WO2018127372A1

WO2018127372A1 - Detection of transient troponin peaks for diagnosis of subjects at high risk of cardiovascular disease

Info

Publication number: WO2018127372A1
Application number: PCT/EP2017/082719
Authority: WO
Inventors: Johanna Cornelia Maria WITTEMAN
Original assignee: Witteman Johanna Cornelia Maria
Priority date: 2016-12-13
Filing date: 2017-12-13
Publication date: 2018-07-12

Abstract

The present invention provides a method to assess troponin peaks in a subject to estimate CHD risk, said method comprising a) fine-temporal serial testing in body fluid samples obtained from the subject the concentration of troponin; b) assessment of troponin peaks in a subject, wherein preferably an accumulative peak score is computed, and whereby peak height and peak frequency are indicative for the risk of CHD in the subject; c) the troponin peaks are assessed and corresponding CHD risk computed in an ongoing fashion; d) a clinical algorithm is used to determine an increased CHD risk in the subject. The present invention also provides a method to use above method to select a suitable therapy against CHD for the subject, to monitor treatment effectiveness and to guide treatment course in a subject. In an alternative, the assessment of troponin peaks will be performed in combination with the assessment of NT-proBNP peaks to estimate CHD risk.

Description

Detection of transient troponin peaks for diagnosis of subjects at high risk of cardiovascular disease

Field of the invention

The present invention relates to the field of medicine, specifically cardiovascular medicine and diagnosis of cardiovascular disease. In particular, the invention pertains to the use of biomarkers such as cardiac troponins for identification of subjects at high risk of cardiovascular disease.

Background art

In primary and secondary cardiovascular disease prevention, biomarkers are used to identify subjects at high risk of cardiovascular disease with the aim to tailor treatment and thereby reduce disease risk. Because the rate of cardiovascular disease remains high, finding suitable biomarkers is an important and long-standing objective.

Cardiac troponins T and I are components of the contractile apparatus of cardiomyocytes and are released into the blood stream after cell injury. Cardiac troponins T and I are 100% specific for cardiomyocytes. Troponins have been firmly established as diagnostic biomarkers for myocardial infarction (Ml) in patients with suspected acute coronary syndrome (ACS).

Elevated troponin levels in the acute setting do not only have diagnostic relevance but also are an established prognostic marker of recurrent ischemic events. Elevated levels of troponin have also been observed in patients with heart failure (HF) where they are an established prognostic marker for heart failure, hospitalization or death.

With the introduction of a high-sensitive troponin assay in 2009, much lower troponin levels became detectable. Unexpectantly, low-elevated levels of troponin using the high-sensitive assay were observed in patients with stable CAD and in presumably healthy subjects. This gave rise to the concept of chronic low-elevated levels of troponin outside the acute setting of ACS. Low- elevated levels of troponin in patients with stable CAD, measured shortly or longer in time after the original ACS, were found to be predictive of heart failure and cardiovascular death but were not (or to a much lower extend) predictive of ischemic events. Similar results were found in presumably healthy populations.

The sources of low-level elevations of troponin in patients with stable CAD and in presumably healthy populations are unknown. Mechanisms underlying troponin release from cardiomyocytes in these populations include transient episodes of clinically silent ischemia due to plaque rupture with small vessel occlusions, apoptosis, demand versus supply imbalance, increased myocardial strain due to pressure or volume overload, inflammatory processes and reduced renal clearance.

Atherosclerotic plaque rupture in the culprit lesion during ACS is sometimes associated with more or less simultaneous plaque ruptures in other vessels indicating a state of overall coronary instability.

NT-proBNP is another well-known cardiac marker. BNP is synthesized and released from cardiomyocytes in response increased wall stress NT-proBNP is known as an indicator of wall stress and is used to aid in diagnosis of heart failure. NT-proBNP also is an established predictor of death in HF. NT-proBNP is also increased after myocardial infarction.

Biomarkers are dynamic and changes in biomarkers levels may have additive prognostic value to level. Studies on temporal change in troponin and NT-proBNP have shown that increases in these cardiac markers are associated with increased risks of cardiovascular disease.

It is an object of the instant invention to provide further improvements in the cardiac marker- based prognosis and diagnosis of cardiovascular disease.

Summary of the invention

In a first aspect, the invention relates to a method of detecting the presence of troponin peaks in a subject to determine whether a subject is at increased risk of coronary heart disease (CHD). The method preferably comprises the steps of: a) fine-temporal serial testing of the concentration of troponin in body fluid samples obtained from the subject; and, b) assessment of troponin peaks in a subject whereby the presence of troponin peaks is indicative for a high risk of CHD in the subject, and wherein preferably a cumulative measure of peaks is computed in an ongoing fashion.

In the method of the invention, the serial testing preferably comprises measuring at least three samples obtained at a time interval of less than 7 days, more preferably, the serial measuring comprises measuring serial samples obtained at a time interval of less than 7 days, most preferably every two days or daily.

In the method of the invention, a troponin peak preferably is detected when consecutive samples are obtained with intervals of no more than 2 days and the concentration of troponin increases by at least 5 ng/L from baseline in 2 days and returns to baseline in no more than 3 days, more preferably, a troponin peak is detected when consecutive samples are obtained with intervals of at least one day and the concentration of troponin increases by at least 5 ng/L from baseline in 1 day and returns to baseline in no more than 3 days.

In one embodiment of the method of the invention, the method comprising the (further) steps of: a) fine-temporal serial testing of the concentration of NT-proBNP in body fluid samples obtained from the subject; and b) assessment of NT-proBNP peaks in a subject whereby the presence of NT-proBNP peaks is indicative for a high risk of CHD in the subject, and wherein preferably a cumulative measure of peaks is computed in an ongoing fashion.

Preferably in the methods according to the invention the troponin is at least one of troponin I, troponin T and a variant thereof, of which troponin I or a variant thereof is preferred, and the NT- proBNP can be replaced by a BNP-type peptide or a variant thereof.

The subject in the methods of the invention preferably is a subject selected from the group consisting of: a) a subject diagnosed with stable coronary artery disease (CAD); b: a subject having suffered from an acute coronary syndrome (ACS), c) an asymptomatic subject from the general population; and d) an asymptomatic subject from the general population belonging to a risk group for coronary heart disease. In a further embodiment, the method according to the invention comprises the further step of determining whether a subject is at high risk of coronary heart disease where other risk factors or a risk score is added to improve risk assessment.

In yet a further embodiment, the method according to the invention further comprises the step of determining the susceptibility of the subject to a therapy against coronary heart disease. Preferably, in this embodiment, the a subject determined to be at high CHD risk as based on the assessment of troponin or NT-proBNP peaks is determined susceptible to a therapy against coronary heart disease.

In another embodiment of the method of the invention, the method further comprises the step of determining the effectiveness of a therapy against CHD in the subject, wherein a decrease in CHD risk as based on the assessment of (reduced occurrence of) troponin or NT-proBNP peaks during treatment is indicative that the treatment is effective and no change or an increase in CHD risk as based on the assessment of troponin or NT-proBNP peaks during treatment is indicative that the treatment is not effective.

In yet another embodiment of the method of the invention, the method further comprises the step of determining the treatment strategy of a subject regarding a therapy against CDH, wherein a decrease in CHD risk as based on the assessment of troponin or NT-proBNP peaks during treatment is indicative that the treatment strategy does not need adjustment and no change or an increase in CHD risk as based on the assessment of troponin peaks during treatment is indicative that the treatment strategy needs adjustment.

In one embodiment of the method of the invention, the method further comprises any one of the steps of determining CHD risk, determining the suitability of a treatment and treatment guiding in a subject, where the method of detection of troponin peaks is combined with a method for detection of NT-proBNP peaks.

In one further embodiment of the method of the invention, the method further comprises the step of referring a subject for clinical evaluation of the cause of a troponin or NT-proBNP peak other than one that puts the subject at risk for coronary heart disease.

Preferably in the methods of the invention, the concentrations of troponin and NT-proBNP in the body fluid samples are measured at the point of care.

In again another embodiment of the method of the invention, an algorithm is used to compute at least one of: a) a cumulative troponin or NT-proBNP peak measure from the troponin or NT- proBNP test data; b) risk of CHD from the troponin or NT-proBNP test data; and, c) suitability for a treatment, treatment efficiency and treatment strategy from the troponin or NT-proBNP test data.

In a second aspect the invention pertains to a computer comprising a processor and memory, the processor being arranged to read from said memory and write into said memory, the memory comprising data and instructions arranged to provide said processor with the capacity to perform a method according to the invention. Preferably, the computer has an input connected to a sample analyzer for receiving analysis data signals of troponin and NT-proBNP in body fluid samples obtained from a subject and wherein the processor is arranged for determining from said analysis data signals at least for a diagnosis, risk, susceptibility to therapy or treatment monitoring in accordance with a method of the invention.

In a third aspect, the invention relates to a sample analyzer comprising a computer according to the second aspect, wherein the sample analyzer comprises means for measuring the concentration of at least one of a cardiac troponin and a BNP-type peptide or variants thereof in a body fluid sample.

In a fourth aspect, the invention relates to a computer program product comprising data and instructions and arranged to be loaded in a memory of a computer, the data and instructions being arranged to provide said computer with the capacity to perform a method according to the invention.

In a fifth aspect, the invention relates to a data carrier provided with a computer program product according to the fourth aspect.

Description of the invention

Coronary microembolization

Mild forms of rupture of an atherosclerotic plaque in an epicardial coronary artery may result in the washout of atherothrombotic debris in the coronary microcirculation and its subsequent microembolization. (Heusch et al. Circulation. 2009; 120: 1822-1836.)) Troponin may be released from necrotic cells during ischemia or from the cytosolic pool of myocytes in the situation of reversible injury caused by microembolization. (White, JACC 201 1 57;2406-8). Microembolization may also cause transient depression of the left ventricle (transient LV dysfunction).. This is mediated by activation of cytokines (TNF-alpha, interleukin-6) which via a negative inotropic effect on myocytes may increase the levels of BNP and NT-proBNP by increasing BNP gene expression. Because interleukin-6 also regulates the production of CRP, this mechanism may also cause upregulation of the production of CRP.(Thielmann et al. Circ Res 2002;90:807-13.)

LV structural and functional alterations

LV structural and functional alterations relate to abnormalities in LV mass, LV dimensions or LV function as can be determined by echocardiography or other imaging methods. Silent plaque rupture and transient LV dysfunction due to coronary microembolization may occur in subjects without pre-existing LV structural and functional alterations or in subjects at any level of such LV alterations.

Use of individual patterns of markers

The principle of the method of the invention is that individual patterns of markers will be recorded. These are graphs of the biomarker against time for each subject. From these patterns a number of summary measures can be obtained. A summary measure is a single numbers which summarizes an aspect of that subject's pattern. The use of summary measures to describe and analyze serial measurements is potentially a useful and simple tool in medical research. Two main ways are distinguished in which a marker can change over time (1 ) the biomarker starts from a baseline (sometimes zero), rises to a peak, and then returns to baseline. This is displayed as a peaked curve (2). The biomarker steadily increases or decreases with time and does not start to return to its initial value during the period of measurement. This is displayed as a growth curve. Both peak and growth curves are described by Matthews et al. (Br. Med. J. 1990, 300:230-235), which is incorporated by reference herein. Usual summary measures for a peaked curve pattern are maximum peak height, peak width, and the number of peaks. Usual summary measures for a growth pattern are the rate at which the biomarker changes. A good measure of this rate will often be the slope of a line fitted to the data. In some circumstances, for example when the data tend to level off over the last few values, the difference from baseline provides a good summary measure. Description of the embodiments

In a first aspect, the invention relates to a method of diagnosing silent cardiac impairment in a subject. The method preferably comprises the steps of: a) continual measuring in body fluid samples obtained from the subject the concentration of at least one cardiac troponin or a variant thereof; b) continual measuring in body fluid samples obtained from the subject the concentration of a BNP-type peptide or a variant thereof; and, c) diagnosing silent cardiac impairment in a subject. In the method of the invention, preferably the silent cardiac impairment can be diagnosed in a subject as due to a silent coronary plaque rupture by detection of a peak in the concentration of at least one cardiac troponin or a variant thereof. In addition, in the method of the invention, preferably the silent cardiac impairment can be diagnosed as due to transient left ventricular (LV) dysfunction by detection of a peak in the concentration of a BNP type peptide or a variant thereof. In addition, in the method of the invention, preferably the silent cardiac impairment can further be diagnosed in a subject as due to a (subclinical) alteration in at least one of the left ventricular (LV) structure and function, by detection of a gradual increase in the concentration of a BNP-type peptide or a variant thereof, preferably by detection of an increase from baseline level of the concentration of a BNP- type peptide or a variant thereof, whereby the concentration does not return to its initial baseline value. Preferably, the BNP-type peptide concentration does not return to its initial baseline value until after at least 2 or 4 weeks or at least 2, 3, 4, or 8 months.

Continual is herein understood to have its ordinary meaning, i.e. Occurring frequently, with intervals between'. The continual measuring of biomarkers in body fluid samples obtained from the subject in the method of the invention thus means that the concentration of the biomarkers is measured in more than one different samples, which samples are frequently obtained from the subject, with intervals between the obtaining of the different samples. The different sample are thus obtained at different time points. Preferably, in the method of the invention, the continual measuring comprises measuring at least two samples obtained at a time interval of less than 7, 6, 5, 4, 3, 2 or 1 day(s). More preferably, the continual measuring comprises measuring serial samples obtained at a time interval of less than 7, 6, 5, 4, 3, 2 or 1 day(s). Most preferably two or 1 days.

In one embodiment the continual measuring comprising serial samples obtained at a time interval of less than 1 day, for example 2, 3, 4, 5, 6 or more times per day.

Preferably, in the method of the invention, the continual measuring of the biomarkers is carried out over a period of at least 1 , 2 or 4 weeks, or over a period of at least 1 , 2, 4, 8 months, or over a period of at least 1 , 2, 4 or 8 years or more. In one embodiment, the continual measuring of the biomarkers in the method of the invention is carried out at least for a period of at least 1 , 2 or 4 weeks, or over a period of at least 1 , 2, 4, 8 months, or over a period of at least 1 , 2, 4 or 8 years or more that the subject has not been diagnosed with a silent cardiac impairment, whereby preferably the absence of a silent cardiac impairment is diagnosed in accordance with the method of the invention.

In one embodiment the frequency of testing is more than once a day. In another alternative embodiment the time interval between testing is 7 days or more. In one embodiment the testing is continual meaning fine-temporal testing with periods without testing in between. These periods can be 1 , 3, 6, or 9 months or 1 , 2, 3 or more years.

Diagnosis of a silent cardiac impairment as due to a silent coronary plaque rupture

In the method of the invention, the silent cardiac impairment is preferably diagnosed in a subject as due to a silent coronary plaque rupture by detection of a peak in the concentration of at least one cardiac troponin or a variant thereof. It is understood herein that the silent cardiac impairment diagnosed in a subject as due to a silent coronary plaque rupture is a minor myocardial injury, which causes and is thereby detected by means of a peak in the concentration of at least one cardiac troponin or a variant thereof. Diagnostic and classification criteria for minor myocardial injury due to silent plaque rupture are based on levels and patterns in troponin. Biomarker changes for minor myocardial injury due to silent plaque rupture are considered to follow a peaked pattern. Peaks are considered to occur as single peaks. However they may occur in a cluster, sometimes superimposed on another reflecting microembolizations from a single plaque rupture. Detection of peaks is primarily based on the pattern of troponin values. In the method of the invention, silent plaque rupture is diagnosed to be present when at least one peak is detected with a height of at least 10, 20, 30, 40, or 50 ng/L. A peak is defined by the presence of a steep rising and somewhat slower falling pattern. The rising pattern should be seen within one day if this is the frequency of testing. Otherwise, the rise should take place in two days. The falling pattern is somewhat slower and should be seen within in one or two days after maximum peak.

In one embodiment, a troponin peaks is described in the following way. In the method of the invention, a troponin pattern is a temporal troponin value sequence. A troponin peaked curve is considered to be present when the troponin level starts from a baseline or initial value, rises to a maximum peak value within one day, and then returns to the baseline or initial value in 1 , 2, or 3 days. Preferably, a peaked curve is detected when consecutive samples are obtained with intervals of no more than 2 days and the concentration of troponin increases by at least 5, 10, 20, 30, 40, 50, or 100 ng/L, from baseline in 2 days and returns to baseline in no more than 3 days. More preferably, a peak is detected when consecutive samples are obtained with intervals of no more than 1 day and the concentration of troponin increases by at least 5, 10, 20, 30, 40 or 50, or 100 ng/L from baseline in 1 day and returns to baseline in no more than 1 , 2 or 3 days.

Preferably, a peaked curve is detected when consecutive samples are obtained with intervals of no more than 3 days and the concentration of troponin increases by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50 or 100 ng/L from baseline in 3 days or less.

Preferably, a peaked curve is detected when consecutive samples are obtained with intervals of no more than 7 days and the concentration of troponin increases by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 17, 16, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50 or 100 ng/L from baseline in 7 days or less.

Therefore in the method of the invention, a peak is detected when the concentrations of at least one cardiac troponin or a variant thereof in 2 or 3 consecutive samples differ each by at least 5, 10, 20, 30, 40 or 50 ng/L. Preferably, a peak is detected when the concentrations of at least one cardiac troponin or a variant thereof in 2 consecutive samples differ by at least 1 , 2, 3, 4, 5, 6, 7, 8,

9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50 or 100 ng/L.

Preferably, a peak is detected when consecutive samples are obtained with intervals of no more than 2 days and the concentration of at least one cardiac troponin or a variant thereof increases by at least 5, 10, 20, 30, 40 or 50 ng/L from baseline in 2 days and returns to baseline in no more than 4 days. More preferably, a peak is detected when consecutive samples are obtained with intervals of no more than 1 day and the concentration of at least one cardiac troponin or a variant thereof increases by at least 5, 10, 20, 30, 40 or 50 ng/L from baseline in 1 day and returns to baseline in no more than 2 days. Baseline level is defined as the level of troponin measured at least two successive points in time where these successive points represent close values, e.g. differing by no more than 2, 5, 10, 20 or 30%. Baseline and reference amounts may further be determined and used as described below or in WO 2015/171989.

In one embodiment the baseline level is computed in the following way. The baseline level can for example be measured as the level of troponin measured at at least two successive points in time where these successive points represent close values, e.g. differing by no more than 2, 5,

10, 20, or 30%. Alternatively a long-term or longitudinal baseline level of a person may be computed based on more measurements of the subject. This is especially useful when successive peaks are present and observed levels do not immediately go back to baseline (but do eventually return to baseline and do not constitute a gradual increasing pattern) or when peaks may be superimposed on one another. It is to be understood that the baseline level of an individual does not necessary need to be a low level but can be any level at which a subject sustains for some period of time. E.g. a subject with left ventricular structural alterations or abnormalities may have a high baseline troponin level.

In one embodiment, the method of the invention further comprises classification of (the diagnosis of) minor myocardial injury due to plaque rupture. In the classification, classes of severity, duration and/or frequency of the peaks of the peaks are made with peaks based on the concentration of the cardiac troponin as detected in the method of the invention. Severity is defined as the maximum peak height. Peak height is measured as the difference from baseline level to the maximum level obtained. Severity is classified in the following categories: 10-20, 20-30, 30-40, 40- 50, 50-100, 100-200, >200 ng/l. Duration is the peak width. When peaks are superimposed on each other it is the width of the cluster of peaks. Duration is classified in the following categories: 1 , 2, 3 or 4 days, or for clusters of peaks 1 week or 2 weeks. Frequency is the number of peaks for a unit of time. When peaks form clusters or are superimposed on each other it is the number of peak clusters. Frequency is classified in the following categories: once per week, once per month, once per 6 months or once per year. It is understood that the more severe, the longer the duration and the more frequent, the higher the classification and that the higher classification, the higher the risk of coronary heart disease and cardiovascular disease, and more a subject is susceptible for therapy against coronary heart disease. In one embodiment cumulative measures of troponin peaks are computed using classes of severity, duration and frequency.

It is understood that the more severe, the longer the duration and the more frequent, the higher the classification and that the higher classification, the higher the risk of coronary heart disease and cardiovascular disease, and the more a subject is susceptible for therapy against coronary heart disease. Frequency is the number of peaks per week or the number of peaks per month.

An important way to quantitatively assess troponin peaks is to use information from peak height, for example maximum peak height. Another example is the mean or median of peak heights. In one embodiment, peak height is measured as the difference from baseline level to the maximum level reached in a peaked curve, which is scored in the following categories: 5-10, 10-20, 20-30, 30-40, 40-50, 50-100, 100-200, 200-500, 500-1000 and >1000 ng/L. However, as troponin is not measured continuously, the maximum level observed may reflect a point on the upslope or downslope of a peaked curve rather than the true maximum of a peaked curve. Therefore, another important way to quantitatively assess the extent of troponin peaks is to measure peak frequency, which is the number of peaks per unit of time. In one embodiment, the features of peak height and peak frequency are used alone or combined to compose a rating scale. Using such a scale, a subject is determined to have an increased risk of CHD if the rating exceeds a threshold level. The use of the scale at this threshold should identify subjects with an increased risk of CHD with the highest accuracy.

Troponin peaks should be distinguished from the well-known rise and fall pattern in troponin during an acute myocardial infarction. The method of the present invention relates to the detection of troponin peaks outside the acute setting of an ACS. In most situations therefore troponin peaks detected by the method of this invention will be clinically silent and not be accompanied by symptoms of chest pain..

Measurement of high-sensitivity troponin is preferred in the method of this invention, but the method is not limited to use of high-sensitivity troponin. Measurement of troponin-l is preferred in the method of this invention because of its better performance in earlier presenters of myocardial infarction. However, measurement of troponin-T in the method of this invention is not excluded. Diagnosis of a silent cardiac impairment as due to transient left ventricular (LV) dysfunction

In the method of the invention, the silent cardiac impairment is preferably diagnosed in a subject as due to transient LV dysfunction by detection of a peak in the concentration of a BNP type peptide or a variant thereof.

In the method of the invention, transient LV dysfunction is diagnosed to be present when at least one peak is detected with a height of at least 50, 100, 200, 300, 400, or 500 ng/L. A peak is defined by the presence of a steep rising and somewhat slower falling pattern. The rising pattern should be seen within one day if this is the frequency of testing. Otherwise, the rise should take place in two days. The falling pattern is somewhat slower and should be seen within in one or two days after maximum peak.

Therefore in the method of the invention, a peak is detected when the concentrations of a BNP type peptide or a variant thereof in 2 or 3 consecutive samples differ each by at least 50, 100, 200, 300, 400 or 500 ng/L. More preferably, a peak is detected when the concentrations of a BNP type peptide or a variant thereof in 2 consecutive samples differ by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 ng/L. Preferably, a peak is detected when consecutive samples are obtained with intervals of no more than 2 days and the concentration of a BNP type peptide or a variant thereof increases by at least 50, 100, 200, 300, 400 or 500 ng/L from baseline in 2 days and returns to baseline in no more than 4 days. More preferably, a peak is detected when consecutive samples are obtained with intervals of no more than 1 day and the concentration of a BNP type peptide or a variant thereof increases by at least 50, 100, 200, 300, 400 or 500 ng/L from baseline in 1 day and returns to baseline in no more than 2 days.

A peaked curve preferably is detected when consecutive samples are obtained with intervals of no more than 2 days and the concentration of NT-proBNP increases by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 ng/L from baseline in 2 days or less. More preferably, a peaked curve is detected when consecutive samples are obtained with intervals of no more than 7 days and the concentration of NT-proBNP increases by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 ng/L from baseline in 7 days or less.

Baseline level is defined as the level of BNP type peptide measured at least two successive points in time where these successive points represent close values, e.g. differing by no more than 2, 5, 10, 20 or 30%. Baseline and reference amounts may further be determined and used as described below or in WO 2015/171989.

In one embodiment, the method of the invention further comprises classification of (the diagnosis of) minor myocardial injury due to plaque rupture.

In one embodiment, the method of the invention further comprises classification of (the diagnosis of) minor myocardial injury due to transient ventricular dysfunction. In the classification, classes of severity and/or duration and/or frequency of the peaks are made with peaks based on the concentration of the BNP type peptide or a variant thereof as detected in the method of the invention. Severity is defined as the maximum peak height. Peak height is measured as the difference from baseline level to the maximum level obtained. Severity is classified in the following categories: 50-100, 100-200, 200-300, 300-400, 400-500, 500-1000, >1000 ng/L. Duration is the peak width. When peaks are superimposed on each other it is the width of the cluster of peaks. Duration is classified in the following categories: 1 , 2, 3 or 4 days, or for clusters of peaks 1 week or 2 weeks. Frequency is the number of peaks for a unit of time. When peaks form clusters or are superimposed on each other it is the number of peak clusters. Frequency is classified in the following categories: once per week, once per month, once per 6 months or once per year. It is understood that the more severe, the longer the duration and the more frequent, the higher the classification and that the higher classification, the higher the risk of coronary heart disease and cardiovascular disease, and the more a subject is susceptible for therapy against coronary heart disease and cardiovascular disease. In one embodiment cumulative measures of NT-proBNP peaks are computed using classes of severity, duration and/or frequency.

It is understood that the more severe, the longer the duration and the more frequent, the higher the classification and that the higher classification, the higher the risk of heart failure and cardiac death, and more a subject is susceptible for therapy against heart failure and cardiac death. In one embodiment cumulative measures of NT-proBNP peaks are computed using classes of severity, duration and frequency.

In the Examples herein it was shown that the troponin peaks and the troponin peaks combined with NT-proBNP peaks as could be characterized in the study were present more frequently in stable CAD patients that developed a new ACS event as compared to stable CAD patients that did not develop a new ACS event. Therefore, in the method of the invention, a silent cardiac impairment is a silent myocardial injury preceding coronary heart disease. Thus, with a silent myocardial injury referred to in this way in the application is meant a silent myocardial injury that is associated with an increased risk of coronary heart disease.

In another embodiment a silent myocardial injury preceding coronary heart disease is detected when the troponin level exceeds a first predetermined threshold and troponin change exceeds a second predetermined threshold , and at least one measured NT-proBNP concentration exceeds a third predetermined threshold and NTproBNP change exceeds a fourth predetermined threshold . In this embodiment the following thresholds can be used:

The first predetermined threshold is at least 1 , 2, 3, 4, 5, 10, 20, 30, 40 or 50 ng/l.

The second predetermined threshold is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 ng/L.

The third predetermined threshold is at least 10, 25, 50,100, 200, 300 ,400 ,500, 600, 700, 800, 900 or 1000 ng/l.

The fourth predetermined threshold is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400 or 500ng/L. Diagnosis of a silent cardiac impairment as due to a subclinical alteration in the left ventricular (LV) structure and/or function

In addition, in the method of the invention, a silent cardiac impairment is preferably diagnosed in a subject as due to a subclinical alteration in at least one of the left ventricular (LV) structure and function. The alteration in at least one of the left ventricular (LV) structure and function can be a clinical but will usually be a subclinical alteration. Preferably, a silent cardiac impairment is diagnosed in a subject as due to an alteration in left ventricular (LV) structure and/or function, by detection of a gradual increase in the concentration of a BNP-type peptide or a variant thereof. Preferably, the detection of an increase in the concentration of a BNP-type peptide or a variant thereof is detection of an increase from baseline level of the concentration of a BNP-type peptide or a variant thereof, the concentration does not return to its initial baseline value. The subclinical alteration in the left ventricular (LV) structure and/or function will usually be a gradual decrease of the left ventricular (LV) structure and/or function.

Detection of an increase in the concentration the BNP-type peptide means that preferably an increase is detected greater than 10, 15, 20, 25, 30, 40, 50, 60 or 75% from baseline. Alternatively, detection of an increase in the concentration of the BNP-type peptide means that preferably an increase is detected greater than 10, 15, 20, 25, 30, 40, 50, 60 or 75% compared a reference amount of the BNP-type peptide in healthy individuals, preferably compared to age and gender matched controls. Baseline and reference amounts may further be determined and used as described herein or in WO 2015/171989.

Preferably, in the method of the invention, a silent cardiac impairment is diagnosed in a subject as due to a subclinical alteration in left ventricular (LV) structure and/or function, by detection of an increase from baseline in the concentration of a BNP-type peptide or a variant thereof of at least 50 ng/L, or the cut-off can be a concentration of a BNP-type peptide or a variant thereof of at least 50, 75, 100, 150, 200 or 300 ng/L. It is understood herein that for the diagnosis of a silent cardiac impairment as due to a subclinical alteration in left ventricular (LV) structure and/or function, the increase from baseline level of the concentration of a BNP-type peptide preferably is a gradual increase from baseline, e.g. an gradual increase that occurs over the course of at least 1 , 2, 4 weeks or 2, 4, or 8 months or longer. However, in order to distinguish an gradual increase from a peak pattern, the increase from baseline level of the concentration of a BNP-type peptide or a variant thereof is preferably only used for the diagnosis of a subclinical alteration in left ventricular (LV) structure and/or function if the concentration of the BNP-type peptide or a variant thereof does not return to its initial baseline value, or at least does not return to its initial baseline value within at least 2 or 4 weeks or at least 2, 3, 4, or 8 months or longer after the onset of the increase.

Diagnostic methods

The term "sample" herein 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 without limitation, samples of blood, plasma, serum, urine, samples of blood, plasma or serum. It is to be understood that the sample depends on the marker to be determined. Therefore, it is encompassed that the polypeptides as referred to herein are determined in different samples. Cardiac troponins and BNP-type peptides are preferably determined in body fluid samples, such as e.g. a blood, blood serum or blood plasma sample.

The methods of the present invention, in some embodiments, are in vitro methods. In some embodiments, the methods can 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 methods of the present invention may be also used for monitoring, confirmation, subclassification and differentiation of (a) subject(s) in need of therapy and/or cardiac intervention. The method may be carried out manually or assisted by automation. In some embodiments, one or more steps of the methods may in total or in part be assisted by automation, e.g., by a suitable robotic and sensory equipment for any obtaining results steps or a computer-implemented step to compare the levels of the biomarkers values to control or reference values and/or to assess their behavior in time.

The term "diagnosing" as used herein means identifying the cause of the silent myocardial impairment, i.e. the underlying disorder or disease condition resulting in the apparent silent myocardial impairment and preferably differentiating between underlying disorder or disease condition being silent coronary plaque rupture, transient LV dysfunction or left ventricular (LV) structural or functional alterations

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.

Cardiac biomarkers

The method of the present invention makes use of so-called "markers", "biomarkers" or "molecular markers". These terms are known to the person skilled in the art and refer to polypeptides or proteins which are expressed in the body of the subject. On the one hand, the expression or elevated expression can be the consequence of a pathophysiological state which has occurred or is occurring in the subject, and an elevated concentration, in respect to "normal" values (which, as the case may be, can be zero) measured in a physiologically healthy subject, is indicative of the pathophysiological state (or the "disease") occurring in the subject. On the other hand, the protein can be expressed in certain concentrations in physiologically healthy subjects, and the expression is raised in consequence of a pathophysiological state which has occurred or is occurring in the subject. In the context of the present invention, the markers which are measured usually belong to the first group, i.e. they are present in higher concentrations than normal if the subject suffers from a pathophysiological state or disease. All the marker types and markers employed in the present invention are known to the person skilled in the art.

Cardiac troponins

The present invention makes use of cardiac troponins and variants thereof. It is known that patients suffering from myocardial infarction (Ml) can be diagnosed using cardiac troponins, preferably troponin T or I, most preferably troponin I.

The term "cardiac Troponin" refers to all Troponin isoforms expressed in cells of the heart and, preferably, the subendocardial cells. These isoforms are well characterized in the art as described, e.g., in Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44; 487-493. Preferably, cardiac Troponin refers to Troponin T and/or Troponin I, and, most preferably, to Troponin T. It is to be understood that isoforms of Troponins may be determined in the method of the present invention together, i.e. simultaneously or sequentially, or individually, i.e. without determining the other isoform at all. Amino acid sequences for human Troponin T and human Troponin I are disclosed in Anderson, (1995, supra) and Ferrieres (1998, supra). The term "cardiac Troponin" encompasses also variants of the aforementioned specific Troponins, i.e., preferably, of Troponin T, and more preferably, of Troponin I. Such variants have at least the same essential biological and immunological properties as the specific cardiac Troponins. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification, e.g., by ELISA Assays using polyclonal or monoclonal antibodies specifically recognizing the said cardiac Troponins. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition wherein the amino acid sequence of the variant is still, preferably, at least 50, 60, 70, 80, 85, 90, 92, 95, 97, 98, 99 or 100% identical with the amino sequence of the specific Troponin. "Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical" or "essentially similar" when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program "needle" (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for 'needle' and for 'water' and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred.

Variants may be allelic variants or any other species specific homologs, paralogs, or orthologues. Moreover, the variants referred to herein include fragments of the specific cardiac Troponins or the aforementioned types of variants as long as these fragments have the essential immunological and biological properties as referred to above. Preferably, the cardiac troponin variants have immunological properties (i.e. epitope composition) comparable to those of human troponin T or troponin I. Thus, the variants shall be recognizable by the aforementioned means or ligands used for determination of the concentration of the cardiac troponins. Thus, the variants shall be recognizable by the aforementioned means or ligands used for determination of the concentration of the cardiac troponins. Such fragments may be, e.g., degradation products of the Troponins. Further included are variants which differ due to posttranslational modifications such as phosphorylation or myristylation. Preferably the biological property of troponin I and its variant is the ability to inhibit actomyosin ATPase or to inhibit angiogenesis in vivo and in vitro, which may e.g. be detected based on the assay described by Moses et al. 1999 PNAS USA 96 (6): 2645- 2650). Preferably the biological property of troponin T and its variant is the ability to form a complex with troponin C and I, to bind calcium ions or to bind to tropomyosin, preferably if present as a complex of troponin C, I and T or a complex formed by troponin C, troponin I and a variant of troponin T.

Brain natriuretic peptide (BNP)-type peptides

The present invention further makes use of Brain natriuretic peptide (BNP)-type peptides and variants thereof. The term "brain natriuretic peptide (BNP)-type peptide" or "BNP-type peptide" relates to pre-proBNP, proBNP, NT-proBNP, and BNP and variants thereof (see e.g. Bonow, 1996, Circulation 93: 1946-1950). Specifically, the aforementioned pre-pro peptide of the brain natriuretic peptide (having 134 amino acids in length) comprises a short signal peptide, which is enzymatically cleaved off to release the pro peptide (108 amino acids). The pro peptide is further cleaved into an N-terminal pro peptide (NT-pro peptide, 76 amino acids) and the active hormone (32 amino acids). BNP is metabolized in the blood, whereas NT-proBNP circulates in the blood as an intact molecule and as such is eliminated renally. The in-vivo half-life of NT-proBNP is 120 min longer than that of BNP, which is 20 min (Smith 2000, J Endocrinol. 167: 239-46.). Preanalytics are more robust with NT-proBNP allowing easy transportation of the sample to a central laboratory (Mueller 2004, Clin Chem Lab Med 42: 942-4.). Blood samples can be stored at room temperature for several days or may be mailed or shipped without recovery loss. In contrast, storage of BNP for 48 hours at room temperature or at 4° Celsius leads to a concentration loss of at least 20 % (Mueller loc.cit.; Wu 2004, Clin Chem 50: 867-73.). Therefore, depending on the time-course or properties of interest, either measurement of the active or the inactive forms of the natriuretic peptide can be advantageous. The most preferred natriuretic peptides according to the present invention are NT- proBNP or variants thereof. As briefly discussed above, the human NT-proBNP, as referred to in accordance with the present invention, is a polypeptide comprising, preferably, 76 amino acids in length corresponding to the N-terminal portion of the human NT-proBNP molecule. The structure of the human BNP and NT-proBNP has been described already in detail in the prior art, e.g., WO 02/089657 and WO 02/083913. Preferably, human NT-proBNP as used herein is human NT- proBNP as disclosed in EP 0 648 228 B1. These prior art documents are herewith incorporated by reference with respect to the specific sequences of NT-proBNP and variants thereof disclosed therein. The NT-proBNP referred to in accordance with the present invention further encompasses allelic and other variants of said specific sequence for human NT-proBNP discussed above. Specifically, envisaged are variant polypeptides which are on the amino acid level preferably, at least 50, 60, 70, 80, 85, 90, 92, 95, 97, 98, 99 or 100% identical to human NT-proBNP, preferably over the entire length of human NT-proBNP. Variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologues. Substantially similar and also envisaged are proteolytic degradation products which are still recognized by the diagnostic means or by ligands directed against the respective full-length peptide. Also encompassed are variant polypeptides having amino acid deletions, substitutions, and/or additions compared to the amino acid sequence of human NT-proBNP as long as the said polypeptides have NTproBNP properties. NT-proBNP properties as referred to herein axe immunological and/or biological properties. Preferably, the NT-proBNP variants have immunological properties (i.e. epitope composition) comparable to those of human NT-proBNP. Thus, the variants shall be recognizable by the aforementioned means or ligands used for determination of the amount of the natriuretic peptides. Biological and/or immunological NT-proBNP properties can be detected by the assay described in Karl et al. (Karl 1999, Scand J Clin Lab Invest 230: 177-181 ), Yeo et al. (Yeo 2003, Clinica Chimica Acta 338: 107-1 15). Variants also include posttranslationally modified peptides such as glycosylated peptides. Further, a variant in accordance with the present invention is also a peptide or polypeptide which has been modified after collection of the sample, for example by covalent or noncovalent attachment of a label, particularly a radioactive or fluorescent label, to the peptide.

Assays for measuring the concentration of cardiac biomarkers

Obtaining the results of an assay measuring or monitoring levels of a BNP-type peptide, such as NT-proBNP, and/or a cardiac troponin, such as troponin I and/or T, referred to in this specification encompasses measuring the amount or concentration, preferably semi-quantitatively or quantitatively. In some embodiments, the step of obtaining the results of an assay measuring or monitoring levels of the BNP-type peptide and/or cardiac troponin can encompass ordering a third party to perform the assay or using the results obtained from a third party without directing the third party to perform the measurements, for example, if the results are already available. 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 disclosure, determining the amount of a peptide or polypeptide can be achieved by all known means for determining the amount of a peptide or polypeptide 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, in some embodiments, can 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, in some embodiments, 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 (available for example on Elecsys™ analyzers), CBA (an enzymatic Cobalt Binding Assay, available for example on Roche-Hitachi™ analyzers), and latex agglutination assays (available for example on Roche- Hitachi™ analyzers).

In some embodiments, the levels of cardiac troponin T and I are measured with a high sensitive (hs) assay (hs cTnT or hs cTnl) as e.g. described by Sherwood and Newby (J Am Heart Assoc. 2014 Jan 27;3(1 ):e000403. doi: 10.1 161/JAHA.1 13.000403). In some embodiments, the levels of cardiac troponin T, I and /or NT-proBNP are measured with an antibody-based assay. In some embodiments, cardiac troponin T, I and /or NT-proBNP are measured using a sandwich immunoassay. In some embodiments, cardiac troponin T, I and /or NT-proBNP can be measured on the Elecsys 2010 analyzer (Roche Diagnostics, Indianapolis, Indiana). deFilippi et al. J Am Coll Cardiol. 2010;55:441-450; deFilippi et al. Jama. 2010;304:2494-2502; Giannitsis et al. Clin Chem. 2010;56:254-261 . In some embodiments, cardiac troponin T, I and /or NT-proBNP can be measured on the Cobas e 41 1 (e 41 1 ) analyzer and the Cobas e 601 (e601 ) module (Roche Diagnostics, Indianapolis, Indiana). The 2010, e601 and e601 measure cardiac troponin T, I and NT-proBNP using the principle of sandwich immunoassay and ElectroQieiniLuminescence (ECL) technology. The principle for these systems is formation of a 'sandwich' immunoassay complex in which antigen analyte (either cardiac troponin T, I or NT-proBNP) is bound by two monoclonal antibodies, each targeting a different epitope location on the antigen analyte molecule. One of the monoclonal antibodies is bound to the substance biotin; the other analyte specific monoclonal antibody to a different epitope location is labeled with a Ruthenium complex for detection. During an initial incubation period, these two monoclonal antibodies are mixed with a sample containing the analyte (cardiac troponin T, I or NT-proBNP). Because each antibody has high affinity for a different epitope on. the analyte molecule, a <biotinylated antibody-analyte-Ruthenium antibody> sandwich complex is formed during the initial incubation. In a second step, streptaviden coated paramagnetic beads are added to the same measurement cell and incubated. Because streptavidin and biotin form one of the strongest, most resilient non covalent bond in nature, the paramagnetic beads serve to capture the immune complex sandwich containing analyte (cardiac troponin T, I or NT-proBNP). The reaction mixture is aspirated into a reaction, cell where a magnetic field is applied, which causes the magnetic beads to bind to the surface of the measurement cell. Unbound substances are then removed by treatment with a solution (ProCell/ProCeil M). The solution also provides tri-n- propylamine, which is essential for the ECL reaction. Application of a voltage to the electrode induces chemiluminescent emission, which is measured by a photomultiplier tube. The concentration of each analyte (cardiac troponin T, I or NT-proBNP) in samples is determined from a calibration curve which is instrument-specifically generated by a two point calibration a master curve provided by the reagent barcode.

Determining the amount of cardiac troponin T, I and/or NT-proBNP peptides may, in some embodiments, comprise 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 disclosure includes both covalent and non-covalent binding. A ligand according to the present disclosure can be any compound, e.g., a peptide, polypeptide, nucleic acid, aptamer or small molecule, binding to the cardiac troponin T, I and/or NT-proBNP peptides described herein. Suitable ligands include antibodies, nucleic acids, aptamers, peptides or polypeptides such as receptors or binding partners for the cardiac troponin T, I and/or NT-proBNP peptides 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 disclosure also includes single chain antibodies, camelid antibodies and VHH fragments thereof, chimeric antibodies and humanized antibodies as can be prepared by methods well known in the art. Suitably, the ligand or agent binds specifically to the cardiac troponin T, I and/or NT-proBNP peptides. Specific binding according to the present disclosure 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. Suitably, the specifically bound cardiac troponin T, I and/or NT-proBNP peptides should be bound with at least 3 times higher, in some embodiments, at least 10 times higher and even in some embodiments 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. In some embodiments, said method is semi-quantitative or quantitative. Suitable methods are described in the following.

The term "amount" as used herein encompasses the absolute amount of a cardiac troponin T, I and/or NT-proBNP peptide, the relative amount or concentration of the said 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 subject to be diagnosed

The terms "subject" or "patient" as used herein refer to a mammal, preferably a human. In the method of the invention, the subject can be a male or a female. Preferably, in the method of the invention, the subject is selected from the group consisting of: a) an asymptomatic subject; b) an asymptomatic subject belonging to a risk group for coronary heart disease, heart failure or cardiovascular disease; c) a subject diagnosed with stable coronary artery disease (CAD); and, d) a subject having sufferered from an acute coronary syndrome (ACS).

In the method of the invention, the subject can be an asymptomatic subject, however, preferably the subject is an asymptomatic subject belonging to a risk group for coronary heart disease, heart failure or cardiovascular disease, including heart failure and coronary heart disease. The risk group preferably is a risk group which includes at least subjects suffering from hypertension, diabetes, obesity, metabolic syndrome, high cholesterol and subjects having a history of smoking. The risk group can e.g. be a risk group including subjects that have has been determined to be at risk for cardiovascular disease based on risk factors, such as, but not limited to, Framingham risk factors. The risk group can further include subjects that have been determined to be at risk for cardiovascular disease using risk factors, such as, but not limited to, Framingham risk factors and/or guidelines jointly issues by the American Heart Association and American College of Cardiology. Most preferably the risk group is as defined in accordance with prevailing European and/or US guidelines (e.g. 2013 ACC/AHA Guideline on the Assessment of Cardiovascular Risk, http://dx.doi.org/10.1 161/01.cir.0000437741.48606.98).

In another embodiment of the method of the invention, the subject is a subject diagnosed with stable coronary artery disease (CAD) or a subject having suffered from an acute coronary syndrome (ACS).

Coronary artery disease or coronary heart diseases is herein understood to include stable CAD, acute coronary syndrome (unstable angina), myocardial infarction, and sudden cardiac death. In the method of the invention, a subject determined to have an increased risk of coronary heart disease is thus a subject having an increased risk of at least one of stable CAD, acute coronary syndrome, myocardial infarction, and sudden cardiac death.

In the method of the invention, a subject in the condition of a stable CAD is typically a subject with angiographically proven CAD, i.e. with affected coronary arteries, with plaques on the inner walls of the coronary artery (atherosclerosis) and stenosis in a major coronary artery. CAD is considered a serious cardiac risk. CAD patients are considered as "stable" if the CAD does not manifest itself in the form of acute cardiovascular events.

In the method of the invention, a subject can also be a subject having suffered from an acute coronary syndrome (ACS). The term "acute coronary syndrome" encompasses various phases of coronary heart disease which are immediately life-threatening. This concerns in particular emergency medical care, specifically, acute myocardial infarction and/or angina pectoris, as well as sudden cardiac death. In addition to acute myocardial infarction, which according to WHO criteria (WHO (1979): Nomenclature and criteria for diagnosis of ischemic heart disease, Report of the Joint International Society and Federation of Cardiology/World Health Organization task force on standardization of clinical nomenclature, Circulation 59 (3): 607- 609) is defined as an acute chest pain event lasting longer than 20 minutes in conjunction with ST segment elevation and/or an increase in myocardial enzymes, the term "unstable angina pectoris" (AP) has become established, which according to the invention is interpreted as "acute coronary syndrome" (Hamm C. W.: Leitlinien: Akutes coronary syndrome (ACS)— Teil 1 : ACS ohne persistierende ST-Hebung [Guidelines: Acute coronary syndrome (ACS)-Part 1 : ACS without persistent ST elevation], Z Kardiol (2004) 93: 72-90; see also: Pschyrembel, De Gruyter, Berlin 2004). Preferably, in the method of the invention, the subject having suffered from an ACS is a subject that has suffered from an ACS at least 30, 40, 50 or 60 days ago and/or subject having suffered from an ACS after having been discharged from hospital (where the subject was treated for the ACS).

Methods for determining the risk of at least one of coronary artery disease and heart failure

In a further aspect, the invention relates to a method for determining the risk of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death in a subject. In this aspect, the above method of the invention of diagnosing a silent cardiac impairment in a subject are used for determining the risk of the subject of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death in the subject. In one embodiment of the above method of the invention of diagnosing a silent cardiac impairment in a subject, the method further comprises the step of determining the risk of the subject of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death. In this method, when a subject is diagnosed with at least one of silent coronary plaque rupture and transient LV dysfunction, the subject is determined to have an increased risk of coronary heart disease and cardiovascular disease. Coronary heart disease is as herein defined above. In view of the imminent risk of future cardiovascular events it is desirable to be able to distinguish within the group of patients with stable CAD between different groups according to their personal cardiac risk such that an "individual state of alert" can be determined for a particular patient in accordance with the risk group to which he has been allotted. Such grouping of patients is usually called "stratification". Distinguishing high risk patients from patients at moderate or low risk would allow a better selection of the most appropriate therapeutic strategy for a particular patient, avoiding, for example, underestimation of the cardiac risk and undermedication of high risk patients on the one hand and unnecessary therapeutic interventions, and the associated costs, with low risk patients on the other. In the method of the invention, when a subject is diagnosed with silent coronary plaque rupture, the subject is allotted to a higher risk group concerning the future incidence of cardiovascular events. The method of the invention thus provides a new method by which patients with stable CAD can be stratified in accordance with their personal cardiac risks, i.e. with respect to their individual risks future incidence of cardiovascular events.

However, when in this method a subject is diagnosed with an alteration in LV structure and/or function, the subject is determined to have an increased risk of at least one of heart failure and cardiac death. Heart failure may be symptomatic or asymptomatic. It is known some subjects with asymptomatic heart failure progress more rapidly to chronic heart failure, and thus are at elevated risk of hospitalization due to heart failure and/or death (Neeland et al, Journal of the American College of Cardiology Vol. 61 , No. 2, 2013). It is important to identify these subjects as early as possible since this would allow for therapeutic measures that prevent or delay the progression to chronic heart failure. The terms "heart failure," "HF," "congestive heart failure," or "CHF" as used herein, refer to the complex clinical syndrome that impairs the ability of the ventricle to fill with or eject blood. Any structural or functional cardiac disorder can cause HF, with the majority of HF patients having impaired left ventricular (LV) myocardial function. Symptoms of HF include dyspnea (shortness of breath), fatigue, and fluid retention. The American Heart Association (AHA) has identified 4 stages in the progression or development of HF. Patients in stages A and B show clear risk factors but have not yet developed HF. Patients in stages C and D currently exhibit or in the past have exhibited symptoms of HF. For example, Stage A patients are those with risk factors such as coronary artery disease, hypertension or diabetes mellitus who do not show impaired left ventricular (LV) function. Stage B patients are asymptomatic, but have cardiac structural abnormalities or remodeling, such as impaired LV function, hypertrophy or geometric chamber distortion. Stage C patients have cardiac abnormalities and are symptomatic. Stage D patients have refractory HF in which they exhibit symptoms despite maximal medical treatment. They are typically recurrently hospitalized or unable to leave the hospital without specialized intervention. In another embodiment of the above method of the invention of diagnosing a silent cardiac impairment in a subject, the method further comprises the step of determining the risk of CAD or of cardiovascular disease in at least one of a subject diagnosed with stable CAD and a subject having suffered from an ACS, wherein a diagnosis of silent coronary plaque rupture is indicative of increased risk of CAD or of cardiovascular disease. An increased risk of CAD or of cardiovascular disease includes an increased risk of the future, or recurrent as the case may be, incidence of cardiovascular events, including ACS. In a subject having suffered from an ACS, increased risk of CAD or of cardiovascular disease thus includes increased risk of recurrent events.

In another embodiment, peak detection of troponin may be performed in combination with peak detection of NT-proBNP to identify subjects at high risk of coronary heart disease. The method preferably comprises one or more of the steps of: a) fine-temporal serial testing in body fluid samples obtained from the subject the concentration of NT-proBNP; b) assessment of NT-proBNP peaks in a subject, wherein preferably an cumulative peak measure is computed, and whereby the presence of peaks is indicative for whether a subject is at high risk CHD; c) the NT-proBNP peaks are assessed in an ongoing fashion; In this method of the invention, preferably, a peak of NT- proBNP is detected when consecutive samples are obtained with intervals of no more than 2 days and the concentration of NT-proBNP increases by at least 25, 50, 100, 200, 300, 400, 500 ng/L, preferably by at least 100 ng/L from baseline in 2 days and returns to baseline in 1 , 2, or 3 days.

More preferably, a peak is detected when consecutive samples are obtained with intervals of no more than 1 day and the concentration of NT-proBNP increases by at least 25, 50, 100, 200, 300, 400, 500 ng/L, preferably by at least 100 ng/L from baseline in 1 day and returns to baseline in no more than 1 , 2, or 3 days. Alternatively, a NT-proBNP peak can be defined as described above for the alternative of defining a troponin peak. Incorporation of risk scores in for risk stratification

In one embodiment, the subject may have stable CAD or may have suffered from ACS. In this method of the invention, the method relates to further incorporating person information (age, sex), and risk factors or clinical risk scores designed for subjects with ACS in risk assessment and risk stratification of CHD with troponin peaks. These risk scores are preferably in accordance with prevailing guidelines, e.g. 2014 ACC/AHA/AATS/PCNA/SCAI/STS focused update of the guideline for the diagnosis and management of patients with stable ischemic heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines, and the American Association for Thoracic Surgery, Preventive Cardiovascular Nurses Association, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons. J Am Coll Cardiol. 2014 Nov 4;64(18):1929-49 and 2014 AHA/ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014 Dec 23;64(24):e139-228. In one embodiment, the subject is asymptomatic and may or may not exhibit risk factors for coronary heart disease. In this method of the invention, the method relates to further incorporating person information (age, sex), and risk factors or risk scores designed for asymptomatic subjects for risk stratification of CHD with troponin peaks. Such a risk score preferably is a risk score in accordance with prevailing European and/or US guidelines (e.g.2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014 Jul 1 ;63(25 Pt B):2889-934).

In one embodiment the identification of subjects at high risk of coronary heart disease is complemented by the identification of subjects at high risk of atherosclerotic cardiovascular disease or by the identification of subjects at high risk of cardiovascular disease.

The method of the invention should not be implemented in isolation. Therefore, in one embodiment, the method of the invention further comprises clinical evaluation of the subject for evaluation of possible causes of a troponin peak other than reasons that put the subject at an increased risk of coronary heart disease. Such an evaluation takes place prior to treatment of the subject. Other such reasons include left ventricular structural alterations, left ventricular dysfunction or heart failure.

Methods for determining susceptibility to therapy

In yet a further aspect, the invention relates to a method for determining the susceptibility of the subject to therapy or further testing. In this aspect, the above method of the invention of diagnosing a silent cardiac impairment in a subject are used for determining the susceptibility of the subject to therapy or further testing, preferably cardiac therapy.

In one embodiment of the above method of the invention of diagnosing a silent cardiac impairment in a subject, the method further comprises the step of determining the susceptibility of the subject to therapy or further testing, preferably cardiac therapy. In this method, a subject diagnosed with at least one of silent coronary plaque rupture and transient LV dysfunction is determined susceptible to therapy for coronary heart disease. In this method, a subject is also determined susceptible to therapy for coronary heart disease, if the subject is determined to have an increased risk of CAD, or if the subject is at least one of a subject diagnosed with stable CAD and a subject having suffered from an ACS and is determined to have an increased risk of CAD or of cardiovascular disease, or for a subject having suffered from an ACS if the subject is determined to have an increased risk of recurrent events.

CAD can be treated by medical treatment, by bypass of at least one large coronary artery, by bypass of small coronary vessels or by percutaneous coronary intervention (PCI). If, on the other hand, in this method, a subject diagnosed with LV dysfunction is determined susceptible to therapy against heart failure. Such therapy can include administering to the patient an effective amount of one or more ACE inhibitors, angiotensin receptor blockers, beta-blockers, angiotensin receptor neprilysin inhibitors, aldosterone receptor antagonists, life style modification (inclusive of increasing physical activity), and specialty consultation with a cardiovascular specialist.

In this method, alternatively a subject diagnosed with alterations in LV structure and/or function is determined to be a candidate to undergo (further) testing for determination of LV hypertrophy and/or LV dysfunction, e.g. by echography or other imaging techniques and optionally therapy for the prevention of heart failure and/or cardiac death.

A method of troponin peak detection to guide treatment strategy

The method disclosed herein can also be used to determine whether a treatment against

CHD is suitable for a subject. In some embodiments, methods are provided for evaluating the effectiveness of a treatment or for evaluation of treatment strategy. The assessment of troponin peaks is described above.

CAD can be treated by drug treatment, by bypass of at least one large coronary artery, by bypass of small coronary vessels or by percutaneous coronary intervention (PCI). Pharmaceutical drug treatment includes any therapy for atherosclerosis designed to reverse or slow the progression of atherosclerosis, including but not limited to treatment with statins, niacin or other cholesterol- lowering agents, anti-inflammatory agents, aspirin, anti-platelet agents, anticoagulant agents, blood pressure lowering medications, agents for smoking cessation, or any other pharmaceutical compound.

A method to determine if a treatment against CHD is suitable for a subject

In one embodiment, the method preferably comprises one or more of the steps of: a) fine- temporal serial testing in body fluid samples obtained from the subject the concentration of troponin; b) assessment of troponin peaks in a subject, wherein preferably an cumulative peak measure is computed, and whereby the presence of peaks is indicative for whether a subject is at increased risk of CHD; c) the troponin peaks are assessed in an ongoing fashion; and, d) to determine whether a treatment against CHD is suitable for a subject. In step d) herein preferably a treatment against CHD is determined to be suitable for a subject determined to be at increased risk of CHD based on the presence of troponin peaks.

A method for determining the efficiency of a treatment against CHD in a subject

In another embodiment, the method preferably comprises one or more of the steps of: a) fine- temporal serial testing in body fluid samples obtained from the subject the concentration of troponin during treatment; b) assessment of troponin peaks in a subject, wherein preferably an cumulative peak measure is computed, and whereby the presence of peaks is indicative that a subject is at high risk of CHD; c) the troponin peaks are assessed in an ongoing fashion; and d) to determine whether a treatment against CHD is effective for a subject. In step d) herein preferably the treatment against CHD is determined to be effective in a subject if CHD risk based on the assessment of troponin peaks decreases during treatment and is determined not to be effective in a subject if the CHD risk based on the assessment of troponin peaks does not decrease or increases during treatment. A method for determining the treatment strategy of a treatment against CHD in a subject

In yet a further embodiment, the method preferably comprises one or more of the steps of: a) fine-temporal serial testing in body fluid samples obtained from the subject the concentration of troponin during treatment; b) assessment of troponin peaks in a subject, wherein preferably an cumulative peak measure is computed, and whereby the presence of peaks is indicative whether a subject is a high risk of CHD; c) the troponin peaks are assessed in an ongoing fashion; and d) to determine the treatment strategy of a treatment against CHD in a subject. In step d) herein preferably the treatment strategy (type of drug, continuation, dosing) of a therapy against CHD is not adjusted in a subject if CHD risk based on the assessment of troponin peaks decreases during treatment and is adjusted in a subject if CHD risk based on the assessment of troponin peaks does not decrease or increases during treatment.

In a preferred embodiment of the methods of the invention, peak detection of troponin as described above, is performed preferably in combination with peak detection of at least NT-proBNP to improve identification and stratification of subjects at high risk of coronary heart disease as well as for at least one of: a) to select a suitable therapy against CHD for the subject, b) to monitor treatment effectiveness and c) to guide treatment strategy in a subject. In other embodiments of the methods of the invention, peak detection of troponin as described above, is performed combination with peak detection of other markers, in addition to NT-proBNP, to identify subjects at high risk of coronary heart disease, as well as for or at least one of: a) to select a suitable therapy against CHD for the subject, b) to monitor treatment effectiveness and c) to guide treatment strategy in a subject.. Such additional markers may be selected from the group of inflammatory markers, e.g. C-reactive protein (CRP).

Automation of parts of the method

In some embodiments, one or more steps of the methods may in total or in part be assisted by automation, e.g. computer-implemented steps for algorithm-based peak assessment and assessment of their behavior and accumulation in time and/or for algorithm-based computation of CHD risk and/or for algorithm-based determination of treatment susceptibility and treatment guidance.

In this embodiment the invention provides methods wherein an algorithm is used in which a growing body of information is accumulated . Herein, information on troponin peaks is summarized in a cumulative measure reflecting the aggregate burden of peaks in an ongoing fashion. The algorithm will take into account information from the pattern such as peaks superimposed on one another and other peak features beyond peak height and peak frequency that may be important for CHD risk such as area under the curve, defined as area under the troponin curve, isolated peaks versus clustered peaks, and duration of clustered peaks. In this method of the invention the frequency of testing and the length of the interval between testing is determined based on accumulated peak information. On the other hand , peak definition is dependent on the frequency of testing . For example, when the frequency of testing is at least every day, a point in a troponin pattern is regarded as a peak if it takes the maximum value within one day from the previous, initial measurement, if its value is higher than and differs by at least 5, 10, 20, 30, 40 or 50, or 100 ng/L from the previous value, if its value is higher than the value of the subsequent measurement, and if the subsequent values show a decreasing trend back to the initial value in 1 , 2, 3, or 4 days. When the frequency of testing is more than 3 days, a point in a troponin pattern is regarded as a peak if it is higher than the previous value, if its value is higher than and differs by at least 5, 10, 20, 30, 40 or 50, or 100 ng/L from the previous value, if its value is higher than the value of the subsequent measurement, and if the subsequent value is close to the baseline or initial value.

Point of care testing

In one aspect, the invention pertains to the methods of the invention for diagnosing a silent cardiac impairment in a subject, for determining the risk of the subject of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death and/or for determining the susceptibility of the subject to therapy, wherein the concentrations of at least one of the cardiac troponin or a variant thereof and the BNP-type peptide in the body fluid samples are measured on- site rather than in a laboratory. The measurements are thus performed at the point-of-care, e.g. using a point-of-care system or device.

In one aspect, the point of care testing involves the following. The invention pertains to the methods of the invention for determining CHD risk wherein the concentrations of troponin and NT- proBNP in the body fluid samples are measured on-site rather than in a laboratory. The measurements are thus performed at the point-of-care, e.g. using a point-of-care system or device. In one embodiment, the testing is performed at home and the point-of-care device is used by the subject him or herself. In this embodiment, a home-based point-of-care device for troponin needs to allow for a finger-stick procedure of blood taking. Although a finger-stick point of care device for troponin is yet on the market (i-STAT point of care device, Abbott, Princeton NJ) troponin testing in this device is not based on a high-sensitivity assay. The point-of-care device may be used to monitor troponin values and to adjust the frequency of testing and interval between testing if necessary. For example the frequency of testing may need to be increased after a first troponin peak is detected. The point-of-care device enables to perform multiple troponin tests and enables to integrate these troponin tests in a testing algorithm for obtaining an cumulative peak measure as well as in clinical algorithms for simultaneous computation of CHD risk and for determination of treatment susceptibility and treatment guiding.

System or device

In a further aspect, the invention relates to a system or device for performing a method methods of the invention for diagnosing a silent cardiac impairment in a subject, for determining the risk of the subject of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death and/or for determining the susceptibility of the subject to therapy.

The system or device for performing a method methods of the invention preferably comprises a computer. In one aspect therefore the invention relates to a computer comprising a processor and memory, the processor being arranged to read from said memory and write into said memory, the memory comprising data and instructions arranged to provide said processor with the capacity to perform a method according to the invention as herein defined above. The computer preferably has an input connected to a sample analyzer for receiving analysis data signals of the concentration of at least one of a cardiac troponin and a BNP-type peptide or variants thereof in body fluid samples obtained from a subject and wherein the processor is arranged for determining from said analysis data signals at least one a diagnosis, risk or susceptibility to therapy in accordance with a method of the invention as herein defined above.

The system or device for performing a method methods of the invention preferably comprises a computer

The system or device for performing a method methods of the invention sample analyzer that comprises means for measuring the concentration of at least one of a cardiac troponin and a BNP-type peptide or variants thereof in a body fluid sample. Preferably the sample analyzer further comprises a computer as defined above.

In a further aspect the invention relates to a computer program product comprising data and instructions and arranged to be loaded in a memory of a computer, the data and instructions being arranged to provide said computer with the capacity to perform a method of the invention as herein defined above.

In yet another aspect, the invention pertains to a data carrier provided with a computer program product as defined above.

Use of the methods of the invention

In a further aspect, the invention pertain to the use of a diagnostic method according to the invention, for determining the risk of the subject of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death.

In another aspect, the invention pertain to the use of a diagnostic method according to the invention for determining the susceptibility of the subject to at least one of therapy for coronary artery disease and therapy against heart failure. Preferred embodiments of the invention

FIG. 1 is a block diagram of an operating environment 100 that supports a method and system for detecting cardiac ischemia in a subject, according to an example embodiment as e.g. described in US20160345912, which is herein incorporated reference in its entirety. The environment 100 may comprise at least two computing devices 140, 1 12 and a mobile device102, which may communicate via a communications network 106. The computing devices 140, 1 12, 102 may be connected either wirelessly or in a wired or fiber optic form to the communications network 106. The at least one sensor 1 12 may be communicatively coupled, either wirelessly or in a wired or fiber optic form to the mobile device102. Communications network 106 may be a packet switched network, such as the Internet, or any local area network, wide area network, enterprise private network, cellular network, phone network, mobile communications network, or any combination of the above.

Sensor 1 12 and computing devices 140, 102 may each comprise a computing device 400, described below in greater detail with respect to FIG. 4. In one embodiment, at least one sensor 1 12 may be a point of care device that records data from a subject 1 10. In another embodiment, at least one sensor 1 12 may include functions that record other information of subject 1 10, such as blood pressure and heart rate. Further, sensor 1 12 and computing device140 may each comprise mobile computing devices such as cellular telephones, smart phones, tablet computers, wearable devices, or other computing devices such as a desktop computer, laptop, game console, etc. In one embodiment, the at least one sensor 1 12 may be integrated with mobile device 102.

Mobile device 102, and computing devices 1 12, 140 may each include program logic comprising computer source code, scripting language code or interpreted language code that perform various functions. In one embodiment, the aforementioned program logic may comprise program module 407 in FIG. 4.

FIG. 1 further shows that mobile device 102 contains a database 104. Computing devices

1 12, 140 may also each include databases.

Environment 100 may be used when the disclosed computing devices transfer data to and from database 104 of mobile device 102 as e.g. described in US20160345912, which is herein incorporated reference in its entirety. Various types of data may be stored in the database 104 of mobile device 102. For example, the database 104 may store a medical history 204, data on risk factors, demographic data of the subject, or other clinical data 204 of the subject 1 10of the subject.

Note that although mobile device102 is shown as a single and independent entity, in one embodiment, the functions of mobile device 102 may be integrated with another entity, such as the computing device 1 12.

In a preferred embodiment, mobile device 102 is a cellular telephone, smart phone or tablet computer that is wirelessly connected to sensor 1 12. An app is installed on the mobile device 102 for processing the data that it receives from sensor 1 12. Different wireless communication standards such as Bleutooth, ZigBee, RFID and WiFi can be used for the transmission. The main functions that the mobile device 102 performs in this embodiment are data signal acquisition, peak detection, ischemia detection, risk stratification, graphical user interface, save record of subject in database, wireless transmission to third parties including health professionals 144, remote medical or service center, or server 142. In case of an emergency it can automatically inform the concerned health care professional. A database 104 is created in the mobile device 102 that stores records of the subject so that at any time it can be analysed or resend. In one embodiment, an interface may be used to identify the device and initiate a secure data connection. It can prompt for customer permission to submit diagnostic information to remote diagnostic service, can submit a diagnostic file and can receive information or guidance from a remote diagnostic service.

FIG. 3 is a flow diagram of a method 300 for detecting cardiac ischemia in a subject, according to an example embodiment. The method 300 is described with reference to FIG. 2, which is a diagram 200 showing the data flow of the process for facilitating treatment of patients, according to an example embodiment.

In an optional preliminary step, the method 300 begins with the database 104 receiving (such as via network 106 or via the subject 1 10) and storing predefined values 210 and clinical data 212 from, for example, a data provider 140, which may be a third party provider of data. Clinical data 212 refers to data that may be gathered from a clinical experiment or study that establishes parameters, ranges and/or normal values that are then used as a benchmark to measure other tested subjects. The clinical data may refer to clinical values or ranges for variables such as peak height (defined in greater detail below) from a tested group. In one alternative, clinical data 212 may also represent one or more ranges of values for one variable or feature. For example, clinical data for peak height may indicate a range of values. In another alternative, clinical data 210 may also represent multiple ranges. For example, clinical data of peak height may indicate a first range of values .which indicate a normal range, a second range of values which indicate a borderline range and a third range of values that indicate an high-risk range. Predefined values 210 may refer to predefined values (for variables such as peak height— defined in greater detail below) that, according to research or empirical data, correspond to certain diagnosis, including ischemia due to large vessel occlusion, ischemia due to cardiac microembolism.

FIG. 3 is a flow diagram of a method 300 for detecting peaks in the serial marker data to determine ischemia classes risk of ischemic cardiac events in a subject, according to an example embodiment. Method 300 describe the steps that occur when a subject undergoes serial cardiac marker testing, such as a asymptomatic subject, a subject with multiple risk factors, a patient with coronary artery disease (CAD) or patient having suffered from acute coronary syndrome (ACS), wherein the detection and determination process is facilitated by the use of environment 100. The method 300 is described with reference to FIG. 2 which is a diagram 200 showing the data flow of the process for facilitating self-testing of subjects according to an example embodiment.

Before taking a first blood sample, the subject enters subject data 204 in the data base of the mobile device 102. The subject data 204 are described in detail below. In one embodiment, during the marker monitoring, the subject enters data on chest pain in the data base 104 of mobile device 102 each time chest pain is present using a short, simple and standardized chest pain questionnaire based on the Canadian Cardiovascular Society grading score or the Rose questionnaire.

The method 300 begins with the process of blood marker monitoring in order to detect and diagnose cardiac ischemia. The first step 302 may be a step wherein a subject 1 10 may visit a healthcare professional or doctor. During the visit, which may be a conventional, in-person visit or a virtual visit using teleconferencing technology, the doctor, and/or another healthcare professional working under the direction of the doctor, may interact with the subject 1 10 and suggest to perform blood marker monitoring in order to diagnose cardiac ischemia or the subject consults the doctor or health profession about blood marker monitoring. In another embodiment the subject 1 10 may decide independently to start blood marker monitoring in order to detect and diagnose cardiac ischemia. Before starting with the monitoring, clinical/history/risk factor data 204 of subject 1 10 are entered in database 104 of the mobile device. In the method of the invention the blood marker monitoring is performed at the point of care and using a point of care device. In step 304, certain information is generated and entered into the database 104 of mobile device 102. Said information may include marker data 202 coming from point of care device 1 12.

Point-of-care testing is a form of testing in which the analysis is performed where healthcare is provided close to or near the patient. One definition of point-of-care testing is: "testing that is performed near or at the site of a patient with the result leading to possible change in the care of the patient" (International standard ISO 22870, Point-of-care testing (POCT) - Requirements for quality and competence). In general, point-of-care testing encompasses any tests that are performed at or near a patient and at the site where care or treatment is provided. Results are typically available relatively quickly so that they can be acted upon without delay. The instrument can be used by non-specialists to detect and diagnose disease, and can enable the selection of optimal therapies through patient screening and monitoring of a patient's response to a chosen treatment. Preferably, in the method of the invention, the point of care testing is performed in the home and/or work setting.

It should be understood that herein the expression measurement of concentrations of biochemical markers generally means the whole process of blood sampling and measurements. In one embodiment this can be taking blood samples and analyzing the samples using the point of care device to determine the concentration levels of the biochemical markers. Both steps 302 and 304 are part of the point of care system.

Point of care devices are typically comprised of a credit-card sized plastic single-use

(disposable) cassette that hosts a microfluidic network of channels, conduits, filters, reaction and reagent chambers to process clinical specimens such as blood, saliva, urine, or environmental samples such as drinking water, food, air. The cassette or 'chip' is mated with a small, portable instrument that provides the cassette with controlled heating, fluidic actuation and flow control, and detection capabilities. Most commonly, the test result is determined by measuring an optical signal such as fluorescence. Ideally, the system is self-contained, can be operated by non-technical users. Within minutes the levels of blood markers will be shown on the display.

The point of care device in the method of the invention is a biosensor. Biosensors are defined (Turner, A.P.F., 1989. Sens. Actuators 17, 433-450) as analytical devices incorporating a biological material (e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, natural products etc.), a biologically derived material (e.g. recombinant antibodies, engineered proteins, aptamers etc.) or a biomimic (e.g. synthetic receptors, biomimetic catalysts, combinatorial ligands, imprinted polymers etc.) intimately associated with or integrated within a physicochemical transducer or transducing microsystem, which may be optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical. Biosensors usually yield a digital electronic signal which is proportional to the concentration of a specific analyte or group of analytes. While the signal may in principle be continuous, devices can be configured to yield single measurements to meet specific market requirements. Examples of biosensors include immunosensors, enzyme-based biosensors, organism- and whole cell-based biosensors. In step 302 the blood collection takes place. In one example, the subject takes a first blood sample using a fingerstick device to prick the finger, such as a lancet. The finger stick device is used to puncture the skin to obtain small blood specimens/samples for testing markers in the blood. The subject may also use a lancing device, which is a device that holds a lancet firmly, and when triggered, moves the lancet linearly ahead to prick the skin in a controlled manner. A reusable fingerstick device can be used that resembles a pen and have the means to remove and replace the lancet (endcap) after each use. In a preferred embodiment, a device is used that optimizes the reduction of pain associated with finger pricking.

Thereafter, the subject places the blood on a chemically active disposable test strip and inserts the test strip in the meter device. The test strip can be e.g. a disposable strip, chip or a cartridge, that contains the necessary reagents, and possibly also fluidics, optics, electrodes etc. In the method of the invention the test strip is preferably a test strip for multiple tests, meaning that multiple test reagents are embedded on a single strip. In a preferred embodiment, multiple tests can be performed with one drop of blood or with a single finger prick sample. In step 304, the electronic device 1 12 determines the level of blood markers in the blood.

In another embodiment, the device 1 12 is a fully printed biosensing instrument. In one example, this is a printed instrument developed by a collaboration between Linkoping University and Swedish ICT non-profit Acreo. (The paper potentiostat. Turner, A. In: 4th International Conference on Bio-Sensing Technology, 10-13 May 2015, Lisbon, Portugal., Elsevier, 2015, Turner, A.P.F., Beni, V., Gifford, R., Norberg, P., Arven, P., Nilsson, D., Ahlin, J., Nordlinder, S. and Gustafsson, G. (2014). Printed Paper- and Plastic-based Electrochemical Instruments for Biosensors. 24th Anniversary World Congress on Biosensors - Biosensors 2014, 27-30 May 2014, Melbourne, Australia. Elsevier).The instrument has the size of a credit card that can analyse blood and saliva samples. Componentes are sensors, display, push button, battery, chip for measurements, chip for communication, printed inter-connects and resistors. It is simple to use: it is switched on by pressing a button, then the subject applies a blood sample to a circle in the bottom right corner and waits for a digital reading to be displayed and sent to the subject's mobile phone. The mobile Phone lights up and dispays the medical results, which it also sends to the online diagnosis database. Near-field communication uses magnetic induction between two loop antennas located within each other's near field, effectively forming an air-core transformer. It operates within the globally available and unlicensed radio frequency ISM band of 13.56 MHz and provides short- range, reasonably secure transmission to a mobile device.

In one embodiment, the device is a medical implant and biomarker measurement takes place e.g. every 10 minutes, such as a device developed at the Ecole Polytechnique Federale de Lausanne (EPFL) (Carrara et al. International Symposium on Circuits and Systems (ISCAS) in Lisbon 2015).

In one embodiment, the device is a E-skin device that is modified to measure biomarkers in blood (Mechanisms and Materials of Flexible anStretchable Skin Sensors. Yicong Zhao and Xian Huang. Micromachines 2017, 8, 69; doi: 10.3390/mi8030069). In one example, the method for measuring troponin is a on/off-switchable LSPR nano- immunoassay for troponin-T (on/off-switchable LSPR nano-immunoassay for troponin-T. (On/off- switchable LSPR nano-immunoassay for troponin-T (on/off-switchable LSPR nano-immunoassay for troponin-T Ashaduzzaman Md. Scientific Reports; 7:44027; 6 april 2017).

In step 304, the electronic device 1 12 determines the level of blood markers in the blood.

Examples of biochemical markers are cardiac troponin-l, NT-proBNP and CRP.

Results of a recent study comparing the predictive properties of troponin T and troponin I found that troponin I is a better diagnostic marker for early presenters with myocardial infarction (Gimenez et al. Eur Heart J, 20 May 19, 2014). These results indicate that early changes in troponin may be better detectable with troponin I compared to troponin T. Results from Shi et al. suggest that cTnl exists in serum mainly in a complexed and C-terminal degraded form that may be resistant to proteolytic digestion (Shi at al. Clinical Chemistry 45:71018-1025 (1999). Katrukha et al. also demonstrated that the main part of cTnl in serum collected from acute myocardial infarction patients is presented in the complex form. They measured free and complexed Tnl for at least 80 to 100 h during myocardial infarction. They found that the ratio cTnl total /cTnl free was changed during the time of observation. At the beginning of observation this ratio was low; then it increased to its maximal value and at the end of the observation it returned to its initial value. (Katrukha AG et al. Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form. Clinical Chemistry 43:81379-1385 (1997).

The opposite seems to be the case for cTnT. For cTnT, Michielsen et al found that the intact cTnT protein was detected only during the first 12 h after the cTnT concentration started to increase above the AMI cut-off value of 0.03 μg/L. Thereafter only fragments with molecular weights ranging from 10 to 30 kDa were detected, with two fragments being most prominent (15 and 25 kDa) and which had a longer stay in blood. (Michielsen EC(1 ), Diris JH, Kleijnen W, Wodzig WK, Van Dieijen- Visser MP. Investigation of release and degradation of cardiac troponin T in patients with acute myocardial infarction. Clin Biochem. 2007 Aug;40(12):851-5. Epub 2007 Apr 19).

These results suggest that in the early phase of myocardial infarction when troponin is released from the cytosolic pool (reflecting ischemia), troponin I in blood is relatively stable while troponin T disappears quickly, while in the later phase when troponin is released from structurally bound troponin (reflecting necrosis), the opposite is the case. This agrees with the patterns of troponin I and troponin T found in myocardial infarction patients where not only troponin I peaks earlier and downslopes faster than troponin T but maximum peak height of troponin I is a multitude higher compared to troponin T.

In one embodiment, troponin I is measured using antibodies (natural, semisynthetic, synthetic) that recognize epitopes on the specific forms of cTnl that originate from the cytosolic pool in the early phase of myocardial infarction.

In one embodiment, troponin I and/or troponin T is measured using antibodies (natural, semisynthetic, synthetic) that recognize epitopes on the specific forms of troponin I or troponin T that originate from troponin structurally bound in the myocyte in the later phase of myocardial infarction. In one embodiment, troponin-T is used in one or more steps of the method 300 in stead of or additional to troponin-l.

In steps 308 to 320, the steps illustrates the sequence of steps beginning with the acquisition (A/D conversion) by the mobile device 102 of the data produced by the point of care device 1 12. These steps are detection of a possible peak, upslope detection, pattern recognition and feature measurements, diagnostic classification, risk stratification and determination of treatment susceptibility.

In the method of the invention, the approach for peak detection is based on the assumption that a peak pattern in serial marker data reflective of cardiac ischemia is a composite entity composed of a troponin peak complex (T-peak complex), characterized by a steep T-1 peak reflecting troponin released from the cytosolic pool during ischemia and a broad T-2 peak, reflecting release of troponin that was structurally bound during myocyte necrosis; a NT-proBNP peak reflecting regional or global transient ventricular wall dysfunction and optionally a CRP peak, reflecting increased inflammation. Collectively, these peaks form a multi marker peak complex. Complexes which are overlapping or close to each other are combined to form a peak cluster.

In the method of the invention, a further assumption is that there are two distinct types of cardiac ischemia whereby various marker peaks constitute a typical multi marker pattern. One is a multi-marker peak pattern reflecting cardiac ischemia due to epicardial or large vessel occlusion. This peak pattern is characterized by a high troponin peak and a relatively lower NT-proBNP peak. The other is a multi-marker peak pattern reflecting cardiac ischemia due to microembolization. This pattern is characterized by a low troponin peak, a very high and very broad NT-proBNP peak and a very broad CRP peak. Cardiac ischemia due to microembolization is characterized by a lower maximum peak height of troponin compared to cardiac ischemia due to epicardial vessel occlusion. Typically, maximum peak height of troponin-l during an episode of microembolization is below 1000 ng/L, often below 500 ng/L, whereas maximum peak height for troponin I after epicardial vessel occlusion followed by myocardial infarction typically reaches values of 10.000 ng/L and higher. Further, the NT-proBNP level after cardiac ischemia due to microembolization is a multitude higher than the NT-proBNP level after cardiac ischemia due to epicardial vessel occlusion.

Coronary microembolization is a potential cause of regional myocardial contractile dysfunction in the absence of an atherosclerotic obstruction of an epicardial coronary artery that could account for such dysfunction. (Coronary Microembolization. Erbel R, Heusch G. JACC Vol. 36, No. 1 , 2000:22-24). This may account for myocardial infarction in the presence of non- obstructed epicardial coronary vessels. Such a type of myocardial infarction is seen in what is called Takotsubo syndrome.

Myocardial microcirculatory dysfunction is predicted by an adverse risk factor profile, including diabetes, high cholesterol, hypertension, and poor renal function while Takotsubo cardiomyopathy is particularly seen in older women in which this risk factor profile prevails. Furthermore, Takotsubo syndrome is characterized by a low troponin over NT-proBNP ratio: the troponin T over NT-proBNP ratio in Takotsubo, STEMI and NSTEMI patients was 0.2, 5.3 and 1.0 respectively (Frohlich GM et al, IJC-D-1 1-01099 July 19th, 201 1 ). The method of the invention is derived from the idea that there exists a type of cardiac ischemia that is characterized by detectable low-height troponin peaks that go along with high- height NT-proBNP peaks and that this pattern is caused by micro-em bolizations (see also drawings previous related patent application submitted Dec 13, 2016). Since the same marker pattern seems to be found in the syndrome called Takotsubo cardiomyopathy, both may have the same origin.

In the method of the invention, a real-time peak detection algorithm is developed that takes into account the known characteristics of the T-1 peak and the NT-proBNP peak, being their steep upslope. The method successively involves height thresholding, increasing the sampling frequency after a possible peak is detected, and subsequent slope determination for estimating the rate of change in the marker. From the slope also peak onset can be computed which can be used as a reference point for recognizing the remaining features of the peak pattern. The main aim of the latter is to detect and estimate concomitant upslopes in the other markers. This is because the height and slope of the upslope of the peak can be obtained most accurately and most early in the development of a peak and will be used for quick classification and risk stratification. The algorithm only uses known thresholding techniques and does not require high CPU which makes the detection method suitable for application in a low-powered (battery-powered) environment such as in a mobile device.

The peak detection method uses two detection criteria. The first criterion evaluates whether or not the height of the signal exceeds the height threshold. To this end, each new data point is compared to the baseline value. Once a "break through point" is detected, the sampling frequency is increased in 312. At the same time, the height value serves as input in the classification algorithm in step 318. Then, the second criterion evaluates whether the slope exceeds the slope threshold in 314. Because the upslope of the troponin peak has a characteristic shape, the steep upslope, which approximates a straight line, the slope is determined by fitting a straight line to the data-points of the upslope using least square estimation. If the slope exceeds the slope threshold, it is confirmed as the slope of a T-1 peak. To preserve the benefits of the real-time approach, the fitting is performed on the first part of the upslope before the entire set of data characterizing the upslope is acquired. The slope values are used to obtain an estimate of the rate of change of the marker that, like the height values, serve as input in the classification algorithm in step 318. Once the upslope is confirmed as the upslope of a T-1 peak, the computed slope from the fitted curve is used to determine a rough estimate of the onset of the T-1 peak. This is the point where the fitted curve crosses the baseline. For this purpose a baseline is created as a horizontal line starting from the last computed baseline moving average. The line approximates the true baseline to the extent that the true baseline does not change over a short period of time. The onset of the upslope of the T-1 peak then serves as the reference point for recognition of the remaining parts of the multi marker peak complex.

All measured biomarkers are tracked in the same way as described above for troponin. All biomarkers have their own settings for all steps in method 300, like thresholds for noise, height, and slope. If NT-proBNP exceeds its height threshold level at an earlier point in time than troponin-l, than in the above description troponin is replaced by NT-proBNP. If CRP exceeds the height threshold at an earlier point in time than troponin-l, than in the above description troponin is replaced by CRP. This however is not likely since the CRP peak start is expected to start at a later point in time than the troponin and NT-proBNP peak.

In one embodiment the T-1 peak and the T-2 peak of troponin are measured by different assays reflecting (predominantly) respectively release of troponin from the cytosolic pool and release of troponin from structurally bound troponin in the myocyte.

Recognition of the other parts of the multi marker peak complex involves several steps. Most important for classification of the peak pattern is detection of a concomitant upslopes of NT-proBNP (or of troponin in case NT-proBNP is the first to pass the height threshold). In this situation the following applies. A height criterion evaluates whether or not the height of the NT-proBNP signal exceeds the threshold level. To this end, each new data point is compared to the baseline NT- proBNP value. If such a "break through point is detected, the slope criterion evaluates whether the NT-proBNP slope exceeds the slope threshold by fitting a straight line to the data-points of the upslope using least square estimation. If the slope exceeds the slope threshold, it is confirmed as the slope of a NT-proBNP peak. Next, the ratio of troponin peak height to NT-proBNP peak height is computed at each data point on a peak and the maximum ratio is used for classification. A similar approach is taken for CRP.

In a preferred embodiment of the method of the invention, provided that an appropriate sampling frequency is applied, when after the time of passing the height threshold in step 310, data points are found to be on a downslope, only the height value will be used for quantification in the classification step 318 and the presence of a negative change could optionally be used in the classification as a qualitative rather than quantitative information.

The selection of an assay reflecting troponin released during the early phase from the cytosolic pool and having a long stay in blood (such as troponin-l) will facilitate the measurement of an upslope in the method of the invention since even though the release of troponin is fast, its level will only slowly disappear from the blood and its measurement will not be disturbed by the concomitant (but delayed) release of troponin from the structurally bound troponin.

In one embodiment the rate of change of a downslope will be estimated using a straight line or more advanced fitting functions.

Recognition of the other parts of the peaks in step 316 involve determination of maximum peak height of the detected peaks, measured by using the height of the tallest point in the peak, thereby comparing each peak point to its neighbors. It also involves detection of the T-2 peak. When only one assay for troponin is used, at best, the T-2 peak can be seen as a shoulder on the T-1 peak. Shoulders peaks are sensitive to changes that affect the separation of peaks and therefore shoulder peaks are useful as qualitative rather than quantitative information.

When troponin release is low, in marathon runners followed long enough two separated peaks were seen in some runners (JACC Vol. 52, No. 22, 2008 November 25, 2008: 1813-6). This supports the idea that the T-1 and T-2 peak can be completely separated when troponin release is low, and that also in this situation the ischemia phase may be followed by a phase of myocyte necrosis. Using the least-square curve-fitting to new T-1 upslopes, deconvolution of overlapping peaks can be performed and the slope of each new T-1 peak can be obtained. Thereby the peak heights and number of T-1 peaks can be computed. The same approach will be used for new NT-proBNP peaks. In one embodiment, this approach is also used for new CRP peaks.

The end of a peak is defined when the baseline is reached. For this purpose a baseline is created as a horizontal line starting from the last computed baseline moving average. The line approximates the true baseline to the extent that the true baseline does not change over a short period of time. Since noise prevents determining the exact point where the downslope crosses the baseline, a few percentages of the baseline value is added to the baseline value. For example 1 ,2,3,4, or 5%.

The accuracy of the algorithm is dependent on accurate estimation of the baseline level. For this purpose, the baseline is constructed using a moving average. In one embodiment this is the exponentially weighted average. In one embodiment this is a zero lag exponential moving average (ZLEMA). This method removes a selected amount of lag from an exponential moving average. (Rocket science for traders, Ehlers J. John Wiley & Sons, New York.). To be accepted as a baseline point, a data point must lie within a defined distance from the moving average. Examples of such a distances are 1 ,2,3,4 or 5 ng/L for troponin and 1 , 2, 3, 4, or 5 ng/l for NT-proBNP. In this way larger values are ignored for computing the baseline. The number of data points in the moving time window depend on the sampling frequency. In one embodiment, the window contains all data point over the past 1 ,2, 3,4,5,6,7, 8,9, 10, 1 1 , 12, 13,or 14 days.

The algorithm is designed such that quick decisions, as early as possible, can be based on provisional determinations and prior to the end of data point collection. This means that peak feature measurements will be provisionally until confirmed after collection of more data points. To present and display results, the device reports graphical plots, peak feature values, diagnosis and risk scores periodically. Each time a report is provided in step 324 (e.g. at each time a new data point comes in) classification in 318 and risk stratification in 320 will be performed and reported using the currently available data independent of whether or not the peak cluster has ended.

It is desirable that the detected peaks from the collected blood samples reflect the real peaks as accurately as possible within realistic limits. In the method of the invention blood collection preferably takes place 1 to 4 times per day, depending on the risk of the subject. E.g. a patient with recent acute coronary syndrome (ACS) (up to 6 months after onset of ACS) is at high risk of recurrent ACS and in order to detect a recurrent ACS in the earliest stages a frequency of 3 to 6 times per day may be appropriate and feasibly for a short period of time. On the other hand, an older female with CVD risk factors (diabetes, high cholesterol, high blood pressure) may be at high risk of microembolizations. Such a person may benefit from detection of cardiac ischemia due to microembolizations which comprises a non-acute risk of repeated events. In this situation, troponin monitoring may be used for risk prediction and given the persistence of troponin I in blood after an episode (24 to 48 hours) a sampling frequency of once per day may be adequate. Testing practices are dependent on the schedule of the subject. The tests should be conducted with intervals of equal time in between as far as this is practical. E.g. when the frequency is three times per day, a logical schedule would be one test taken in the morning, one test taken after lunch break at two am and one test taken in the evening at 8 pm. Chest pain may be a reason for immediate blood sampling independent of the predefined sampling schedule.

Besides quick decision based on the occurrence of peaks that should be acted on immediately it is also important to compute cumulative measures of peaks that do not harbor an immediate risk. Such a cumulative measure will be an indicator of the risk of coronary heart disease. A cumulative measure could include information of the frequency of peaks, the height values of peaks and the duration of peak clusters. This information can be collected for a period from 1 day to 1 month or longer before analysis is started provided that no peaks occur that indicate an immediate risk. It is also possible that cumulative measures are computed on a continuous basis and a subject is alerted when a cumulative measure exceeds a predefined threshold level.

The noise threshold (the level below which a peak is considered to be a noise peak) may be adapted to the characteristics of the incoming signal during the ongoing processing as described in method 300. Also other thresholds used in the method of the invention may be adapted during the process based on the characteristics of the incoming signal and characteristics of the subject.

In 310, height thresholds for the measured biomarkers are used. The height threshold is the difference between the new data point and the baseline value (the moving average value).

Examples of the height thresholds are: the height threshold for troponin-l: 2.5, 5, 10, 20, 50 ng/L; the height threshold for NT-proBNP: 5, 10, 20, 30, 40 ng/L; the height threshold for CRP: 2.5, 5, 10, 20, 30, 40, or 50 mg/dl.

In 314, slope thresholds for the measured biomarkers are used. The slope threshold is expressed as the increase in the marker value per unit of time.

Examples of the slope thresholds are: the slope threshold for troponin-l: 1 ,2,3,4,5,6,7,8,9,10,1 1 ,12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/L increase per hour; the slope threshold for NT-proBNP: 1 ,2,3,4,5,6,7,8,9,10,1 1 ,12,13,14, 15, 16, 17, 18, 19, or 20 ng/L increase per hour; the slope threshold for CRP: 1 ,2,3,4,5,6,7,8,9,10, 1 1 ,12,13, 14,15, 16,17, 18,19, or 20 mg/dl increase per hour.

In 312, after a first marker exceeds the height threshold, the sampling frequency is increased for determination of the upslope in 314. The sampling frequency is initially increased to "taking a blood sample every 30 minutes". In other examples this may be 10, 20, 30, 40, 50 or 60 minutes up to once a day or less. After the peak maximum is reached, or after other directions are provided through the report in the classification and risk stratification steps, the sampling frequency is reduced to for example every 2 hours. In other examples this may be 1 ,3,4,5,6,7 ,8,9, 10, 1 1 , or 12 hours. The sampling frequency may be further reduced with time if no new peaks appear. The sampling frequency returns to normal after the end of a peak cluster. The end of a peak cluster is reached after the last peak has ended and when no new peaks appear in a time period after the end of the last peak. This period may be 1 ,2,3,4,5,6,7,8,9, 10, 1 1 , 12, 18, or 24 hours or 2,3,4,5,6,7, 10 or 14 days.

In step 318, peak pattern classification takes place. In this step, peak features that are measured from the detected and recognized peaks in the previous steps are compared to predefined values 210 that correspond to certain classes. Examples of algorithms for defining these classes are given below.

The class Cardiac ischemia due to large vessel occlusion

The class Cardiac ischemia due to large vessel occlusion can be defined as:

If troponin level exceeds a height classification level threshold of 100 ng/L OR troponin increases with a classification slope threshold of 10 ng/L per hour then the assigned class is "cardiac ischemia due to large vessel occlusion".

In one embodiment, an additional classification ratio threshold for assigning this class is the presence of a troponin/NT-proBNP ratio greater than 1.

In other embodiments, these classification thresholds can be:

Examples of the classification level threshold for troponin: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 1000 ng/L.

Examples of the classification slope threshold for troponin: 1 ,2,3,4,5,6,7,8,9,10,1 1 ,12,13,14,15,16,17,18,19,20,21 ,22,23,24,25, 30, 40, 50, 100 ng/ L per hour.

Examples of the classification ratio threshold for troponin/NT-proBNP: 0.5, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5.

The class Cardiac ischemia due to microembolization

The class Cardiac ischemia due to microembolization can be defined as:

IF

troponin level exceeds a classification level threshold of 10 ng/L OR troponin increases with a classification slope threshold of 2 ng/L per hour

AND

NT proBNP level exceeds a classification level threshold of 50 ng/L OR NT-proBNP increases with a classification slope threshold of 10 ng/L per hour

THEN

the assigned class is "cardiac ischemia due to microembolization".

In one embodiment, an additional classification ratio threshold for assigning this class is a troponin/NT-proBNP ratio smaller than 0.5.

In one embodiment, a level of CRP exceeding a classification level threshold (e.g. 5, 10, 20 or 30 mg/dl) or exceeding a classification slope threshold (e.g. 2,3,4,5,6,7,8,9,10 or 15 mg/dl per hour) is an additional or supportive criterion for assigning the category "cardiac ischemia due to m icroem bol ization" .

In other embodiments, these classification thresholds for cardiac ischemia due to microembolization can be:

Examples of the classification level threshold for troponin: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 ng/L. Examples of the classification slope threshold for troponin: 1 ,2,3,4,5,6,7,8,9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19,20,21 ,22,23,24, or 25 ng/ L per hour.

Examples of the classification level threshold for NT-proBNP: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 200, 300, 400, 500, or 1000 ng/L.

Examples of the classification slope threshold for NT-proBNP:

1 ,2,3,4,5,6,7,8,9, 10,1 1 ,12,13, 14, 15,16,17, 18, 19,20,21 ,22,23,24,25, 30, 40, 50, 100 ng/ L per hour.

Examples of the classification ratio threshold for troponin/NT-proBNP: 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

It needs to be understood that peak height is underestimated when the sampling frequency is less than optimal. Therefore, the use of a slope criterion in these definitions is useful.

In one embodiment cumulative measures of peaks are computed over a period of time. In step 320, risk stratification takes places. In this step the risk of cardiac death and non-fatal ischemic cardiac events is estimated. An estimation of the level of risk in the method of the invention should set the pace of the initial evaluate and treatment and should be used for (1 ) selection of the site of care (coronary care unit, monitored step-down unit, or outpatient setting) and (2) selection of therapy, especially platelet glycoprotein (GP) llb/llla inhibitors and coronary revascularization.

A risk score is calculated based on the class as assigned in step 318 and stored clinical and risk factor data of the subject. The risk score should be used for be used for (1 ) selection of the site of care (coronary care unit, monitored step-down unit, or outpatient setting) and (2) selection of therapy, especially platelet glycoprotein (GP) llb/llla inhibitors and coronary revascularization.

In the method of the invention ECG information that reflects ischemia, which is present in most questionnaires for estimating cardiac event risk in patients with chest pain (GRACE etc.) is replaced by information from the measurement of marker peaks.

The principle behind the risk score in the method of the invention is that CHD history increases risk of cardiac death and non-fatal ischemic cardiac events in subjects assigned the class "cardiac ischemia due to large vessel occlusion" and that risk factors (high age, diabetes, high cholesterol, high blood pressure, poor renal function) increase the risk of cardiac death and nonfatal ischemic cardiac events in subjects assigned the class "cardiac ischemia due to microembolism".

Examples or risk scores in the method of the invention are:

Five severity categories (from low to high) of the class "cardiac ischemia due to large vessel occlusion":

8, 10, 12, 14, 16 points*

CHD history (three categories from low to high): 0, 2, 4 points

Risk factors (three categories from low to high): 0, 1 , 2 points

Five severity categories (from low to high) of the class "cardiac ischemia due to m icroem bol ization" :

8, 10, 12, 14, 16 points**

Risk factors (three categories from low to high): 0, 2, 4 points

CHD history (three categories from low to high): 0, 1 , 2 points *Examples of categories for the five severity categories of the class "cardiac ischemia due to large vessel occlusion" are:

Category 1 :

A troponin height level of 10-14 ng/L or a troponin slope level 3-4 ng/L per hour

Category 2:

A troponin height level of 15-29 ng/L or a troponin slope level of 5-9 ng/L per hour

Category 3:

A troponin height level of 30 to 49 ng/L or a troponin slope level of 10 to 14 ng/L per hour Category 4:

A troponin height level of 50 to 99ng/L or a troponin slope level of 15 to 19 ng/L per hour

Category 5:

A troponin height level of at least 100 ng/L or a troponin slope level of at least 20 ng/L per hour

If a subject can be assigned to more than one categories, the highest category will be assigned.

In other embodiments these categories may be defined using other cut-off points. Examples of such cut-off point points are:

For category 1 :

Categories of troponin height level: 5-9, 10-14, 15-19, 20-24, 25-29, 30-34, 35-39, 40-44, 45- 49, 50-59, 60-69, 70-79, 80-89, 90-99, 100-149, 150-199, 200-499,500-999, 1000-1999,2000-4999 ng/L.

Categories of troponin slope: 1-2, 3-4, 5-6,7-8,9-10, 1 1-12, 13-14, 15-16, 17-18, 19-20, 10-14, 15- 19,20-29,30-39,40-49,50-59,60-69,70-79,80-89,90-99,100-149, 150-199,200-499 ng/L.

For category 2:

Categories of troponin height level: 5-9, 10-14, 15-19, 20-24, 25-29, 30-34, 35-39, 40-44, 45-

49, 50-59, 60-69, 70-79, 80-89, 90-99, 100-149, 150-199, 200-499,500-999, 1000-1999,2000-4999 ng/L.

For category 3:

For category 4:

Categories of troponin height level: 5-9, 10-14, 15-19, 20-24, 25-29, 30-34, 35-39, 40-44, 45- 49, 50-59, 60-69, 70-79, 80-89, 90-99, 100-149, 150-199, 200-499,500-999, 1000-1999,2000-4999 ng/L. Categories of troponin slope: 1-2, 3-4, 5-6,7-8,9-10, 1 1-12, 13-14, 15-16, 17-18, 19-20, 10-14, 15- 19,20-29,30-39,40-49,50-59,60-69,70-79,80-89,90-99,100-149, 150-199,200-499 ng/L.

For category 5:

Categories of troponin slope: 1-2,3-4,5-6,7-8,9-10, 1 1-12, 13-14, 15-16, 17-18, 19-20, 10-14, 15- 19,20-29,30-39,40-49,50-59,60-69,70-79,80-89,90-99,100-149, 150-199,200-499 ng/L.

In other embodiment some examples values as given in the examples above for level and slope of troponin may be combined, e.g. category 1 for troponin slope may combine the example values range 3-4 and 5-6 to form a range 3-6 ng/L per hour.

**Examples of categories for the five severity categories of the class "cardiac ischemia due to microembolizations" are:

Category 1 :

A troponin height level of at least 10 ng/L or a troponin slope level of at least 5 ng/L per hour AND

A NT-proBNP height level of 25-49 ng/L or a NT-proBNP slope level of 3 -4 ng/L per hour Category 2:

A troponin height level of at least 10 ng/L or a troponin slope level at least 5 ng/L per hour AND

A NT-proBNP height level of 50 to 99 ng/L or a NT-proBNP slope level of 5-9 ng/L per hour Category 3:

A NT-proBNP height level of 100 to 149 ng/L or a NT-proBNP slope level of 10-14 ng/L per hour

Category 4:

A NT-proBNP height level of 150 to 299 ng/L or a NT-proBNP slope level of 14-20 ng/L per hour

Category 5:

A NT-proBNP height level of at least 300 ng/L or a NT-proBNP slope level of at least 20 ng/L per hour

If a subject can be assigned to more than one categories, the highest category will be assigned. In other embodiments these categories may be defined using other cut-off points. Examples of such cut-off point points are:

For category 1 :

Thresholds for troponin height level: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,

80, 85, 90, 95, 100 ng/L.

Thresholds for troponin slope level: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 ng/ L per hour.

The values of the thresholds for troponin height level and troponin slope are used in the OR function as defined above.

Categories of NT-proBNP height level: 5-9, 10-14, 15-19, 20-24, 25-29, 30-34, 35-39, 40-44, 45-49, 50-59, 60-69, 70-79, 80-89, 90-99, 100-149, 150-199, 200-499,500-999, 1000-1999,2000- 4999 ng/L

Categories of NT-proBNP slope: 1-2, 3-4,5-6,7-8,9-10, 1 1-12, 13-14, 15-16, 17-18, 19-20, 10- 14, 15-19,20-29,30-39,40-49,50-59,60-69,70-79, 80-89, 90-99, 100-149, 150-199, 200-499 ng/L.

For category 2:

Thresholds for troponin height level: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ng/L.

Categories of NT-proBNP height level: 5-9, 10-14, 15-19, 20-24, 25-29, 30-34, 35-39, 40-44, 45-49, 50-59, 60-69, 70-79, 80-89, 90-99, 100-149, 150-199, 200-499, 500-999, 1000-1999, 2000- 4999 ng/L

Categories of NT-proBNP slope: 1-2, 3-4, 5-6, 7-8, 9-10, 1 1-12, 13-14, 15-16, 17-18, 19-20, 10-14, 15-19, 20-29, 30-39, 40-49, 50-59, 60-69, 70-79, 80-89, 90-99, 100-149, 150-199, 200-499 ng/L.

For category 3:

80, 85, 90, 95, 100 ng/L.

Categories of NT-proBNP height level: 5-9, 10-14, 15-19, 20-24, 25-29, 30-34, 35-39, 40-44, 45-49, 50-59, 60-69, 70-79, 80-89, 90-99, 100-149, 150-199, 200-499, 500-999, 1000-1999, 2000- 4999 ng/L Categories of NT-proBNP slope: 1-2, 3-4, 5-6, 7-8, 9-10, 1 1-12, 13-14, 15-16, 17-18, 19-20, 10-14, 15-19, 20-29, 30-39, 40-49, 50-59, 60-69, 70-79, 80-89, 90-99, 100-149, 150-199, 200-499 ng/L.

For category 4:

80, 85, 90, 95, 100 ng/L.

The value of the thresholds for troponin height level and troponin slope are used in the OR function as defined above.

For category 5:

Thresholds for troponin slope level: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18,

19, 20, 21 , 22, 23, 24, or 25 ng/ L per hour.

In other embodiment some examples values as given in the examples above for level and slope of troponin may be combined, e.g. category 1 for NT-proBNP slope may combine the example values range 3-4 and 5-6 to form a range 3-6 ng/L per hour.

In one embodiment an additional criterion of a troponin over NT-proBNP ratio below one is an additional reguirement for each of the severity categories for "cardiac ischemia due to microembolization"". In other embodiments this ratio may be different, e.g. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 1 , 4, 1 ,6,1 ,8 or 2.0. In other embodiments, this ratio may be used to also define the severity categories for "cardiac ischemia due to microembolisation".

In other embodiments other cut-offs may be chosen based on increasing experience with the method.

Herein the risk factors are diabetes mellitus, high cholesterol, hypertension and poor renal function. The three categories for risk factors are e.g. 0 risk factors, 1 or 2 risk factors, 3 or 4 or 5 risk factors. Another example is 0 or 1 risk factor, 2 or 3 risk factors, 4 or 5 risk factors. Another example is 0 or 1 risk factor, 2 risk factors, 3 or more risk factors. Also other combinations can be used to define the categories.

The three categories for CHD history are e.g. absent, angina pectoris, history of PTCA,

CABG, ACS or myocardial infarction. Also other definitions of these categories scan be used, corresponding to those in known risk estimation functions.

In the method of the invention, the subject is his or her own control because baseline levels are taken during a stable phase. Therefore, once the level of troponin has crossed the threshold level, a changing pattern is immediately confirmed.

The risk score of a subject is the sum of the obtained points by the subject. The higher the sum the higher the risk. In one embodiment the sum is categorized in categories of low, intermediate and high risk. For example a score of 12 points or higher indicates high risk, a score of 8 to 1 1 points indicates intermediate risk and a score below 8 points indicates low risk. In another example a score of 14 points or higher indicates high risk, a score of 10 to 13 points indicates intermediate risk and a score below 10 points indicates low risk. In other embodiments the cut-off points for the categories may differ as well as the points attributed to each of the variables in the risk score, while keeping the general principle behind attributing the points (heaviest weight on the marker class obtained in 318, CHD history higher weight than risk factors for cardiac ischemia due to large vessel occlusion, risk factors higher weight than CHD history for cardiac ischemia due to microembolisation). In one embodiment the risk score is aligned with known risk scores for subjects with possible ACS such as the GRACE score to appropriately divide subjects in categories of low, intermediate and high risk.

The interpretation of the categories low, intermediate and high corresponds to those in the guidelines for ACS, for example in: 2014 AHA ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. This means that the risk score should be used for be used for (1 ) selection of the site of care (coronary care unit, monitored step-down unit, or outpatient setting) and (2) selection of therapy, especially platelet glycoprotein (GP) llb/llla inhibitors and coronary revascularization.

A diagnosis of cardiac ischemia due to large vessel disease can set a need for immediate contact with a physician. In this situation reperfusion therapy may be most suitable. In this situation, the subject will be informed to contact a physician immediately in 324.

Also a diagnosis of cardiac ischemia due to microembolisation can set a need for immediate contact with a physician in order to reduce an ongoing stream of microembolizations. In this situation optimal anti-platelet therapy and anti-thrombotic therapy may be most suitable. In this situation, the subject will be informed to contact a physician immediately in 324.

When no immediate contact with a physician is required, subjects with an intermediate risk score may be advised to go to a monitored step down unit to be further evaluated and receive appropriate treatment. No need for such action is necessary for subjects with a low risk score which may be advised that no action is needed or to consult a physician at their earliest convenience.

When based on the level or pattern of troponin, NT-proBNP and CRP another diagnosis is made by the device 102, the subject will be informed to contact a physician in 324.

In one embodiment a remote diagnostic center can be contacted by the subject to evaluate the marker pattern and provide therapeutic advice.

Use of the method for treatment monitoring and determination of treatment strategy

The method disclosed herein can also be used to determine whether a treatment against CHD is suitable for a subject, whether treatment is efficient or effective, which treatment strategy is needed and whether the treatment strategy needs to be adjusted.

The method of the invention further involves determining the suitability of a therapy against coronary heart disease in the subject, wherein the presence of cardiac ischemia due to large vessel occlusion as based on the assessment of troponin and NT-proBNP peaks is indicative that treatment against coronary heart disease is suitable for the subject and wherein the presence of cardiac ischemia due microembolization as based on the assessment of troponin and NT-proBNP peaks is indicative that treatment against microembolization is suitable for the subject.

The method of the invention further involves determining the efficacy or effectiveness of a therapy against coronary heart disease in the subject, wherein a decrease in risk of cardiac events as based on the assessment of (reduced occurrence of) troponin or NT-proBNP peaks during treatment is indicative that the treatment is effective and no change or an increase in risk of cardiac events as based on the assessment of troponin or NT-proBNP peaks during treatment is indicative that the treatment is not effective.

The method on the invention further involves determining the treatment strategy of a subject regarding a therapy against CDH, wherein a decrease in cardiac events as based on the assessment of troponin or NT-proBNP peaks during treatment is indicative that the treatment strategy does not need adjustment and no change or an increase in cardiac events as based on the assessment of troponin peaks during treatment is indicative that the treatment strategy needs adjustment

FIG. 4 is a block diagram of a system including an example computing device 400 and other computing devices. Consistent with the embodiments described herein, the aforementioned actions performed by mobile device 102, and devices 112, 140 may be implemented in a computing device, such as the computing device 400 of FIG. 4. Any suitable combination of hardware, software, or firmware may be used to implement the computing device 400. The aforementioned system, device, and processors are examples and other systems, devices, and processors may comprise the aforementioned computing device. Furthermore, computing device 400 may comprise an operating environment for method 300 as described above. Method 300 may operate in other environments and is not limited to computing device 400.

With reference to FIG. 4, a system consistent with an embodiment may include a plurality of computing devices, such as computing device 400. In a basic configuration, computing device 400 may include at least one processing unit 402 and a system memory 404. Depending on the configuration and type of computing device, system memory 404 may comprise, but is not limited to, volatile (e.g. random access memory (RAM)), non- volatile (e.g. read-only memory (ROM)), flash memory, or any combination or memory. System memory 404 may include operating system 405, and one or more programming modules 406. Operating system 405, for example application program and is not limited to any particular application or system. This basic configuration is illustrated in FIG. 4 by those components within a dashed line 420.

Computing device 400 may have additional features or functionality. For example, computing device 400 may also include additional data storage devices (removable and/or non-removable) such as, for example, magnetic disks, optical disks, or tape. Such additional storage is illustrated in FIG. 4 by a removable storage 409 and a non-removable storage 410. Computer storage media may include volatile and nonvolatile, removable and non- removable media implemented in any method or technology for storage of information, such as computer readable instructions, data structures, program modules, or other data. System memory 404, removable storage 409, and nonremovable storage 410 are all computer storage media examples (i.e. memory storage.) Computer storage media may include, but is not limited to, RAM, ROM, electrically erasable read-only memory (EEPROM), flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store information and which can be accessed by computing device 600. Any such computer storage media may be part of device 400. Computing device 400 may also have input device(s) 412 such as a keyboard, a mouse, a pen, a sound input device, a camera, a touch input device, etc. Output device(s) 414 such as a display, speakers, a printer, etc. may also be included. The aforementioned devices are only examples, and other devices may be added or substituted.

Computing device 400 may also contain a communication connection 416 that may allow device 400 to communicate with other computing devices 418, such as over a network in a distributed computing environment, for example, an intranet or the Internet. Communication connection 416 is one example of communication media. Communication media may typically be embodied by computer readable instructions, data structures, program modules, or other data in a modulated data signal, such as a carrier wave or other transport mechanism, and includes any information delivery media. The term "modulated data signal" may describe a signal that has one or more characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media may include wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, radio frequency (RF), infrared, and other wireless media. The term computer readable media as used herein may include both computer storage media and communication media. As stated above, a number of program modules and data files may be stored in system memory 404, including operating system 405. While executing on processing unit 402, programming modules 406 (e.g. program module 407) may perform processes including, for example, one or more of method 300's steps as described above.

The aforementioned processes are examples, and processing unit 402 may perform other processes. Other programming modules that may be used in accordance with embodiments may include electronic mail and contacts applications, word processing applications, spreadsheet applications, database applications, slide presentation applications, drawing or computer-aided application programs, etc.

Generally, consistent with the embodiments, program modules may include routines, programs, components, data structures, and other types of structures that may perform particular tasks or that may implement particular abstract data types. Moreover, embodiments may be practiced with other computer system configurations, including hand-held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. Embodiments may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

Furthermore, embodiments may be practiced in an electrical circuit comprising discrete electronic elements, packaged or integrated electronic chips containing logic gates, a circuit utilizing a microprocessor, or on a single chip (such as a System on Chip) containing electronic elements or microprocessors. Embodiments may also be practiced using other technologies capable of performing logical operations such as, for example, AND, OR, and NOT, including but not limited to mechanical, optical, fluidic, and quantum technologies. In addition, embodiments may be practiced within a general purpose computer or in any other circuits or systems.

Embodiments, for example, are described above with reference to block diagrams and/or operational illustrations of methods, systems, and computer program products according to said embodiments. The functions/acts noted in the blocks may occur out of the order as shown in any flowchart. For example, two blocks shown in succession may in fact be executed substantially concurrently or the blocks may sometimes be executed in the reverse order, depending upon the functionality/acts involved.

While certain embodiments have been described, other embodiments may exist. Furthermore, although embodiments have been described as being associated with data stored in memory and other storage mediums, data can also be stored on or read from other types of computer-readable media, such as secondary storage devices, like hard disks, floppy disks, or a CD-ROM, or other forms of RAM or ROM. Further, the disclosed methods' stages may be modified in any manner, including by reordering stages and/or inserting or deleting stages, without departing from the claimed subject matter.

Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing the claims

Specific embodiments

The invention also pertains to the following numbered embodiments:

1. A method of diagnosing a silent cardiac impairment in a subject, the method comprising the steps of:

a) continual measuring in body fluid samples obtained from the subject the concentration of at least one cardiac troponin or a variant thereof;

b) continual measuring in body fluid samples obtained from the subject the concentration of a

BNP-type peptide or a variant thereof; and,

c) diagnosing silent cardiac impairment in a subject:

i) as due to a silent coronary plaque rupture by detection of a peak in the concentration of at least one cardiac troponin or a variant thereof;

ii) as due to transient left ventricular (LV) dysfunction by detection of a peak in the concentration of a BNP type peptide or a variant thereof; or,

iii) as due to a subclinical alteration in the left ventricular (LV) structure and/or function, by detection of an increase from baseline level of the concentration of a BNP-type peptide or a variant thereof, whereby the concentration does not return to its initial baseline value.

2. A method according to embodiment 1 , wherein the continual measuring comprises measuring at least two samples obtained at a time interval of less than 7 days, or wherein preferably, the continual measuring comprises measuring serial samples obtained at a time interval of less than 7 days, more preferably every two days or daily.

3. A method according to any one of the preceding embodiments, wherein a silent cardiac impairment in a subject is diagnosed as due to a silent coronary plaque rupture by detection of a peak when the concentrations of at least one cardiac troponin or a variant thereof in 2 consecutive samples differ by at least 5 ng/L from each other or in 3 consecutive samples 2 of the samples differ each by at least 5 ng/L from the third sample, and wherein a silent cardiac impairment in a subject is diagnosed as due to transient LV dysfunction by detection of a peak when the concentrations of a BNP type peptide or a variant thereof in 2 consecutive samples differ by at least 50 ng/L from each other or in 3 consecutive samples 2 of the samples differ each by at least 50 ng/L from the third sample.

4. A method according to embodiment 4, wherein a cardiac troponin peak is detected when consecutive samples are obtained with intervals of no more than 2 days and the concentration of at least one cardiac troponin or a variant thereof increases by at least 5 ng/L from baseline in 2 days and returns to baseline in no more than 4 days and wherein wherein a BNP type peptide peak is detected when consecutive samples are obtained with intervals of no more than 2 days and the concentration of a BNP type peptide or a variant thereof increases by at least 50 ng/L from baseline in 2 days and returns to baseline in no more than 4 days. 5. A method according to any one of the preceding embodiments, wherein silent cardiac impairment in a subject is diagnosed as due to a subclinical alteration in the left ventricular (LV) structure and/or function, when an increase of at least 100 ng/L from baseline level of the concentration of a BNP-type peptide or a variant thereof is detected, which increase does not return to its initial value within at least 2 weeks.

6. A method according to any one of the preceding embodiments, wherein the cardiac troponin is at least one of troponin I, troponin T and a variant thereof, of which troponin I or a variant thereof is preferred, and wherein the BNP-type peptide or a variant thereof is NT-proBNP or a variant thereof.

7. A method according to any one of the preceding embodiments, wherein the subject is selected from the group consisting of:

a) an asymptomatic subject;

b) an asymptomatic subject belonging to a risk group for cardiovascular disease, heart failure or cardiovascular disease;

c) a subject diagnosed with stable coronary artery disease (CAD); and,

d) a subject having suffered from an acute coronary syndrome (ACS).

8. A method according to any one of the preceding embodiments, wherein the method further comprises the step of determining the risk of the subject of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death, wherein a subject diagnosed with at least one of silent coronary plaque rupture and transient LV dysfunction is determined to have an increased risk of coronary heart disease and cardiovascular disease and a subject diagnosed with an alteration in LV structure and/or function is determined to have an increased risk of heart failure and/or cardiac death.

9. A method according to any one of the preceding embodiments, wherein the method further comprises the step of determining the susceptibility of the subject to therapy, wherein a subject diagnosed with at least one of silent coronary plaque rupture and transient LV dysfunction is determined susceptible to therapy for coronary heart disease and a subject diagnosed with alterations in LV structure and/or function is determined to be a candidate to undergo further echo or other imaging testing for determination of LV hypertrophy and/or LV dysfunction and optionally therapy against heart failure and/or cardiac death.

10. A method according to any one of the preceding embodiments, wherein the concentrations of at least one of the cardiac troponin or a variant thereof and the BNP-type peptide in the body fluid samples are measured at the point of care.

1 1. A computer comprising a processor and memory, the processor being arranged to read from said memory and write into said memory, the memory comprising data and instructions arranged to provide said processor with the capacity to perform a method according to any one of the preceding embodiments.

12. A computer according to embodiment 12, wherein the computer has an input connected to a sample analyzer for receiving analysis data signals of the concentration of at least one of a cardiac troponin and a BNP-type peptide or variants thereof in body fluid samples obtained from a subject and wherein the processor is arranged for determining from said analysis data signals at least one a diagnosis, risk or susceptibility to therapy in accordance with a method of any one of the preceding embodiments.

13. A sample analyzer comprising a computer in accordance with embodiment 1 1 or 12, wherein the sample analyzer comprises means for measuring the concentration of at least one of a cardiac troponin and a BNP-type peptide or variants thereof in a body fluid sample.

14. A computer program product comprising data and instructions and arranged to be loaded in a memory of a computer, the data and instructions being arranged to provide said computer with the capacity to perform a method according to any one of the preceding embodiments.

15. A data carrier provided with a computer program product as defined in embodiment 14.

16. Use of a method according to any one of embodiments 1 - 7, for determining the risk of the subject of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death.

17. Use of a method according to any one of embodiments 1 - 7, for determining the susceptibility of the subject to at least one of therapy for coronary artery disease and therapy against heart failure.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Description of the drawings

Figure 1 . Block diagram showing the general architecture of the method according to an embodiment of the invention.

Figure 2. Diagram showing the data flow of the method according to an embodiment of the invention.

Figure 3. Flow chart of the method according to an embodiment of the invention.

Figure 4. Block diagram of a system including a computing device according to an embodiment of the invention. Figures 5-12. Individual patient data on concentration of high-sensitivity cardiac troponin-l (hs-cTnl) and NT-proBNP by time of follow-up in patients with stable coronary artery disease (CAD) after acute coronary syndrome (ACS). In Figures 6-8, the y-as represents concentration of hs-cTnl in ng/L on a 2log scale and the x-as represents time in days since ACS. In ). In Figures 9-12, the y-as represents concentration of NT-proBNP in pmol/L on a 2log scale and the x-as represents time in days since ACS. PE cases are patients who developed a primary endpoint (PE) during follow-up. The primary endpoint (PE) was defined as cardiac death, non-fatal myocardial infarction, or unplanned coronary revascularisation for progressive angina. Non-cases are patients who did not develop a primary endpoint during follow-up (n=142). Figure 6, 8, and 1 1 are an enlargements of parts of respectively Figures 5, 7 and 9 wherein individual patients are indicated with numbered arrows. Figure 1 1 shows only the part of Figure 10 after 45 days.

Figure 13. Figures 13A-E display individual patient data on concentration of hs-cTnl by time of follow-up. In the first 57 graphs (45 in PE cases, 12 in non-PE cases), the y-as represents concentration of hs-cTnl in ng/L on a log schale and the x-as represents time in days since ACS on a log scale. The vertical dotted line reflects the timepoint of the recurrent event.

Figure 14. Figures 14A-D show patterns of the same first 45 graphs of the earlier series (PE-cases) where now the y-axis represents concentration of hs-cTnl in ng/L and the x-as represents time in days (without log scales).

Examples

Example 1

Figures 5 to 12 displays data from a study of 844 patients with stable coronary artery disease after acute coronary syndrome (ACS) who were followed for recurrent events for one year after ACS in the BIOMArCS study. BIOMArCS is a multicenter, prospective follow-up study of patients presenting with ACS who were enrolled in 18 hospitals in the Netherlands between March 2008 and January 2015. Levels of high sensitivity troponin-l (hs-cTnl) and NT-proBNP were measured every fortnight in the first half year and monthly thereafter during one year of follow-up.

Figures 5 to 12 displays individual patient data on concentration of hs-cTnl and NT-proBNP by time of follow-up. In the two upper graphs, the y-as represents concentration of hs-cTnl in ng/L on a 2log scale and the x-as represents time in days since index ACS. In the two bottom graphs, the y-as represents concentration of NT-proBNP in pmol/L on a 2log scale and the x-as represents time in days since index ACS. PE cases are patients who developed a primary endpoint (PE) during follow-up (n=45). The primary endpoint (PE) was defined as cardiac death, non-fatal myocardial infarction, or unplanned coronary revascularisation for progressive angina. Non-cases are patients who did not develop a primary endpoint during follow-up (n=142). Because troponin-l reached stable levels only after 45 days (van den Berg et al. European Heart Journal (2017) 38 (Supplement), 787 Abstract: P3648) subjects were included when they had at least two measurements after day 45. Figures 5 to 8 show troponin levels in PE cases and non-cases. PE cases had a significantly higher level of troponin-l during the stabilized phase compared to non-cases (24 ng/L and 7 ng/l respectively, p=0.000). A peak in troponin-l was defined to be present when troponin-l level exceeded a height level of 30 ng/l and the subject exhibited a change defined as the difference between two consecutive measurements of at least 10 ng/l. Seven patients from the group of PE cases (7 out of 22) and 10 patients from the group of non-cases (10 out of 1 17) exhibited a peak in troponin-l. These results indicate that subjects with a troponin-l peak had a 3.7 increased risk of developing a recurrent ACS event compared to subjects without a troponin-l peak (odds ratio =3.7; 95% confidence interval 2.37 - 10.1 ).

Figures 9 to 12 show NT-proBNP levels in PE cases and non-cases. The level of NT-proBNP was extremely high in all subject, in the PE cases as well as in the non-cases. Please note that the scale is not only on a 2log scale but also is in pmol/l (conversion factor pmol/l to ng/l is 8.45). E.g., in PE cases all but 8 measurements were above 200 ng/l and also in non-cases, only a small fraction of the measurements was below 200 ng/l. The upper limit of normal as used in one study was 190 ng/l (Defillipi et al JACC 2010;55:441-50). Even though both groups had extremely high NT-proBNP levels, the mean NT-proBNP level during the stabilized phase was significantly higher PE cases compared to the non-cases (108 versus 47 pmol/l, p=0.001 ). Also remarkable is the high intra-individual variation in NT-proBNP as can be seen in these graphs. Approximately half of the PE cases had at least one measurement above the mean of 108 pmol/l (912 ng/l). Of those, 10 subjects had a peak indicating a change in NT-proBNP that was substantial (Figure 1 1 ). This indicates that most PE cases probably reached this very high level through a transient increase in NT-proBNP. Please note that only 2.4% of subjects in the BIOMArCS study had heart failure. The high levels of NT-proBNP are therefore not related to structural changes in the left ventricle and are likely to be transient.

The data indicate that troponin peaks and NT-proBNP peaks are present in patients outside the acute setting of an ACS and are indicative of an increased risk of coronary heart disease. Troponin levels were increased only moderately and peaks were found more often in PE cases compared to non-cases. Levels of NT-proBNP were higher in PE cases compared to non-cases and were associated in almost all subjects with a substantive change in NT-proBNP.

The combination of a transient moderately elevated levels of troponin and a transient extremely elevated level of NT-proBNP may indicate a silent myocardial injury due to microembolisms. The higher mean CRP levels in PE cases versus non-cases supports this (3.5 mg/l and 2.2 mg/l respectively (p=0.05).

Graphs are based on data from the BIOMArCS study of Erasmus MC, Rotterdam, The Netherlands. The graph was presented as a slide during a presentation on August 28 at the ESC congress, Rome, August 27-31 , 2016, in the Hotlines session Registries scores and outcomes. Akkerhuis M. 2016.12.13 Serial Biomarkers to identify patients vulnerable for myocardial infarction. FP nr. 2017. Example 2

Figures 13 and 14 display data from a study of 844 patients with stable coronary artery disease who were followed for recurrent events for one year after acute coronary syndrome (ACS) (BIOMArCS study). Levels of troponin-l (hs-cTnl) were measured every fortnight in the first half year and monthly thereafter.

Figure 13A-E display individual patient data on concentration of hs-cTnl by time of follow-up. In the first 57 graphs (45 in PE cases, 12 in non-PE cases), the y-as represents concentration of hs-cTnl in ng/L on a log scale and the x-as represents time in days since ACS on a log scale. The vertical dotted line reflects the time point of the recurrent event. Figure 14A-D show patterns of the same first 45 graphs of the earlier series (PE-cases) where now the y-axis represents concentration of hs-cTnl in ng/L and the x-as represents time in days (without log scales). PE cases are patients who developed a primary endpoint (PE) during follow-up. The primary endpoint (PE) was defined as cardiac death, non-fatal myocardial infarction (Ml), or unplanned coronary revascularisation for progressive angina. Non-PE cases are patients who did not develop a primary endpoint during follow-up. Subjects 1-12 from non-PE cases are randomly selected from all non-PE cases. For detection of peaked curves, only patients with 2 or more measurements after 45 days were considered eligible. This resulted in 22 eligible patients among PE cases and 9 eligible non-cases.

A peak in troponin-l was defined to be present when troponin-l level exceeded a height level of 30 ng/l and the subject exhibited a change defined as the difference between two consecutive measurements of at least 10 ng/l. Patients 25, 26, 28, 29, 30, 31 and 45 from the set of PE patients (7/22) and 1 patient, patient 1 (1/9) from the group of non-cases had at least one peak in troponin- I (odds ratio = 2.9, 95% confidence interval 0.34-26.8). Although the confidence interval is wide due to the small number of PE non-cases, the result supports that peaks are more frequent in post-ACS patients who develop a recurrent ACS event compared to patients who do not develop a recurrent ACS event.

Graphs are based on data from the BIOMArCS study of Erasmus MC, Rotterdam, The Netherlands. BIOMArCS is a multicenter, prospective follow-up study of patients presenting with ACS who were enrolled in 18 hospitals in the Netherlands between March 2008 and January 2015. Graphs were made available to the applicant for the purpose of a patent application.

Claims

1. A method of detecting a silent myocardial injury preceding coronary heart disease in a subject, the method comprising the steps of:

b) detecting silent myocardial injury preceding coronary heart disease in a subject by detection of a peak in the concentration of at least one cardiac troponin or a variant thereof.

2. The method according to claim 1 , wherein the continual measuring in body fluid samples obtained from the subject further comprises the concentration of NT-proBNP, and wherein the method further comprises:

a) continual measuring in body fluid samples obtained from the subject the concentration of a BNP-type peptide or a variant thereof; and,

b) detecting silent myocardial injury preceding coronary heart disease based on additional detection in the subject of a peak in the concentration of at least one BNP type peptide or a variant thereof. 3. A method according to claim 1 , wherein the continual measuring comprises measuring at least two samples obtained at a time interval of 7 days or less days, or wherein preferably, the continual measuring comprises measuring serial samples obtained at a time interval of less than 7 days, more preferably every two days or daily or twice a day. 4. A method according to any one of the preceding claims, wherein a silent myocardial injury preceding coronary heart disease in a subject is detected by detection of a peak when the concentrations of at least one cardiac troponin or a variant thereof in 2 consecutive samples differ from each other by a predetermined amount . 5. A method according to claim 4 wherein the predetermined value is at least 1 , 2,

3,

4,

5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 ng/L.

6. A method according to any one of the preceding claims, wherein a silent myocardial injury preceding coronary heart disease in a subject is detected by detection of a BNP-type peptide peak when the concentrations of a BNP type peptide or a variant thereof in 2 consecutive samples differ from each other by a predetermined amount .

7. A method according to claim 6, wherein the predetermined amount is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or 500 ng/L.

8. A method according to any one of the preceding claims, wherein a silent myocardial injury preceding coronary heart disease in a subject is detected by:

detection of a troponin peak when the concentrations of troponin in 2 consecutive samples differ from each other by a first predetermined amount and

by detection of a BNP-type peptide peak when the concentrations of a BNP type peptide or a variant thereof in 2 consecutive samples differ from each other by a second predetermined amount. 9. The method according to claim 8, wherein:

the first predetermined amount is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30ng/L; and

wherein the second predetermined amount is at least 1 , 2, 3, 4, 5, 6, 7, 8,

9, 10, 1 1 , 12, 13, 14, 15, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or 500 ng/L.

10. A method according to any preceding claim, further comprising the step of calculating a troponin baseline and/or an NT-proBNP baseline.

1 1. The method according to claim 10, wherein at least one of the troponin baseline and the NT-proBNP baseline is calculated according to moving average baseline method.

12. A method according to any preceding claim, wherein a silent myocardial injury preceding coronary heart disease is detected by detection of a troponin peak when consecutive samples are obtained within a time period, the time period being preferably no more than 7 days, more preferably no more than 2 days, more preferably less than 24 hours, and the concentration of at least one cardiac troponin or a variant thereof increases by a predetermined amount from a calculated baseline in the time period.

13. A method according to claim 12 wherein the predetermined amount is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 ng/L.

14. A method according to any preceding claim, wherein a silent myocardial injury preceding coronary heart disease is detected by detection of a BNP-type peak when consecutive samples are obtained within a time period, the time period being preferably no more than 7 days, more preferably no more than 2 days, and more preferably less than 24 hours, and the concentration of a BNP type peptide or a variant thereof increases by a predetermined amount from a calculated baseline in the time period.

15. A method according to claim 14, wherein the predetermined amount is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or 500 ng/L. 16. A method according to any preceding claim wherein a silent myocardial injury preceding coronary heart disease is detected when consecutive samples are obtained within a time period, the time period being preferably no more than 7 days, more preferably no more than 2 days, more preferably less than 24 hours; and

a troponin peak is detected when the concentration of at least one cardiac troponin or a variant thereof increases by a first predetermined amount from a calculated baseline within the time period and

a BNP type peptide peak is detected when consecutive samples are obtained with the time period and the concentration of a BNP type peptide or a variant thereof increases by a second predetermined amount from a calculated baseline.

17. A method according to claim 16, wherein:

the first predetermined amount is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15,

16,

17. 18, 19, 20, 25, or 30ng/L; and

wherein the second predetermined amount is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or 500 ng/L.

18. A method according to any preceding claim, wherein a silent myocardial injury preceding coronary heart disease is detected when the troponin level exceeds a first predetermined threshold and troponin change exceeds a second predetermined threshold , and at least one measured NT- proBNP concentration exceeds a third predetermined threshold and NTproBNP change exceeds a fourth predetermined threshold .

19. A method according to claim 18, wherein:

the first predetermined threshold is at least 1 , 2, 3, 4, 5, 10, 20, 30, 40 or 50 ng/L;

the second predetermined threshold is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14,

15, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 ng/L;

the third predetermined threshold is at least 10, 25, 50, 100, 200, 300, 400 ,500, 600, 700, 800, 900, 1000 ng/L;

the fourth predetermined threshold is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400 or 500ng/L.

20. A method according to any one of the preceding claims, wherein the cardiac troponin is at least one of troponin I, troponin T and a variant thereof, of which troponin I or a variant thereof is preferred, and wherein the BNP-type peptide or a variant thereof is NT-proBNP or a variant thereof.

21. A method according to any one of the preceding claims, wherein the subject is selected from the group consisting of:

a) an asymptomatic subject;

c) a subject diagnosed with stable coronary artery disease (CAD); and,

d) a subject having suffered from an acute coronary syndrome (ACS).

22. A method of detecting a peak in troponin for detecting a silent myocardial injury preceding coronary heart disease in a subject, the method comprising the steps of:

(i) receiving a data set comprising a plurality of data points corresponding to a measured concentration of cardiac troponin or variant thereof in a plurality of body fluid samples obtained from a subject at spaced apart time intervals;

(ii) comparing the measured troponin concentration to a predetermined troponin height threshold;

(iii) identifying measured troponin concentrations that exceed the predetermined height threshold;

(iv) determining a change in measured troponin concentrations that exceed the predetermined height threshold, the determined change representing the rate of change of troponin levels in the subject over time;

(v) comparing the determined change in measured troponin concentration to a predetermined threshold change value;

(vi) classifying the data set as indicative of a subject having a silent myocardial injury preceding coronary heart disease based on the comparison of a measured maximum troponin height and/or the calculated troponin change with respective predetermined troponin classification threshold values.

23. The method according to claim 22, wherein the data set further comprises measured concentration of NT-proBNP for each of the plurality of samples, wherein the method further comprises:

(i) comparing the measured NT-proBNP concentration in each of the plurality of samples to a predetermined NT-proBNP height threshold;

(ii) identifying measured NT-proBNP values that exceed the predetermined NT- proBNP height threshold;

(iii) determining a change in measured NT-proBNP values that exceed the NT-proBNP height threshold, the change representing the change of NT-proBNP over time;

(iv) comparing the determined change of the measured NT-proBNP values to a threshold change value;

wherein the step of classification of the data set as indicative of the subject having a silent myocardial injury preceding coronary heart disease is further based on the comparison of a maximum measured NT-proBNP height and the calculated NT-proBNP change with respective predetermined NT-proBNP classification threshold values.

24. The method according to claim 22 or claim 23, further comprising the step of calculating a troponin and/or an NT-proBNP baseline, preferably according to a moving average baseline, and wherein each of the predetermined troponin and NT-proBNP height thresholds is greater than the respective calculated moving average baseline.

25. The method according to any one of claims 22 to 24, wherein the plurality of data points are supplied to the processing means simultaneously as a complete data set.

26. The method according to any one of claims 22 to 25, wherein the data set is an evolving data set, wherein the plurality of data points are supplied to the processing means at spaced apart time intervals, e.g. in real-time as the measurements are taken or with a short delay after taking the measurements.

27. The method according to any of claims 22 to 26, wherein the measured change in troponin concentration is calculated as a difference between two or more measured troponin concentrations, preferably two or more consecutive measured troponin concentrations.

28. A method according to claim 27, wherein the troponin change is based on the difference in level between two or three consecutive samples.

29. A method according to any one of claims 22 to 27 wherein the change in troponin concentration is calculated by comparing at least one measured troponin concentration to a calculated troponin baseline level.

30. The method according to claim 29, wherein the baseline troponin level is calculated as a moving average baseline.

31. The method according to any one of claims 23 to 30, wherein the measured change in NT- proBNP concentration is calculated as a different between two or more measured troponin concentrations.

32. The method according to any of claims 23 to 31 , wherein the NT-proBNP change is based on the different between two or three consecutive samples.

33. The method according to any of claims 23 to 30, wherein the change in NT-proBNP concentration is calculated by comparing at least one measured NT-proBNP concentration to a calculated NT-proBNP baseline level.

34. The method according to claim 33, wherein the baseline NT-proBNP baseline level is calculated as a moving average baseline.

35. The method according to any one of claims 22 to 27, wherein the change in troponin concentration is calculated as a slope of troponin concentration over time.

36. The method according to any one of claims 23 to 30, wherein the change in NT-proBNP concentration is calculated as a slope of NT-proBNP concentration over time.

37. The method according to claim 35 or claim 36, wherein the troponin slope and/or the NT- proBNP slope is calculated according to a least square estimation.

38. The method according to any one of claims 35 to 37, further comprising the step of determining an onset of the troponin slope and/or the NT-proBNP slope by estimating a point at which the calculated slope intersects its respective baseline.

39. The method according to any of claims 22 to 38, further comprising the step of decreasing the time interval between sample collection if at least one measured troponin level and/or one measured NT-proBNP level exceeds the predetermined height threshold.

40. The method according to any one of claims 22 to 39, further comprising:

determining a ratio of measured troponin peak height and measured NT-proBNP peak height for each of the plurality of samples and determining a maximum ratio;

wherein the step of classifying the data set is further based on the calculated maximum ratio.

41. A method according to any of claims 22 to 40, wherein the step of determining the change in troponin values and/or NT-proBNP values includes the last value preceding the exceeding of the height threshold.

42. A method according to any of claims 22 to 41 , wherein:

the troponin detection height threshold is 30 ng/l; and

the troponin detection change threshold is 10 ng/l.

43. A method according to any of claims 22 to 42, wherein a silent myocardial injury preceding coronary heart disease is detected when:

the troponin level exceeds a threshold of 30 ng/l and troponin change exceeds a level of 10 ng/l, and

at least one measured NT-proBNP concentration exceeds a threshold of 900 ng/l and NTproBNP change exceeds a level of 200 ng/l.

44. A method according to any of claims 22 to 43, wherein:

a) the troponin classification height threshold is 5, 10, 20, 30, 40 ,50, 100 ng/l

b) the troponin classification change threshold is 2, 5, 10, ng/l per

c) the NT-proBNP classification height threshold is 50, 100, 200, 500, 1000, 2000 ng/l, d) the NT-proBNP classification change threshold is 25, 50, 100, 200, 500, 1000 ng/l.

45. The method of any of claims 22 to 44 wherein the troponin classification height threshold is an amount at least 20% greater than a concentration of the cardiac troponin marker of a population of healthy individuals.

46. The method of any of claims 22 to 45, wherein the NT-proBNP classification height threshold value is an amount at least 20% greater than a concentration of the cardiac troponin marker of a population of healthy individuals.

47. A method according to any one of the claims 22 to 46, further comprising risk stratification of the subject based on the assigned classification and stored clinical and risk factor data to select the site of care and appropriate therapy.

48. A method according to any preceding claim, further comprising the step of computation of a risk score representing the risk of coronary heart disease in subjects with a detected silent myocardial injury preceding coronary heart disease.

49. A method according to claim 48, wherein the risk score additionally includes risk factors associated with the subject (e.g. high age, diabetes, high cholesterol, high blood pressure and poor renal function).

50. A method according to any one of the preceding claims wherein a plurality of peaks is detected and a cumulative measure of the peaks is used to determine a risk score and wherein a higher frequency and/or duration and/or maximum peak height, is associated with a higher risk of coronary heart disease.

51. A method according to any preceding claim, wherein the method further comprises the step of determining whether a subject is at high risk of coronary heart disease where other risk factors are added or a risk score is used to improve risk prediction.

52. A method according to any one of the preceding claims, wherein the method further comprises the step of risk stratification of the subject wherein a subject in which a silent myocardial injury preceding coronary heart disease is detected is determined to have an increased risk of coronary heart disease and cardiovascular disease.

53. A method according to any one of the preceding claims, wherein the method further comprises the step of determining the susceptibility of the subject to therapy, wherein a subject in which a silent myocardial injury preceding coronary heart disease is detected is determined susceptible to a predetermined therapy for coronary heart disease based on a determination of risk which incorporates the determined classification of silent myocardial injury in the subject.

54. A method according to any one of the preceding claims, wherein the method further comprises the step of determining the effectiveness of a therapy against coronary heart disease in the subject, wherein a decrease in coronary heart disease risk as based on the assessment of (reduced occurrence) of a silent myocardial injury preceding coronary heart disease during treatment is indicative that the treatment is effective and no change or an increase in coronary heart disease risk as based on the assessment of the occurrence of silent myocardial injury preceding coronary heart disease during treatment is indicative that the treatment is not effective.

55. A method according to any one of the preceding claims, wherein the method further comprises the step of determining the treatment strategy of a subject regarding a therapy against coronary heart disease wherein a decrease in coronary heart disease risk as based on the assessment of the occurrence of silent myocardial injury preceding coronary heart disease during treatment is indicative that the treatment strategy does not need adjustment and no change or an increase in coronary heart disease risk as based on the assessment of the occurrence of silent myocardial injury preceding coronary heart disease during treatment is indicative that the treatment strategy needs adjustment.

56. A method according to any one of the preceding claims, wherein the concentrations of at least one of the cardiac troponin or a variant thereof and the BNP-type peptide in the body fluid samples are measured at the point of care.

57. A method according to any of the preceding claims, wherein the step of classification and/or risk stratification is repeated each time a new data point is received.

58. The method according to claim 57, wherein the step of classification and/or risk stratification is independent of whether or not a peak cluster has ended

59. Use of a method according to any one of claims 1 to 58, for determining the risk of the subject of at least one of coronary heart disease, heart failure, cardiovascular disease and cardiac death.

60. Use of a method according to any one of claims 1 to 59, for determining the susceptibility of the subject to at least one of therapy for coronary artery disease and therapy against heart failure.

61. A system comprising processing means for carrying out the following steps:

(i) receiving a data set comprising a plurality of data points each corresponding to a measured concentration of troponin in a plurality of body fluid samples collected from a subject at spaced apart time intervals;

(iii) identifying measured troponin concentrations that exceed the predetermined troponin height threshold;

(iv) determining a change of measured troponin values that exceed the predetermined troponin detection height threshold, the determined change representing the rate of change of measured troponin concentration over time,

(v) comparing the change in the measured troponin values to a predetermined detection change threshold;

(vi) classifying the data set as indicative of the subject having a silent myocardial injury preceding coronary heart disease based on the comparison of a measured maximum troponin height and/or the calculated troponin change with respective predetermined troponin classification threshold values.

62. The system according to claim 61 , wherein the processing means is further configured to carry out the following steps:

comparing the measured NT-proBNP level in each of the plurality of samples to a predetermined NT-proBNP baseline threshold;

identifying measured NT-proBNP values that exceed the predetermined NT-proBNP baseline threshold;

determining a slope of the plurality of measured NT-proBNP levels that exceed the NT- proBNP baseline threshold, the slope representing the change of NT-proBNP over time; and

comparing the slope of the measured NT-proBNP values to a threshold slope value; wherein classification of the data set is further based on the comparison of the calculated NT-proBNP slope and the measured NT-proBNP peak height with the predetermined NT-proBNP threshold values.

63. The system according to any of claims 61 to 62, wherein the processing means is provided as part of a point of care testing system.

64. The system according to claim 63, wherein the point of care testing system comprises at least one biosensor configured to measure levels of troponin and/or NT-proBNP in a sample acquired from a subject.

65. A system according to claim 64, wherein the biosensor is configured to measure both troponin I and troponin T.

66. A system according to claim 64 or 65 wherein the biosensor is configured to measure troponin I using antibodies that recognize epitopes on the specific forms of troponin

I that originate from the cytosolic pool in the early phase of myocardial infarction.

67. A system according to claim 64, 65 or 66, wherein the biosensor is configured to measure troponin T using antibodies that recognize epitopes on the specific forms of troponin T that originate from the release of structurally bound troponin T in the later phase of myocardial infarction.

68. The system according to any of claims 64 to 67, wherein the processing means is provided in the biosensor.

69. The system according to any one of claims 62 to 68, wherein the processing means is provided in a mobile device, e.g. a mobile telephone.

70. The system according to claim 67, wherein the biosensor is configured for wireless communication with the mobile device.

71. The system according to any one of claims 61 to 70, wherein the system further comprises a memory configured to store additional clinical data.

72. The system according to any one of claims 61 to 71 , wherein the system is configured to alert a subject and/or a healthcare provider to adapt the time interval between sample collection in response to at least one of a measured troponin value or a measured NT-proBNP value exceeding the respective predetermined height threshold.

73. The system according to any one of claims 61 to 72, further comprising communication means configured to allow communication with a system of a healthcare provider, wherein the system is configured to provide alert information to the healthcare provider based on the classification of the data.

74. A computer program product comprising instructions which, when the program is executed by a computer, cause the computer to carry out the method of any one of claims 22 to 60.

75. Computer-readable medium comprising instructions which, when executed by a computer, cause the computer to carry out the steps of any one of claims 22 to 60.

76. A computer comprising a processor and memory, the processor being arranged to read from said memory and write into said memory, the memory comprising data and instructions arranged to provide said processor with the capacity to perform a method according to any one of the preceding claims.

77. A computer according to claim 76, wherein the computer has an input connected to a sample analyzer for receiving analysis data signals of the concentration of at least one of a cardiac troponin and a BNP-type peptide or variants thereof in body fluid samples obtained from a subject and wherein the processor is arranged for determining from said analysis data signals at least one a diagnosis, risk or susceptibility to therapy in accordance with a method of any one of the preceding claims.

78. A sample analyzer comprising a computer in accordance with claim 76 or 77, wherein the sample analyzer comprises means for measuring the concentration of at least one of a cardiac troponin and a BNP-type peptide or variants thereof in a body fluid sample.

79. A computer program product comprising data and instructions and arranged to be loaded in a memory of a computer, the data and instructions being arranged to provide said computer with the capacity to perform a method according to any one of the preceding claims.

80. A data carrier provided with a computer program product as claimed in claim 79.