Means and methods for assessing the risk of patients presenting to emergency departments based on very low concentrations of troponin I or T or using a combination of markers

The present invention relates to a method of risk stratification in a subject. Preferably, the method according to the present invention permits identifying if a subject is to be admitted to the hospital or an intensive care unit or can be discharged to home. In most cases, the subject presents to the emergency department. The method is based on the determination of very low concentrations of troponin T or troponin I in a sample of the said subject. Also encompassed by the present invention are devices and kits for carrying out the aforementioned methods.

When subjects present to an emergency department with any kind of discomfort, a rapid diagnosis of the pathological state of the subject is mandatory in order to identify the cause underlying his discomfort and avoid consequences to the subject's health. A highly relevant topic is the decision if the patient will be admitted to the hospital - for further, time consuming analysis and/or intensive care treatment, or if the patient can be discharged to home.

For example, in the case of acute cardiovascular events, a decision for a certain treatment regimen must be made, usually, within a short period of time. Cardiovascular complications, particularly heart diseases, are the leading cause of morbidity and mortality in the Western hemisphere. Cardiovascular complications can remain asymptomatic for long periods of time. However, they may have severe consequences once an acute cardiovascular event, such as myocardial infarction, as a cause of the cardiovascular complication occurs. Therefore, guidelines exist for the rapid diagnosis of patients presenting to a physician, generally in an emergency station, and being suspected of suffering from an acute coronary syndrom ACS (i.e. unstable angina pectoris UAP or myocardial infarction MI), see J.Am.Coll. Cardiol. 2000; 36, pages 959-969. In a subject suspected to have MI, an electrocardiogram is recorded, and the level of Troponin T or Troponin I is determined. Further, it is analysed if the suspected subject shows evident syndroms like chest pain, palpitation, nausea, vomitting and further syndroms known to the person skilled in the art. If the subject is positive in 2 of the 3 criteriae, then he or she is admitted to the hospital for further examination.

However, the composition of the subjects presenting to an emergency station is heterogenous, comprising about 35% of subjects suffering from cardiovascular complications (including both acute, i.e. ischemic complications and non-ischemic complications), 10% of subjects suffering from pulmonary complications and 56% subjects suffering from other complications, e.g. tumors, and which may additionally suffer from cardiovascular complications. The conventional diagnostic techniques, specifically for emergency situations, usually do not allow for a reliable diagnosis and/or risk assessment covering these various pathological states. A further drawback is the often occuring lack of personnel and the varying occupancy in emergency stations.

At present, there does not exist a standardized diagnosis procedure covering the various dieseases a physicain may encounter in an emergency department, preferably in an medical emergency department. Thus, a rapid and acurate diagnosis allowing a decision if the subject can be discharged to home or has to be admitted to the hospital for further examination or intensive care treatment (which can be live saving) cannot be carried out in the emergency station with sufficient accuracy. As a consequence thereof, many patients will either be admitted or discahrged in to home where the opposite would have been the appropriate measure.

In some cases, so-called molecular markers permit to establish rapid and sufficiently accurate diagnosis of the pathological state of a subject. A prominent example is troponin T and/or troponin I for the diagnosis of MI, as mentioned beforehand, or natriuretic peptides, in particular BNP and NT-proBNP for various non-ischemic heart diseases, e.g. heart failure. Blood levels of cardiac troponins are sensitive and specific markers of myocardial injury that are routinely used for the diagnosis of acute myocardial infarction. With regard for the determination of troponin, as an example of an ischemic marker, reference is made to Katus et al. (MoI Cell Cardiol 1989; 21 :1349-53), Hamm et al. (NEJM 1992; 327: 146-50), Ohmann et al. (NEJM 1996; 335: 1333-34), Christenson et al. (ClinChem 1998; 44: 494-501), and to EP-A-O 394 819. Particularly preferred test for detection of troponin T are elektrochemiluminescence immunoassays and for detection of troponin I fluorescence polarization immunoassays, respectively, reference is made to

James et al. (ClinChem 2006; 52: 6832-37), and numerous other publications.

Troponin levels may also be elevated in some patients with heart failure, but blood levels are shown to be much lower compared with patients with acute myocardial infarction when currently available assays are used.

Recently, previously undetectable very low concentrations of troponin I measured by the second-generation Stratus CS® with reagents provided by Dade Behring suggest that troponin values below the 99th percentile could contain diagnostic information in patients with heart diseases. The limit of detection for this assay is 0.02 μg/L, the 99th percentile is 0.07 μg/L, and the lowest concentration with a CV <10% has been reported as both 0.06 μg/L and 0.10 μg/L, respectively. In addition, very low concentrations of troponin T measured by using a new high sensitive troponin T assay (hsTnT, Roche Diagnostics Elecsis Troponin T 5 ^th Generation) have been suggested to be an indicator for adverse outcomes in chronic heart failure (Latini et al. Circulation 2007; 116: 1242-49) In this issue of Circulation, Latini and colleagues report results of an investigation in which they measured circulating troponin T in 4053 patients with heart failure using a high-sensitivity troponin (hsTnT) assay. Whereas circulating troponins were detectable in 10% of the patients with heart failure when conventional assays were used, 90% of the patients had detectable troponin T levels using the hsTnT assay. The authors report that higher hsTnT was a marker of greater severity of heart failure, was associated with greater risk of adverse outcomes, and added incremental value to prediction models for death. Similar results were obtained by using new high sensitive troponin I fluorescence polarization immunoassays. These data demonstrate the prognostic utility in heart failure patients of measurement of very low circulating troponins at levels that are not detectable with conventional assays, emphasizing the potential importance of the high sensitive troponin assays.

Therefore, there is a need for diagnostic or prognostic measures which allow an assessment and/or an individual risk stratification for a subject presenting to the emergency department. The measures should permit this assessment/risk strtification also in patients not presenting with cardiovascualr diseases, in particular not with acute cardiovascular diseases.

The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs.

The technical problem is solved by the embodiments characterized in the claims and herein below.

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Accordingly, the present invention relates to a method of identifying if a subject is to be admitted to the hospital or to an intensive care unit, the method comprising

a) determining the amount of very low concentrations of troponin I or T in a sample of the subject; and b) comparing the amount of very low concentrations of troponin I or T determined in step a) to a reference amount, whereby a subject to be admitted to the hospital or to an intensive care unit is to be identified.

It has been surprisingly found that determining very low concentrations of troponin I or T alone and comparing it with a respective reference amount of troponin I or T provides a high predictive value for identifying if a subject is to be admitted to the hospital, for deciding about admitting a subject to the hospital, or for predicting the risk of mortality for a subject. This is even more surprising as a remarkably high positive predictive value is achieved independent and irrespective of a diagnosis and a disease status of the subject, even with subjects having no sign or apparent symptom of an acute cardiovascular event or a myocardial infarction.

Alternatively, the method according to the invention comprises the determination of at least three markers, whereby at least one neurohormonal marker, at least one ischemic cardiac marker, at least one marker of the transforming growth factor-β cytokine superfamily, and/or at least one inflammatory marker are determined, whereafter the determined amounts are compared to reference amounts of the determined markers, respectively. According to the present invention, it has been further found surprisingly that when at least three markers, selected from at least one neurohormonal marker, at least one ischemic cardiac marker, at least one marker of the transforming growth factor-β cytokine superfamily, and/or at least one inflammatory marker, are determined in the same or different sample of the subject, an even higher predictive value is achieved for identifying if a subject is to be admitted to the hospital, for deciding about admitting a subject to the hospital, or for predicting the risk of mortality for a subject. Again, such a remarkably high positive predictive value is achieved independent and irrespective of a diagnosis and a disease status of the subject, even with subjects having no sign or apparent symptom of an acute cardiovascular event or a myocardial infarction.

Preferably, the subject is a subject presenting to an emergency department, preferably in a hospital, even more preferably a medical emergency department.

In the context of the present invention, the term "emergency department" refers to any location where individuals feeling uncomfortable present, in order to consult a person having a medical background, preferably a physician, to have an analysis of their pathological state and the cause underlying their discomfort. Typical examples are emergency departments or emergency rooms in hospitals, emergency ambulances, doctor's offices and other institutions suitable for treatement of critical ill patients.

The method of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to sample pre-treatments or evaluation of the results obtained by the method. The method may be carried out manually or assisted by automation. Preferably, step (a) and/or (b) may in total or in part be assisted by automation, e.g., by a suitable robotic and sensory equipment for the determination in step (a) or a computer-implemented comparison in step (b).

The term "identifying" as used herein means assessing whether a subject is in a pathological state necessitating to admission to the hospital for further examination, and/or to an intensive care treatment, or intervention. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for all (i.e. 100%) of the subjects to be identified. The term, however, requires that a statistically significant portion of subjects can be identified (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p- value determination, Student's t-test, Mann- Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.

Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the subjects of a population can be properly identified by the method of the present invention.

The term "subject" as used herein relates to animals, preferably mammals, and, more preferably, humans.

However, it is envisaged in accordance with the aforementioned method of the present invention that the subject shall not be suffering from an acute cardiovascular event as defined by the American College of Cardiology (see above), e.g. chest discomfort, dyspnoea, ECG changes and others as described above. More preferably, the subject shall not exhibit one or more episodes of angina lasting at least 5 min within the preceding 24 h, and not have either a positive cardiac troponin I or T value when conventional assays are used or at least 0-5 mm of transient or persistent ST-segment depression not known to be preexisting and not attributable to coexisting disorders. Alternatively, the subject shall not exhibit symptoms of ischaemia that were increasing or occurring at rest, or that warranted the suspicion of acute myocardial infarction, with the last episode within the preceding 48 h. Myocardial ischaemia has to be verified by electrocardiography (ST depression =0 1 mV or T- wave inversion =0 1 mV) or by raised biochemical markers (creatine kinase [CK]-MB >6 ug/L, troponin-T >0.01 ng/ml, qualitative troponin I or T test positive, or catalytic activity of CK, CK-B, or CK MB higher than the local diagnostic limit for myocardial infarction).

Acute cardiovascular events are, preferably, acute coronary syndromes (ACS). ACS patients can show unstable angina pectoris (UAP) or myocardial infarction (MI). MI can be an ST-elevation MI (STEMI) or a non-ST-elevated MI (NSTEMI). The occurring of an ACS can be followed by a left ventricular dysfunction (LVD) and symptoms of heart failure.

The term "sample" refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ. Samples of body fluids can be obtained by well known techniques and include, preferably, samples of blood, plasma, serum, or urine, more preferably, samples of blood, plasma or serum. Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy. Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting. Preferably, cell-, tissue- or organ samples are obtained from those cells, tissues or organs which express or produce the peptides referred to herein.

The term "very low concentrations of troponin I or T" refers to concentrations of troponin I or T below the corresponding 99 ^th percentile cutoff established on the basis of apparently healthy reference populations for the commercial available troponin I and T methods.

Reference is made to "Joint European Society of Cardiology/American College of Cardiology Committee. Myocardial infarction redefined - a consensus document of the Joint: European Society of Cardiology/ American College of Cardiology Committee for the redefinition of myocardial infarction. J Am Coll Cardiol 2000; 36: 959-69.

It has been found that neurohormonal markers, ischemic cardiac markers, markers of the transforming growth factor-β cytokine superfamily, and inflammatory markers represents four groups of independent risk indicators, so that, combining at least three of these marker groups according to the invention, an additively increased sensitivity and specificity and, therefore, higher predictive values and higher diagnostic effectiveness result when it comes to identifying patients with elevated risk and/or unfavourable prognosis. In the case of the patients tested, this, in turn, makes it possible to establish an improved indication for suitable therapeutic measures.

The method according to the invention according to this preferred embodiment comprises the determination of at least three markers, whereby one neurohormonal marker, one ischemic cardiac marker, one marker of the transforming growth factor-β cytokine superfamily, and/or one inflammatory marker are determined.

The neurohormonal marker can be selected , for example, from atrial natriuretic peptide (ANP), brain (B-type) natriuretic peptide (BNP), or N-terminal fragments of the respective propeptides NT-proANP, proANP and NT-proBNP, ProBNP is preferably preferred as a neurohormonal marker.

Troponin T or troponin I, preferably very low concentrations of troponin I or T, for example can be determined as ischemic cardiac markers.

The inflammatory marker can be selected, for example, from C-reactive protein (CRP), interleukins, particular IL-6, and adhesion molecules such as VCAM and ICAM. CRP or IL-6 are preferably determined as the inflammatory marker.

The marker of the transforming growth factor-β cytokine superfamily can be selected, for example from TGF-β isoforms, preferably MIC-I or GDF-15 can be determined.

The combination determination according to the invention is preferably performed so that parallel determinations of the markers are performed in one or more samples from a patient

to be investigated. Preferably, one or more samples collected from the patient, e.g., whole blood samples, or plasma samples, or serum samples, are investigated in one or more tests simultaneously or immediately sequentially. The determinations are performed particularly preferably in a single patient sample. The combined determination of markers can be performed, in principle, based on any known method using common commercial assays. Automated analyzers can be used for the determination, for example. As an alternative, rapid assays, or assays of Point of Care (POC) analysis systems for use in the emergency room, in the hospital ward or intensive care station, in the ambulance or doctor's office, or as a patient self-test can also be used.

Determining the amount of troponin I or T or any other peptide or polypeptide (marker as used herein) referred to in this specification relates to measuring the amount or concentration, preferably semi-quantitatively or quantitatively. Measuring can be done directly or indirectly. Direct measuring relates to measuring the amount or concentration of the peptide or polypeptide based on a signal which is obtained from the peptide or polypeptide itself and the intensity of which directly correlates with the number of molecules of the peptide present in the sample. Such a signal - sometimes referred to herein as intensity signal -may be obtained, e.g., by measuring an intensity value of a specific physical or chemical property of the peptide or polypeptide. Indirect measuring includes measuring of a signal obtained from a secondary component (i.e. a component not being the peptide or polypeptide itself) or a biological read out system, e.g., measurable cellular responses, ligands, labels, or enzymatic reaction products.

In accordance with the present invention, determining the amount of a peptide or polypeptide (marker as used herein) can be achieved by all known means for determining the amount of a peptide in a sample. Said means comprise immunoassay devices and methods which may utilize labeled molecules in various sandwich, competition, or other assay formats. Said assays will develop a signal which is indicative for the presence or absence of the peptide or polypeptide. Moreover, the signal strength can, preferably, be correlated directly or indirectly (e.g. reverse- proportional) to the amount of polypeptide present in a sample. Further suitable methods comprise measuring a physical or chemical property specific for the peptide or polypeptide such as its precise molecular mass or NMR spectrum. Said methods comprise, preferably, biosensors, optical devices coupled to

immunoassays, biochips, analytical devices such as mass- spectrometers, NMR- analyzers, or chromatography devices. Further, methods include micro-plate ELISA-based methods, fully-automated or robotic immunoassays (AccuTnl, Beckmann-Coulter Diagnostics; Architect cTnl and AxSYM cTnl, Abbott Diagnostics; ADVIA Centaur XP cTNI, Bayer Healthcare; Immulite 2500 cTnl, Diagnostics Product Corporation; Elecsys cTnT, Roche Diagnostics), CBA (an enzymatic Cobalt Binding Assay, available for example on Roche- Hitachi analyzers), and latex agglutination assays (available for example on Roche- Hitachi ¹ ^ analyzers).

Preferably, determining the amount of a peptide or polypeptide (marker as used herein) comprises the steps of (a) contacting a cell capable of eliciting a cellular response the intensity of which is indicative of the amount of the peptide or polypeptide with the said peptide or polypeptide for an adequate period of time, (b) measuring the cellular response. For measuring cellular responses, the sample or processed sample is, preferably, added to a cell culture and an internal or external cellular response is measured. The cellular response may include the measurable expression of a reporter gene or the secretion of a substance, e.g. a peptide, polypeptide, or a small molecule. The expression or substance shall generate an intensity signal which correlates to the amount of the peptide or polypeptide.

Also preferably, determining the amount of a peptide or polypeptide (marker as used herein) comprises the step of measuring a specific intensity signal obtainable from the peptide or polypeptide in the sample. As described above, such a signal may be the signal intensity observed at an m/z variable specific for the peptide or polypeptide observed in mass spectra or a NMR spectrum specific for the peptide or polypeptide.

Determining the amount of a peptide or polypeptide (marker as used herein) may, preferably, comprises the steps of (a) contacting the peptide with a specific ligand, (b) (optionally) removing non-bound ligand, (c) measuring the amount of bound ligand. The bound ligand will generate an intensity signal. Binding according to the present invention includes both covalent and non-covalent binding. A ligand according to the present invention can be any compound, e.g., a peptide, polypeptide, nucleic acid, or small molecule, binding to the peptide or polypeptide described herein. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides such as receptors or binding partners for the peptide or polypeptide and fragments thereof comprising the binding domains for the peptides, and aptamers, e.g. nucleic acid or peptide aptamers. Methods to prepare such

ligands are well-known in the art. For example, identification and production of suitable antibodies or aptamers is also offered by commercial suppliers. The person skilled in the art is familiar with methods to develop derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced into the nucleic acids, peptides or polypeptides. These derivatives can then be tested for binding according to screening procedures known in the art, e.g. phage display. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab) ₂ fragments that are capable of binding antigen or hapten. The present invention also includes single chain antibodies and humanized hybrid antibodies wherein amino acid sequences of a non-human donor antibody exhibiting a desired antigen- specificity are combined with sequences of a human acceptor antibody. The donor sequences will usually include at least the antigen-binding amino acid residues of the donor but may comprise other structurally and/or functionally relevant amino acid residues of the donor antibody as well. Such hybrids can be prepared by several methods well known in the art. Preferably, the ligand or agent binds specifically to the peptide or polypeptide. Specific binding according to the present invention means that the ligand or agent should not bind substantially to ("cross-react" with) another peptide, polypeptide or substance present in the sample to be analyzed. Preferably, the specifically bound peptide or polypeptide should be bound with at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher affinity than any other relevant peptide or polypeptide. Non-specific binding may be tolerable, if it can still be distinguished and measured unequivocally, e.g. according to its size on a Western Blot, or by its relatively higher abundance in the sample. Binding of the ligand can be measured by any method known in the art. Preferably, said method is semi-quantitative or quantitative. Suitable methods are described in the following.

First, binding of a ligand may be measured directly, e.g. by NMR or surface plasmon resonance.

Second, if the ligand also serves as a substrate of an enzymatic activity of the peptide or polypeptide of interest, an enzymatic reaction product may be measured (e.g. the amount of a protease can be measured by measuring the amount of cleaved substrate, e.g. on a Western Blot). Alternatively, the ligand may exhibit enzymatic properties itself and the "ligand/peptide or polypeptide" complex or the ligand which was bound by the peptide or polypeptide, respectively, may be contacted with a suitable substrate allowing detection by

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the generation of an intensity signal. For measurement of enzymatic reaction products, preferably the amount of substrate is saturating. The substrate may also be labeled with a detectable lable prior to the reaction. Preferably, the sample is contacted with the substrate for an adequate period of time. An adequate period of time refers to the time necessary for an detectable, preferably measurable, amount of product to be produced. Instead of measuring the amount of product, the time necessary for appearance of a given (e.g. detectable) amount of product can be measured.

Third, the ligand may be coupled covalently or non-covalently to a label allowing detection and measurement of the ligand. Labeling may be done by direct or indirect methods. Direct labeling involves coupling of the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding (covalently or non-covalently) of a secondary ligand to the first ligand. The secondary ligand should specifically bind to the first ligand. Said secondary ligand may be coupled with a suitable label and/or be the target (receptor) of tertiary ligand binding to the secondary ligand. The use of secondary, tertiary or even higher order ligands is often used to increase the signal. Suitable secondary and higher order ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The ligand or substrate may also be "tagged" with one or more tags as known in the art. Such tags may then be targets for higher order ligands. Suitable tags include biotin, digoxygenin, His-Tag, Glutathion-S- Transferase, FLAG, GFP, myc-tag, influenza A virus haemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and/or C-terminus. Suitable labels are any labels detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatically active labels include e.g. horseradish peroxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT- BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl -phosphate, available as ready-made stock solution from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), ECF™ (Amersham Biosciences). A suitable enzyme-substrate combination may result in a colored reaction product, fluorescence or chemoluminescence, which can be measured according to methods known in the art (e.g. using a light-sensitive film or a suitable camera system). As for measuring the enyzmatic reaction, the criteria given above

apply analogously. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes (e.g. Alexa 568). Further fluorescent labels are available e.g. from Molecular Probes (Oregon). Also the use of quantum dots as fluorescent labels is contemplated. Typical radioactive labels include ³⁵ S, ¹²⁵ I, ³² P, ³³ P and the like. A radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager. Suitable measurement methods according the present invention also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electro-generated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immune tests, electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA), turbidimetry, nephelometry, latex- enhanced turbidimetry or nephelometry, or solid phase immune tests. Further methods known in the art (such as gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamid gel electrophoresis (SDS-PAGE), Western Blotting, and mass spectrometry), can be used alone or in combination with labeling or other dectection methods as described above.

The amount of a peptide or polypeptide (marker as used herein) may be, also preferably, determined as follows: (a) contacting a solid support comprising a ligand for the peptide or polypeptide as specified above with a sample comprising the peptide or polypeptide and (b) measuring the amount peptide or polypeptide which is bound to the support. The ligand, preferably chosen from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, is preferably present on a solid support in immobilized form. Materials for manufacturing solid supports are well known in the art and include, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc. The ligand or agent may be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. Suitable methods for fixing/immobilizing said ligand are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like. It is also contemplated to use "suspension arrays" as arrays according to the present invention (Nolan 2002, Trends Biotechnol. 20(l):9-12). In such

suspension arrays, the carrier, e.g. a microbead or microsphere, is present in suspension. The array consists of different microbeads or microspheres, possibly labeled, carrying different ligands. Methods of producing such arrays, for example based on solid-phase chemistry and photo-labile protective groups, are generally known (US 5,744,305).

The term "amount" as used herein encompasses the absolute amount of a polypeptide or peptide (marker as used herein), the relative amount or concentration of the said polypeptide or peptide as well as any value or parameter which correlates thereto or can be derived therefrom. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said peptides by direct measurements, e.g., intensity values in mass spectra or NMR spectra. Moreover, encompassed are all values or parameters which are obtained by indirect measurements specified elsewhere in this description, e.g., response levels determined from biological read out systems in response to the peptides or intensity signals obtained from specifically bound ligands. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations.

The term "comparing" as used herein encompasses comparing the amount of the peptide or polypeptide (marerk as used herein) comprised by the sample to be analyzed with an amount of a suitable reference source specified elsewhere in this description. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from a test sample is compared to the same type of intensity signal of a reference sample. The comparison referred to in step (b) of the method of the present invention may be carried out manually or computer assisted. For a computer assisted comparison, the value of the determined amount may be compared to values corresponding to suitable references which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format. Based on the comparison of the amount determined in step a) and the reference amount, it is possible to assess whether a subject is susceptible for a cardiac intervention, i.e. belonging to the group of subjects which can be successfully treated by the cardiac intervention. Therefore, the reference amount is to be chosen so that either a difference or a similarity in the compared amounts allows identifying those the test subject which belong into the group of subjects susceptible for

cardiac intervention or identifying those test subjects which are not susceptible for a cardiac intervention.

Accordingly, the term "reference amount" as used herein refers to an amount of the respective marker as used herein which allows assessing whether a subject is to be admitted to the hospital or to an intensive care unit, or can be discharged to home. Accordingly, the reference may e.g. be derived from (i) a subject known to have been successfully admitted to the hospital, i.e. who has been subject to further examination and subsequent treatment or intensive care treatment based on the results of the further investigation without the occurrence of adverse effects such as mortality or side effects caused by unadapted treatment regimen, or (ii) a subject known to have not been admitted to the hospital and which died or developed side effects caused by unadapted treatment regimen. Moreover, the reference amount may define a threshold amount, whereby an amount larger than the threshold shall be indicative for a subject which should be admitted to the hospital for further examination and/or intensive treatment, while an amount lower than the threshold amount shall be an indicator for a subject which can not be discharged to home. The reference amount applicable for an individual subject may vary depending on various physiological parameters such as age, gender, or subpopulation, as well as on the means used for the determination of the polypeptide or peptide referred to herein (marker as used herein). A suitable reference amount may be determined by the method of the present invention from a reference sample to be analyzed together, i.e. simultaneously or subsequently, with the test sample. A preferred reference amount serving as a threshold may be derived from the upper limit of normal (ULN), i.e. the upper limit of the physiological amount to be found in a population of apparently healthy subjects. The ULN for a given population of subjects can be determined by various well known techniques. A suitable technique may be to determine the median of the population for the peptide or polypeptide amounts to be determined in the method of the present invention. A preferred threshold (i.e. reference amount) for very low concentrations of troponin I or T is at least one to two times the lower detection limit. The lower detection limit of the high sensitive hsTnT assay referred to in this context is, preferably, 0,002 ng/ml.

Thus, the reference amount defining a threshold amount for hsTnT as referred to in accordance with the present invention is 0,002 ng/ml and below the 99 ^th percentile of an apparently healthy population.

An amount of very low concentration of troponin I or T larger than the reference amount is, more preferably, indicative for a subject which should be admitted to hospital.

Advantageously, it has been found in the study underlying the present invention that very low concentration of troponin T is a reliable prognostic biomarker for assessing the success of cardiac interventions for subjects in need thereof, i.e. subjects which suffer from cardiovascular complications and, in particular, those which are affected by acute cardiovascular events or heart failure. Thanks to the present invention, a risk/success stratification can be easily performed before subjecting a patient to a cardiac intervention. In case the patient turns out to be not susceptible for a cardiac intervention, said dangerous, time and/or cost intensive therapy can be avoided. Thus, besides preventing a subject from the adverse and severe side effects accompanying a cardiac intervention, the method of the present invention will be beneficial for the health system in that resources will be saved. It is to be understood that according to the method of the present invention described herein above and below, the amount of very low concentration of troponin I or T or means for the determination thereof can be used for the manufacture of a diagnostic composition for identifying a subject being susceptible for a cardiac intervention.

In addition or alternatively, the above method of the present invention may be used to identify a subject susceptible to intensive care treatement in an intensive care unit. The intensive care treatment may comprise additional diagnostic procedures, e.g. transthoratic echocardiography (TTE), transesophageal echocardiography (TEE), abdominal ultrasound examination, CT-scan of the chest, chest radiography, high-resolution CT-scan, cardiac catheterisation (left 6 right heart), perfusion/inhalation scintigraphy, compression ultrasound examination, treadmill exercise ECG, bronchoscopy, phlebography, and angiography. In case of cardiac diseases, thromboembolic complications, stroke or pulmonary embolismen, cancer and other extracardiac diseases a therapy comprises PCI, different surgical procedures, e.g. embolectomy, and specific medication e.g. oxygen, oral anticoagulation with vitamin K antagonists, anticoagulation with unfractionated heparins, LMW heparins, other anti-thrombotica, thrombolytic drugs, acetylsalicyclic acid, clopidigrel, loops diuretics and other diuretics, beta-blockers, ACE inhibitors, digitalis, calcium antagonists, nitrates, steroids, theophyllin, beta-2 mimetics, other bronchodilators, NSAIDs, opiates, antibiotics etc.. Therapeutic intervention comprises all kinds of surgery and medications.

It is to be understood that the definitions and explanations of the terms made above and below apply accordingly for all embodiments described in this specification and the accompanying claims.

The present invention further relates to a method of deciding about admitting a subject to the hospital, the method comprising

a) determining the amount of very low concentration of troponin I or T in a sample of the subject ; and b) comparing the amount of very low concentration of troponin I or T determined in step a) to a reference amount; and c) deciding whether the subject is to be admitted to the hospital or to an intensive care unit.

Preferably, the said therapy to be selected for a subject by the method of the present invention said therapy is a drug-based therapy. More preferably, the said medicament is an ACE inhibitor, preferably captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, or trandolapril, an AT-I receptor blocking agent, preferably, candesartan, losartan, or valsartan, a β-recpetor blocking agent, preferably, bisoprolol, carvedilol, metoprolol or succinate, or an an aldosterone antagonist, preferably, spironolacton or eplerenone.

Another preferred therapy to be selected for a subject in accordance with the present invention is an interventional therapy. An interventional therapy as referred to herein is a therapy which is based on physical interventions with the subject, e.g., by PCI, surgery, and/or electrophysiological interventions. More preferably, said interventional therapy is cardiac ^synchronisation therapy (CRT) or based on implantation of a cardioverter defibrillator (ICD).

The present invention, furthermore, relates to a method for predicting the risk of mortality for a subject comprising

a) determining the amount of very low concentration of troponin I or T in a sample of the subject ; and

b) comparing the amount of very low concentration of troponin I or T determined in step a) to a reference amount; and c) predicting the risk of mortality based on the result of steps a) and b).

Preferably, the subject is a subject presenting to the emergency station.

The term "predicting" used herein refers to assessing the probability according to which a subject will die within a defined time window (predictive window) in the future. The predictive window is an interval in which the subject will die according to the predicted probability. The predictive window may be the entire remaining lifespan of the subject upon analysis by the method of the present invention. Preferably, however, the predictive window is an interval of one month, six months or one, two, three, four, five or ten years after appearance of the cardiovascular complication (more preferably and precisely, after the sample to be analyzed by the method of the present invention has been obtained). As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be analyzed. The term, however, requires that the assessment will be valid for a statistically significant portion of the subjects to be analyzed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probability envisaged by the present invention allows that the prediction will be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort.

The term "mortality" as used herein relates to mortality which is caused by cardiovascular complications, lung diseases, pulmonary embolism, thrombosis, thromboembolic complications, stroke, tumors and malignant diseases, sepsis, septic shock, bleeding disorders, organ failure, acute kidney disease, infectious diseases, gastrointestinal complications, pancreatits, rheumatic disorders and others.

The term "cardiovascular complication" as used herein refers to any chronic disorder of the cardiovascular system or any acute cardiovascular event. Preferably, a chronic disorder of

the cardiovascular system as used herein encompasses coronary heart diseases, stable angina pectoris (SAP) or heart failure, preferably chronic heart failure. Acute cardiovascular events are, preferably, acute coronary syndromes (ACS). ACS patients can show unstable angina pectoris (UAP) or myocardial infarction (MI). MI can be an ST- elevation MI (STEMI) or a non-ST-elevation MI (NSTEMI). NSTE-ACS as used herein encompasses UAP and NSTEMI. The occurring of an MI can be followed by a left ventricular dysfunction (LVD) or development of heart failure. Further preferred cardiovascular complications encompass cardiac brady- or tachyarrhythmias including sudden cardiac death and stroke (cerebrovascular events or accidents). Most preferably, the said cardiovascular complication is ACS or heart failure.

The expression "predicting the risk of mortality" as used herein means that it the subject to be analyzed by the method of the present invention is allocated either into the group of subjects of a population having a normal, i.e. non-elevated, risk for mortality or into a group of subjects having a significantly elevated risk. An elevated risk as referred to in accordance with the present invention means that the risk of mortality within a predetermined predictive window is elevated significantly for a subject with respect to the average risk for mortality in a population of subjects. Preferably, for a predictive window of one year, the average risk is within the range of 0.5 and 3.0 %, preferably, 1.5%. An elevated risk as used herein, preferably, relates to a risk of more than 3.0 %, preferably, more than 5.0 %, and, most preferably within 3.0 % and 8.0 % with respect to a predictive window of one year. The time horizon over which such 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 from 1 month to 1 year.

Encompassed by the present invention is, further, a device for identifying a subject to be admitted to the hospital, adapted to carry out the method of the present invention, comprising means for determining the amount of very low concentration of troponin I or T in a sample of the subject and means for comparing said amount to a reference amount, whereby a subject to be admitted to the hospital is identified.

The term "device" as used herein relates to a system of means comprising at least the aforementioned means operatively linked to each other as to allow the prediction. Preferred means for determining the amount of very low concentration of troponin I or T and means for carrying out the comparison are disclosed above in connection with the method of the

invention. How to link the means in an operating manner will depend on the type of means included into the device. For example, where means for automatically determining the amount of the peptides are applied, the data obtained by said automatically operating means can be processed by, e.g. a computer program in order to obtain the desired results. Preferably, the means are comprised by a single device in such a case. Said device may accordingly include an analyzing unit for the measurement of the amount of the peptides or polypeptides in an applied sample and a computer unit for processing the resulting data for the evaluation. Alternatively, where means such as test stripes are used for determining the amount of the peptides or polypeptides, the means for comparison may comprise control stripes or tables allocating the determined amount to a reference amount. The test stripes are, preferably, coupled to a ligand which specifically binds to the peptides or polypeptides referred to herein. The strip or device, preferably, comprises means for detection of the binding of said peptides or polypeptides to the said ligand. Preferred means for detection are disclosed in connection with embodiments relating to the method of the invention above. In such a case, the means are operatively linked in that the user of the system brings together the result of the determination of the amount and the diagnostic or prognostic value thereof due to the instructions and interpretations given in a manual. The means may appear as separate devices in such an embodiment and are, preferably, packaged together as a kit. The person skilled in the art will realize how to link the means without further ado. Preferred devices are those which can be applied without the particular knowledge of a specialized clinician, e.g., test stripes or electronic devices which merely require loading with a sample. The results may be given as output of raw data which need interpretation by the clinician. Preferably, the output of the device is, however, processed, i.e. evaluated, raw data the interpretation of which does not require a clinician. Further preferred devices comprise the analyzing units/devices (e.g., biosensors, arrays, solid supports coupled to ligands specifically recognizing the natriuretic peptide, Plasmon surface resonace devices, NMR spectrometers, mass- spectrometers etc.) or evaluation units/devices referred to above in accordance with the method of the invention.

The term "peptide or polypeptide" or "marker" as used herein means troponin I or T or other suitable predictive marker described herein, wherein according to a particular embodiment at least three markers are determined, including but not limited toat least one neurohormonal marker, at least one ischemic cardiac marker, at least one marker of the transforming growth factor-β cytokine superfamily, and/or at least one inflammatory

marker as respectively explained above. The term "peptide or polypeptide" or "marker" may additionally mean another peptide known in the art as risk marker.

Moreover, the present invention also relates to a device for predicting the risk of mortality or a further acute cardiovascular event for a subject adapted to carry out the method of the present invention comprising means for determining the amounts of the peptide or polypeptide (marker as used herein) in a same or different sample of a subject and means for comparing said amounts to reference amounts, whereby it is predicted whether a subject is at risk of mortality or a acute cardiovascular event.

As to a use of inflammatory marker according to a particular embodiment, an amount of C- reactive protein (CRP), in particular high-sensitivity C-reactive protein (hs-CRP) is determined in the same or different sample of the subject in addition to determining the amount of troponin I or T, which preferred embodiment provides a greatly enhanced sensitivity of determination results and specifity of discriminating between subjects to be admitted to the hospital and eventually being subjected to intervention or subjects at risk of mortality, and subjects not considered further for clinical or medical treatments. C-reactive protein can be assayed by known methods (Roberts et al., Clin Chem 2001, 47: 418-425 [Erratum, Clin Chem 2001;47:980.] ), for example by rate nephelometry (e.g. Dade Behring NA latex CRP; Dade Behring BN II analyzer) or by monoclonal-antibody, solid- phase, sandwich-type enzyme immunoassay (e.g. IMχ, Abbott Laboratories, North Chicago), calibrated with the World Health Organization's International Reference Standard for CRP Immunoassay (85/506) (WHO Expert Committee on Biological Standardization. Thirty-seventh report. World Health Organ Tech Rep Ser 1987; 760:21- 22.). The range of values detected by the assay typically is 0.05 to 30 μg /ml. The median normal value for C-reactive protein typically is 0.8 μg/ml, with 90 percent of normal values <3 μg/ml and 99 percent <10.0 μg/ml. A reference amount, i.e. level for C-reactive protein of > 4 μg/ml, and especially a level C-reactive protein of >10 μg/ml should preferably be the threshold for being admitted to the hospital.

Similarly, other inflammatory marker can be determined and compared to a corresponding reference amount, including but not limited to interleukins such as preferably IL-6, and adhesion molecules such as VCAM and ICAM.

Similarly, the marker of the transforming growth factor-β cytokine superfamily, preferably selected from the group consisting of TGF-β isoforms and more preferably from MIC-I and GDF-15, can be determined, and the reference amount of the marker of the transforming growth factor-β cytokine superfamily is preferably about 1.0 ng/ml.

In the multi-marker approach, the ischemic cardiac marker is preferably TnT or TnI and more preferably hsTnT or hsTnl, and the reference amount of the ischemic cardiac marker is preferably about 8 pg/ml.

Further envisaged is a device for deciding about admitting a subject to the hospital, adapted to carry out the method of the present invention, comprising means for determining the amount of very low concentration of troponin I or T in a sample of the said subject and means for comparing said amount to a reference amount, whereby it is decided whether the subject is to be admitted to the hospital.

The present invention also relates to a device for predicting the risk of mortality in a subject, adapted to carry out the method of the present invention, comprising means for determining the amount of very low concentration of troponin I or T in a sample of the subject and means for comparing said amounts to reference amounts, whereby it is predicted whether the subject is at risk of mortality.

Furthermore, a kit for carrying out the methods of the present invention, for identifying a subject to be admitted to the hospital, deciding about admitting a subject to the hospital, or predicting the risk of mortality in a subject is envisaged by the present invention. Said kit comprising means for determining the amount of very low concentration of troponin I or T in a sample of a subject and means for comparing said amounts to reference amounts, wherein a subject to be admitted to the hospital is identified, a decision about admitting the subject to the hospital or to an intensive care unit is made, or the risk of mortality in the subject is predicted.

The term "kit" as used herein refers to a collection of the aforementioned means, preferably, provided in separately or within a single container. The container, also preferably, comprises instructions for carrying out the method of the present invention.

AIl references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

The present invention will be described in the following by illustrative embodiments and examples, with the present invention however not being limited thereto.

The figures show:

Fig. 1 shows admission and discharge rates depending on hsTnT values above or below the hsTnT median value;

Fig. 2 shows admission and discharge rates depending on hsTnT values falling in different quartiles of hsTnT levels;

Fig. 3 shows the results of ROC analysis for prediction discharge or admission by using hsTnT values; Fig. 4 shows the ROC curve of GDF- 15 ;

Fig. 5 shows the ROC curve of NT-proBNP; and Fig. 6 shows the ROC curve of hsCRP.

Examples

EXAMPLE 1

We tested the hypothesis that use of very low concentrations of troponin improves decision making in the emergency room and the overall management of patients presenting to emergency departments (EDs) by evaluating for incremental diagnostic and prognostic value of very low concentrations of troponin, and prospectively examining the clinical impact of very low concentrations of troponin guided decision making regarding discharge, admission to hospital, and intensive care treatment.

METHODS: A total of 255 patients presenting to the emergency department (ED) of an university hospital were studied. Blood samples were obtained in the ED from all patients admitted. High sensitive troponin T (hsTnT) was determined in 255 unselected consecutive patients. Follow-up at discharge included the assessment of clinical course and treatment. The

variables discharge, admission, and intensive care treatment were studied and associated to the baseline hsTnT values. Cut-off thresholds of hsTnT suitable for risk stratification and medical decision making (discharge or admissions to ICU or general care units), were calculated using ROC analysis.

RESULTS:

Presenting diagnoses were grouped into 4 categories:

1. Confirmed or suspected ischemic heart disease, including acute coronary syndrome and ischemic heart failure: 31(12,1%) 2. Nonischemic heart disease, including arrhythmias without acute ischemic trigger, vascular heart disease, and cardiomyopathies: 28 (11,0%)

3. Lung disorders, including asthma, chronic obstructive disease, and pulmonary embolism: 26 (10,2%)

4. All other disorders: 170 (66,6%)

In summary, 66,6% of the patients presented at the ED with non-cardiovascular or nonrespiratory disorders whereas 23,1% presented with ischemic and non-ischemic heart diseases.

Statistical analysis 1. Descriptive statistic

Table 1 shows the values of arithmetic mean, median, and percentils of hsTnT concentrations and Table 2 and Fig. 1 show admission and discharge rates depending on hsTnT values above or below the hsTnT median value. Patients with hsTnT values above the median of 6.60 pg/ml were admitted more often (Admission/Discharge rate = 2,26) compared to those with hsTnT values below the median (Admission/Discharge rate = 0,52).

2. Quartiles

The quartile ranges are displayed in Table 3 and Figure 2 showing that patients with increasing hsTnT values were admitted more often to the hospital. Patients in the lrst

quartile revealed an admission/discharge rate of 0,45 compared with an admission/discharge rate of 3,57 of patients in the 4 ^th quartile.

3. ROC analysis Results of ROC analysis for prediction discharge or admission by using hsTnT values are shown in Table 4 and Figure 3. The area under the curve (AUC) of 0,722 demonstrates the high discrimination power of hsTnT between discharge and admission. The hsTnT cut off value with optimal sensitivity/negative predictive value (62,9%/65,7%) and optimal specifity/ positive predictive value (76,46%/74,l) was estimated as 8,71 pg/ml (indicated by an asterisk in Table 4).

4. "Rule in" hsTnT cut off value for admission

In order to identify patients presenting at the emergency department who have to be admitted to hospital a hsTnT "rule in" cut off value of 30,26 pg/ml could be obtained from the ROC analysis. The corresponding specifity and positive predictive value is 91,1% and 78,8%, respectively. Patients with GDF- 15 values > 30,26 pg/ml should be admitted to hospital (and not be discharged) independent of diagnosis and underlying disease.

CONCLUSION Very low concentration of troponin is a marker for decision making and a suitable tool for risk stratification of patients presenting at the emergency department. Moreover, by using hsTnT "rule in" cut off value patients could be identified who should be admitted to hospital and not be discharged independent of diagnosis and disease status of the patient.

Table 1: Descriptive statistic variables

Variable hsTnT (pg/ml)

Sample size 255

Lowest value 0,0000

Highest value 4190,2000

_e n

- 25 -

Percentiles 95% Confidence Interval

2,5 0,0000 0,0000 to 0,0000

0,0000 0,0000 to 0,0000

10 0,0000 0,0000 to 0,0000

25 0,8025 0,0000 to 1 ,4850

75 22,1875 15,2502 to 33,3516

90 72,6600 46,3710 to 101 ,4934

95 142,4800 73,7694 to 244,8465

97,5 243,5238 119,8511 to 512,4172

Table 2: Admission - discharge below and above hsTnT median

Table 2: Frequency table & Chi-square test

Codes Y 0 1

0 84 44 0 52

1 39 88 2 26

(48,2%) (51 ,8%)

Chi-square 29,744

DF 1

Significance level P < 0,0001

Contingency coefficient 0,323

Table 3: Admission - discharge between quartiles of hsTnT levels

Frequency table & Chi-square test

Codes X Admission = 1 , Discharge = 0 Codes Y hsTnT quartiles

Codes X hsTnT (pg/ml)

Codes Y 0 1

1 44 20 < 0 75 2 40 24 0 96 - 6 60 3 25 38 6 69 - 21 82 4 14 50 22 31 - 4190 20

(48,2%) (51 ,8%)

Chi-square 35,659 DF 3 Significance level P < 0,0001

Chi-square test for trend

Chi-square (trend) 34,207 DF 1

Significance level P < 0,0001

Table 4: ROC analysis

ROC curve

Variable hsTnT (pg/ml)

Classification variable Admission

Positive group

Admission = 1

Sample size 132

Negative group

Discharge = 0 Sample size 123 ¹

Disease prevalence (%) 51,8

Area under the ROC curve 0,721

- - - Standard error 0,032

95% Confidence interval j 0,661 tc ) 0,775 i

Significance level P (Area=0.5) 0,0001 I

Criterion Sensitivity 95% Cl i Specificity 95% Cl ' +LR ; -LR +PV -PV

>=0 100,0 97,2-100,0 0,0 0,0-3,0 1,00 51,8 >0 86,4 79,3-91,7 32,5 24,4-41,6 1,28 0,42 57,9 69,0

>0,22 86,4 i 79,3-91,7 33,3 25,1 -42,4 1,30 0,41 58,2 69,5 ^'

>0,3 85,6 i 78,4-91,1 33,3 25,1 -42,4 1,28 0,43 57,9 68,3 >0,31 85,6: 78,4-91,1 34,1 25,8-43,2 1,30 0,42 58,2 68,9!

>0,57 85,6 ^■ 78,4-91,1 35,0 26,6-44,1 1,32 0,41 58,5 69,4:

>0,72 84,8 77,6 - 90,5 35,0 26,6-44,1 1,30 0,43 58,3 68,3 >0,75 84,8 77,6 - 90,5 35,8 27,3 - 44,9 1,32 0,42 58,6 68,7

>0,96 84,8 77,6 - 90,5 36,6 28,1 -45,7 1,34 0,41 58,9 69,2

>0,98 84,8 77,6 - 90,5 37,4 28,8 - 46,6 1,36 0,41 59,3 69,7 >1,01 84,1 76,7 - 89,9 37,4 28,8-46,6 1,34 0,43 59,0 68,7 ^'

>1,04 83,3 75,9 - 89,2 37,4 28,8-46,6 1,33 0,45 58,8 67,6 _

>1,14 83,3 75,9 - 89,2 38,2 29,6 - 47,4 1,35 0,44 59,1 68,1 \ >ϊ,17 ^" 83,3 75,9 - 89,2 39,0 30,4 - 48,2 1,37 0,43 59,5 68,6 >T,21 ^" 83,3 ^{" "} 75,9-89,2 ^{" "} 39 ^" 8 ^" 31,1 -49/1 ^" 1,39 0,42 ^{* "} 59,8 ^" 69,0 ^" j

>1,25 83,3 75,9 - 89,2 40,7 31,9-49,9 1,40 0,41 60,1 69,4! >1,26 83,3 75,9 - 89,2 4i ;s 32,7 - 50,7 1,42 0,40 60,4 69,9 ^'

>1,28 83,3' 75,9 - 89,2 42,3 33,4-51,5 1,44 0,39 60,8 70,3 >1,38 81,8 ^' 74,2 - 88,0 42,3 33,4-51,5 1,42 0,43 60,3 68,4 ^; >1,43 81,8 ^" 74,2 - 88,0 43,1 34,2 - 52 ^~ 3 1,44 0,42 60,7 68,8,

>1,44 81,8 74,2 - 88,0 43,9 35,0-53,1 1,46 0,41 61,0 69,2 >1,47 81,1 73,3 - 87,4 43,9 35,0-53,1 1,44 0,43 60,8 68,4 >1,5 81,1 ^* 73,3 - 87^4 ^~ 44J ^{" "} 35,7-53,9 1 ,47 ^" 0,42 ^* 61,1 68,8

>1,71 80,3 72,5 - 86,7 44,7 35,7 - 53,9 1,45 0,44 60,9 67,9 >1,79 80,3 72,5-86,7 45,5 36,5 - 54,8 1,47 0,43 61,3 68,3 ^' >1,93 ^" 80,3 ^' 72,5 - 86,7 46,3 37,3 - 55,6 1,50 0,43 61,6 68,7 j

>2 80 ,3 72,5 - 86,7 47,2 38,1 -56,4 1,52 0,42 62,0 69,0

>2,02 80 ,3 72,5 - 86,7 48,0 38,9 - 57,2 1,54 0,41 62,4 69,4

>2,32 80,3 72,5 - 86,7 48,8 39,7 - 58,0 1,57 0,40 ^' 62,7 69,8

>2,35 i 79,5 71,7-86,1 48,8 39,7 - 58,0 1,55 0,42 j 62,5' 69,0 >2,42 i 78,8 70,8 - 85,4 48,8 39,7 - 58,0 1,54 0,43 ^' 62,3 68,2

>2,52 78,0 70,0 - 84,8 48,8 39,7 - 58,0 1,52 0,45 62,0 67,4

>2,72 i 77,3 69,2-84,1 48,8 39,7 - 58,0 1,51 0,47' 61,8 66,7 >2,73 77,3 69,2-84,1 49,6 40,5 - 58,7 1,53 0,46 62,2 67,0

>2,79 77,3 69,2-84,1 50,4 41,3-59,5 1,56 0,45 62,6 67,4

>2,88 77,3 69,2-84,1 51,2 42,0 - 60,3 1,58 0,44 63,0 67,7

>3,04 76,5 68,4 - 83,4 51,2 42,0 - 60,3 1,57 0,46 62,7 67,0

>3,05 75,8 67,5 - 82,8 51,2 42,0 - 60,3 1,55 0,47 62,5 66,3 _

>3,27 75,0 66,7-82,1 51,2 42,0 - 60,3 1,54 0,49 62,3 65,6 >3,36 75,0 66,7-82,1 52,0 42,8-61,1 1,56 0,48 ^* 62,7 66,0 ]

>3,81 74,2 65,9-81,5 52,0 42,8-61,1 1,55 0,50 62,4 65,3:

>3,83 74,2 65,9-81,5 53,7 44,4 - 62,7 1,60 0,48 63,2 66,0 >3;88 73,5 65,1 -80,8 53,7 44,4 - 62,7 1,59 0,49 63,0 65,3 ^;

>3,9 73,5 65,1 -80,8 54,5 45,2 - 63,5 1,61 0,49 63,4 65,7. >4,36 73,5 65,1 -80,8 55,3 46,1-64,3 1,64 ^" 0,48 ^* 63,8 66,0 ^' >4,45 73,5 ^{" ~} 65 ^~ 1 - 80,8 56 A 46,9-65,0 1,67 ^" 0,47 64,2 66,3 ^'

>4,52 72,7 64,3-80,1 56,1 46,9 - 65,0 1,66 0,49 64,0 65,7' >4,69 72,7 64,3-80,1 56,9 47,7 - 65,8 1,69 0,48 64,4 66,0 ^;

>4,76 72,7 64,3-80,1 57,7 48,5 - 66,6 1,72 0,47 64,9 66,4 '

>4,85 72,7 64,3 - 80,1 58,5 49,3-67,3 1,75 0,47 65,3 66,7 >4,86 72,0 63,5 -79 ^~ 4 58,5 49,3-67,3 1,74 0,48 65,1 66,1 ! >4,93 ^~ 72,0 63,5-79,4 59,3 50,1-68,1 ^" 1,77 0,47 65,5 ^" 66,4 '

>4,96 72,0 63,5 - 79,4 60,2 50,9-68,9 1,81 0,47 66,0 66,7. >4,98 71,2 62,7 - 78,8 60,2 50,9 -68,9 ^" 1,79 ^" 0,48 ^" 65,7 66,1 ,

>5 70,5 61,9-78,1 60,2 50,9 - 68,9 1,77 0,49 65,5 65,5

>5,01 70,5 61,9-78,1 61,0 51,8-69,6 1,81 0,48 _, 66,0 65,8 >5,16 70,5 61,9-78,1 61,8 52,6 - 70,4 1,84 0,48 66,4 66,1

>5,4 69,7 61,1 -77,4 61,8 52,6 - 70,4 1,82 0,49 66,2 65,5,

>5,49 68,9 60,3 - 76,7 61,8 52,6 - 70,4 1,80 0,50 65,9 65,0 >5,53 68,9 60,3 - 76,7 62,6 53,4-71,2 1,84 0,50 66,4 65,3

>5,54 68,9 60,3 - 76,7 63,4 54,3-71,9 1,88 0,49 66,9 65,5 i

>5,61 68,9 60,3 - 76,7 64,2 55,1 -72,7 1,93 0,48 67,4 65,8 >5,62 68,2 59,5 - 76,0 64,2 55,1 -72,7 1,91 0,50 67,2 65,3 ^!

>6,22 68,2 59,5 - 76,0 65,0 55,9 - 73,4 1,95 0,49 67,7 65,6' >6,3ϊ ^{" " "} 68,2 59,5-76,0 ^{" "} 65,9 ^" 56,8-74,2 2,00 0,48 ^* 68,2 ^~~ 65^9 I >6,34 68,2 59,5 - 76,0 66,7 57,6 - 74,9 2,05 0,48 68,7 66,1

>6,38 68,2 59,5 - 76,0 67,5 58,4 - 75,6 2,10 0,47 69,2 66,4

>6,45 68,2 59,5 - 76,0 68,3 59,3 - 76,4 2,15 0,47 69,8 66,7 >6,58 67,4 58,7 -75,3 68,3 59,3 - 76,4 2,13 0,48 69,5 66,1

>6,6 66,7 57,9 - 74,6 68,3 59,3 - 76,4 2,10 0,49 69,3 65,6 I

>6,69 65,9 57,2 - 73,9 68,3 59,3 - 76,4 2,08 0,50 69,0 65,1 >6,98 65,9 57,2 - 73,9 69,1 60,1 - 77,1 2,13 0,49 69,6 65,4!

>7,01 65,9 57,2 - 73,9 69,9 61,0-77,9 2,19 0,49 70,2 65,6 I

>7,28 65,2 56,4 - 73,2 69,9 61,0-77,9 2,17 0,50 69,9 65,2 I >7,32 65,2 56,4 - 73,2 70,7 61 ,8 - 78,6 2,23 0,49 70,5 65,4 ;

>7,37 65,2 56,4 - 73,2 71,5 62,7 - 79,3 2,29 0,49 71,1 65,7' >7,63 64,4 55,6 - 72,5 71,5 62,7 - 79,3 2,26 0,50 70,8 65,2 ^' >7,77 64,4 55,6 - 72,5 72,4 63,6 - 80,0 2,33 0,49 71,4 65,4

>7,82 64,4 55,6 - 72,5 73,2 64,4 - 80,8 2,40 0,49 72,0 65,7 j >7,97 63,6 54,8-71,8 73,2 64,4 - 80,8 2,37 0,50 71 ,8 ^" 65,2' >8,02 62,9 54,0-71,1 73,2 64,4 - 80,8 2,34 0,51 ^' 71,6 ^* 64,7!

>8,05 62,9 54,0-71,1 74,0 65,3-81,5 2,42 0,50 72,2 65,Oi

>8,07 62,9, 54,0-71,1 74,8 i 66,2 - 82,2 2,49 0,50 72,8 65,2

>8,13 62,9 54,0-71,1 75,6, 67,0 - 82,9 2,58 0,49 73,5 65,5

>8,71 * 62,9 54,0-71,1 76,4, 67,9 - 83,6 2,67 0,49 74,1 65,7

>8,72 62,1 53,3 - 70,4 76,4 ' 67,9 - 83,6 2,63 0,50 73,9 65,3

>9,06 61,4 52,5 - 69,7 76,4 67,9 - 83,6 2,60 0,51 73,6 64,8

>9,1 60,6 51,7-69,0 76,4 67,9 - 83,6 2,57 0,52 73,4 64,4

>9,27 60,6 51,7-69,0 77,2 68,8 - 84,3 2,66 0,51 74,1 64,6

>9,28 59,8 51,0-68,3 78,0, 69,7 - 85,0 2,73 0,51 74,5 64,4

>9,3 59,1 50,2 - 67,6 78,0 69,7 - 85,0 2,69 0,52 74,3 64,0

>9,41 59,1 50,2 - 67,6 78,9 70,6 - 85,7 2,80 0,52 75,0 64,2

>9,92 58,3, 49,4 - 66,8 78,9 70,6 - 85,7 2,76 0,53 74,8 63,8

>10,11 57,6 48,7-66,1 78,9 70,6 - 85,7 2,72 0,54 74,5 63,4

> 10,56 57,6 48,7-66,1 79,7 71,5-86,4 2,83 0,53 75,2 63,6

>10,97 57,6 48,7-66,1 80,5 72,4-87,1 2,95 0,53 76,0 63,9

>11,03 56,8 47,9 - 65,4 80,5 72,4-87,1 2,91 0,54 75,8 63,5

>11,1 56,1 47,2 - 64,7 80,5 72,4-87,1 2,87 0,55 75,5 63,1

>11,2 55,3 46,4 - 64,0 80,5 72,4-87,1 2,83 0,56 75,3 62,7

>11,28 54,5 45,7 - 63,2 80,5 72,4-87,1 2,80 0,56 75,0 62,3

>11,85 53,8 44,9 - 62,5 80,5 72,4-87,1 2,76 0,57 74,7 61,9

>11,91 53,8 44,9 - 62,5 81,3 73,3 - 87,8 2,88 0,57 75,5 62,1

>12,26 53,0 44,2-61,8 81,3 73,3 - 87,8 2,84 0,58 75,3 61,7

>12,45 52,3 43,4-61,0 81,3 73,3 - 87,8 2,80 0,59 75,0 61,3

>12,56 51,5 42,7 - 60,3 81,3 73,3 - 87,8 2,75 0,60 74,7 61,0

>12,88 50,8 41,9-59,6 81,3 73,3 - 87,8 2,71 0,61 74,4 60,6

>13,05 50,0 41,2-58,8 81,3 73,3 - 87,8 2,67 0,62 74,2 60,2

>13,07 49,2 40,4-58,1 81,3 73,3 - 87,8 2,63 0,62 73,9 59,9

>13,28 49,2 40,4-58,1 82,1 74,2 - 88,4 2,75 0,62 74,7 60,1

>13,31 48,5 39,7 - 57,3 82,1 74,2 - 88,4 2,71 0,63 74,4 59,8

>13,47 47,7 39,0 - 56,6 82,1 74,2 - 88,4 2,67 0,64 74,1 59,4

>14,03 47,7 39,0 - 56,6 82,9 75,1 -89,1 2,80 0,63 75,0 59,6

>14,15 47,7 39,0 - 56,6 83,7 76,0 - 89,8 2,94 0,62 75,9 59,9

>14,52 47,0 38,2 - 55,8 83,7 76,0 - 89,8 2,89 0,63 75,6 59,5

>14,95 46,2 37,5 - 55,1 83,7 76,0 - 89,8 2,84 0,64 75,3 59,2

>15,01 45,5 36,8 - 54,3 83,7 76,0 - 89,8 2,80 0,65 75,0 58,9

>15,18 44,7 36,0 - 53,6 83,7 76,0 - 89,8 2,75 0,66 74,7 58,5

>15,32 43,9 35,3 - 52,8 83,7 76,0 - 89,8 2,70 0,67 74,4 58,2

>15,46 43,9 35,3 - 52,8 84,6 76,9 - 90,4 2,84 0,66 75,3 58,4

>16,34 43,2 34,6-52,1 84,6 76,9 - 90,4 2,80 0,67 75,0 58,1

>16,98 42,4 33,9-51,3 84,6 76,9 - 90,4 2,75 0,68 74,7 57,8,

> 17,46 42,4 33,9-51,3 85,4 77,9-91,1 2,90 0,67 75,7 58,0

>18,1 41,7 33,2 - 50,6 85,4 77,9-91,1 2,85 0,68 75,3 57,7,

>18,44 41,7 33,2 - 50,6 86,2 78,8-91,7 3,01 0,68 76,4 57,9

>18,52 40,9 32,4 - 49,8 86,2 78,8-91,7 2,96 0,69 76,1 57,6

>18,77 40,9 32,4 - 49,8 87,0 79,7 - 92,4 3,14 0,68 77,1 57,8

>20,24 40,2 31,7-49,0 87,0 79,7 - 92,4 3,09 0,69 76,8 57,5

>20,28 40,2 31,7-49,0 87,8 80,7 - 93,0 3,29 0,68 77,9 57,8

>20,7 39,4 31,0-48,3 87,8 80,7 - 93,0 3,23 0,69 77,6 57,4

>21,11 38,6 30,3-47,5 87,8 80,7 - 93,0 3,17 0,70 77,3 57,1

>21,12 38,6 30,3-47,5 88,6 81,6-93,6 3,39 0,69 78,5 57,4

>21,82 37,9 29,6 - 46,7 88,6 81,6-93,6 3,33 0,70 78,1 57,1

>22,31 37,1 28,9-46,0 88,6 81,6-93,6 3,26 0,71 77,8 56,8

>23,68 37,1 28,9-46,0 89,4 82,6 - 94,2 3,51 0,70 79,0 57,0

>24 35,6 27,5 - 44,4 89,4 82,6 - 94,2 3,37 0,72 78,3 56,4

>24,78 34,8 26,8-43,6 89,4 82,6 - 94,2 3,30 0,73 78,0 56,1

>25,39 34,1 26,1 - 42,8 89,4 82,6 - 94,2 3,23 0.74 ¹ 77,6 55,8

>26,81 33,3 25,4-42,1 89,4 82,6 - 94,2 3,15 0,75, 77,2 55,6 >27,43 32,6 24,7-41,3 89,4 82,6 - 94,2 3,08 0,75 ^■ 76,8 55,3

>28,79 31,8 24,0 - 40,5 89,4 82,6 - 94,2 3,01 0,76 76,4 55,0

>28,88 31,1 23,3 - 39,7 89,4 82,6 - 94,2 2,94 0,77 ' 75,9 54,7 >29,09 31,1 23,3 - 39,7 90,2 83,6 - 94,9 3,18 0,76 77,4 55,0

>30,26 31,1 23,3 - 39,7 91,1 84,6 - 95,4 3,47 0,76 78,8 55,2

>31,8 31,1 23,3 - 39,7 91,9 85,6 - 96,0 3,82 0,75 80,4 55,4 >32,29 30,3 22,6 - 38,9 91,9 85,6 - 96,0 3,73 0,76 ^' 80,0 55,1

>34,46 30,3 22,6 - 38,9 92,7 86,6 - 96,6 4,14 0,75 81,6 55,3 ^~ >34,48 ^" 2975 21,9-38,1 92,7 86^6-96,6 ^" 4,04 ^"" 0,76 ^*" 81,2 ^{" "} 55,1 >36,02 ^" 29^5 21,9-38,1 93,5 87,6 ^" - 97,1 ^" 4,54 0,75 ^" 83,0 ^" 55,3

>37,5 29,5 21,9-38,1 94,3 88,6 - 97,7 5,19 0,75 84,8 55,5

>38,11 28,8 21,2-37,3 94,3 88,6 - 97,7 5,06 0,76 84,4 55,2 >38,52 28,8 21,2-37,3 95,1 89,7 - 98,2 5,90 0,75 ^' 86,4 55,5

>39,09 28,0 20,6 - 36,5 95,1 89,7 - 98,2 5,75 0,76 86,0 55,2 >42,2 27,3 19,9-35,7 95,1 897 - 98,2 5,59 0,76 85,7 54,9 >45,11 27,3 19,9-35,7 95,9 90,8 ^" - 98,7 ^{" "} 6,71 0,76 87,8 55,1

>45,29 27,3 19,9-35,7 96,7 91,9-99,1 8,39 0,75 90,0 55,3 >45,46 26,5 19,2-34,9 96,7 91,9-99,1 8,15 0,76 89,7 55,1

>45,62 25,8 18,5-34,1 97,6 93,0 - 99,5 10,56 0,76 91,9 55,0

>47,56 25,0 17,9-33,3 97,6 93,0 - 99,5 10,25 0,77 91,7 54,8 >47,74 ^" 24,2 17,2-32,5 97,6 93,0-99,5 9,94 0,78 91,4 54,5 >48,24 23,5 ^" ϊ6 ^~ 6-31, ^~ 6 ^" 97,6 93,0-99,5 ^" 9,63 ^"" 0,78 ^* 91,2 54,3 ^"

>50,54 22,7 15,9-30,8 97,6 93,0 - 99,5 9,32 0,79 90,9 54,1 >50,81 22,0 15,2-30,0 97,6 93,0 - 99,5 9,01 0,80 90,6 53,8

>55,16 21,2 14,6-29,2 97,6 93,0 - 99,5 8,70 0,81 90,3 53,6

>56,56 20,5 13,9-28,3 97,6 93,0 - 99,5 8,39 0,82 90,0 53,3 >58,11 19,7 13,3-27,5 97,6 93,0 - 99,5 8,08 0,82 89,7 53,1

>58,96 18,9 12,6-26,7 97,6 93,0 - 99,5 7,77 0,83 89,3 52,9

>68,9 18,2 12,0-25,8 97,6 93,0 - 99,5 7,45 0,84 88,9 52,6 >72,59 18,2 12,0-25,8 98,4 94,2 - 99,8 11,18 0,83 92,3 52,8

>72,66 17,4 11,4-25,0 98,4 94,2 - 99,8 10,72 0,84 92,0 52,6

>73,14 16,7 10,8-24,1 98,4 94,2 - 99,8 10,25 0,85 91,7 52,4 >73,34 15,9 10,1 -23,3 98,4 94,2 - 99,8 9,78 0,85 91,3 52,2

>73,66 15,9 10,1 -23,3 99,2 95,5 - 99,9 19,57 0,85 95,5 52,4 ^" >73,89 15,2 9,5-22,4 99,2 95,5- 99,9 18,64 0,86 ^* -95,2 52,1 >79,51 14,4 8,9-21,6 99,2 95,5 - 99,9 17,70 0,86 95,0 51,9

>80,32 13,6 8,3 - 20,7 99,2 95,5 - 99,9 16,77 0,87 94,7 51,7

>85,38 12,9 7,7-19,8 99,2 95,5 - 99,9 15,84 0,88 94,4 51,5 >93,49 12,1 7,1 -18,9 99,2 95,5 - 99,9 14,91 0^89 94,1 51,3

>101,23 11,4 6,5-18,1 99,2 95,5 - 99,9 13,98 0,89 93,7 51,0 ^" >i ^" θ3 10,6 5,9-17,2 ^{""" """} 99,2 ^" 95,5-99> ^" ϊ3.05 ^" O ^~ 9θ ^{" ""} 93,3 50,8 >119,18 9,8 5,4-16,3 99,2 95,5 - 99,9 12,11 0,91 ^' 92,9 50,6

>120,28 9,1 4,8-15,3 99,2 95,5 - 99,9 11,18 0,92 92,3 50,4 ^" >ϊ49,88 8,3 4,2-14,4 99,2 95,5 - 99,9 10,25 0,92 ^{' ""} 91 ,7 ^" 50 ^~ 2 >162,56 7,6 3,7-13,5 99,2 95,5 - 99,9 9,32 0,93 90,9 50,0 > 177,14 6,8 3,2 - ^' te ^' fi 99> 95 ^~ 5 -99,9 8,39 0^94 ^{" "} 90 ^" θ 49,8 >186,83 6,1 2,7-11 ^~ 6 99,2 95,5-99,9 7,45 0,95 ^' 88,9 49,6 >194,27 6,1 2,7-11,6 100,0 97,0-100,0 0,94 100,0 49,8

>239,19 5,3 2,2-10,6 100,0 97,0-100,0 0,95 100,0 49,6 >240,79 4,5 1,7-9,6 100,0 97,0-100,0 0,95 100,0 49,4 >262,66 3,8 ^" 1^3 -8,6 ^{"" "} i ^~ od,o ^~ 97,0 - ϊ00,θ ^" 0,96 ^{" ~} 100,0 ^" 49,2

>351,13 3,0 0,9 - 7,6 100,0 97,0-100,0 0,97 100,0 49,0

>427,9 2,3 0,5 - 6,5 100,0 97,0 - 100,0 0,98 ' 100,0 48,8

>454,65 J 1 ,5 0,2 - 5,4 100,0 97,0 - 100,0 0,98 100,0 48,6 >560,89 0,8 0,1 - 4,2 100,0 97,0 - 100,0 0,99 100,0 48,4

>4190,2002 ! o,o L 0,0 - 2,8 100,0 97,0 - 100,0 1 ,00 48,2

+LR : Positive likelihood ratio

-LR : Negative likelihood ratio +PV : Positive predictive value -PV : Negative predictive value

EXAMPLE 2

A 56 years old female was admitted to the emergency room with suspected gastrointestinal bleeding or acute gastritis. Case history:

Chronic hepatitis B, liver cirrhosis

Physical examination: Weight: 62 Kg

Size: 162 cm Heart rate: 60/min

Blood pressure: 70/138 mmHg

Clinical signs and symptoms:

• No clinical signs for cardiovascular impairment. • No clear clinical signs related to gastrointestinal bleeding or gastritis.

Laboratory: hsTnT value: 0,0 pg/ml; < (rule in cut off value for admission to hospital; < optimized

ROC cut off value

Result:

The patient could be discharged to home!

EXAMPLE 3

A 16 years old male was admitted to the emergency room with suspected gastrointestinal complication in an unclear clinical situation. Case history: Stable coronary heart disease, kidney disease

Physical examination:

Weight: 105 Kg

Size: 188 cm Temperature: 39 °C

Heart rate: 65/min

Blood pressure: 94/120

Clinical signs and symptoms: • Diarrhoe

• Fever

• Gastrointestinal pain

Laboratory: hsTnT value: 58,96 ng/ml; > rule in cut off value for admission to hospital.

Result:

The patient was admitted to the hospital.

EXAMPLE 4

A 27 years old male was admitted to the emergency department with suspected acute gastritis. Case history:

Patient has never been hospitalized, and takes no medication. She exhibited no signs of health problems.

Physical examination: Weight: 85 Kg

Size: 180 cm

Temperature: 36 °C Heart rate: 65/min

Blood pressure: 85/126 mmHg

Clinical signs and symptoms:

• Tickle/itch paresthesia

• Hperventilation

Laboratory: hsTnT value: 0,31 ^λ pg/ml; < rule in cut off value for admission to hospital; < optimized ROC cut off value

Result: The patient was discharged to home.

EXAMPLE 5

A 69 years old male was admitted to the emergency room with dyspnea and suspected ventricular dysfunction in an unclear clinical situation.

Physical examination:

Weight: 87 Kg

Size: 178 cm

Temperature: 36,5 °C

Heart rate: 101/min Blood pressure: 56/172 mmHg

Clinical signs and symptoms:

• Dyspnea, atypical for myocardial infarction

• Signs of COPD • No signs of myocardial infartion

Laboratory: hsTnT value: 80,32 pg/ml; > rule in cut off value for admission to hospital

Result: The patient was admitted to the intensive care unit.

EXAMPLE 6

The additional biomarkers high sensitivity C-reactive protein (hsCRP), NT-proBNP and GDF- 15 were determined in parallel with high sensitive troponin T (hsTnT) in the 255 unselected consecutive patients presenting at the ermergency department. Follow-up at discharge included the assessment of clinical course and treatment. The variables discharge, admission, and intensive care treatment were studied and associated to five different marker combinations, 1 : hsTnT + GDF- 15 + hsCRP, 2: hsTnT + NT-proBNP + GDF-15, 3: hsTnT + NT-proBNP + hsCRP, and 4: NT-proBNP + GDF-15 + hsCRP, 5: hsTnT + NT-proBNP + GDF-15 + hsCRP. Cut-off thresholds of each marker suitable for risk stratification and medical decision making (discharge or admissions to ICU or general care units), were calculated using ROC analysis (see Figs. 4-6; Tabl. 1).

Tabl. 1 : Cut off thresholds for hsCRP, GDF-15, hsTnT, and NT-proBNP from ROC analysis

Sensitivity Specificity Cut off value hsCRP 59,1 73,3 >4,9558 μg/ml

GDF-15 65,9 65,9 >l,5508 ng/ml hsTnT 62,9 75,6 >8, 13 pg/ml

NT-proBNP 53,0 83,7 >239,42 pg/ml

For the different marker combinations positive predictive values (PPV) and negative predictive values (NPV) for admission to hospital were estimated based on the ROC analysis derived cutoff thresholds.

The cut off thresholds indicated in Table 1 above are significant for hsCRP, GDF-15, hsTnT, and NT-proBNP, respectively. For other marker peptides selected from respective groups of neurohormonal markers, ischemic cardiac markers, markers of the transforming growth factor-β cytokine superfamily, and/or inflammatory markers, corresponding ROC analysises can be performed to derive respective cutoff thresholds therefrom. Typically, reference amounts are below the 99 ^th percentile of an apparently healthy population cut off value of the respectively used methods for determining the corresponding marker.

EXAMPLE 7: The diagnostic performance data of the marker combination hsTnT + GDF- 15 + hsCRP is displayed in Table 2.

Table 2: Multi marker approach hsTnT + GDF- 15 + hsCRP

(hsTnT AND GDF AND CRP) Hospitalization

Yes No Sums

Yes 46 3 49

No 86 120 206 Sums 132 123 255

Sensitivity: true positive rate: A / (A+B) = 34.8% Specificity: true negative rate: D / (C+D) = 97.6% Prevalence (hospitalization) : 51.8% Positive predictive value: 94.0% Negative predictive value: 58.2%

Fisher's exact test

0 120 86 (80,8%)

1 3 ' 46 (19,2%)

(48,2%) (51 ,8%)

P < 0,000000001

EXAMPLE 8

The diagnostic performance data of the marker combination hsTnT + NT-proBNP + hsCRP is displayed in Table 3.

Tabl. 3: Multimarker approach hsTnT + NT-proBNP + hsCRP

(hsTnT AND BNP AND CRP) Hospitalization

Yes No Sums

Yes 45 3 48

No 87 120 207 Sums 132 123

Sensitivity: true positive rate: A / (A+B) = 34.0% Specificity: true negative rate: D / (C+D) = 97.6% Prevalence (hospitalization) : 51.8% Positive predictive value: 93.8% Negative predictive value: 57.9%

Fisher's exact test

Classification X a_Aufnahme Classification Y I TNT BNP CRP

Classification X

Classification Y 0 1

0 120 87 (81 ,2%)

1 3 45 (18,8%)

(48,2%) (51 ,8%)

P < 0,000000001

EXAMPLE 9

The diagnostic performance data of the marker combination hsTnT + NT-proBNP + GDF- 15 is displayed in Table 4.

Tabl. 4: Multimaker approach hsTnT + NT-proBNP + GDF-15

(hsTnT AND BNP AND GDF) Hospitalization Yes No Sums

Yes 53 5 58

No 79 118 197 Sums 132 123

Sensitivity: true positive rate: A / (A+B) = 40.2% Specificity: true negative rate: D / (C+D) = 95.9% Prevalence (hospitalization): 51.8% Positive predictive value: 91.3% Negative predictive value: 59.9%

Fisher's exact test

Classification X a_Aufnahme Classification Y I_TNT_BNP_GDF

Classification X

Classification Y 0 ' 1

0 118 79 (77,3%)

1 5 53 (22,7%)

(48,2%) (51 , 3%)

P < 0,000000001

EXAMPLE 10

The diagnostic performance data of the marker combination NT-proBNP + GDF- 15 + hsCRP is displayed in Table 5.

Tabl. 5: Multimarker appraoch NT-proBNP + GDF- 15 + hsCRP

(BNP AND GDF AND CRP) Hospitalization Yes No Sums

Yes 43 4 47

No 89 119 208 Sums 132 123

Sensitivity: true positive rate: A / (A+B) = 32.6% Specificity: true negative rate: D / (C+D) = 96.7% Prevalence (hospitalization): 51.8% Positive predictive value: 91.4% Negative predictive value: 57.2%

Fisher's exact test

Classification X a_Aufnahme Classification Y I_BNP_GDF_CRP

Classification X

Classification Y o ; 1

0 119 89 (81 ,6%) 1 4 43 (18,4%)

(48,2%) (51 ,8%)

EXAMPLE 11

The diagnostic performance data of the marker combination NT-proBNP + GDF- 15 + hsTnT + hsCRP is displayed in Table 6.

Tabl. 6: Multimarker approach NT-proBNP + GDF- 15 + hsTnT + hsCRP

BNP, GDF, hsTnT, CRP Hospitalization

Yes No Sums

Yes 40 2 42

No 92 121 213 Sums 132 123

Sensitivity: true positive rate: A / (A+B) = 30.3% Specificity: true negative rate: D / (C+D) = 98.4% Prevalence (hospitalization) : 51.8% Positive predictive value: 95.3% Negative predictive value: 56.8%

Fisher's exact test