and patients were classified as having the disease if two markers were elevated. In addition, a 4-marker panel, consisting of BNP, S-100p, MMP-9 and vWF Al-iitegrin was evaluated in the same context as the 6-marker panel, with specificity set to 9 7% using the same threshold levels. The 4-marker panel is used as a model for selecting a subset of markers from a l:irger panel of markers in order to improve sensitivity or specificity for the disease, as described earlier. The data presented in Tables 3-7 indicate that both panels are useful in the diagnosis of all stroke types, especially at early times form onset. Use of the 4-marker panel provides higher specificity than the 6-marker panel, with equivalent sensitivities for hemorrhagic strokes within the first 48 hours from onset. The 6-marker panel demonstrates higher sensitivity for ischemic stroke at all time points than the 4-marker panel, indicating that the 6-marker approach is useful to attain high sensitivity (i.e. less false negatives), and the 4-marker panel is useful to attain high specificity (i.e. less false positives). Sensitivity Analysis — Ischemic; Stroke (Table Removed) Sensitivity Analysis - Subarachnoid Hemorrhage (Table Removed) Sensitivity Analysis - Intracerebral Hemorrhage (Table Removed) Sensitivity Analysis - All Hemorrhagic Stroke (Table Removed) Sensitivity Analysis - All Stroke (Table Removed) [0371 ] The 6-marker and 4-marker panels were also evaluated for their ability to identify patients with transient is^hemic attacks (TIAs). By nature, TIAs are ischemic events with short duration that do not cause permanent neurological damage. TIAs may be characterized by the localized release of markers into the bloodstream that is interrupted with the resolution of the event. Therefore, it is expected that the sensitivity of the panel of markers would decrease over time. Both the 6-marker panel, with specificity set to 92%, and*vhs 4-marker panel, with specificity set to 97%, exhibit significant decreases in sensitivity within the first 24 hours of the event, as described in Table 8. These decreases are not observed in any of the stroke populations described in Tables 3-7. The data indicate that the collection of data from patients at successive time points may allow the differentiation of patients with TIAs from patients with other stroke types. The identification of patients with TIAs is beneficial because these patients are at increased risk for a future stroke. Sensitivity Analysis - TIA (Table Removed) [0372] Example 9. Markers for cerebral vasosoasm in patients presenting with subarachnoid hemorrhage. [0373] 45 consecutive patients, 38 admitted to a hospital with aneurysmal subarachnoid hemorrhage (SAH), and 7 control patients admitted for elective aneurysm clipping, were included in this study. In all patients with SAH, venous blood samples were taken by venipuncture at time of hospital admission and daily thereafter for 12 consecutive days or until the onset of vasospasm. Development of cerebral vasospasm was defined as the onset of focal neurological deficits 4-12 days after SAH or transcranial doppler (TCD) velocities > 190 cm/s. In patients undergoing elective aneurysm clipping, 3 ±1 venous blood samples were taken per patient over the course of a median of 13 days after surgery. Collected blood was centrifuged (10,000g), and the resulting supernatant was immediately frozen at -70°C until analysis was completed. Measurements of vWF, VEGF, and MMP-9 were performed using immunometric enzyme irimunoassays. [0374] To determine if any changes hi plasma vWF, VEGF, and MMP-9 observed in a pre-vasospasm cohort were a result of pre-clinical ischemia or specific to the development of cerebral vasospasm, these markers were also measured in the setting of embolic or thrombotic focal cerebral ischemia. A single venous blood sample was taken by venipuncture at the time of admission from a consecutive series of 59 patients admitted within 24 hours of the onset of symptomatic focal ischemia. Forty-two patients admitted witf» symptomatic focal ischemia subsequently demonstrated MRI evidence of cerebral infarction. Seventeen patients did not demonstrate radiological evidence of cerebral infarction, experienced symptomatic resolution, were classified as transient ischemic attack, and therefore were not included in analysis. [0375] Three cohorts were classified as non-vasospasm (patients admitted with SAH and not developing cerebral vasospasm), pre-vasospasm (patients admitted with SAH and subsequently developing cerebral vasospasm), and focal ischemia (patients admitted with symptomatic focal ischemia subsequently defined as cerebral infarction on MRI). Mean peak plasma vWF, VEGF, and MMP-9 levels were compared between cohorts by two-way ANOVA. The alpha error was set at 0.05. When the distribution had kurtosis, significant skewing, or the variances were significantly different, the non-parametric Mann Whitney U statistic for inter-group comparison was used. Correlations between Fisher grade and plasma markers were assessed by the Spearman Rank correlation coefficient. Logistic regression analysis adjusting for patient age, gender, race, Hunt and Hess, and Fisher grade was used to calculate the odds ratio of developing vasospasm per threshold of plasma marker. [0376] Thirty eight patients were admitted and yielded their first blood sample 1 ±1 days after SAH. Of these, 22 (57%) developed cerebral vasospasm a median seven days (range, 4-11 days) after SAH. Eighteen (47%) developed focal neurological deficits and four (10%) demonstrated TCD evidence of vasospasm only. Three patients in the SAH, non-vasospasm cohort were Fisher grade 1 and were not included in inter-cohort plasma marker comparison. Patient demographics, clinical characteristics, and Fisher grades for the non-vasospasm and pre-vasospasm cohorts are given in the following table. Demographics, clinical presentation, and radiographical characteristics of 38 patients admitted with SAH. (Table Removed) [0377] In the non-vasospasm cohort, mean peak plasma vWF (p=0.974), VEGF (p=0.357), and MMP-9 (p=0.763) were unchanged versus controls (Table 10). Plasma vWF, VEGF, and MMP-9 v/ere increased in the pre-vasospasm versus non-vasospasm cp?£!t (Table 10). Increasing Fisher grade correlated to greater peak plasma vWF (p [0378] Additionally, twenty males and 22 females (age: 59 ± 15 years) presented within 24 hours of symptomatic focal ischemia with a mean NIH stroke scale score of 6.7 ± 6.6. In the focal ischemia cohort, mean peak plasma vWF (p=0.864), VEGF (p=0.469), md MMP-9 (p=0.623) were unchanged versus controls (Table 10). Plasma vWF, VEGF, and MMP-9 were markedly increased in the pre-vasospasm versus focal ischemia cohort, as shown in the following table. Mean peak plasma markers in the non-vasospasm, pre-vasospasm, and focal ischemia cohorts. Control group given as reference. (Table Removed) [0379] Following SAH, elevated plasma vWF, VEGF, and MMP-9 independently increased the odds of subsequent vasospasm 17 to 25 fold with positive predictive values ranging from 75% to 92%, as «hown in the following table. Positive/negative predictive values and odds ratio for subsequent onset of vasospasm associated with various levels of plasma vWF, VEGF, and MMP-9 by logistic regression analysis. (Table Removed) [0380] [0381 ] The following tables demonstrate the use of methods of the present invention for the diagnosis of stroke. T le "analytes panel" represents the combination of markers used to analyze test samples obtained from stroke patients and from non-stroke donors (NHD indicates normal healthy donor; NSD indicates non-specific disease donor). The time (if indicated) represents the interval between onset of symptoms and sample collection. ROC curves were calculated for the sensitivity of a particular panel of markers versus 1-(specificity) for the panel at various cutoffs, and the area under the curves determined. Sensitivity of the diagnosis (Sens) was determined at 92.5% specificity (Spec); and specificity of the diagnosis was also detennined at 92.5% sensitivity. 3-Marker Analyte Panel - Analytes: Caspase-3, MMP-9, GFAP. (Table Removed) 4-Marker Panel - Analytes: C aspase-3, MMP-9, vWF-Al and BNP. (Table Removed) 6-Marker Panels: Analytes as indicated. (Table Removed) 7-Marker Panel - Analytes: Caspase-3, NCAM, MCP-1, S100-P, MMP-9, vWF-integrin andBNP. (Table Removed) * - Recognizes all forms of MMP-9 * - Recognizes all forms of MMP-9 except active MMP-9 * - Recognizes all forms of MMP-9 excspt MMP-9/TIMP complexes 8-Marker Panel - Analytes: Caspase-3, NCAM, MCP-1, SlOO-p, MMP-9, vWF-Al, BNP and GFAP. (Table Removed) [0382] Additional stroke panels may be provided using 3,4, 5,6,7, 8, or more markers selected from the group consisting of IL-lra, C-reactive protein, von Willebrand factor (vWF), Tweak, creatine kinase-BB, c-Tau, D-dimer, thrombus precursor protein, vascular endothelial growth factor (VEGF), matrix metalloprotease-9 (MMP-9), neural cell adhesion molecule (NCAM), BNP, SI000, and caspase-3. The following exemplary panels are provided for thye diagnosi.*: of ischemic stroke, using normal healthy donor samples as a "control" group. [0383] (Table Removed) [0384] Example 11. Exemplary panels for differentiating ischemic stroke versus hemorrhagic stroke [0385] The following table demonstrates the use of methods of the present invention for the differentiation of different types of stroke, in this example ischemic stroke versus hemorrhagic stroke. The "anaJyte panel" represents the combination of markers used to analyze test samples obtained arom ischemic stroke patients and from hemorrhagic stroke patients. Sensitivity of the diagnosis (Sens) was determined at 92.5% specificity (Spec); and specificity of the diagnosis; was also determined at 92.5% sensitivity. , [0386] (Table Removed) [0387] Example 12. Hxemolarv panels for diagnosing acute stroke [0388] The primary endpo categories based on the latency from symptom onset to blood draw: less than six hours (16 samples), and 6-24 hours (38 samples). Control patients initially suspected of having a stroke but not meeting the clinical criteria served as controls. These 21 included'patients v^_ wit}. TIA (13 patients); syncope (n=l ), and other (n=7 ). The control group was enriched with patients without vascular disease (n= 157). [0389] Following obtaining informed consent, phlebotomy was performed and collected blood was centrifuged (10,000g), and the resulting supernatant immediately frozen at -70°C until analysis was completed as described previously (Grocott et al., 2001, McGirt et al., 2002). Measurements of biochemical markers were performed by Biosite Diagnostics (San Diego, CA) using a Genesis Robotic Sample Processor 200/8 (Tecan; Research Triangle park, NC).. Vll assays were performed in a 10-uL reaction volume in 384-well microplates, with the amount of bound antigen detected by means of alkaline phosphatase-conjugated secondary antibodies and AttoPhos substrate (JBL Scientific, San Luis Obispo, CA). [0390] Descriptive statistic s, including frequencies and percentages for categorical data, as well as the mean and standard deviation, median, 1st and 3rd quartiles, and the minimum and maximum value: for continuous variables, were calculated for all demographic and sample assay data. Demographic variables were compared by Wilcoxon test (age) or Chi-Squared test fr categorical variables. Distributions of marker values were examined for outliers and non-normality. The ability to distinguish stroke by marker levels at a given sample period was tested in stages in this exploratory study in order to minimize overtesting. First, each marker was tested as the single predictor in a univariate logistic regression. Based on these results, on the clinical characteristics of the markers, and on correlation with other markers, a set of 3 markers was selected for testing in a multivariable logistic model. Non-significant markers were removed from this model and up to 2 more markers were tested additionall / to arrive at a final model providing the greatest stability of estimates and predictive utility. Correlations among the included markers were checked to avoid collinearity, and influence statistics (change in Chi-Square) were examined to guard against undue influence of any one observation. Finally the validity of the model was checked by bootstrapping. Fifty test datasets of the same size as the analysis dataset were generated by random selection with replacement from the analysis dataset. Then the model was fit on each "bootstrapped" dataset, and the results inspected for consistency. In this manner separate models were developed for two time periods of marker sampling at which sufficient numbers of stroke samples were available, 0-6 hours and 6-24 hours. Multiple samples from the same patient were not used in the same analysis, preserving in dependence in each analysis. Where multiple samples were available from the same patient within the same time period, only the sample closest to the start of the time period was used in the analysis. To -nvestigate the association of time after onset of symptoms with the level of serum markers, a dataset was prepared including all samples from 0-24 hours after onset for all patients with stroke. The time association was initially inspected for each marker using a Spearman rank correlation; correlations with p [0391 ] The patient demographics from the acute (0-6 hours from symptom onset to blood collection), and subacute (6-24 hours from symptom onset to blood collection were comparable. Male patients wtre less likely to be diagnosed with clinical stroke in both data sets, whereas prior history of myocardial infarct and African American race were associated with increased incidence of stroke. Patient demographics for the data set in which blood was collected acutely (within six hours of symptom onset), and subacutely (between six and twenty four hours after symptom onset. There was no significant difference in age between patients with clinical stroke and patients without stroke in either data set (age expressed as mean ± standard deviation). There was an increased proportion of male patients in both subacute and acute patients without stroke. An increased proportion of stroke patients in both data sets were African American, and had a prior incidence of myocardial infarction. (Table Removed) [0392] Twenty six biochemical markers involved in pathogenesis of stroke and neuronal injury were prospectively defined and divided into one of six categories: markers of gttal activation, non-specif c mediators of inflammation; markers of thrombosis or impaired hemostasis, markers of cellular injury; markers of peroxidized lipid/myelin breakdown; markers of apoptosis/ miscellaneous. The univariate logistic analysis demonstrated four markers that were highly correlated with stroke (pO.OOl) at both time periods. These included one marker of glial activation (S100P), two markers of inflammation (vascular cell adhesion molecule, IL-6), and Won Willebrand factor (vWF). In addition, several markers were differentially upregulated as a function of time. Specifically, caspase 3, a marker of apoptosis, increased as a function of time (over a 24 hour period from symptom onset to blood draw), suggesting an increasing volume of irreversibly damaged tissue. [0393] Two data sets were created representing serum collected from patients that presented acutely (blood drawn within six hours) and subacute stroke (blood drawn between six and twenty four hours). Markers of glial activation and inflammation were assayed in the blood of patients presenting with suspected cerebral ischemia, and univariate logistic regression performed for each marker. Given the non-normal distribution of many of the assays, data is presented as median ± interquartile range; signifacance represents unadjusted p value from each univariate logistic model. P>0.05 is assumed to be nonsignificant (NS). (Table Removed) [0394] Two data sets were created representing serum collected from patients that presented acutely (blood drawn within six hours) and subacute stroke (blood drawn between six and twenty four hours). Markers of acute cerebral ischemia, including apoptosis, myelin breakdown and peroxidation, thrombosis, and cellular were assayed in the blood of patients presenting with suspected cerebral ischemia, and univariate logistic regression performed for each marker. Given the non-normal distribution of many of the assays, data is presented as me dian ± interquartile range; sigmfacance represents unadjusted p value from each anivariate logistic model. P>0.05 is assumed to be nonsignificant (NS). (Table Removed) [0396] To maximize the se: isitivity and sensitivity of a diagnostic test utilizing these markers, we next created a thres variable panel of stroke biomarkers using multivariable logistic regression as described above. For acute patients (time from symptom onset to blood draw less than or equa to six hours), sensitivity and specificity was optimized using the variables of MMP9, vWf, and VCAM; wherein the concentration of a marker is direu-tiy related to a predicted probability of stoke. Each of these variables contributed to the model significantly and independently (Table 21). The overall model Likelihood ratio chi-square for this logistic model was 71.4 (p 94%. MMP-9 was significant (p [0397] Confidence interval for odds ratios, in units of 1 standard deviation of predictor. A logistic regression model was created from the data set of all patients in which blood was drawn within six hours from symptom onset. The odds ratio for each of the three covariates (MMP9, vWF, and VCAM) is presented per unit of one standard deviation. (Table Removed) [0398] In similar fashion, a logistic regression model was developed for patients with snbacute symptoms (6-24 noun: elapsed from symptom onset to blood draw). For this time period, sensitivity and specificity was optimized using the variables of SlOOb, VCAM, and vWFal. Each of which contributed to the model significantly and independently (Table 22). The overall model Likelihood ratio chi-square for this logistic model was 95.1 (p concordance indexes >89%. SlOOb was significant (pO.OS) in 47 samples out of 50, VCAM in 45/50, and vWFal in 49/50. Confidence inten al for odds ratios, in units of 1 standard deviation of predictor. A logistic regression model was created from the data set of all patients in which blood was drawn between six and twenty four hours from symptom onset. The odds ratio for each of the three covariates (SlOOp, vWF, and VCAM) is presented per unit of one standard deviation. (Table Removed) [0400] Example 13. Exemplary panels for differentialing between acute a"d non-acute stroke [0401] Using the methods described in U.S. Patent Application No. 10/331,127, entitled METHOD AND SYSTEM FOR DISEASE DETECTION USING MARKER COMBINATIONS (attorney -locket no. 071949-6802), filed December 27,2002, exemplary panels for differentiating between acute and non-acute stroke was identified. Starting with a large number of potential markers (e.g., 19 different markers) an iterative procedure was applied. In this procedure, individual threshold concentrations for the markers are not used as cutoffs per se, but are used as values to which the assay values for each patient are compared and normalized. A window factor was used to calculate the minimum and maximum valut%; above and below the cutoff. Assay values above the maximum are set to the maxinum and assay values below the minimum are set to the minimum. The absolute values of the weights for the individual markers adds up to 1. A negative weight for a marker implies that the assay values for the control group are higher than those for the diseased group. A "panel response" is calculated using the cutoff, window, and weighting factors. The panel responses for the entire population of patients and controls are subjected to ROC analysis and a panel response cutoff is selected to yield the desired sensitivity and specificity for the panel. After each set of iterations, the weakest contributors to the equation art: eliminated and the iterative process starts again with the reduced number of markers. This process is continued until a minimum number of markers that will still result in acceptable sensitivity and specificity of the panel is obtained. [0402] The panel composition for identifying acute stroke (0-12 hours) comprised the following markers: BNP, GFAP, IL-8,0-NGF, vWF-Al, and CRP, while the panel composition for identifying -ion-acute stroke (12-24 hours) comprised the following markers: BNP, GFAP, IL-8. CK-BB, MCP-1, and IL-lra. A positive result was identified as being at least 90% sensitivity at 94.4% specificity. As shown below, the markers employed can provide panels to identify acute stroke, identify non-acute stroke, and/or differentiate between acute and non-acute stroke. [0403] 0-12 hour panel results (Table Removed) [0404] 12-24 hour panel results (Table Removed) [0405] Alternative exemplary panels for differentiating between a 0-6 time of stroke onset and post-6 hour stroke onset were also identified. The panel composition for identifying acute stroke (0-6 hours) comprised the following markers: BNP, GFAP, CRP, [0406] 0-6 hour panel results (Table Removed) [0408] Example 14. Markers and marker panels for predicting cerebral vasosoasm after subarracbnoid hemorrhage [0409] Delayed ischemic neurological deficits (DIND) tesulting from cerebral vasospasm is a major cause of morbidity and mortality following aneurysmal subarachnoid hemorrhage (SAH). Despite ntensive efforts to reveal its pathogenesis, the biological processes underlying DIND remains unclear. To identify exemplary markers and marker panels predictive of cerebral vasospasm, daily blood samples were drawn 48 hours after symptom onset in 52 patients presenting with aneurismal subarrachnoid hemorrhage. 23 patients (45%) developed clinical cerebral vasospasm, and only blood samples drawn prior to onset of clinical manifestations of cerebral vasospasm were considered. Univariate logistic regression was performed using peak marker levels, and the most significant variables were entered into a multiple logistic regression model. [041 1] The final logistic modsl included VEGF (p=0.002), NCAM (p=0.004), and caspase-3 (p=0.009). with an overall p value of [0412] Recently, Sviri et al. (Stroke 31:118-122, 2000) identified a correlation between serum BNP levels and DIND. Sviri demonstrated a 6-fold elevation in serum BNP 7-9 days after SAH only in patients developing symptomatic cerebral vasospasm, whereas no elevation occurred in the serum BNP of patients without symptomatic vasospasm [18]. However, the temporal relationship between rising BNP and onset of DIND was not reported, raising the question as to whether serum BNP may precipitate DIND, serving as a predictive serum marker for impending DIND. [0413] Thus, in a second study, 40 consecutive patients admitted with aneurysmal SAH were enrolled. The patient's Clinical condition at admission was graded according to the Hunt and Hess classifications. The severity of SAH was classified from the initial CT appearance Diagnostic cerebral angiography was performed during the first 24 hours after admission. All patients underv ent craniotomy and aneurysm clipping week and at the onset of suspected DIND. The significance of differences for continuous variables was determined usrng Student's t-test. Non-parametric data were compared using the Mann Whitney test. Percentages were compared using the chi-squared test. Multivariate V... logistic regression analyses adjusting for Hunt and Hess grade and Fisher grade were used to assess the independent association between BNP and onset of DIND [0414] 16 (40%) patients developed symptomatic cerebral vasospasm after SAH. A >3-fold increase in admission serum BNP was associated with the onset of hyponatremia (p [0415] Increasing serum BNP levels were independently associated with hyponatremia, did not significantly increase until the first 24 hours after onset of DIND, and predicted 2-week GCS. Increasing BNP may exacerbate blood flow reduction due to cerebral vasospasm and serve as a marker to determine aggressiveness of diagnostic and therapeutic management. [0416] While the invention has been described and exemplified hi sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. [0417] Example 15. Marker^ and marker panels for distinguish'" p intracram'al [0418] The early management of acute ischemic stoke involves excluding the presence of intracranial hemorrhage (ICH). Blood was drawn from 113 patients who were diagnosed with either ischemic stroke or 1CH. All patients presented within 48 hours from onset of symptoms. The primary clinical outcome was the presence of ICH verified by CT or the clinical diagnosis of ischemic stroke, defined as focal neurological symptoms of vascular origin persisting for greater than 24 hours with consistent radiographic findings. Univariate logistic regression was performed on each variable and the most significant ones were e%u?red into a multiple legist c regression model. Collinearity was examined, and a final model with three variables was generated. [0419] 34 patients (30%) were diagnosed with ICH and 79 (70%) with ischemic stroke. The final logistic model included C-reactive protein (P = 0.0 1 3), vascular endothelial growth factor (P = 0.045), and BMP (P = 0.030), with an overall P value of [0420] Example 16. Market's and marker panels for predicting cerebral vasospasm after subarrachnoid hemorrhage [0421] Delayed ischemic neuiological deficits (DIND) resulting from cerebral vasospasm is a major cause of morbidity and mortality following aneurysmal subarachnoid hemorrhage (SAH). Despite intensive efforts to reveal its pathogenesis, the biological processes underlying DIND remains unclear. [0422] To identify exemplary markers and marker panels predictive of cerebral vasospasm, daily blood samples were drawn 48 hours after symptom onset in 52 patients presenting with aneurismal subarrachnoid hemorrhage. 23 patients (45%) developed clinical cerebral vasospasm, and only blood samples drawn prior to onset of clinical manifestations of cerebral vasospasm were considered. Univariate logistic regression was performed using peak marker levels, and the me st significant variables were entered into a multiple logistic regression model. [0423] The final logistic mode, included VEGF (p=0.002), NCAM (p=0.004), and caspase-3 (negative predictive value of 95%) and a specificity of 91% (positive predictive value of JO/ |£4/i4] Recently, Sviri et al. (Stroke 31:118-122,2000) identified a correlation between serum BNP levels and DIND. Sviri demonstrated a 6-fold elevation in serum BNP 7-9 days after SAH only in patients developing symptomatic cerebral vasospasm, whereas no elevation occurred in the serum BNP of patients without symptomatic vasospasm [18]. However, the temporal relationship between rising BNP and onset of DIND was not reported, raising the question as to whether serum BNP may precipitate DIND, serving as a predictive serum marker for impending DIND. [0425] Thus, in a second study, 40 consecutive patients admitted with aneurysmal SAH were enrolled. The patient's clinical condition at admission was graded according to the Hunt and Hess classifications. The severity of SAH was classified from the initial CT appearance Diagnostic cerebral angiography was performed during the first 24 hours after admission. All patients underwent craniotomy and aneurysm clipping [0426] 16 (40%) patients developed symptomatic cerebral vasospasm after SAH. A >3-fold increase in admission serum BNP was associated with the onset of hyponatremia (p grade (OR, 1 .28; 95%CI, 1 . 1 -1 .6). In patients developing vasospasm, mean serum BNP increased 5.4-fold within 24, hours after vasospasm onset, and 11.2-fold the first 3 days after vasospasm onset. Patients with increasing BNP levels from admission demonstrated . ' no chaage (0 +/-3) in Glascow Coma Score (GCS) two weeks after SAH versus a 3.0 +/-2 (p [0427] Increasing serum BNP levels were independently associated with hyponatremia, did not significantly increase until the first 24 hours after onset of DIND, and predicted 2-week GCS. Increasing BNP may exacerbate blood flow reduction due to cerebral vasospasm and serve as a marker to determine aggressiveness of diagnostic and therapeutic management. I [0428] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. [04291 Example 17. Markers end marker panels hemorrhage from ischemic stroke [0430] The early management of acute ischemic stoke involves excluding the presence of intracranial hemorrhage (ICH). Blood was drawn from 113 patients who were diagnosed with either ischemic stroke or ICH. All patients presented within 48 hours from onset of symptoms. The primary clinical outcome was the presence of ICH verified by CT or the clinical diagnosis of ischemic stroke, defined as focal neurological symptoms of vascular origin persisting for greater than 24 hours with consistent radiographic findings. Univariate logistic regression was performed on each variable and the most significant ones were entered into a multiple logistic regression model. Collinearity was examined, and a final model with three variables was generated. [0431] 34 patients (30%) were diagnosed with ICH and 79 (70%) with ischemic stroke. The final logistic model included C-reactive protein (P = 0.0 1 3), vascular endothelial growth factor (P = 0.045), and BNP (P = 0.030), with an overall P value of panel of three biomarkers was able to rule out ICH with high sensitivity in patients presenting with stroke. Such a panel may prove useful as a point-of-care test to rule out ICH in patients with suspected ischemic stroke prior to therapeutic intervention. [0432] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. [0433] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. .The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims. [0434] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. [0435] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. [0436] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitatk n, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and. vacations are considered to; be within the scope of this invention asdefined by the appended claims. [043 7] Other embodiments are ret forth within the following claims. We Claim: 1. A kit for differential diagnosis comprising: a device containing a plurality of diagnostic zones on a test surface; one of said diagnostic zones having one or more antibodies that bind to B-type natriuretic peptide (BNP) or a marker related to BNP; one of said diagnostic zones having one or more antibodies that bind to at least one cardiac troponin form selected from the group consisting of free cardiac troponin I, free cardiac troponin T, cardiac troponin I in a complex with one or both of troponin T and troponin C, cardiac troponin T in a complex with one or both of troponin I and troponin C, total cardiac troponin I and total cardiac troponin T; and one of said diagnostic zones having one or more antibodies that bind to D-dimer; said plurality of antibodies being provided to enable assessment of ratio of concentration of said markers for differential diagnosis to determine presence or absence of congestive heart failure, myocardial infarction or pulmonary embolism. 2. A kit for differential diagnosis as claimed in claim 1, wherein said markers relate to B-type natriuretic peptide (BNP), NT-pro BNP or pro-BNP. 3. A kit for differential diagnosis as claimed in claim 1, wherein said markers are detected in a plurality of sandwich immunoassays. 4. A kit for differential diagnosis as claimed in claim 1, wherein said test surface consists of said antibodies immobilized on solid support. 5. A kit for differential diagnosis as claimed in claim 4, wherein said solid support is selected from the group consisting of magnetic or chromatographic matrix particles, the surface of an assay place, a solid substrate material or membrane. 6. A kit for differential diagnosis as claimed in claim 5, wherein said membrane is preferably plastic, nylon or paper. 7. A kit for differential diagnosis, substantially as hereinbefore described with reference to the foregoing examples.

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3018-delnp-2005-abstract.pdf

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