Myocardial injury assessment method and device, sample analysis system and application thereof

Abstract

The invention relates to a myocardial injury assessment method and device, a sample analysis system and application thereof. According to the method, the device and the sample analysis system, individual myocardial injury pathogenesis and risk can be rapidly and accurately evaluated, and diagnosis decisions and treatment decisions can be guided.

Description

Myocardial injury assessment method, device, sample analysis system and use thereof

[0001] This application is based on the application with CN application number 202311024062.1 and application date August 14, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present application belongs to the field of disease diagnosis, and specifically relates to a method for evaluating myocardial damage in a subject in vitro, a device for obtaining characteristic parameters for evaluating myocardial damage in a subject, a sample analysis system, and a reagent for quantitative detection of a large-size cardiac troponin ternary complex and / or a reagent for quantitative detection of a total cardiac troponin ternary complex in the preparation of a kit. Background Art

[0003] Cardiovascular diseases pose a serious threat to human health. In China, the morbidity and mortality rates of cardiovascular diseases are increasing year by year. Chest pain is a common symptom of various cardiovascular diseases, accompanied by various clinical manifestations, and is often associated with dyspnea. The risk of acute fatal chest pain is extremely high. Accurate risk stratification and diagnosis of patients with chest pain in the emergency department and the establishment of rapid and reasonable diagnostic procedures are crucial for correct management and treatment decision-making. Distinguishing the causes of chest pain, especially acute and non-acute myocardial injury, is of great clinical significance and urgent clinical need.

[0004] Cardiac troponin (cTn) is a highly specific and sensitive biomarker of myocardial injury and is widely used to detect myocardial injury after or during myocardial infarction.

[0005] Cardiac troponin consists of three subunits: cardiac troponin I (cTnI), cardiac troponin T (cTnT), and troponin C (TnC). Serum concentrations of cTnI and cTnT are highly correlated with the severity of myocardial injury. Troponin typically binds to actin filaments in the form of a ternary complex (cTnITC). Upon myocardial injury, troponin is degraded from the myofilaments and released into the blood. Within cells or in the circulation, cTnITC is degraded into different forms by proteases. Studies have shown that the form of troponin present in the blood may be associated with an individual's physiological and pathological status.

[0006] A deep understanding of the relationship between different forms of troponin and diseases, the correct interpretation of the causes of elevated cTn, and the distinction between acute and non-acute myocardial injury are crucial for the rapid stratification and accurate clinical diagnosis of patients with chest pain.

[0007] Summary of the Invention

[0008] In order to solve the above technical problems, in a first aspect, the present application provides a method for assessing myocardial damage in a subject in vitro, comprising:

[0009] detecting the level of one or more myocardial injury markers in a sample from the subject;

[0010] obtaining characteristic parameters for assessing myocardial injury based on the content of the one or more myocardial injury markers;

[0011] comparing the characteristic parameter with a reference value of the characteristic parameter;

[0012] Based on the results of the comparison, assessing myocardial damage in the subject;

[0013] The one or more myocardial injury markers include a large-size cardiac troponin ternary complex (large-size cTnITC, large-size ITC complex) and / or a total cardiac troponin ternary complex (total cTnITC, total ITC complex). The large-size cardiac troponin ternary complex may also be referred to as a long cardiac troponin ternary complex (long cTnITC, long ITC complex).

[0014] In a second aspect, the present application provides a device for obtaining characteristic parameters for assessing myocardial damage in a subject, comprising:

[0015] a data receiving module configured to receive levels of one or more myocardial injury markers obtained from a sample from a subject, wherein the one or more myocardial injury markers include a large cardiac troponin ternary complex and / or a total cardiac troponin ternary complex;

[0016] a data processing module configured to process the content data of one or more myocardial injury markers received by the receiving module to obtain characteristic parameters for evaluating myocardial injury;

[0017] And, an output module is configured to output the characteristic parameters.

[0018] In a third aspect, the present application provides a sample analysis system, comprising:

[0019] A sample holding portion, used for holding a container containing a sample of a subject;

[0020] The sample dispensing part is used to draw the sample of the subject from the sample holding part and discharge it into the reaction cup to be loaded with the sample;

[0021] A reagent carrying portion, used for carrying detection reagents;

[0022] The reagent dispensing part is used to draw the detection reagent from the reagent carrying part and discharge it into the reaction cup to be added with the reagent;

[0023] The reaction part is used to place the reaction cup so as to incubate the test solution obtained by the reaction of the subject's sample and the detection reagent in the reaction cup;

[0024] a detection unit having a signal detector for detecting a signal of a test solution in a reaction cup to measure and output the content of one or more myocardial injury markers in a sample of a subject, wherein the one or more myocardial injury markers include a large-sized cardiac troponin ternary complex and / or a total cardiac troponin ternary complex;

[0025] The data processing unit includes a processor and a computer-readable storage medium, wherein the computer-readable storage medium stores computer-readable instructions, wherein when the computer-readable instructions are executed by the processor, the processor is caused to perform the following steps:

[0026] receiving and processing the content of the one or more myocardial injury markers to obtain characteristic parameters for assessing myocardial injury; and

[0027] The characteristic parameters are output.

[0028] In a fourth aspect, the present application provides the use of a reagent for quantitatively detecting a large-sized cardiac troponin ternary complex and / or a reagent for quantitatively detecting a total cardiac troponin ternary complex in a sample in the preparation of a kit, wherein the kit is used to assess myocardial damage in a subject.

[0029] In a fifth aspect, the present application provides a reagent for quantitatively detecting a large-sized cardiac troponin ternary complex and / or a reagent for quantitatively detecting a total cardiac troponin ternary complex in a sample, for use in assessing myocardial damage in a subject.

[0030] In various aspects of the present application, characteristic parameters obtained based on the content of large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex can be used to assess myocardial damage more quickly and accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0032] FIG1 is a schematic block diagram of an apparatus for obtaining characteristic parameters for evaluating myocardial injury in a subject, provided in an embodiment of the present application.

[0033] FIG2 shows a schematic diagram of a sample analysis system provided in an embodiment of the present application.

[0034] FIG3 is an analysis of the signal-to-noise ratio of the large-size cTnITC detection kit. The signal-to-noise ratio data of each group of samples correspond to kits 1 to 8 from left to right.

[0035] FIG4 is an analysis of the signal-to-noise ratio of the total cTnITC detection kit. The signal-to-noise ratio data of each group of samples correspond to kits 1 to 12 from left to right.

[0036] FIG5 shows the specificity of the large-scale cTnITC detection kit validated using serum samples.

[0037] FIG6 shows the specificity of the total cTnITC detection kit validated using serum samples.

[0038] FIG7 shows the linearity analysis of the large-size cTnITC detection kit 3.

[0039] FIG8 shows the linearity analysis of large-scale cTnITC detection kit 4.

[0040] FIG9 shows the linearity analysis of total cTnITC detection kit 3.

[0041] FIG10 shows the linearity analysis of the total cTnITC detection kit 4.

[0042] Figure 11 shows the enrollment and testing process for patients with early myocardial infarction.

[0043] Figure 12 shows the ratios of large cTnITC / total complexes, total cTnITC / total complexes, and cTnT / total complexes in patients with different chest pain durations. P values ​​between groups were compared using the Kruskal-Wallis test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significantly different.

[0044] Figure 13 shows the ratios of large cTnITC / total complexes, total cTnITC / total complexes, cTnT / total complexes, large cTnITC / cTnT, and total cTnITC / cTnT in patients with type 1 and type 2 myocardial infarction. P values ​​between type 1 and type 2 myocardial infarction were compared using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ns, not significant.

[0045] FIG14 shows the enrollment and testing process of patients with acute myocardial infarction (type 1) in Example 3-1.

[0046] FIG15 shows the enrollment and testing process for patients with chronic cardiac events (cardiomyopathy or chronic heart failure) in Example 3-1.

[0047] FIG16 shows the enrollment and testing process of patients with chronic cardiac events (pneumonia) in Example 3-1.

[0048] Figure 17 shows the ratios of large cTnITC / total complexes, total cTnITC / total complexes, cTnT / total complexes, large cTnITC / cTnT, and total cTnITC / cTnT in samples from acute myocardial infarction (type 1) and chronic cardiac events. Values ​​are presented as median ± interquartile range. P values ​​between patients with acute myocardial infarction (type 1), cardiomyopathy or chronic heart failure, and pneumonia were compared using the Kruskal-Wallis test. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

[0049] Figure 18 shows the patient enrollment and testing process for invasive procedures.

[0050] Figure 19 shows the enrollment and testing process for patients with chronic cardiac events.

[0051] Figure 20 shows the ratios of large cTnITC / total complexes, total cTnITC / total complexes, cTnT / total complexes, large cTnITC / cTnT, and total cTnITC / cTnT in patients with invasive myocardial injury and chronic cardiac events. Values ​​are presented as median ± interquartile range. P values ​​for patients with chronic cardiac events, surgical procedures, and interventional procedures were compared using the Kruskal-Wallis test. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

[0052] Figure 21 shows the patient enrollment and testing process for cardiac surgery.

[0053] FIG22 shows the survival curves of cardiac surgery patients in different troponin fragment concentration groups.

[0054] Figure 23 shows the enrollment and testing process for patients with acute myocardial infarction (type 1).

[0055] FIG24 shows the survival curves of patients with acute myocardial infarction (type 1) in different troponin fragment concentration groups.

[0056] Figure 25 shows the enrollment and testing process for patients with chronic cardiac events (cardiomyopathy or chronic heart failure).

[0057] FIG26 shows the survival curves of patients with chronic cardiac events (cardiomyopathy or chronic heart failure) in different troponin fragment concentration groups.

[0058] Figure 27 shows the enrollment and testing process for patients with acute chest pain suspected of coronary syndrome.

[0059] The technical solutions in the embodiments will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all. The following description of the embodiments is merely illustrative and in no way limits the present invention. All other embodiments obtained by ordinary technicians in this field based on the embodiments without creative work are within the scope of protection of the present invention.

[0060] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the immunology laboratory procedures used herein are conventional procedures widely used in the relevant fields. To better understand the embodiments of the present invention, definitions and explanations of relevant terms are provided below.

[0061] As used herein, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a method or apparatus comprising a series of elements includes not only the elements explicitly recited, but also other elements not explicitly listed, or elements inherent to the practice of the method or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other related elements in the method or apparatus comprising the element.

[0062] As used herein, the term "at least one" means 1 or more than 1 under reasonable conditions, such as 2, 3, 4, 5 or 10, etc.

[0063] As used herein, the terms "first" and "second" merely distinguish similar objects and do not represent a specific ordering of the objects. It is understood that "first" and "second" can be interchanged to indicate a specific order or precedence where appropriate. It should be understood that the objects distinguished by "first" and "second" can be interchanged where appropriate so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein.

[0064] As used herein, the terms "individual" and "subject" are preferably mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats), preferably humans. In some embodiments, it specifically refers to patients with specific clinical symptoms, such as patients with chest pain.

[0065] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its antigen. The binding affinity between two molecules can be expressed as K. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as a molar concentration (M). K D The smaller the value, the tighter the two molecules bind and the higher the affinity. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Bind to the antigen. Corresponding methods for analyzing antibody specificity are described, for example, in Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press and Harlow & Lane (1999) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Non-limiting examples of suitable studies are, for example, binding studies, blocking and competition studies using structurally and / or functionally closely related molecules. These studies can be performed using methods such as fluorescence activated cell sorting (FACS) analysis, flow cytometry titration (FACS titration) analysis, surface plasmon resonance (SPR, for example using), isothermal titration calorimetry (ITC), fluorescence titration or radiolabeled ligand binding assays. More methods include, for example, immunoblotting (Western Blot), ELISA (including competitive ELISA) tests, RIA tests, ECL tests and IRMA tests.

[0066] As used herein, the term "diagnosis" refers to methods by which it can be estimated and / or determined whether a patient has a particular disease or condition.

[0067] As used herein, the term "prognosis" is generally determined by one or more markers or characteristic parameters that indicate the probability that a particular outcome or process will occur.

[0068] As used herein, the terms "sample" and "specimen" refer to a body fluid or tissue sample of a subject, such as whole blood, serum, plasma (including lithium heparin plasma, EDTA plasma), urine, saliva, biological tissue or cells, preferably whole blood, serum or plasma.

[0069] As used herein, the terms "troponin" and "Tn" refer to proteins on myosin within myocytes that regulate the calcium-mediated interaction between actin and myosin. They are present in cardiac and skeletal muscle and are composed of three subunits: troponin T (TnT), troponin I (TnI), and troponin C (TnC). TnT is the tropomyosin-binding subunit that interacts with actin and tropomyosin; TnI is the inhibitory subunit that inhibits the ATPase activity of actomyosin; and TnC is the only subunit that binds to calcium and enables skeletal or cardiac muscle contraction. The terms "cardiac troponin" and "cTn" refer to all troponin isoforms expressed in cardiac cells, preferably subendocardial cells. These isoforms have been well characterized in the art, for example as described in Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493. The term "cardiac troponin" also includes variants of a particular cardiac troponin that have at least the same basic biological and immunological properties as that particular cardiac troponin. In particular, if they are detected by the same specificity as mentioned herein, they share the same basic biological and immunological properties. It will be understood that the isoforms of troponin can be assayed together (simultaneously or sequentially) or separately (i.e., without assaying the other isoforms at all).

[0070] As used herein, the terms "cardiac troponin T" and "cTnT" refer to the cardiac troponin T subunit, the amino acid sequence of which is disclosed in the UniProt database under accession number P45379.

[0071] As used herein, the terms "cardiac troponin I" and "cTnI" refer to the cardiac troponin I subunit, the amino acid sequence of which is disclosed in the UniProt database under accession number P19429.

[0072] As used herein, the terms "troponin C" and "TnC" refer to the troponin C subunit, the amino acid sequence of which is disclosed in the UniProt database under accession number P63316.

[0073] As used herein, the term "cardiac troponin binary complex" includes a binary complex consisting of a full-length troponin C protein or any amino acid fragment thereof and a full-length cardiac troponin I protein or any amino acid fragment thereof.

[0074] As used herein, the terms "large-size cardiac troponin" or "large-size cTnITC (large-size ITC complex)" are used interchangeably herein and are intended to include a complex formed by any of the full-length protein or fragments of TnC, the full-length protein or fragments of cTnI, one or more stretches of amino acid residues 223-287 of cTnT, and one or more stretches of amino acid residues 1-222 of cTnT.

[0075] As used herein, the terms "total cardiac troponin ternary complex" or "total cTnITC (total ITC complex)" are used interchangeably herein and are intended to include complexes formed by all full-length proteins or fragments of TnC, full-length proteins or fragments of cTnI, and one or more stretches of amino acid residues 223-287 of cTnT, and optionally one or more stretches of amino acid residues 1-222 of cTnT.

[0076] As used herein, the terms "total cardiac troponin complex" and "total complex" include the total cardiac troponin ternary complex, the binary complex consisting of the full-length protein of troponin C or any amino acid fragment thereof and the full-length protein of cardiac troponin I or any amino acid fragment thereof.

[0077] Differential assessment and diagnostic methods

[0078] Cardiac troponin is a highly specific and sensitive biomarker of myocardial injury and is widely used to detect myocardial injury after or during myocardial infarction. However, troponin analysis currently cannot distinguish between different types of myocardial injury. This field still lacks effective tools to assess myocardial injury, distinguish between different types of myocardial injury, and assist clinicians in making rapid and accurate diagnoses of individual pathological conditions, thereby guiding prognosis.

[0079] The first aspect of the present application provides a method for assessing myocardial damage in a subject in vitro, comprising:

[0080] detecting the level of one or more myocardial injury markers in a sample from the subject;

[0081] obtaining characteristic parameters for assessing myocardial injury based on the content of the one or more myocardial injury markers;

[0082] comparing the characteristic parameter with a reference value of the characteristic parameter;

[0083] Based on the results of the comparison, assessing myocardial damage in the subject;

[0084] Wherein, the one or more myocardial injury markers include large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex.

[0085] The term "myocardial injury" is known in the art and refers to pathological changes in heart cells, which lead to abnormal cardiac performance. Patients may experience chest pain, palpitations, shortness of breath, palpitations, etc. th URL is diagnosed as myocardial injury. If it is accompanied by an increase or decrease, it is considered acute myocardial injury. If it remains elevated with an increase of less than 20%, it may be chronic myocardial injury. Myocardial injury can be seen in a variety of cardiac and non-cardiac diseases.

[0086] In this article, the assessment of myocardial injury includes diagnosis (eg, diagnosis of etiology or classification), grading (eg, grading the severity or stage of myocardial injury), or monitoring (eg, prognosis) of myocardial injury.

[0087] Differential assessment or diagnosis refers to a method of diagnosing a specific disease and / or specific condition in a particular individual based on a comparison of characteristic properties observable in that individual with characteristic properties of the underlying disease that underlies the symptoms of that particular individual. Depending on the range of diseases and conditions that must be considered in a differential assessment or diagnosis, the type and number of laboratory analyses that a physician must perform can be very diverse. For example, in the case of chest pain, a physician may choose to perform a range of laboratory analyses, including a physical examination, an echocardiogram, an electrocardiogram recording, a high-sensitivity troponin I test, a high-sensitivity troponin T test, and a coronary angiogram. However, the physician must integrate the information obtained from the suite of tests to arrive at a clinical diagnosis that most closely represents the range of symptoms and / or diagnostic test results from the subject.

[0088] This application describes markers and characteristic parameters that can be used or used to assist in the assessment of myocardial injury. By detecting the level of one or more myocardial injury markers in a sample, and further obtaining a characteristic parameter for assessing myocardial injury, the value of the characteristic parameter is compared with the value of the characteristic parameter in individuals suffering from a specific condition or at risk of a specific condition, or with the value of the characteristic parameter in individuals known not to suffer from the specific condition. The results of such comparison are then linked to an assessment or diagnosis, thereby generating a response.

[0089] Myocardial injury markers and characteristic parameters

[0090] In the present application, the markers used to assess myocardial injury include at least large cardiac troponin ternary complex and / or total cardiac troponin ternary complex. Depending on the diagnostic scenario of application, the markers may further include one or more of cTnI, cTnT, TnC, cardiac troponin binary complex, and total cardiac troponin complex.

[0091] The content of the marker is particularly preferably a concentration, which can be determined using an immunoassay. Examples of such immunoassays are enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence. In some embodiments, the quantitative detection of each marker is particularly preferably performed using an ELISA method, for example, using a commercially available ELISA kit for detection.

[0092] Comparison with reference values

[0093] "Comparison" refers to comparing the characteristic parameter obtained from the experimenter with its reference value. It should be understood that comparison used herein generally refers to the comparison of the value of the corresponding characteristic parameter. The comparison can be performed manually or computer-assisted. Therefore, the comparison can be implemented by a computer device (such as a device or analysis system disclosed herein). The characteristic parameter and the reference value can be, for example, compared with each other and the comparison can be automatically implemented by a computer program executing an algorithm for comparison. The computer program implementing the evaluation will provide the desired evaluation in a suitable output form. For computer-assisted comparison, the characteristic parameter can be compared with a suitable reference value, and the reference value is stored in a database by a computer program. The computer program can further evaluate the comparison result, i.e., automatically provide the desired evaluation in a suitable output form.

[0094] Reference values ​​can be used to define and establish threshold values. Threshold values ​​are preferably used to assess / diagnose subjects as described herein. The diagnosis or assessment can be provided by a data processing module of a device or system described herein based on a calculated "value" and a reference value or threshold value. For example, the data processing module of the system can provide an indicator in the form of text, symbols or numerical values ​​that indicates the diagnosis or assessment. The reference value applicable to the subject can vary, depending on the marker selected and its determination method. Suitable reference values ​​can be determined from a reference sample to be analyzed together with the test sample (i.e., simultaneously or sequentially).

[0095] In principle, the reference level of a patient group suffering from a specific disease or at least one abnormality or not suffering from the above-mentioned disease or abnormality can be calculated by applying standard statistical methods based on the mean or median value of a specific marker. In some embodiments, statistical analysis is performed using Graphpad Prism, SPSS and Excel software. The statistical differences between different subgroups are analyzed by Mann-Whitney U test (for comparison between two groups) and Kruskal-Wallis test (for comparison between multiple groups) to determine statistical significance. A P value <0.05 is considered to be significant.

[0096] Specifically, the accuracy of a test method (such as for a diagnostic event) is preferably described by a receiver operator characteristic curve (ROC) (see Zweig 1993, Clin. Chem. 39: 561-577). The ROC curve is a curve drawn based on a series of different binary classification methods (cutoff thresholds), with the true positive rate (TPR) as the vertical axis and the false positive rate (FPR) as the horizontal axis. The ROC_AUC (area under the curve) represents the area enclosed by the ROC curve and the horizontal axis. In some embodiments, the ROC curve is drawn using SPSS software to obtain the area under the curve AUC. Different TPRs and FPRs can be obtained on the ROC curve by adjusting the threshold. The larger the threshold, the smaller the FPR and the larger the TPR. Conversely, the larger the FPR, the smaller the TPR. Youden's J statistic is a statistic for evaluating the performance of a classifier. It is equal to TPR-FPR, that is, sensitivity. The larger its value, the better the performance of the classifier. On the ROC curve, the point where Youden's J statistic reaches its maximum value is called the Youden point, and the corresponding threshold is called the Youden threshold. In some embodiments, the Youden threshold is used as the optimal threshold point (CUTOFF value).

[0097] In some embodiments, the present invention provides a method for determining the confidence interval of a patient's disease or condition. The method may be used to determine the confidence interval of the patient's disease or condition. The method may be used to determine the confidence interval of the patient's disease or condition. The method may be used to determine the confidence interval of the patient's disease or condition. The method may be used to determine the confidence interval of the patient's disease or condition. The method may be used to determine the confidence interval of the patient's disease or condition. The method may be used to determine the confidence interval of the patient's disease or condition. The method may be used to determine the confidence interval of the patient's disease or condition. The method may be used to determine the confidence interval of the patient's disease or condition.

[0098] The effectiveness of a diagnostic method and the predictive value of marker parameters for primary endpoints are described by the receiver operating characteristic (ROC) curve. The ROC curve is plotted based on the sensitivity and specificity obtained by continuously varying the judgment threshold within the observed data range. The Y-axis represents sensitivity and the X-axis represents 1 minus specificity. The closer the curve is to the upper left corner, the higher the diagnostic accuracy. The area under the receiver operating characteristic (AUC) indicates the effectiveness or correctness of the diagnosis. A P value < 0.05 is considered significant. Because the ROC curve is composed of multiple critical values ​​representing their respective sensitivity and specificity, the ROC curve can be used to select the optimal diagnostic limit for a diagnostic method. The closer the ROC curve is to the upper left corner, the higher the test sensitivity, the lower the false positive rate, and the better the performance of the diagnostic method. It can be seen that the point on the ROC curve closest to the upper left corner has the highest sum of sensitivity and specificity. This point or the value corresponding to its adjacent points is often used as a diagnostic reference value (also called a diagnostic threshold, judgment threshold, preset condition, or preset range).

[0099] In one embodiment, the term "reference value" can be a predetermined value. As will be appreciated by the skilled person, a reference value is predetermined and is arranged on, for example, specificity and / or sensitivity, to meet conventional requirements. These requirements can, for example, change between administrative departments. It can, for example, measure sensitivity or specificity and must be arranged on certain limits, for example, 80%, 90%, 95% or 98% respectively. These requirements can also be limited in terms of positive or negative predictive value. Nevertheless, based on the teachings of the present invention, reaching a reference value that meets these requirements is easy to accomplish. The reference value is derived from a patient suffering from a disease or abnormality as mentioned herein, or suffers from a patient with its severe or mild form.

[0100] In some embodiments, the reference value has been predetermined in the reference sample of the disease suffered by the subject. In some embodiments, the reference value can, for example, be set to any percentage between 25% and 75% of the overall distribution of the characteristic parameter in the disease under study. In other embodiments, the reference value can, for example, be set to, determined according to the overall distribution of the disease reference sample under study, the median, tertiles or quartiles. In some embodiments, the reference value can be set to the median determined from the overall distribution of the characteristic parameter in the disease under study. In some embodiments, continuous variables are presented as medians (25-75% quartiles); categorical variables are presented as digital (percentage) representations.

[0101] In the present application, the characteristic parameter for assessing myocardial injury can be selected from the content of large-size cardiac troponin ternary complex, the content of total cardiac troponin ternary complex, and a combination of the content of large-size cardiac troponin ternary complex and / or the content of total cardiac troponin ternary complex and the content of other myocardial injury markers (e.g., cTnI, cTnT or total cardiac troponin complex).

[0102] In some embodiments, characteristic parameters for assessing myocardial injury are obtained based on the levels of the detected markers, including:

[0103] determining the content of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; or

[0104] obtaining characteristic parameters for assessing myocardial damage based on the content of the large-sized cardiac troponin ternary complex and the content of the total cardiac troponin ternary complex;

[0105] For example, the characteristic parameter for assessing myocardial damage is determined based on the content change (including difference and ratio) of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex before and after the event.

[0106] In some embodiments, the one or more myocardial injury markers preferably further include at least one of cTnI, cTnT, and total cardiac troponin complex.

[0107] In some embodiments, based on the content of one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including: inputting the content of the multiple myocardial injury markers into a preset function model to obtain the output of the preset function model as a characteristic parameter for assessing myocardial injury. The input preset function model can be, for example, a linear function model, a nonlinear function model, or a machine learning model. In some embodiments, a binary logistic regression model is used to construct a combination of variables for establishing a combination parameter. In some embodiments, a binary logistic regression analysis is used to analyze the correlation between marker parameters and the occurrence of primary endpoint events. A P value of <0.05 is considered to indicate that the variable is significant. Cox regression analysis is used to determine the relationship between marker parameters or their combination and the outcome of the study subjects, and the hazard ratio (HR) is calculated based on the Cox proportional hazard model to analyze the risk level of different risk groups of marker parameter values, compared to the multiple of the risk of an endpoint event in the baseline group. A P value of <0.05 is considered to indicate that the variable is significant.

[0108] In one example, the contents of multiple myocardial injury markers can be directly input as variables into a preset function model, for example, the content of large-size cardiac troponin ternary complex or total cardiac troponin ternary complex and the content of at least one of cTnI, cTnT and total cardiac troponin complex can be input as variables into the preset function model to obtain the output of the preset function model as a characteristic parameter for evaluating myocardial injury.

[0109] In another example, the ratio between the contents of multiple myocardial injury markers can also be input as a variable into a preset function model, for example, the ratio of the content of large-size cardiac troponin ternary complex and / or total cardiac troponin ternary complex to the content of at least one of cTnI, cTnT and total cardiac troponin complex can be input as a variable into the preset function model to obtain the output of the preset function model as a characteristic parameter for evaluating myocardial injury.

[0110] Herein, when describing the ratio of a certain myocardial injury marker level to another myocardial injury marker level, it includes the ratio of the myocardial injury marker level to the other myocardial injury marker level, and the ratio of the other myocardial injury marker level to the myocardial injury marker level.

[0111] In some embodiments, the characteristic parameter is obtained by the ratio between the levels of multiple myocardial injury markers, that is, based on the levels of the one or more myocardial injury markers, obtaining the characteristic parameter for assessing myocardial injury comprises: determining one of the following ratio parameters as the characteristic parameter for assessing myocardial injury, or obtaining the characteristic parameter for assessing myocardial injury based on at least two of the following ratio parameters, or obtaining the characteristic parameter for assessing myocardial injury based on at least one of the following ratio parameters and the ratio of the level of cTnT to the level of total cardiac troponin complex;

[0112] The ratio parameters include: the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnI, the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnT, the ratio of the content of large-size cardiac troponin ternary complex to the content of total cardiac troponin complex, the ratio of the content of total cardiac troponin ternary complex to the content of cTnI, the ratio of the content of total cardiac troponin ternary complex to the content of cTnT, the ratio of the content of total cardiac troponin ternary complex to the content of total cardiac troponin complex, and the ratio of the content of large-size cardiac troponin ternary complex to the content of total cardiac troponin ternary complex.

[0113] In some embodiments, the ratio parameter is the ratio of the content of large-sized cardiac troponin ternary complex to the content of cTnI, cTnT or total cardiac troponin complex; optionally, the ratio parameter further includes the ratio of the content of total cardiac troponin ternary complex to the content of cTnI, cTnT or total cardiac troponin complex, and / or the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complex.

[0114] For example, the ratio of the content of large-sized cardiac troponin ternary complex or total cardiac troponin ternary complex to the content of one of cTnI, cTnT and total cardiac troponin complex can be determined as a characteristic parameter for assessing myocardial damage.

[0115] Based on the above information, this application can be used for the assessment of myocardial injury.

[0116] Large-sized cardiac troponin ternary complex or total cardiac troponin ternary complex is used to diagnose myocardial injury. It can be used in the following clinical scenarios.

[0117] (1) Diagnosis of early acute myocardial infarction disease status (staging of myocardial infarction patients)

[0118] Regarding the presence of troponin complexes in patients with acute myocardial infarction, literature has reported that ternary complexes are present at higher concentrations in patients with early MI symptoms, representing a greater proportion of total troponin I. However, no studies have yet investigated the analysis of large or total cardiac troponin ternary complexes to aid in the diagnosis of myocardial infarction. Large or total cardiac troponin ternary complexes, either alone or in combination with other clinical markers of myocardial injury, can provide information for the acute phase of myocardial infarction, rapidly identify high-risk patients, and aid in customized clinical decision-making.

[0119] Herein, markers for diagnosing early acute myocardial infarction include large cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or more of cTnI, cTnT, TnC, cardiac troponin binary complex and total cardiac troponin complex.

[0120] In some embodiments, markers for diagnosing early acute myocardial infarction include large cardiac troponin ternary complex, or a combination thereof with cTnI, cTnT, or total cardiac troponin complex.

[0121] In some embodiments, markers for diagnosing early acute myocardial infarction include large cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or more of their complexes with cTnI, cTnT, TnC, cardiac troponin binary complex, and total cardiac troponin, in particular, large cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or both of their complexes with cTnT and total cardiac troponin.

[0122] In some embodiments, based on the levels of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including:

[0123] determining the content of the large-sized cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; or

[0124] Obtaining a characteristic parameter for assessing myocardial injury based on the content of the large-sized cardiac troponin ternary complex and the content of at least one of cTnI, cTnT, the total cardiac troponin ternary complex, and the total cardiac troponin complex, preferably calculating the characteristic parameter for assessing myocardial injury based on the content of the total cardiac troponin ternary complex and the content of cTnT;

[0125] A characteristic parameter for assessing myocardial damage is obtained based on the ratio of the content of the large-size cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex to the content of at least one of cTnI, cTnT and total cardiac troponin complexes, and optionally the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complexes. For example, the ratio of the content of the large-size cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex to the content of cTnT is determined as the characteristic parameter for assessing myocardial damage.

[0126] In some embodiments, a feature parameter based on a combination of multiple myocardial injury markers is constructed based on a logistic regression algorithm, such as a combination of the content of large-sized cardiac troponin ternary complex and the content of total cardiac troponin ternary complex, the content of cTnT, or the content of total cardiac troponin complex, or a combination of the content of large-sized cardiac troponin ternary complex, the content of total cardiac troponin ternary complex, the content of cTnT, and the content of total cardiac troponin complex; another example is a combination of the ratio of the content of large-sized cardiac troponin ternary complex to the content of total cardiac troponin complex, the ratio of the content of total cardiac troponin ternary complex to the content of total cardiac troponin complex, and the ratio of the content of cTnT to the content of total cardiac troponin complex, or a combination of the ratio of the content of large-sized cardiac troponin ternary complex to the content of cTnT, and the ratio of the content of total cardiac troponin ternary complex to the content of cTnT.

[0127] In some embodiments, a ROC curve is generated to establish a prediction model for the characteristic parameter, and an optimal cutoff is calculated as a reference value.

[0128] In some embodiments, based on the results of the aforementioned comparison, assessing myocardial damage in the subject comprises:

[0129] Based on the results of the comparison, the subjects are assigned a myocardial infarction stage.

[0130] In some embodiments, when the characteristic parameter is higher than the reference value of the characteristic parameter, the subject is judged to be in the early stage of acute myocardial infarction, such as myocardial infarction within 72 hours, 30 hours to 72 hours, 10 hours to 30 hours, 10 hours to 72 hours, or within 10 hours of the onset of chest pain.

[0131] In one embodiment, the method further comprises admitting the subject who is determined to be in the early stage of acute myocardial infarction to the hospital.

[0132] The term "above" a reference value means that the level of such a parameter in a sample is higher compared to a reference value or the level of such a parameter in a reference sample. For example, a marker may be detected in a higher amount, or at an elevated level, in a sample from an individual with a given disease than in the same sample from an individual without the disease.

[0133] (2) Differentiating between acute myocardial infarction (type 1) and chronic cardiac events

[0134] Currently, troponin I and troponin T are specific markers of myocardial injury. When myocardial injury occurs, marker levels rise, making it difficult to distinguish between acute and chronic injuries. To identify acute myocardial injury, continuous monitoring of troponin concentrations is often necessary, making it difficult to determine with a single test, potentially delaying diagnosis and treatment.

[0135] In this article, "acute cardiac event" refers to an acute condition of the heart, a disease or a malfunction, particularly acute heart failure, such as myocardial infarction (MI) or arrhythmia. Depending on the extent of the MI, it may be followed by LVD and CHF. A "chronic cardiac event" is a weakening of cardiac function, such as due to ischemia of the heart, coronary artery disease or a previous, particularly small, myocardial infarction (which may be followed by progressive LVD). It may also be a weakening due to inflammatory diseases, heart valve defects (such as mitral valve defects), dilated cardiomyopathy, hypertrophic cardiomyopathy, heart rhythm defects (arrhythmias) and chronic obstructive pulmonary disease. Therefore, it is clear that chronic cardiac events may also include such patients who have suffered from acute coronary syndromes such as MI, but have not currently suffered from acute cardiac events.

[0136] Distinguishing between acute and chronic cardiac events is important because they may require completely different treatments. For example, early treatment with reperfusion may be crucial for a patient presenting with an acute myocardial infarction. However, reperfusion in a patient with chronic heart failure may, at best, cause no or minimal harm to the patient.

[0137] Herein, markers for distinguishing acute myocardial infarction from chronic cardiac events include large cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or more of cTnI, cTnT, TnC, cardiac troponin binary complex and total cardiac troponin complex.

[0138] In some embodiments, the marker for distinguishing acute myocardial infarction from chronic cardiac events comprises large cardiac troponin ternary complex or total cardiac troponin ternary complex, or a combination of large cardiac troponin ternary complex and cTnT.

[0139] In some embodiments, markers for distinguishing acute myocardial infarction from chronic cardiac events include a combination of large cardiac troponin ternary complex and one or more of total cardiac troponin ternary complex, cTnT, and total cardiac troponin complex.

[0140] In some embodiments, based on the levels of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including:

[0141] determining the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; or

[0142] Calculating a characteristic parameter for assessing myocardial damage based on the content of the large-sized cardiac troponin ternary complex and the content of the total cardiac troponin ternary complex; or

[0143] A characteristic parameter for evaluating myocardial injury is obtained based on the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex and the content of cTnT or the total cardiac troponin complex.

[0144] In one embodiment, a characteristic parameter for assessing myocardial damage is obtained based on the ratio of the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex to the content of at least one of cTnI, cTnT and total cardiac troponin complexes, and optionally the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complexes.

[0145] In one example, the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex and the content of cTnT or the total cardiac troponin complex can be directly used as variables to calculate characteristic parameters for assessing myocardial injury.

[0146] In another example, the content of the large-sized cardiac troponin ternary complex and / or the ratio of the content of the total cardiac troponin ternary complex to the content of cTnT or total cardiac troponin complex can be used as variables to calculate characteristic parameters for assessing myocardial damage.

[0147] In some embodiments, a feature parameter based on a combination of multiple myocardial injury markers is constructed based on a logistic regression algorithm, such as a combination of the content of large-sized cardiac troponin ternary complex and the content of total cardiac troponin ternary complex, the content of cTnT, or the content of total cardiac troponin complex, or a combination of the content of large-sized cardiac troponin ternary complex, the content of total cardiac troponin ternary complex, the content of cTnT, and the content of total cardiac troponin complex; another example is a combination of the ratio of the content of large-sized cardiac troponin ternary complex to the content of total cardiac troponin complex, the ratio of the content of total cardiac troponin ternary complex to the content of total cardiac troponin complex, and the ratio of the content of cTnT to the content of total cardiac troponin complex, or a combination of the ratio of the content of large-sized cardiac troponin ternary complex to the content of cTnT, and the ratio of the content of total cardiac troponin ternary complex to the content of cTnT.

[0148] In some embodiments, a ROC curve is generated to establish a prediction model for the characteristic parameter, and an optimal cutoff is calculated as a reference value.

[0149] In some embodiments, performing the aforementioned comparison and assessing myocardial damage in the subject based on the results of the comparison comprises:

[0150] Based on the result of the comparison, it is determined whether the subject has suffered type I myocardial infarction or a chronic cardiac event.

[0151] In some embodiments, when the characteristic parameter is higher than a reference value of the characteristic parameter, the subject is judged to have suffered type I myocardial infarction.

[0152] In some embodiments, the method further comprises admitting the subject diagnosed with type I myocardial infarction to a hospital.

[0153] The use of large-size cardiac troponin ternary complex and / or total cardiac troponin ternary complex can more quickly differentiate the type of myocardial injury through a single time point test, speed up the treatment of patients with acute injury, quickly rule out patients with chronic injury, and speed up clinical turnover.

[0154] (3) Excluding myocardial damage

[0155] Current research focuses on using continuous monitoring of troponin I and troponin T to exclude patients without myocardial injury. For patients with chest pain lasting less than 3 hours, continuous monitoring of troponin concentrations is necessary, even if the troponin levels are very low. The use of large-scale cardiac troponin ternary complexes and / or total cardiac troponin ternary complexes can eliminate patients without myocardial injury with a single-point test, such as those with chest pain onset within 24 hours, for example, within 12 hours, thereby optimizing the diagnostic process.

[0156] In this article, markers used to exclude that myocardial damage has not occurred in subjects with chest pain include large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or more of cTnI, cTnT, TnC, cardiac troponin binary complex and total cardiac troponin complex; preferably large-sized cardiac troponin ternary complex.

[0157] Based on the aforementioned method, characteristic parameters for differentiation are obtained, and based on the aforementioned comparison results, patients without myocardial injury (such as myocardial infarction, especially NSTEMI) are excluded.

[0158] In some embodiments, when the characteristic parameter is lower than the reference value of the characteristic parameter, the subject with chest pain is excluded from a myocardial injury event.

[0159] In some embodiments, the step further includes notifying the patient that myocardial injury has been ruled out to leave the emergency room.

[0160] In some embodiments, based on the levels of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including:

[0161] determining the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; or

[0162] A characteristic parameter for evaluating myocardial damage is obtained based on the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex and the content of the total cardiac troponin complex.

[0163] In some embodiments, a ROC curve is generated to establish a prediction model for the characteristic parameter, and an optimal cutoff is calculated as a reference value.

[0164] (4) Differentiating between type I and type II myocardial infarction

[0165] Differentiating between type I and type II myocardial infarction is crucial; early differentiation can inform subsequent treatment decisions. Currently, differentiation between type 1 and type 2 myocardial infarction is primarily achieved through imaging studies, and no biomarker can effectively differentiate between type I and type II myocardial infarction. Large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex can rapidly differentiate between type I and type II myocardial infarction, supporting customized clinical decision-making and reducing unnecessary medical testing.

[0166] Herein, markers for distinguishing type I from type II myocardial infarction include large cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or more of cTnI, cTnT, TnC, cardiac troponin binary complex, and total cardiac troponin complex.

[0167] Based on the aforementioned method, characteristic parameters for differentiation are obtained, and based on the aforementioned comparison results, the myocardial damage of the subject is evaluated to determine whether the subject has type I myocardial infarction or type II myocardial infarction.

[0168] In some embodiments, when the characteristic parameter is higher than a reference value of the characteristic parameter, the subject is judged to have suffered type I myocardial infarction.

[0169] In some embodiments, subjects diagnosed with type I myocardial infarction are given thrombolytic therapy, while subjects diagnosed with type II myocardial infarction are not given thrombolytic therapy and are given supplemental oxygen.

[0170] (5) Distinguishing between myocardial injury caused by invasive procedures and chronic cardiac events

[0171] Currently, troponin I and troponin T are specific markers of myocardial injury. When myocardial injury occurs, marker levels rise, making it difficult to distinguish between different types of injury. To identify myocardial injury caused by invasive procedures, continuous monitoring of troponin concentrations is often necessary. Using large-scale cardiac troponin ternary complexes and / or total cardiac troponin ternary complexes allows for more rapid differentiation between different types of injury through a single time point test.

[0172] In this article, markers used to distinguish myocardial injury caused by invasive procedures from chronic cardiac events include large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or more of cTnI, cTnT, TnC, cardiac troponin binary complex and total cardiac troponin complex.

[0173] In some embodiments, based on the levels of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including:

[0174] The content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex is determined as a characteristic parameter for evaluating myocardial damage.

[0175] In some embodiments, a characteristic parameter for assessing myocardial damage is obtained based on the ratio of the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex to the content of at least one of cTnI, cTnT and total cardiac troponin complexes, and optionally the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complexes.

[0176] In some embodiments, a feature parameter based on a combination of multiple myocardial injury markers is constructed based on a logistic regression algorithm, such as a combination of the content of large-sized cardiac troponin ternary complex and the content of total cardiac troponin ternary complex, the content of cTnT, or the content of total cardiac troponin complex, or a combination of the content of large-sized cardiac troponin ternary complex, the content of total cardiac troponin ternary complex, the content of cTnT, and the content of total cardiac troponin complex; another example is a combination of the ratio of the content of large-sized cardiac troponin ternary complex to the content of total cardiac troponin complex, the ratio of the content of total cardiac troponin ternary complex to the content of total cardiac troponin complex, and the ratio of the content of cTnT to the content of total cardiac troponin complex, or a combination of the ratio of the content of large-sized cardiac troponin ternary complex to the content of cTnT, and the ratio of the content of total cardiac troponin ternary complex to the content of cTnT.

[0177] In some embodiments, a ROC curve is generated to establish a prediction model for the characteristic parameter, and an optimal cutoff is calculated as a reference value.

[0178] In some embodiments, the aforementioned comparison is performed, and based on the results of the comparison, the subject's myocardial injury is assessed to determine whether the subject has myocardial injury caused by an invasive procedure or a chronic cardiac event.

[0179] In some embodiments, when the characteristic parameter is higher than a reference value of the characteristic parameter, it is determined that the subject has suffered myocardial injury caused by an invasive procedure.

[0180] In some embodiments, for subjects determined to have suffered myocardial damage as a result of invasive surgery, hospitalization time is extended or cardiac monitoring is strengthened.

[0181] In some embodiments, the method can accurately distinguish between myocardial injury caused by invasive procedures and chronic heart-related diseases (e.g., cardiomyopathy, chronic heart failure, structural heart disease, infiltrative disease, stable coronary artery disease, and sustained arrhythmias). In other embodiments, the method can accurately distinguish between myocardial injury caused by invasive procedures and non-heart-related diseases (e.g., pneumonia and renal insufficiency).

[0182] In addition, large-sized cardiac troponin ternary complexes or total cardiac troponin ternary complexes are also used for the prognosis of myocardial injury. Specifically, they can be used in the following clinical scenarios.

[0183] (6) Assessment of the prognosis of acute myocardial injury

[0184] Current research focuses on using troponin I and troponin T to assess the prognosis of patients with myocardial infarction. The use of large-size cardiac troponin ternary complex and / or total cardiac troponin ternary complex assessment is expected to improve the accuracy of predicting future myocardial injury-related cardiac events or death in patients with acute myocardial injury, such as the prognosis of myocardial injury in patients undergoing cardiac surgery or the prognosis of myocardial injury in patients with myocardial infarction.

[0185] In this article, the markers used to assess the prognostic risk of patients with myocardial infarction include large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or more of cTnI, cTnT, TnC, cardiac troponin binary complex and total cardiac troponin complex.

[0186] Based on the characteristic parameters for differentiation obtained by the aforementioned method, and based on the results of the aforementioned comparison, the myocardial damage of the subject is evaluated to determine the prognosis of the subject with myocardial infarction, such as the prognosis within one year, within six months, or within three months.

[0187] In some embodiments, when the characteristic parameter is higher than a reference value of the characteristic parameter, the prognosis is judged to be poor.

[0188] In some embodiments, based on the levels of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including:

[0189] The content of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex is determined as a characteristic parameter for evaluating myocardial damage.

[0190] In some embodiments, the change in the level of large cardiac troponin ternary complex or total cardiac troponin ternary complex in the plasma of the subject before and after cardiac surgery is determined as a characteristic parameter for assessing the prognostic risk of undergoing surgery.

[0191] To assess the prognosis of myocardial injury in patients undergoing surgical procedures such as coronary artery bypass grafting (CABG) or heart valve replacement

[0192] The characteristic parameter for assessing myocardial injury is determined based on the content of the large cardiac troponin ternary complex or total cardiac troponin ternary complex before and after surgery, or the change therein (e.g., the difference or ratio of the content of the large cardiac troponin ternary complex or total cardiac troponin ternary complex between postoperative and preoperative levels). In some embodiments, a characteristic parameter based on a combination of multiple myocardial injury markers is constructed based on a logistic regression algorithm. For example, a combination of the content of the large cardiac troponin ternary complex and the content of cTnT is used.

[0193] In some embodiments, a ROC curve is generated to establish a prediction model for the characteristic parameter, and an optimal cutoff is calculated as a reference value.

[0194] In some embodiments, binary logistic regression analysis is used to assess the risk of a patient's composite endpoint event (selected from a composite of major clinical events including all-cause death, myocardial infarction, and unplanned coronary revascularization, and any combination of minor clinical events including cardiovascular death, components of major clinical events, stroke, hospitalization for heart failure or emergency department observation for 24 hours or more, cardiac arrest or malignant arrhythmia, and other hospitalization events due to cardiovascular disease).

[0195] In some embodiments, Cox regression is used to assess the risk of a patient's composite endpoint event (selected from a composite of major clinical events including all-cause death, myocardial infarction, and unplanned coronary revascularization, and any combination of minor clinical events including cardiovascular death, components of major clinical events, stroke, hospitalization for heart failure or emergency department observation for 24 hours or more, cardiac arrest or malignant arrhythmia, and other hospitalizations due to cardiovascular disease).

[0196] Assessing the prognosis of myocardial injury in patients with myocardial infarction

[0197] The content of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex is determined as a characteristic parameter for evaluating myocardial damage.

[0198] In some embodiments, a ROC curve is generated to establish a prediction model for the characteristic parameter, and an optimal cutoff is calculated as a reference value.

[0199] In some embodiments, binary logistic regression analysis is used to assess the risk of a patient's composite endpoint event (selected from a composite of major clinical events including all-cause death, myocardial infarction, and unplanned coronary revascularization, and any combination of minor clinical events including cardiovascular death, components of major clinical events, stroke, hospitalization for heart failure or emergency department observation for 24 hours or more, cardiac arrest or malignant arrhythmia, and other hospitalization events due to cardiovascular disease).

[0200] In some embodiments, the risk of a patient experiencing a composite endpoint event is assessed using Cox regression.

[0201] (7) Assessing the prognostic risk of patients with chronic myocardial injury

[0202] Current research focuses on using troponin I and troponin T to assess the prognosis of patients with chronic myocardial injury. The use of large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex is expected to improve the accuracy of prediction.

[0203] In this article, markers for assessing the prognostic risk of patients with chronic myocardial injury include large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex, and one or more of cTnI, cTnT, TnC, cardiac troponin binary complex and total cardiac troponin complex.

[0204] Based on the above method, characteristic parameters for differentiation are obtained, and based on the results of the above comparison, prognostic risk assessment is performed on subjects with chronic myocardial damage (e.g., cardiomyopathy, chronic heart failure, structural heart disease, infiltrative disease, stable coronary heart disease, and persistent arrhythmia).

[0205] In some embodiments, when the characteristic parameter is higher than a reference value of the characteristic parameter, the prognosis is judged to be poor.

[0206] In some embodiments, based on the levels of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including:

[0207] The content of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex is determined as a characteristic parameter for evaluating myocardial damage.

[0208] The content of the large cardiac troponin ternary complex or the total cardiac troponin ternary complex is determined as a characteristic parameter for assessing myocardial injury. In some embodiments, a characteristic parameter based on a combination of multiple myocardial injury markers is constructed based on a logistic regression algorithm. For example, a combination of the content of the large cardiac troponin ternary complex and the content of the total cardiac troponin complex, or a combination of the content of the large cardiac troponin ternary complex and the content of cTnT.

[0209] In some embodiments, a ROC curve is generated to establish a prediction model for the characteristic parameter, and an optimal cutoff is calculated as a reference value.

[0210] In some embodiments, binary logistic regression analysis is used to assess the risk of a patient's composite endpoint event.

[0211] In some embodiments, the risk of a patient's composite endpoint event is assessed using Cox regression.

[0212] The method of the present application is preferably ex vivo or in vitro method. In addition, it can include the step outside the step clearly mentioned above. For example, a further step can relate to sample pretreatment and obtain the evaluation of the result by the method. The method can be manually carried out or assisted by automation. Preferably, the detection step, the calculation step and the comparison step can be assisted by automation in whole or in part, for example, by the calculation algorithm performed by the computer on the data processing device in the suitable robot and sensory equipment, the calculation step or the comparison and / or diagnostic algorithm on the data processing device in the comparison step.

[0213] Device and sample analysis system

[0214] In a second aspect, the present application provides a device for obtaining characteristic parameters for assessing myocardial injury in a subject. As shown in FIG1 , the device 100 includes:

[0215] The data receiving module 110 is configured to receive the content of one or more myocardial injury markers obtained from a sample from a subject, wherein the one or more myocardial injury markers include a large cardiac troponin ternary complex and / or a total cardiac troponin ternary complex;

[0216] The data processing module 120 is configured to process the content data of one or more myocardial injury markers received by the receiving module to obtain characteristic parameters for evaluating myocardial injury;

[0217] And, the output module 130 is configured to output the characteristic parameters.

[0218] In some embodiments, the data processing module processes the content of the one or more myocardial injury markers to obtain characteristic parameters for assessing myocardial injury, including:

[0219] The data processing module determines the content of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial injury; or

[0220] The data processing module obtains characteristic parameters for assessing myocardial damage based on the content of the large-sized cardiac troponin ternary complex and the content of the total cardiac troponin ternary complex.

[0221] In some embodiments, the myocardial injury markers further include one or more of the following: cTnI, cTnT, TnC, cardiac troponin binary complex, and total cardiac troponin complex.

[0222] In some embodiments, the one or more myocardial injury markers further include at least one of cTnI, cTnT, and total cardiac troponin complex.

[0223] In some embodiments, the data processing module processes the content of the one or more myocardial injury markers to obtain characteristic parameters for evaluating myocardial injury, including: the data processing module inputs the content of the multiple myocardial injury markers into a preset function model to obtain the output of the preset function model as the characteristic parameters for evaluating myocardial injury.

[0224] In some embodiments, the data processing module processes the content of the one or more myocardial injury markers to obtain characteristic parameters for assessing myocardial injury, including: the data processing module determines one of the following ratio parameters as the characteristic parameter for assessing myocardial injury, or obtains the characteristic parameter for assessing myocardial injury based on at least two of the following ratio parameters, or obtains the characteristic parameter for assessing myocardial injury based on at least one of the following ratio parameters and the ratio of the content of cTnT to the content of total cardiac troponin complex;

[0225] The ratio parameters include: the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnI, the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnT, the ratio of the content of large-size cardiac troponin ternary complex to the content of total cardiac troponin complex, the ratio of the content of total cardiac troponin ternary complex to the content of cTnI, the ratio of the content of total cardiac troponin ternary complex to the content of cTnT, the ratio of the content of total cardiac troponin ternary complex to the content of total cardiac troponin complex, and the ratio of the content of large-size cardiac troponin ternary complex to the content of total cardiac troponin ternary complex.

[0226] In some embodiments, the ratio parameter is the ratio of the content of large-sized cardiac troponin ternary complex to the content of cTnI, cTnT or total cardiac troponin complex; optionally, the ratio parameter further includes the ratio of the content of total cardiac troponin ternary complex to the content of cTnI, cTnT or total cardiac troponin complex, and / or the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complex.

[0227] In a third aspect, the present application provides a sample analysis system, comprising:

[0228] A sample carrying portion, used for carrying a container containing a sample of a subject;

[0229] The sample dispensing part is used to draw the sample of the subject from the sample holding part and discharge it into the reaction cup to be loaded with the sample;

[0230] A reagent carrying portion, used for carrying detection reagents;

[0231] The reagent dispensing part is used to draw the detection reagent from the reagent carrying part and discharge it into the reaction cup to be added with the reagent;

[0232] The reaction part is used to place the reaction cup so as to incubate the test solution obtained by the reaction of the subject's sample and the detection reagent in the reaction cup;

[0233] a detection unit having a signal detector for detecting a signal of a test solution in a reaction cup to measure and output the content of one or more myocardial injury markers in a sample of a subject, wherein the one or more myocardial injury markers include a large-sized cardiac troponin ternary complex and / or a total cardiac troponin ternary complex; and

[0234] The data processing unit includes a processor and a computer-readable storage medium, wherein the computer-readable storage medium stores computer-readable instructions, wherein when the computer-readable instructions are executed by the processor, the processor is caused to perform the following steps:

[0235] receiving and processing the content of the one or more myocardial injury markers to obtain characteristic parameters for assessing myocardial injury; and

[0236] The characteristic parameters are output.

[0237] FIG2 illustrates a sample analysis system 200 provided in an embodiment of the present application, particularly configured as or including a chemiluminescence analyzer. As shown in FIG2 , sample analysis system 200 includes a sample carrier 210, a sample dispensing unit 220, a reagent carrier 230, a reagent dispensing unit 240, a reaction unit 250, a detection unit 260, and an apparatus 270 according to the second aspect.

[0238] The sample carrier 210 is used to carry a container containing a subject's blood sample. For example, the sample carrier 210 can be configured as a sample tray, which includes multiple sample positions for the containers 10. The sample tray can be rotated to move the container containing the blood sample to a corresponding position, such as a position for the sample dispensing unit 220 to draw the blood sample.

[0239] The sample dispensing unit 220 is used to draw a blood sample, such as serum, from the sample holding unit 210 and discharge it into a cuvette to be loaded. For example, the sample dispensing unit 112 may include a sample needle that can be spatially moved in two or three dimensions by a two-dimensional or three-dimensional drive mechanism, thereby enabling the sample needle to be moved to a position for drawing a blood sample and to a cuvette to be loaded, and to discharge the drawn blood sample into the cuvette.

[0240] The reagent carrier 230 is used to carry detection reagents, such as reagents for measuring the content of one or more myocardial injury markers. For example, the reagent carrier 230 can be configured as a disc-shaped reagent tray with multiple positions for holding reagent containers. The reagent carrier 230 can rotate and drive the reagent containers it carries to rotate, so as to rotate the reagent containers to a specific position, such as a position for the reagent to be drawn by the reagent dispensing unit 240. There can be one or more reagent carriers 230.

[0241] The reagent dispensing unit 240 is used to draw the test reagent from the reagent loading unit 230 and discharge it into the cuvette to be added. For example, the reagent dispensing unit 240 may include a reagent needle that can be spatially moved in two or three dimensions by a two-dimensional or three-dimensional drive mechanism, thereby moving the reagent needle to a position for drawing the reagent and to a cuvette to be added, and then discharging the drawn reagent into the cuvette.

[0242] The reaction section 250 is used to place a reaction cup for incubating the test solution obtained by reacting the subject's blood sample with the detection reagent in the reaction cup. For example, the reaction section 250 can be configured as a disc-shaped reaction tray having one or more placement positions for the reaction cups. The reaction tray can rotate and drive the reaction cups in its placement positions to move the reaction cups within the reaction tray and incubate the test solution in the reaction cups.

[0243] Detection unit 260 includes a signal detector, such as an optical detector, for detecting signals from the test solution in the cuvette to determine and output the levels of one or more myocardial injury markers in the subject's sample. Detection unit 260 is, for example, located outside reaction unit 250. Reaction unit 250 rotates to move the container containing the test solution to detection unit 260 for testing. In some embodiments, detection unit 260 is configured as a photometric device.

[0244] For example, when the sample analysis system is constructed as a chemiluminescence analyzer or includes a chemiluminescence analyzer, the detection unit 260 is constructed as a photometric device. A specific detection process of the chemiluminescence analyzer is as follows: the sample dispensing unit 220 draws the sample to be tested from the sample carrying unit 210 and adds it to the reaction cup; the reagent dispensing unit 240 draws the enzyme-labeled reagent and the magnetic bead reagent from the reagent carrying unit 230 and adds them to the reaction cup to which the sample has been added so as to mix them evenly with the sample; the reaction cup is then placed in the reaction unit 250 for reaction, incubation, and magnetic separation and cleaning; then a luminescent substrate is added to the reaction cup that has completed the reaction, incubation, and magnetic separation and cleaning, and incubated for a period of time; finally, the detection unit 260 detects the photons emitted by the substance to be tested in the sample to be tested under the action of the luminescent substrate, so as to calculate the concentration level of the substance to be tested by the number of photons measured. For other embodiments and advantages of the sample analysis system, reference can be made to the above description of the device 100.

[0245] Detection kits and applications

[0246] In a fourth aspect, the present application provides use of a reagent for the quantitative detection of a large cardiac troponin ternary complex and / or a reagent for the quantitative detection of a total cardiac troponin ternary complex in a sample in the preparation of a kit, wherein the kit is used for the assessment of myocardial injury as described herein. Accordingly, the present application provides a kit containing the reagent for the quantitative detection of a large cardiac troponin ternary complex and / or a reagent for the quantitative detection of a total cardiac troponin ternary complex in a sample.

[0247] In some embodiments, the kit further comprises a reagent for quantitative detection of total cardiac troponin complex, a reagent for quantitative detection of cTnI, and / or a reagent for quantitative detection of cTnT.

[0248] Studies have found that the use of the kit of the present application for detecting large-size cardiac troponin ternary complex and total cardiac troponin ternary complex is beneficial for more quickly and accurately obtaining the content of the myocardial injury marker and then obtaining the characteristic parameters for evaluating myocardial injury.

[0249] In some embodiments, the reagent for quantitative detection of large-sized cardiac troponin ternary complex in a sample comprises a first set of antibodies and a second set of antibodies, wherein,

[0250] The first group of antibodies includes one or more antibodies 1-1, each of which is independently selected from antibodies that specifically bind to any one of the amino acid sequences of positions 67 to 222 of cTnT;

[0251] The second group of antibodies includes one or more antibodies 1-2, and each of the antibodies 1-2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.

[0252] In some embodiments, the first group of antibodies does not include an antibody that specifically binds to any stretch of the amino acid sequence of cTnT at positions 223-287.

[0253] In some embodiments, the second panel of antibodies further comprises:

[0254] one or more antibodies 1-3, each of said antibodies 1-3 being independently selected from an antibody that specifically binds to cTnIC; and / or,

[0255] One or more antibodies 1-4, each of the antibodies 1-4 is independently selected from antibodies that specifically bind to any one of the amino acid sequences at positions 18-210 of cTnI.

[0256] In some embodiments, the first group of antibodies includes one or more of the antibody 1-1, and the second group of antibodies includes one or more of the antibody 1-2 and one or more of the antibody 1-3.

[0257] In some embodiments, each of the antibodies 1-1 is independently selected from an antibody that specifically binds to amino acids 67-86, amino acids 119-138, amino acids 132-151, amino acids 145-164, or amino acids 171-190 of cTnT. In some embodiments, each of the antibodies 1-1 is independently selected from an antibody that specifically binds to amino acids 119-138, amino acids 132-151, or amino acids 171-190 of cTnT.

[0258] In some embodiments, each of the antibodies 1-4 is independently selected from an antibody that specifically binds to amino acids 1-15, amino acids 13-22, amino acids 18-22, amino acids 18-28, amino acids 18-35, amino acids 22-31, amino acids 22-40, amino acids 23-29, amino acids 24-40, amino acids 25-40, amino acids 26-35, amino acids 34-37, amino acids 41-49, amino acids 83-89, amino acids 86-90, amino acids 87-90, amino acids 117-126, amino acids 130-145, amino acids 169-178, amino acids 186-192, amino acids 190-196, or amino acids 195-209 of cTnI. In some embodiments, each of the antibodies 1-4 is independently selected from an antibody that specifically binds to amino acids 22-40, amino acids 41-49, or amino acids 83-89 of cTnI. In some embodiments, each of the antibodies 1-4 is independently selected from an antibody that specifically binds to amino acids 41-49 of cTnI.

[0259] In some embodiments, the first set of antibodies are capture antibodies and the second set of antibodies are detection antibodies.

[0260] In some embodiments, the reagent for quantitative detection of total cardiac troponin ternary complex in a sample comprises a first detection reagent and a second detection reagent, wherein,

[0261] The first detection reagent comprises one or more antibodies 2-1, each of the antibodies 2-1 being independently selected from antibodies that specifically bind to any one of the amino acid sequences of positions 223-287 of cTnT;

[0262] The second detection reagent comprises one or more antibodies 2-2, and each antibody 2-2 is independently selected from an antibody that specifically binds to any segment of the TnC amino acid sequence.

[0263] In some embodiments, the first detection reagent further comprises one or more antibodies 2-3, and each of the antibodies 2-3 is independently selected from antibodies that specifically bind to any one of the amino acid sequences of cTnT at positions 67-222.

[0264] In some embodiments, the second detection reagent further comprises:

[0265] one or more antibodies 2-4, each of said antibodies 2-4 being independently selected from an antibody that specifically binds to cTnIC; and / or

[0266] One or more antibodies 2-5, each of the antibodies 2-5 is independently selected from antibodies that specifically bind to any one of the amino acid sequences at positions 18-210 of cTnI.

[0267] In some embodiments, the first detection reagent includes one or more of the antibodies 2-1 and one or more of the antibodies 2-3; the second detection reagent includes one or more of the antibodies 2-2, and one or more of the antibodies 2-4 and / or one or more of the antibodies 2-5.

[0268] In some embodiments, each of the antibodies 2-1 is independently selected from antibodies that specifically bind to amino acids 223-242 and 262-281 of cTnT. In some embodiments, the antibody 2-1 specifically binds to amino acids 223-242 of cTnT.

[0269] In some embodiments, each of the antibodies 2-3 is independently selected from an antibody that specifically binds to amino acids 67-86, amino acids 119-138, amino acids 132-151, amino acids 145-164, or amino acids 171-190 of cTnT. In some embodiments, each of the antibodies 2-3 is independently selected from an antibody that specifically binds to amino acids 119-138 or amino acids 132-151 of cTnT.

[0270] In some embodiments, each of the antibodies 2-5 is independently selected from an antibody that specifically binds to amino acids 1-15, amino acids 13-22, amino acids 18-22, amino acids 18-28, amino acids 18-35, amino acids 22-31, amino acids 22-40, amino acids 23-29, amino acids 24-40, amino acids 25-40, amino acids 26-35, amino acids 34-37, amino acids 41-49, amino acids 83-89, amino acids 86-90, amino acids 87-90, amino acids 117-126, amino acids 130-145, amino acids 169-178, amino acids 186-192, amino acids 190-196, or amino acids 195-209 of cTnI. In some embodiments, each of the antibodies 2-5 is independently selected from an antibody that specifically binds to amino acids 22-40, amino acids 41-49, or amino acids 83-89 of cTnI. In some embodiments, each of the antibodies 2-5 is independently selected from an antibody that specifically binds to amino acids 41-49 of cTnI.

[0271] In some embodiments, the first detection reagent is a capture reagent and the second detection reagent set is a detection reagent.

[0272] In some embodiments, the kit is used for one or more of the following:

[0273] 1) Myocardial infarction staging, in particular, determining whether the subject is in the early stage of myocardial infarction;

[0274] 2) differentiate between type I myocardial infarction and chronic cardiac events;

[0275] 3) Excluding subjects with chest pain who did not experience myocardial injury events;

[0276] 4) Differentiate between type I and type II myocardial infarction;

[0277] 5) Distinguish between myocardial injury caused by invasive procedures and chronic cardiac events;

[0278] 6) Evaluate the prognosis of acute myocardial injury;

[0279] 7) Evaluate the prognosis of chronic myocardial injury. Beneficial effects

[0280] 1) By detecting troponin complexes and fragments in patient samples, it is possible to assess the cause and risk of myocardial injury in individuals in the presence of elevated troponin concentrations.

[0281] 2) Avoid the difficulties caused by continuous monitoring of troponin, improve clinical turnover rate, and help establish a faster diagnostic process.

[0282] 3) It helps stratify patients with chest pain and guides diagnostic and treatment decisions. DETAILED DESCRIPTION

[0283] Example 1. Detection kit and signal-to-noise ratio analysis

[0284] 1. Construction of the kit

[0285] Those skilled in the art will appreciate that kits for quantitatively detecting the myocardial injury markers, including commercially available kits, can be used to determine the content of the myocardial injury markers. For non-limiting purposes, the following exemplary descriptions include detection kits for cTnI, cTnT, total cardiac troponin complex, large-size cTnITC, and total cTnITC.

[0286] The capture antibody-detection antibody (all antibodies used in this article are from Hytest) was applied to the double antibody sandwich chemiluminescence immunoassay method to construct various kits.

[0287] 1. Construction of a kit for detecting myocardial injury markers cTnI, cTnT, and total cardiac troponin complex

[0288] (1) Total complex detection kit:

[0289] Capture antibody: antibody 19C7cc that specifically binds to the cTnI amino acid 41-49 fragment;

[0290] Detection antibody: Antibody 20C6cc that specifically binds to the cTnIC complex epitope;

[0291] (2) cTnT detection kit:

[0292] Capture antibody: Antibody 329cc that specifically binds to the cTnT amino acid 119-138 fragment;

[0293] Detection Antibody: Antibody 406cc that specifically binds to the cTnT amino acid 132-151 fragment.

[0294] (3) cTnI detection kit:

[0295] Capture antibody: antibody 19C7cc that specifically binds to the cTnI amino acid 41-49 fragment;

[0296] Detection Antibody: RecR33, an antibody that specifically binds to the cTnI amino acid 24-40 fragment.

[0297] 2. Construction of a Large-Size cTnITC Detection Kit

[0298] (1) Large size cTnITC detection kit 1

[0299] The capture antibodies were: Antibody 1-1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0300] The detection antibodies are: Antibody 1-3: Antibody 20C6cc that specifically binds to the cTnIC complex epitope.

[0301] (2) Large size cTnITC detection kit 2

[0302] The capture antibodies were: Antibody 1-1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0303] The detection antibodies are: Antibody 1-4: Antibody 19C7cc that specifically binds to amino acid fragment 41-49 of cTnI.

[0304] (3) Large size cTnITC detection kit 3

[0305] The capture antibodies were: Antibody 1-1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0306] The detection antibodies are: Antibody 1-2: Antibody 7B9cc that specifically binds to TnC; and Antibody 1-3: Antibody 20C6cc that specifically binds to the epitope of the cTnIC complex.

[0307] (4) Large size cTnITC detection kit 4

[0308] The capture antibodies were: Antibody 1-1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0309] The detection antibodies are: Antibody 1-2: antibody 7B9cc that specifically binds to TnC; and Antibody 1-4: antibody 19C7cc that specifically binds to amino acid fragment 41-49 of cTnI.

[0310] (5) Large size cTnITC detection kit 5

[0311] The capture antibodies were: Antibody 1-1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0312] The detection antibodies are: Antibody 1-2: Antibody 7B9cc that specifically binds to TnC.

[0313] (6) Large size cTnITC detection kit 6

[0314] The capture antibodies were: Antibody 1-1: Antibody 1C11cc that specifically binds to amino acid fragment 171-190 of cTnT;

[0315] The detection antibodies are: Antibody 1-2: antibody 7B9cc that specifically binds to TnC; and Antibody 1-3: antibody Tcom8 that specifically binds to the epitope of the cTnIC complex.

[0316] (7) Large size cTnITC detection kit 7

[0317] The capture antibodies were: Antibody 1-1: Antibody 406cc that specifically binds to amino acid fragment 132-151 of cTnT;

[0318] The detection antibodies are: Antibody 1-2: Antibody 7B9cc that specifically binds to TnC; and Antibody 1-3: Antibody 20C6cc that specifically binds to the epitope of the cTnIC complex.

[0319] (8) Large size cTnITC detection kit 8

[0320] The capture antibodies were: Antibody 1-1: 300cc of an antibody that specifically binds to amino acid fragment 119-138 of cTnT;

[0321] The detection antibodies are: Antibody 1-2: Antibody 7B9cc that specifically binds to TnC; and Antibody 1-3: Antibody 20C6cc that specifically binds to the epitope of the cTnIC complex.

[0322] In addition to the above kits, the experiments also used kits obtained by replacing the antibody 1-1 used in the large-size cTn ITC detection kits 3-8 with the following antibodies: 7F4 or 7G7, whose specific binding site is the cTnT amino acid 67-86 fragment; 2F3, 1A11, or 1F11cc, whose specific binding site is the cTnT amino acid 145-164 fragment. The experiments also used kits obtained by replacing the antibody 1-4 used in the large-size cTn ITC detection kit 4 with the following antibodies as antibodies 1-4: M18cc, whose specific binding site is the cTnI amino acid 18-28 fragment; 16A11cc, 16A12cc, or 8E10cc, whose specific binding site is the cTnI amino acid 86-90 fragment; M46, whose specific binding site is the cTnI amino acid 130-145 fragment; and MF4cc, whose specific binding site is the cTnI amino acid 190-196 fragment.

[0323] 3. Construction of a total cardiac troponin ternary complex detection kit

[0324] The capture antibody-detection antibody was applied to the double antibody sandwich chemiluminescence immunoassay method to construct a detection kit for detecting the total ternary troponin complex cTnITC in the sample.

[0325] Total cTnITC Detection Kit 1:

[0326] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 329cc (specifically binds to the cTnT amino acid fragment 119-138).

[0327] The detection antibodies are: Antibody 2-4: 20C6cc (specifically binds to the cTnIC complex epitope).

[0328] Total cTnITC Detection Kit 2:

[0329] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 329cc (specifically binds to the cTnT amino acid fragment 119-138).

[0330] The detection antibodies are: Antibody 2-5: 19C7cc (specifically binds to cTnI amino acid fragment 41-49).

[0331] Total cTnITC Detection Kit 3:

[0332] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 329cc (specifically binds to the cTnT amino acid fragment 119-138).

[0333] The detection antibodies are: Antibody 2-2: 7B9cc (specifically binds to TnC); Antibody 2-4: 20C6cc (specifically binds to the cTnIC complex epitope);

[0334] Total cTnITC Detection Kit 4:

[0335] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 329cc (specifically binds to the cTnT amino acid fragment 119-138).

[0336] The detection antibodies are: antibody 2-2: 7B9cc (specifically binds to TnC); antibody 2-5: 19C7cc (specifically binds to cTnI amino acid fragment 41-49).

[0337] Total cTnITC Detection Kit 5:

[0338] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 329cc (specifically binds to the cTnT amino acid fragment 119-138).

[0339] The detection antibody is: Antibody 2-2:7B9cc (specifically binds to TnC).

[0340] Total cTnITC Detection Kit 6:

[0341] The capture antibody is: Antibody 2-1:7E7 (specifically binds to cTnT amino acid fragment 223-242).

[0342] The detection antibody is: Antibody 2-2:7B9cc (specifically binds to TnC).

[0343] Total cTnITC Detection Kit 7:

[0344] The capture antibody is: antibody 2-1:155 (specifically binds to cTnT amino acid fragment 262-281).

[0345] The detection antibodies are: Antibody 2-2: 7B9cc (specifically binds to TnC); Antibody 2-4: Tcom8 (specifically binds to the cTnIC complex epitope).

[0346] Total cTnITC Detection Kit 8:

[0347] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 406cc (specifically binds to the cTnT amino acid fragment 132-151).

[0348] The detection antibodies are: Antibody 2-2: 7B9cc (specifically binds to TnC); Antibody 2-4: 20C6cc (specifically binds to the cTnIC complex epitope);

[0349] Total cTnITC Detection Kit 9:

[0350] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 300cc (specifically binds to the cTnT amino acid fragment 119-138).

[0351] The detection antibodies are: Antibody 2-2: 7B9cc (specifically binds to TnC); Antibody 2-4: 20C6cc (specifically binds to the cTnIC complex epitope);

[0352] Total cTnITC Detection Kit 10:

[0353] The capture antibodies are: antibody 2-1: 155 (specifically binds to the cTnT amino acid fragment 262-281); antibody 2-3: 406cc (specifically binds to the cTnT amino acid fragment 132-151).

[0354] The detection antibodies are: Antibody 2-2: 7B9cc (specifically binds to TnC); Antibody 2-4: 20C6cc (specifically binds to the cTnIC complex epitope);

[0355] Total cTnITC Detection Kit 11:

[0356] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 7G7 (specifically binds to the cTnT amino acid fragment 67-86).

[0357] The detection antibodies are: Antibody 2-2: 7B9cc (specifically binds to TnC); Antibody 2-4: 20C6cc (specifically binds to the cTnIC complex epitope);

[0358] Total cTnITC Detection Kit 12:

[0359] The capture antibodies are: antibody 2-1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); antibody 2-3: 1C11cc (specifically binds to the cTnT amino acid fragment 171-190).

[0360] The detection antibodies are: Antibody 2-2: 7B9cc (specifically binds to TnC); Antibody 2-4: Tcom8 (specifically binds to the cTnIC complex epitope).

[0361] In addition to the above kits, the experiments also used kits in which the antibody 2-3 used in the total cTn ITC detection kits 3-5 and 8-12 was replaced with the following antibodies: 7F4, whose specific binding site is the cTnT amino acid 67-86 fragment; 2F3, 1A11, or 1F11cc, whose specific binding site is the cTnT amino acid 145-164 fragment. The experiments also used kits in which the antibody 2-5 used in the total cTn ITC detection kit 4 was replaced with the following antibodies: M18cc, whose specific binding site is the cTnI amino acid 18-28 fragment; 16A11cc, 16A12cc, or 8E10cc, whose specific binding site is the cTnI amino acid 86-90 fragment; M46, whose specific binding site is the cTnI amino acid 130-145 fragment; and MF4cc, whose specific binding site is the cTnI amino acid 190-196 fragment.

[0362] Each test kit includes:

[0363] A. Magnetic bead coating working solution, used to capture myocardial injury markers in a sample. The magnetic bead coating working solution comprises: a mixture of superparamagnetic particles coated with capture antibodies.

[0364] B. Enzyme marker working solution, used to detect myocardial injury markers captured by superparamagnetic particles. The enzyme marker working solution includes: alkaline phosphatase-labeled detection antibody.

[0365] 4. Myocardial injury marker detection method

[0366] The detection method is as follows:

[0367] Step 1: Add the sample, magnetic bead coating solution, and enzyme marker solution to a reaction tube. After incubation, the target protein in the sample binds to the antibody coated on the magnetic beads, while the antibody-alkaline phosphatase marker binds to the target protein in the sample. After the reaction is complete, the solid phase is placed in a magnetic field, which attracts the magnetic beads, retaining the bound substances and washing away the unbound substances.

[0368] Step 2: Add the chemiluminescent substrate to the reaction tube. The luminescent substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane, AMPPD) is degraded by alkaline phosphatase, removing a phosphate group to form an unstable intermediate. This intermediate generates a methyl m-oxybenzoate anion through intramolecular electron transfer. When the excited methyl m-oxybenzoate anion returns to the ground state, it produces chemiluminescence. The number of photons generated in the reaction is measured by a photomultiplier tube. The number of photons generated is proportional to the concentration of the target protein in the sample. The amount of analyte in the sample is determined using a calibration curve.

[0369] The above-mentioned detection kit can be used in conjunction with Mindray's fully automatic chemiluminescence analyzers CL2000i, CL6000i, CL8000i and other models.

[0370] 2. Signal-to-noise ratio analysis of the detection kit

[0371] 1. Signal-to-noise ratio analysis of large-size cTnITC detection kit

[0372] Samples containing different antigen concentrations, including two high-concentration samples (high-value samples) and two low-concentration samples (low-value samples), were prepared. The antigen was recombinant cardiac troponin ternary complex (Hytest, 8ITCR). These samples were analyzed using large-format cTnITC assay kits 1-8. The signal from a blank sample without antigen was also recorded, and the signal-to-noise ratio was calculated.

[0373] The test results are shown in Figure 3. As can be seen from Figure 3, large-size cTnITC Assay Kits 1-8 all have good signal-to-noise ratios, meeting clinical needs. The signal-to-noise ratio of large-size cTnITC Assay Kit 3 was significantly higher than that of large-size cTnITC Assay Kit 1 and large-size cTnITC Assay Kit 5, and the signal-to-noise ratio of large-size cTnITC Assay Kit 4 was significantly higher than that of large-size cTnITC Assay Kit 2 and large-size cTnITC Assay Kit 5, indicating that the use of TnC-specific antibody 1-2 in combination with antibody 1-3 or antibody 1-4 can significantly improve the signal-to-noise ratio. Large-size cTnITC Assay Kits 3, 7, and 8 have high signal-to-noise ratios, indicating that antibody 1-1, which specifically binds to cTnT amino acids 119-138 and 132-151, significantly contributes to the improvement of the signal-to-noise ratio.

[0374] In addition, the antibody 1-1 used in the large-size cTnITC detection kits 3-8 was replaced with the following antibodies: antibody 7F4 or 7G7 whose specific binding site is the cTnT amino acid 67-86 fragment, and antibody 2F3, 1A11, or 1F11cc whose specific binding site is the cTnT amino acid 145-164 fragment; the antibody 1-4 used in the large-size cTnITC detection kit 4 was replaced with the following antibodies: antibody M18cc whose specific binding site is the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc, or 8E10cc whose specific binding site is the cTnI amino acid 86-90 fragment, antibody M46 whose specific binding site is the cTnI amino acid 130-145 fragment, and antibody MF4cc whose specific binding site is the cTnI amino acid 190-196 fragment, all of which can effectively reflect the signal differences of the samples.

[0375] 2. Signal-to-noise ratio analysis of the total cTnITC assay kit

[0376] The signal-to-noise ratios of total cTnITC assay kits 1-12 were analyzed with reference to the signal-to-noise ratio analysis method of the large-size cTnITC assay kit.

[0377] The test results are shown in Figure 4. As can be seen, total cTnITC Assay Kits 1-12 all exhibited excellent signal-to-noise ratios, meeting clinical needs. Total cTnITC Assay Kit 5 exhibited a higher signal-to-noise ratio than Total cTnITC Assay Kit 6, indicating that the addition of Antibody 2-3, which specifically binds to the cTnT amino acid fragment 67-222, improved the signal-to-noise ratio. Total cTnITC Assay Kit 3 exhibited a significantly higher signal-to-noise ratio than Total cTnITC Assay Kit 1 and Total cTnITC Assay Kit 5, and Total cTnITC Assay Kit 4 exhibited a significantly higher signal-to-noise ratio than Total cTnITC Assay Kit 2 and Total cTnITC Assay Kit 5, indicating that the combined use of Antibody 2-2, which specifically binds to TnC, with either Antibody 2-4 or Antibody 2-5 significantly improved the signal-to-noise ratio. Total cTnITC Assay Kits 3, 8, and 9 exhibited similar signal-to-noise ratios, indicating that using antibodies specifically binding to different fragments of cTnT amino acids 67-222 as Antibody 2-3 resulted in similar signal-to-noise ratios for the resulting kits. The signal-to-noise ratio of Total cTn ITC Assay Kit 8 was significantly better than that of Total cTn ITC Assay Kit 10, indicating that the antibody that specifically binds to amino acids 223-242 of cTnT as Antibody 2-1 has a better signal-to-noise ratio than the antibody that specifically binds to amino acids 262-281 of cTnT. The signal-to-noise ratio of Total cTn ITC Assay Kit 3 was significantly higher than that of Total cTn ITC Assay Kits 11 and 12, indicating that the antibody that specifically binds to amino acids 119-138 of cTnT as Antibody 2-3 is more advantageous than antibodies that specifically bind to amino acids 67-86 or 171-190 of cTnT.

[0378] In addition, the antibody 2-3 used in the total cTnITC detection kits 3-5 and 8-12 was replaced with the following antibodies: antibody 7F4 with a specific binding site for the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc with a specific binding site for the cTnT amino acid 145-164 fragment; and the antibody 2-5 used in the total cTnITC detection kit 4 was replaced with the following antibodies: antibody M18cc with a specific binding site for the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc with a specific binding site for the cTnI amino acid 86-90 fragment, antibody M46 with a specific binding site for the cTnI amino acid 130-145 fragment, and antibody MF4cc with a specific binding site for the cTnI amino acid 190-196 fragment, all of which can effectively reflect the signal differences of the samples.

[0379] 3. Specificity Analysis of Detection Kits

[0380] 1. Specificity Analysis of Large-Size cTnITC Detection Kit

[0381] Equal concentrations of different antigens were added to the serum of healthy individuals and analyzed by chemiluminescent immunoassay using large-format cTnITC detection kits 1-8. The analyzed antigens included: cTnT (Hytest, 8RTT5), cTnI (Hytest, 8RT17), cTnIC (Hytest, 8ICR3), and cTnITC (Hytest, 8ITCR).

[0382] The experimental results are shown in Figure 5. Large-size cTnITC detection kits 1-8 can only recognize cTnITC antigen, but cannot recognize cTnT, cTnI and binary cTnIC. In addition, the antibody 1-1 used in the large-size cTnITC detection kits 3-8 was replaced with the following antibodies: antibody 7F4 or 7G7 whose specific binding site is the cTnT amino acid 67-86 fragment, and antibody 2F3, 1A11, or 1F11cc whose specific binding site is the cTnT amino acid 145-164 fragment; the antibody 1-4 used in the large-size cTnITC detection kit 4 was replaced with the following antibodies: antibody M18cc whose specific binding site is the cTnI amino acid 18-28 fragment, 16A11cc, 16A12cc, or 8E10cc whose specific binding site is the cTnI amino acid 86-90 fragment, antibody M46 whose specific binding site is the cTnI amino acid 130-145 fragment, and antibody MF4cc whose specific binding site is the cTnI amino acid 190-196 fragment, all of which can effectively recognize the cTnITC antigen.

[0383] 2. Specificity Analysis of Total cTnITC Detection Kit

[0384] The specificity of total cTnITC assay kits 1-9 was analyzed by referring to the specificity analysis method of the large-size cTnITC assay kit.

[0385] The experimental results are shown in Figure 6. The detection kits 1-9 for total cTnITC can only recognize cTnITC antigen, but cannot recognize cTnT, cTnI and binary cTnIC. In addition, the antibody 2-3 used in the total cTnITC detection kits 3-5 and 8-12 was replaced with the following antibodies: antibody 7F4 whose specific binding site is the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc whose specific binding site is the cTnT amino acid 145-164 fragment; the antibody 2-5 used in the total cTnITC detection kit 4 was replaced with the following antibodies: antibody M18cc whose specific binding site is the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc whose specific binding site is the cTnI amino acid 86-90 fragment, antibody M46 whose specific binding site is the cTnI amino acid 130-145 fragment, and antibody MF4cc whose specific binding site is the cTnI amino acid 190-196 fragment, all of which can effectively recognize the cTnITC antigen.

[0386] 4. Establishment of blank limit and detection limit of test kit

[0387] The limit of blank (LoB) and limit of detection (LoD) were established according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (EP-17A2 Protocols for Determination of Limits of Detection and Limits of Quantitation).

[0388] LoB test results are derived from five blank samples, run over four days, with four replicates per test. The general formula is LoB = mean + 1.65 * SD.

[0389] LoD test results are derived from five low-concentration samples, run over four days, with each test repeated four times. The general formula is LoD = LoB + 1.65 * SD.

[0390] The test results are shown in Table 1-1 and Table 1-2.

[0391] Table 1-1: LoB and LoD of large-size cTnITC detection kits

[0392] Table 1-2: LoB and LoD of total cTnITC assay kit

[0393] The blank limits and detection limits of large-size cTnITC assay kits 1-8 all met clinical needs. The blank limit and detection limit of large-size cTnITC assay kit 3 were significantly lower than those of large-size cTnITC assay kit 1 and large-size cTnITC assay kit 5, and the blank limit and detection limit of large-size cTnITC assay kit 4 were significantly lower than those of large-size cTnITC assay kit 2 and large-size cTnITC assay kit 5. This indicates that the addition of TnC-specific antibodies 1-2 and their combined use with antibodies 1-3 or 1-4 significantly improves the sensitivity of the assay kits. In addition, the antibody 1-1 used in the large-size cTnITC detection kits 3-8 was replaced with the following antibodies: antibody 7F4 or 7G7 whose specific binding site is the cTnT amino acid 67-86 fragment, and antibody 2F3, 1A11, or 1F11cc whose specific binding site is the cTnT amino acid 145-164 fragment; the antibody 1-4 used in the large-size cTnITC detection kit 4 was replaced with the following antibodies: antibody M18cc whose specific binding site is the cTnI amino acid 18-28 fragment, 16A11cc, 16A12cc, or 8E10cc whose specific binding site is the cTnI amino acid 86-90 fragment, antibody M46 whose specific binding site is the cTnI amino acid 130-145 fragment, and antibody MF4cc whose specific binding site is the cTnI amino acid 190-196 fragment, all of which have lower LoB and LoD values.

[0394] The blank limits and detection limits of total cTnITC assay kits 1-9 all met clinical requirements. Specifically, the blank limit and detection limit of total cTnITC assay kit 5 were lower than those of total cTnITC assay kit 6, indicating that the addition of antibody 2-3, which specifically binds to amino acid fragment 119-138 of cTnT, improved sensitivity. The blank limit and detection limit of total cTnITC assay kit 3 were significantly lower than those of total cTnITC assay kit 1 and total cTnITC assay kit 5. The blank limit and detection limit of total cTnITC assay kit 4 were significantly lower than those of total cTnITC assay kit 2 and total cTnITC assay kit 5, indicating that the addition of antibody 2-2, which specifically binds to TnC, and its use in combination with antibody 2-4 or antibody 2-5 significantly improved the sensitivity of the assay kits. In addition, the antibody 2-3 used in the total cTnITC detection kits 3-5 and 8-12 was replaced with the following antibodies: antibody 7F4 whose specific binding site is the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc whose specific binding site is the cTnT amino acid 145-164 fragment; the antibody 2-5 used in the total cTnITC detection kit 4 was replaced with the following antibodies: antibody M18cc whose specific binding site is the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc whose specific binding site is the cTnI amino acid 86-90 fragment, antibody M46 whose specific binding site is the cTnI amino acid 130-145 fragment, and antibody MF4cc whose specific binding site is the cTnI amino acid 190-196 fragment, all of which have lower LoB and LoD values.

[0395] 5. Linearity Analysis of the Detection Kit

[0396] 1. Linearity Analysis of Large-Scale cTnITC Detection Kit

[0397] Large Dimension cTnITC Assay Kit 3 and Large Dimension cTnITC Assay Kit 4 were used for linearity analysis.

[0398] A clinical serum sample was selected as a high-concentration sample, and the high-concentration sample was diluted according to a certain ratio to obtain a series of diluted samples, and the concentration range of the serial diluted samples was 0-6000 ng / L.

[0399] Samples were analyzed by chemiluminescent immunoassay using the Large Format cTnITC Detection Kit 3. A linear fit was performed between the mean test concentration and the theoretical concentration, and the correlation coefficient within the linear range was calculated.

[0400] The experimental results are shown in Figure 7. The measured concentration of the diluted sample was linearly proportional to the theoretical concentration. The R² value within the linear range (0-6000 ng / L) was 0.9982.

[0401] Samples were analyzed using a chemiluminescent immunoassay using the Large Format cTnITC Detection Kit 4. A linear fit was performed between the mean test concentrations and the theoretical concentrations, and the correlation coefficient was calculated within the linear range. The results are shown in Figure 8 , showing linearity between the test concentrations of the diluted samples and the theoretical concentrations. The R² value within the linear range (0-6000 ng / L) was 0.9995.

[0402] 2. Linearity Analysis of Total cTnITC Detection Kit

[0403] Detection Kits 3 and 4 were used for linearity analysis.

[0404] Two clinical serum samples with different concentrations were selected as high-concentration samples. The high-concentration samples were diluted in a certain proportion to obtain a series of diluted samples. The concentration range of the series samples was 0-120 ng / L and 0-6000 ng / L. The samples were analyzed by chemiluminescence immunoassay using detection kit 3. The average value of the test concentration results and the theoretical concentration were linearly fitted, and the correlation coefficient was calculated within the linear range. The experimental results are shown in Figure 9. The test concentration of the diluted sample is linear with the theoretical concentration. Within the linear range, the R2 value of the low concentration range (0-120 ng / L) is 0.9990, and the R2 value of the high concentration range (0-6000 ng / L) is 0.9992.

[0405] The samples were analyzed using chemiluminescent immunoassay using Detection Kit 4. A linear fit was performed between the average test concentrations and the theoretical concentrations, and the correlation coefficient was calculated within the linear range. The experimental results are shown in Figure 10 . The test concentrations of the diluted samples showed a linear relationship with the theoretical concentrations. Within the linear range, the R² value was 0.9979 for the low concentration range (0-120 ng / L) and 0.9988 for the high concentration range (0-6000 ng / L).

[0406] In the following examples, the above-mentioned cTnI detection kit, cTnT detection kit, total cardiac troponin complex detection kit, large-size cTnITC detection kit 7, and total cTnITC detection kit 8 were used to detect the concentration of each marker in the sample.

[0407] Example 2. Diagnosis of early myocardial infarction (staging of myocardial infarction patients)

[0408] 1. Patient enrollment

[0409] Patients aged 18 years or older were enrolled in the study. All patients received a diagnosis of myocardial infarction and subsequently underwent interventional therapy. Acute myocardial infarction was independently adjudicated by hospital cardiologists based on the definition of acute myocardial infarction. Adjudication of acute myocardial infarction was performed through clinical examination, including physical examination, echocardiography, electrocardiogram (ECG), high-sensitivity troponin I (HSI) testing, high-sensitivity troponin T (HST) testing, and coronary angiography. Patients were excluded if the final diagnosis was not acute myocardial infarction or if the diagnostic information was incomplete. Patients younger than 18 years were excluded; pregnant women were also excluded. Lithium heparinized plasma samples were collected before interventional therapy for analysis of troponin complex and fragment composition. The patient enrollment process is shown in Figure 11.

[0410] Patient information was recorded, including age, gender, symptoms (chest pain, chest tightness, shortness of breath, etc.) and onset time, medical history, hypertension, diabetes, smoking status, creatinine, and glomerular filtration rate. Samples meeting the inclusion criteria were selected for relevant marker testing. A total of 61 patients with acute myocardial infarction were included, of whom 49 were male, accounting for 80%. Patient information is shown in Table 2-1.

[0411] Table 2-1: Enrollment information of patients with acute myocardial infarction

[0412] Note: Continuous variables are presented as medians (25th-75th percentiles); categorical variables are presented as numbers (percentages).

[0413] 2. Analysis of the relationship between troponin marker concentration and chest pain duration in patients with acute myocardial infarction

[0414] After enrollment, samples were analyzed for troponin markers, including total complexes, large-size cTnITC, total cTnITC, cTnT, and cTnI, in the enrolled patient samples using a Mindray chemiluminescence analyzer. Patients were divided into different subgroups based on the duration of chest pain (≤10 hours, 10-30 hours, 30-72 hours, and >72 hours). The concentrations of total complexes, large-size cTnITC, total cTnITC, cTnT, and cTnI in the patient blood are shown in Table 2-2. Concentrations of total complexes, large-size cTnITC, total cTnITC, cTnT, and cTnI were significantly higher in patients with chest pain duration of 10 to 72 hours than in those with chest pain duration of >72 hours. The values ​​measured by the cTnI kit and total complexes were similar, indicating a certain degree of clinical equivalence between the two.

[0415] Table 2-2: Concentrations of total complex, large-sized cTnITC, total cTnITC, cTnT, and cTnI in patients with different chest pain durations

[0416] Note: Values ​​are shown as median (25-75% interquartile range).

[0417] Figure 12 shows the relationship between the proportions of complexes and fragments of cardiac troponin in patients with different durations of chest pain, including the ratio of large-size cTnITC concentration to total complex concentration, the ratio of total cTnITC concentration to total complex concentration, and the ratio of cTnT concentration to total complex concentration. The proportion of ternary cTnITC (including large-size cTnITC and total cTnITC) was higher in patients with early-stage acute myocardial infarction whose chest pain duration was shorter, while the proportion of cTnT was higher in patients with chest pain duration of less than 10 hours and longer than 72 hours. These results indicate that in patients diagnosed with acute myocardial infarction, the composition of cardiac troponin complexes and fragments is highly correlated with the time from symptom onset to blood draw. The proportion of large-size cTnITC or total cTnITC was higher in patients with early-stage acute myocardial infarction whose chest pain duration was shorter, while the proportion of cTnT was higher in patients with chest pain duration of less than 10 hours and longer than 72 hours. The correlation between the complex and fragment composition of troponin and the duration of chest pain in patients with acute myocardial infarction suggests that using a single marker, such as cTnT, alone is difficult to accurately determine the stage of the disease, especially in patients with subtle chest pain symptoms (such as those taking analgesics). Specific recognition of different troponin fragments can be used to diagnose acute myocardial infarction and determine disease status. In particular, the continuous downward trend of the ternary complex (including large-sized cTnITC and total cTnITC) is more obvious and may be more suitable for early diagnosis.

[0418] 3. Diagnostic efficacy of concentration and concentration ratio characteristic parameters for predicting early myocardial infarction

[0419] Table 2-3 shows the diagnostic efficacy of concentration and concentration ratio characteristic parameters. In this example, patients with chest pain onset within 72 hours were selected as early myocardial infarction patients. The P values ​​for total complex concentration, large cTnITC concentration, total cTnITC concentration, cTnI concentration, the ratio of large cTnITC to total complex concentration, the ratio of total cTnITC to total complex concentration, the ratio of cTnT to total complex concentration, the ratio of large cTnITC to cTnT concentration, the ratio of total cTnITC to cTnT concentration, and the ratio of large cTnITC to cTnI concentration were all less than 0.05, indicating that the variables were significant. Receiver-operating characteristic (ROC) curves were constructed using SPSS software, and the area under the curve (AUC) was obtained. The area under the curve (AUC) for large-size cTnITC concentration, the AUC for the ratio of large-size cTnITC to total complex concentrations, and the AUC for the ratio of large-size cTnITC to cTnT concentrations was 0.833, 0.883, and 0.923, respectively, demonstrating good diagnostic efficacy. The values ​​measured by the cTnI kit and total complex concentrations were similar, demonstrating clinical equivalence. These results suggest that using large-size cTnITC or total cTnITC concentrations alone, or the ratio of large-size cTnITC or total cTnITC concentrations to total complex concentrations, cTnT concentrations, or cTnI concentrations as characteristic parameters, is more effective in predicting early myocardial infarction than using total complex concentrations, cTnT concentrations, or cTnI concentrations alone.

[0420] Table 2-3: Diagnostic efficacy of concentration and concentration ratio characteristic parameters for early myocardial infarction

[0421] The Youden Index was calculated based on the sensitivity and specificity corresponding to different cutoff values ​​using the total complex concentration, large-size cTnITC concentration, total cTnITC concentration, cTnI concentration, the ratio of the concentration of large-size cTnITC to total complex, the ratio of the concentration of total cTnITC to total complex, the ratio of the concentration of cTnT to total complex, the ratio of the concentration of large-size cTnITC to cTnT, the ratio of the concentration of total cTnITC to cTnT, and the ratio of the concentration of large-size cTnITC to cTnI as characteristic parameters. The optimal diagnostic CUTOFF value was determined based on the maximum value of the Youden Index (see Tables 2-4).

[0422] Table 2-4: Cutoff values ​​and corresponding sensitivity and specificity of concentration and concentration ratio characteristic parameters for the diagnosis of early myocardial infarction

[0423] 4. The diagnostic efficacy of combining multiple concentration characteristic parameters to predict early myocardial infarction

[0424] Characteristic parameters of marker concentration include total complex concentration, large-size cTnITC concentration, total cTnITC concentration, cTnT concentration, and cTnI concentration. In this embodiment, patients with chest pain onset within 72 hours are selected as early myocardial infarction patients.

[0425] Large-size cTnITC concentration and cTnT concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 0.0661*(large-size cTnITC concentration) - 0.0012*(cTnT concentration) - 0.4725. The prediction probability (characteristic parameter 1) was calculated as: P = 1 / (1 + e -logit )*100%.

[0426] The large-size cTnITC concentration and total complex concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 0.0313*(large-size cTnITC concentration)-0.0009*(total complex concentration)-0.8964. The prediction probability (characteristic parameter 2) was calculated as: P = 1 / (1+e -logit )*100%.

[0427] Large-size cTnITC concentration and total cTnITC concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 0.0224*(large-size cTnITC concentration) - 0.0013*(total cTnITC concentration) - 1.2990. The prediction probability (characteristic parameter 3) was calculated as: P = 1 / (1 + e -logit )*100%.

[0428] Large-size cTnITC concentration, total cTnITC concentration, total complex concentration, and cTnT concentration were selected as characteristic variables for joint analysis. Characteristic parameters were constructed using a logistic regression algorithm. The coefficients for each characteristic variable were estimated using logistic regression, resulting in the following formula for calculating the characteristic parameters: Logit(P) = 0.0615*(large-size cTnITC concentration) - 0.0002*(total cTnITC concentration) + 0.0003*(total complex concentration) - 0.0013*(cTnT concentration) - 0.4727. The predicted probability (characteristic parameter 4) was calculated as: P = 1 / (1 + e-logit) * 100%.

[0429] The areas under the curve (AUCs) for characteristic parameters 1, 2, 3, and 4 were 0.923, 0.880, 0.858, and 0.920, respectively, demonstrating good diagnostic efficacy (see Table 2-5). The optimal cutoff values, determined based on the maximum Youden index, were 0.3829, 0.2949, 0.2249, and 0.3592, respectively (see Table 2-6).

[0430] Table 2-5: Diagnostic efficacy of combined analysis of multiple concentration characteristic parameters for predicting early myocardial infarction

[0431] Table 2-6: Sensitivity and specificity of the cutoff values ​​corresponding to the combined analysis of multiple concentration characteristic parameters

[0432] In addition, other concentration characteristic parameter combinations, including total cTnITC concentration + total complex concentration, total cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total cTnITC concentration + total complex concentration, large-size cTnITC concentration + total cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total complex concentration + cTnT concentration, and total cTnITC concentration + total complex concentration + cTnT concentration, all showed good diagnostic efficacy when used in combination for the diagnosis of early myocardial infarction. These results show that large-size cTnITC concentration and / or total cTnITC concentration combined with other troponin fragment concentrations have good performance in predicting early myocardial infarction.

[0433] 5. Diagnostic efficacy of combined concentration ratio characteristic parameters for predicting early myocardial infarction

[0434] The concentration ratio characteristic parameter includes the ratio of the concentration of large cTnITC or total cTnITC to the concentration of total complexes, cTnT, or cTnI, i.e., large cTnITC concentration / total complex concentration, total cTnITC concentration / total complex concentration, cTnT concentration / total complex or concentration, large cTnITC concentration / cTnT concentration, total cTnITC concentration / cTnT concentration, large cTnITC concentration / cTnI concentration, and total cTnITC concentration / cTnI concentration. In this example, patients with chest pain onset within 72 hours are selected as patients with early myocardial infarction.

[0435] The large-size cTnITC concentration / total complex concentration, total cTnITC concentration / total complex concentration, and cTnT concentration / total complex concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters: Logit(P) = 27.708*(large-size cTnITC concentration / total complex concentration) + 9.322*(total cTnITC / total complex) - 0.558*(cTnT concentration / total complex concentration) - 1.456. The prediction probability (characteristic parameter 5) was calculated as: P = 1 / (1 + e -logit )*100%.

[0436] Large-size cTnITC concentration / cTnT concentration and total cTnITC concentration / cTnT concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 78.924*(large-size cTnITC concentration / cTnT concentration) + 21.483*(total cTnITC concentration / cTnT concentration) - 3.004. The prediction probability (characteristic parameter 6) was calculated as: P = 1 / (1 + e - logit )*100%.

[0437] The areas under the curve (AUCs) for characteristic parameters 5 and 6 were 0.945 and 0.938, respectively, indicating good diagnostic efficacy (see Table 2-7). The optimal cutoff values ​​determined based on the maximum Youden index were 0.2729 and 0.2031, respectively (see Table 2-8).

[0438] Table 2-7: Diagnostic efficacy of combined analysis of multiple concentration ratio characteristic parameters for predicting early myocardial infarction

[0439] Table 2-8: Sensitivity and specificity of the cutoff values ​​corresponding to the combined analysis of multiple concentration ratio parameters

[0440] In addition, other concentration ratio parameter combinations, including large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration, large-size cTnITC concentration / total complex concentration + cTnT concentration / total complex concentration, total cTnITC concentration / total complex concentration + cTnT concentration / total complex concentration, large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration + large-size cTnITC concentration / cTnT concentration, and large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration + total cTnITC concentration / cTnT concentration, all showed good diagnostic efficacy when used in combination for the diagnosis of early myocardial infarction. These results indicate that the combination of the ratio between large-size cTnITC and other troponin fragments and the ratio between total cTnITC and other troponin fragments, as well as the combination of the ratio between at least one of these and other troponin fragments, has good performance in predicting early myocardial infarction.

[0441] These data suggest that large-size cTnITC concentration and total cTnITC concentration can be used independently as characteristic parameters to predict early myocardial infarction. The ratios of large-size cTnITC concentration or total cTnITC concentration to total complex concentration, cTnT, or cTnI can also be used to predict early myocardial infarction, with the ratios of large-size cTnITC concentration / total complex concentration, large-size cTnITC concentration / cTnT concentration, or large-size cTnITC concentration / cTnI concentration being preferred. In addition, the combined use of multiple parameters, such as the combined use of large-size cTnITC concentration and cTnT concentration, the combined use of large-size cTnITC concentration and total complex concentration, the combined use of large-size cTnITC concentration and total cTnITC concentration, or the combined use of large-size cTnITC concentration, total cTnITC concentration, total complex concentration, and cTnT concentration, or the combined use of large-size cTnITC concentration / total complex concentration, total cTnITC concentration / total complex concentration and cTnT concentration / total complex concentration, or the combined use of large-size cTnITC concentration / cTnT concentration and total cTnITC concentration / cTnT concentration, can further improve the diagnostic efficacy. Although not being bound by theory, it is possible that large-size cTnITC or characteristic parameters derived from large-size cTnITC are more suitable for the diagnosis of early myocardial infarction.

[0442] Example 3. Differentiation between Type 1 and Type 2 Myocardial Infarction

[0443] 1. Patient enrollment

[0444] Patients aged 18 years or older were enrolled in the study if their admission and final diagnoses were type 1 or type 2 acute myocardial infarction. All patients were diagnosed with type 1 or type 2 acute myocardial infarction with symptom onset less than 72 hours. Acute myocardial infarction was independently adjudicated by hospital cardiologists according to the definition of acute myocardial infarction. Adjudication of acute myocardial infarction was performed through clinical evaluation, including physical examination, echocardiography, electrocardiogram (ECG), high-sensitivity troponin I (HSI) testing, high-sensitivity troponin T (HSI) testing, and coronary angiography. Patients were excluded if the final diagnosis was not type 1 or type 2 acute myocardial infarction or if the diagnostic information was incomplete. Patients younger than 18 years of age were excluded, as were pregnant women. Lithium heparinized plasma samples were obtained before treatment for analysis of troponin complex and fragment composition.

[0445] A total of 24 patients with type 1 acute myocardial infarction and 6 patients with type 2 acute myocardial infarction were included.

[0446] 2. Analysis of myocardial injury markers in patients with type 1 and type 2 myocardial infarction

[0447] After enrollment, samples were analyzed for troponin markers, including total complexes, large-size cTnITC, total cTnITC, cTnT, and cTnI, in the enrolled patient samples using a Mindray chemiluminescence analyzer. The differences in troponin composition between patients with acute myocardial infarction and those with cardiomyopathy, chronic heart failure, and pneumonia were analyzed. The concentrations of total complexes, large-size cTnITC, total cTnITC, cTnT, and cTnI in the patient blood are shown in Table 3-1. Concentrations of total complexes, large-size cTnITC, total cTnITC, and cTnI were significantly higher in patients with type 1 myocardial infarction than in patients with type 2 myocardial infarction. The values ​​measured by the cTnI kit and the total complex kit were similar, indicating a certain degree of clinical equivalence between the two.

[0448] Table 3-1: Concentrations of total complex, large-sized cTnITC, total cTnITC, cTnT, and cTnI in patients with type 1 and type 2 myocardial infarction

[0449] Note: Values ​​are shown as medians (25th-75th percentile interquartile range). P values ​​for type 1 and type 2 myocardial infarction were compared using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant difference.

[0450] Figure 13 shows the relationship between the proportions of troponin complexes and fragments in patients with type 1 and type 2 myocardial infarction, including the ratio of large cTnITC concentration to total complex concentration, the ratio of total cTnITC concentration to total complex concentration, the ratio of cTnT concentration to total complex concentration, the ratio of large cTnITC concentration to cTnT concentration, and the ratio of total cTnITC concentration to cTnT concentration. Among them, the ratios of large cTnITC concentration to total complex concentration, total cTnITC concentration to total complex concentration, large cTnITC concentration to cTnT concentration, and total cTnITC concentration to cTnT concentration were higher in patients with type 1 myocardial infarction and lower in patients with type 2 myocardial infarction.

[0451] These data suggest that large-size cTnITC concentration or total cTnITC concentration can be used to differentiate type 1 from type 2 myocardial infarction, with large-size cTnITC being preferred.

[0452] Example 4-1. Differentiation between acute myocardial infarction (type 1) and chronic cardiac events

[0453] 1. Patient enrollment

[0454] (1) Acute myocardial infarction (type 1) patient group: Patients with an admission diagnosis and final diagnosis of type 1 acute myocardial infarction were enrolled in the group, and the patients were 18 years of age or older. All patients were diagnosed with acute myocardial infarction and the onset of symptoms was less than 72 hours. These patients subsequently received interventional treatment. Acute myocardial infarction was independently determined by hospital cardiologists based on the definition of acute myocardial infarction. The determination of acute myocardial infarction was completed through clinical examinations, including physical examination, echocardiography, electrocardiogram recording, high-sensitivity troponin I detection, high-sensitivity troponin T detection, and coronary angiography. Patients were excluded if the final diagnosis was not acute myocardial infarction or the diagnostic information was incomplete. Patients under 18 years of age were excluded; pregnant female patients were excluded. Lithium heparin plasma samples were collected before interventional treatment for analysis of troponin complex and fragment composition. The patient enrollment process is shown in Figure 14.

[0455] (2) Chronic cardiac event patient group: a) Patients with an admission diagnosis and final diagnosis of chronic heart failure or cardiomyopathy were selected for inclusion in the group, and the patients were aged 18 years or older. If the final diagnosis was not chronic heart failure or cardiomyopathy, the patient was excluded. If the patient was also diagnosed with acute myocardial infarction, the patient was excluded. Patients under 18 years of age were excluded; pregnant female patients were excluded. Cardiomyopathy and chronic heart failure were independently determined by hospital clinicians. Acute myocardial infarction was independently determined by hospital cardiologists based on the definition of acute myocardial infarction. The first lithium heparin plasma sample after admission was collected from these patients for analysis of troponin complex and fragment composition. The patient enrollment process is shown in Figure 15.

[0456] b) Patients aged 18 years or older were selected for enrollment with both an admission diagnosis and a final diagnosis of pneumonia. Patients were excluded if the final diagnosis was not pneumonia. Patients were also excluded if they were also diagnosed with acute myocardial infarction. Patients younger than 18 years were excluded; pregnant women were excluded. Pneumonia was independently determined by hospital clinicians. Acute myocardial infarction was independently determined by hospital cardiologists based on the definition of acute myocardial infarction. The first lithium heparin plasma sample after admission was collected from these patients for analysis of troponin complex and fragment composition. The patient enrollment process is shown in Figure 16.

[0457] Patient information was recorded, including age, gender, medical history, hypertension, diabetes, smoking history, creatinine, and glomerular filtration rate. Samples meeting the inclusion criteria were selected for relevant marker testing. A total of 24 patients with acute myocardial infarction (type 1) and 145 patients with chronic cardiac events were included. Samples were collected from 94 patients hospitalized for cardiomyopathy or chronic heart failure, and 51 patients with pneumonia. Patient information is provided in Table 4-1-1.

[0458] Table 4-1-1: Characteristics of enrolled patients.

[0459] Continuous variables are presented as medians (25th-75th percentiles); categorical variables are presented as numbers (percentages).

[0460] 2. Analysis of Troponin Composition in Acute Myocardial Infarction (Type 1) and Chronic Cardiac Event Samples

[0461] After sample enrollment, samples were assayed for troponin markers, including total troponin complexes, large-size cTnITC, total cTnITC, and cTnT, using a Mindray chemiluminescence analyzer and accompanying reagents. Differences in troponin composition were analyzed between patients with acute myocardial infarction (type 1) and those with cardiomyopathy, chronic heart failure, and pneumonia. The concentrations of total troponin complexes, large-size cTnITC, total cTnITC, and cTnT in the blood of these patients are shown in Table 4-1-2.

[0462] Table 4-1-2: Concentrations of total complex, large-sized cTnITC, total cTnITC, and cTnT in patients with acute myocardial infarction (type 1) and chronic cardiac events

[0463] Note: Values ​​are shown as median (25-75% interquartile range).

[0464] Figure 17 shows the relationship between the proportions of troponin complexes and fragments between patients with acute myocardial infarction (type 1) and patients with chronic cardiac events, including the ratios of large cTnITC / total complexes, total cTnITC / total complexes, cTnT / total complexes, large cTnITC / cTnT, and total cTnITC / cTnT. The ratios of large cTnITC / total complexes, large cTnITC / cTnT, and total cTnITC / cTnT were lower in patients with chronic cardiac events (including patients with cardiomyopathy or chronic heart failure and pneumonia) and higher in patients with acute myocardial infarction (type 1); whereas the ratio of cTnT / total complexes was higher in patients with chronic cardiac events and lower in patients with acute myocardial infarction (type 1).

[0465] The correlation between the complex and fragment composition of troponin and disease type suggests that specific recognition of different troponin fragments can be used to diagnose and differentiate acute myocardial infarction (type 1) from chronic cardiac events.

[0466] 3. Diagnostic efficacy of concentration characteristic parameters and concentration ratio characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0467] Table 4-1-3 shows the diagnostic efficacy of the troponin complex and fragment markers used. In this example, patients suffered from acute myocardial infarction (type 1) or chronic cardiac events. Patients with chronic cardiac events suffered from cardiomyopathy, chronic heart failure, or pneumonia. To facilitate analysis of the ratio between complexes, the concentration of total complexes in the patient samples included in the analysis was greater than 0.1 pmol / L.

[0468] The P values ​​for total complex concentration, large-size cTnITC concentration, total cTnITC concentration, cTnT concentration, cTnI concentration, large-size cTnITC / total complex ratio, total cTnITC / total complex ratio, cTnT / total complex ratio, large-size cTnITC / cTnT ratio, total cTnITC / cTnT ratio, and large-size cTnITC / cTnI ratio were all less than 0.05, indicating significance. The area under the curve (AUC) for large-size cTnITC concentration, total cTnITC concentration, and large-size cTnITC / cTnT ratio were 0.929, 0.869, and 0.870, respectively, demonstrating good diagnostic efficacy. The values ​​measured by the cTnI kit and total complex are similar, indicating clinical equivalence between the two. The results showed that the concentration of large-size cTnITC or total cTnITC alone, or the ratio of large-size cTnITC to cTnT, could more effectively distinguish acute myocardial infarction (type 1) from chronic cardiac events than the concentration of total troponin complex, cTnT, or cTnI alone.

[0469] Table 4-1-3: Diagnostic efficacy of concentration characteristic parameters and concentration ratio characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0470] The Youden index was calculated using the sensitivity and specificity corresponding to different cutoff values ​​of the characteristic parameters, and the optimal diagnostic cutoff value was determined based on the maximum value of the Youden index (Table 4-1-4). The CUTOFF value of total complex concentration was 247.7, the CUTOFF value of large-size cTnITC concentration was 1.9, and the CUTOFF value of total cTnITC concentration was 13.6; the CUTOFF value of cTnT concentration was 754.7; the CUTOFF value of cTnI concentration was 278.8; the CUTOFF value of large-size cTnITC / total complex was 0.0498; the CUTOFF value of total cTnITC / total complex was 0.1779; the CUTOFF value of cTnT / total complex was 3.4450; the CUTOFF value of large-size cTnITC / cTnT was 0.0165; the CUTOFF value of total cTnITC / cTnT was 0.0365; and the CUTOFF value of large-size cTnITC / cTnI was 0.0705.

[0471] Table 4-1-4: Cutoff values ​​and corresponding sensitivity and specificity of concentration and concentration ratio characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0472] 4. Diagnostic efficacy of multi-parameter joint analysis of concentration characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0473] Multi-parameter combined analysis using concentration characteristic parameters is used to distinguish the diagnostic efficacy of acute myocardial infarction (type 1) from chronic cardiac events. The characteristic parameters include the concentrations of troponin complexes and fragments, namely, the total complex concentration, the concentration of large-sized cTnITC, the concentration of total cTnITC, the concentration of cTnT, and the concentration of cTnI. In this example, the patients had acute myocardial infarction (type 1) or chronic cardiac events. The chronic cardiac event patients had cardiomyopathy, chronic heart failure, or pneumonia, and the concentration of total complexes in the patient samples was greater than 0.1 pmol / L.

[0474] Large-size cTnITC concentration and cTnT concentration were selected as feature quantities for joint analysis. Combined prediction parameters were constructed based on the logistic regression algorithm. The coefficients of each feature quantity were estimated by logistic regression. The prediction value calculation formula was output. Logit(P) = 0.0484*(large-size cTnITC concentration) - 0.0003*(cTnT concentration) - 2.3441. The prediction probability (prediction parameter 1) was calculated as: P = 1 / (1 + e -logit )*100%

[0475] The large-size cTnITC concentration and total complex concentration were selected as characteristic quantities for joint analysis. Combined prediction parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression. The prediction value calculation formula was output. Logit(P) = 0.0266*(large-size cTnITC concentration) - 0.00004*(total complex concentration) - 2.3382. The prediction probability (prediction parameter 2) was calculated as: P = 1 / (1 + e -logit )*100%

[0476] The large-size cTnITC concentration and total cTnITC concentration were selected as characteristic quantities for joint analysis. Combined prediction parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression. The prediction value calculation formula was output. Logit(P) = 0.0315*(large-size cTnITC concentration)-0.0016*(total cTnITC concentration)-2.3538. The prediction probability (prediction parameter 3) was calculated as: P = 1 / (1+e -logit )*100%

[0477] The large-size cTnITC concentration + total cTnITC concentration + total complex concentration + cTnT concentration were selected as feature quantities for joint analysis. Combined prediction parameters were constructed based on the logistic regression algorithm, and the coefficients of each feature quantity were estimated by logistic regression. The prediction value calculation formula was output. Logit (P) = 0.0523 * (large-size cTnITC concentration) + 0.0221 * (total cTnITC concentration) + 0.0001 * (total complex concentration) - 0.0019 * (cTnT concentration) - 2.0412. The prediction probability (prediction parameter 4) was calculated as: P = 1 / (1 + e-logit) * 100%

[0478] The areas under the curve (AUCs) for prediction parameters 1, 2, 3, and 4 were 0.870, 0.928, 0.845, and 0.960, respectively, demonstrating good diagnostic efficacy (Table 4-1-5). The optimal cutoff values, determined based on the maximum Youden index, were 0.1150, 0.0920, 0.1828, and 0.1077, respectively (Table 4-1-6).

[0479] Table 4-1-5: Diagnostic efficacy of multi-parameter combined analysis of troponin concentration characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0480] Table 4-1-6: Sensitivity and specificity of the cutoff values ​​corresponding to the multi-parameter joint analysis of troponin concentration characteristic parameters

[0481] In addition, other combinations, including total cTnITC concentration + total complex concentration, total cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total cTnITC concentration + total complex concentration, large-size cTnITC concentration + total cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total complex concentration + cTnT concentration, and total cTnITC concentration + total complex concentration + cTnT concentration, showed good diagnostic efficacy when used in combination to distinguish acute myocardial infarction (type 1) from chronic cardiac events. These results show that combined analysis of large-size cTnITC concentration or total cTnITC concentration with other troponin fragment concentrations has good performance in distinguishing acute myocardial infarction (type 1) from chronic cardiac events.

[0482] 6. Diagnostic efficacy of multi-parameter joint analysis of concentration ratio characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0483] The diagnostic efficacy of multi-parameter combined analysis using concentration ratios was used to distinguish between acute myocardial infarction (type 1) and chronic cardiac events. The concentration ratio characteristic parameters included the ratios of troponin complexes and fragments to total troponin complexes, cTnT, or cTnI, i.e., large cTnITC / total complexes, total cTnITC / total complexes, cTnT / total complexes, large cTnITC / cTnT, total cTnITC / cTnT, large cTnITC / cTnI, and total cTnITC / cTnI. In this example, the patients had acute myocardial infarction (type 1) or chronic cardiac events, and the chronic cardiac event patients had cardiomyopathy, chronic heart failure, or pneumonia, and the concentration of total complexes in the patient sample was greater than 0.1 pmol / L.

[0484] Large cTnITC / total complex + total cTnITC / total complex + cTnT / total complex were selected as feature quantities for joint analysis. Combined prediction parameters were constructed based on the logistic regression algorithm, and the coefficients of each feature quantity were estimated by logistic regression. The prediction value calculation formula was output: Logit(P) = 10.532*(large cTnITC / total complex) + 24.346*(total cTnITC / total complex) - 1.076*(cTnT / total complex) - 1.099. The prediction probability (prediction parameter 5) was calculated as: P = 1 / (1 + e -logit )*100%

[0485] Large-size cTnITC / cTnT and total cTnITC / cTnT were selected as feature quantities for joint analysis. Combined prediction parameters were constructed based on the logistic regression algorithm, and the coefficients of each feature quantity were estimated by logistic regression. The output prediction value calculation formula was: Logit(P) = 16.149*(large-size cTnITC / cTnT) + 62.770*(total cTnITC / cTnT) - 4.041. The prediction probability (prediction parameter 6) was calculated as: P = 1 / (1 + e -logit )*100%

[0486] The areas under the curve (AUCs) for prediction parameters 5 and 6 were 0.961 and 0.892, respectively, demonstrating good diagnostic efficacy (Table 4-1-7). The optimal cutoff values, determined based on the maximum Youden index, were 0.1562 and 0.2286, respectively (Table 4-1-8).

[0487] Table 4-1-7: Diagnostic efficacy of multi-parameter combined analysis of concentration ratios for differentiating acute myocardial infarction (type 1) from chronic cardiac events

[0488] Table 4-1-8: Sensitivity and specificity of the cutoff values ​​corresponding to the multi-parameter combined analysis of troponin complex and fragment ratios

[0489] In addition, other combinations, including large-size cTnITC / total complex + total cTnITC / total complex, large-size cTnITC / total complex + cTnT / total complex, total cTnITC / total complex + cTnT / total complex, large-size cTnITC / total complex + total cTnITC / total complex + large-size cTnITC / cTnT, and large-size cTnITC / total complex + total cTnITC / total complex + total cTnITC / cTnT, were used in the above-mentioned combination to distinguish acute myocardial infarction (type 1) from chronic cardiac events, all of which had good diagnostic efficacy. These results show that the combined analysis of the ratio of large-size cTnITC to other troponin fragments and the ratio of total cTnITC to other troponin fragments, as well as the combined analysis of the ratio between at least one of the two and other troponin fragments, has good performance in distinguishing acute myocardial infarction (type 1) from chronic cardiac events.

[0490] These data suggest that large-size cTnITC complexes and total cTnITC complexes alone can be used to differentiate between acute myocardial infarction (type 1) and chronic cardiac events. The ratio of large-size cTnITC complexes or total cTnITC complexes to total complexes, and the ratio of large-size cTnITC concentration or total cTnITC concentration to cTnT concentration can also be used to differentiate between acute myocardial infarction (type 1) and chronic cardiac events. The combined use of multiple marker concentrations, such as the concentration of troponin large-size cTnITC complexes + total complexes, large-size cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total cTnITC concentration, or large-size cTnITC concentration + total cTnITC concentration + total complexes + cTnT concentration, or the combined use of multiple marker ratios, such as the combined use of large-size cTnITC / total complexes, total cTnITC / total complexes, cTnT / total complexes, or the combined use of large-size cTnITC concentration / cTnT concentration, total cTnITC concentration / cTnT concentration, can further improve the diagnostic efficacy.

[0491] Example 4-2. Differentiation between acute myocardial infarction (type 1) and chronic cardiac events (patients under observation)

[0492] 1. Patient enrollment

[0493] Patients with a troponin I value between the 99th percentile and 5 times the 99th percentile at the time of their initial blood draw were included. These patients could not be included or excluded based on the high-sensitivity cardiac troponin 0-3 hour rapid diagnostic algorithm and were designated as observation patients. A total of 84 patients were enrolled, including 9 with type 1 acute myocardial infarction and symptom onset less than 72 hours, and 75 with other chronic cardiac events.

[0494] Patient information is shown in Table 4-2-1.

[0495] Table 4-2-1: Characteristics of enrolled patients.

[0496] Note: Continuous variables are presented as medians (25th-75th percentiles); categorical variables are presented as numbers (percentages).

[0497] After sample enrollment, samples were assayed for troponin markers, including total troponin complexes, large-size cTnITC, total cTnITC, and cTnT, using a Mindray chemiluminescence analyzer and accompanying reagents. The differences in troponin composition between patients with acute myocardial infarction (type 1) and those with chronic cardiac events were analyzed. The concentrations of total complexes, large-size cTnITC, total cTnITC, and cTnT in the blood of these patients are shown in Table 4-2-2.

[0498] Table 4-2-2: Values ​​of concentration or concentration ratio characteristic parameters in patients with acute myocardial infarction (type 1) and chronic cardiac events

[0499] Note: Values ​​are shown as median (25-75% interquartile range).

[0500] The ratios of large-size cTnITC concentration to total complex concentration, large-size cTnITC concentration to cTnT concentration, and total cTnITC concentration to cTnT concentration were lower in patients with chronic cardiac events and higher in patients with acute myocardial infarction (type 1). In contrast, the ratio of cTnT to total complex concentration was higher in patients with chronic cardiac events and lower in patients with acute myocardial infarction (type 1). The association between complex and fragment composition of troponin and disease type suggests that specific recognition of different troponin fragments can be used to distinguish acute myocardial infarction (type 1) from chronic cardiac events in patients with troponin I values ​​between the 99th percentile and 5 times the 99th percentile.

[0501] Table 4-2-3 shows the diagnostic efficacy of the concentrations and concentration ratio characteristic parameters used in distinguishing acute myocardial infarction (type 1) from chronic cardiac events in patients with troponin I measurements between the 99th percentile and 5 times the 99th percentile.

[0502] Table 4-2-3: Diagnostic efficacy of concentration and concentration ratio characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0503] The P values ​​for large-size cTnITC concentration, total cTnITC concentration, cTnT concentration, the ratio of large-size cTnITC concentration to total complex concentration, the ratio of cTnT concentration to total complex concentration, the ratio of large-size cTnITC concentration to cTnT concentration, and the ratio of total cTnITC concentration to cTnT concentration were all less than 0.05, indicating significance. The area under the curve (AUC) for large-size cTnITC concentration, the ratio of large-size cTnITC concentration to total complex concentration, the ratio of large-size cTnITC concentration to cTnT concentration, and the ratio of total cTnITC concentration to cTnT concentration were 0.842, 0.852, 0.875, and 0.842, respectively, demonstrating good diagnostic efficacy. The results showed that using large-size cTnITC concentration or the ratio of large-size cTnITC concentration to total complex concentration, the ratio of large-size cTnITC concentration to cTnT concentration, and the ratio of total cTnITC concentration to cTnT concentration as characteristic parameters can more effectively distinguish acute myocardial infarction (type 1) from chronic cardiac events.

[0504] The Youden Index was calculated using the sensitivity and specificity corresponding to different cutoff values ​​of the characteristic parameters, and the optimal diagnostic CUTOFF value was determined based on the maximum value of the Youden Index (see Table 4-2-4).

[0505] Table 4-2-4: Cutoff values ​​and corresponding sensitivity and specificity of concentration and concentration ratio characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0506] The diagnostic efficacy of a signature parameter derived from combining concentration signature parameters of multiple myocardial injury markers for distinguishing acute myocardial infarction (type 1) from chronic cardiac events in patients with troponin I values ​​between the 99th percentile and 5 times the 99th percentile was analyzed. The concentration signature parameters for myocardial injury markers included the concentrations of troponin complexes and fragments, namely, total complex concentration, large-size cTnITC concentration, total cTnITC concentration, cTnT concentration, and cTnI concentration.

[0507] Large-size cTnITC concentration and cTnT concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, thereby obtaining the calculation formula for the characteristic parameters, namely, Logit(P) = 0.1593*(large-size cTnITC concentration) - 0.0052*(cTnT concentration) - 1.3890. The prediction probability (characteristic parameter 1) was calculated as: P = 1 / (1 + e -logit )*100%.

[0508] The large-size cTnITC concentration and total complex concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 0.1722*(large-size cTnITC concentration) + 0.0066*(total complex concentration) - 3.2247. The prediction probability (characteristic parameter 2) was calculated as: P = 1 / (1 + e -logit )*100%.

[0509] Large-size cTnITC concentration and total cTnITC concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 0.1761*(large-size cTnITC concentration) + 0.0608*(total cTnITC concentration) - 3.2829. The prediction probability (characteristic parameter 3) was calculated as: P = 1 / (1 + e -logit )*100%.

[0510] Large-size cTnITC concentration, total cTnITC concentration, total complex concentration, and cTnT concentration were selected as characteristic variables for joint analysis. Characteristic parameters were constructed using a logistic regression algorithm. The coefficients for each characteristic variable were estimated using logistic regression, resulting in the following formula for calculating the characteristic parameter: Logit(P) = 0.1450*(large-size cTnITC concentration) + 0.2588*(total cTnITC concentration) + 0.0086*(total complex concentration) - 0.0160*(cTnT concentration) - 1.048. The predicted probability (characteristic parameter 4) was calculated as: P = 1 / (1 + e-logit) * 100%.

[0511] The areas under the curve (AUCs) for characteristic parameters 1, 2, 3, and 4 were 0.880, 0.809, 0.803, and 0.899, respectively, demonstrating good diagnostic efficacy (see Table 4-2-5). The optimal cutoff values ​​for diagnosis were determined based on the maximum Youden index (see Table 4-2-6).

[0512] Table 4-2-5: Diagnostic efficacy of combined analysis of multiple concentration characteristic parameters for distinguishing acute myocardial infarction (type 1) from chronic cardiac events

[0513] Table 4-2-6: Sensitivity and specificity of the cutoff values ​​corresponding to the combined analysis of multiple concentration characteristic parameters

[0514] In addition, other concentration parameter combinations, including total cTnITC concentration + total complex concentration, total cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total cTnITC concentration + total complex concentration, large-size cTnITC concentration + total cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total complex concentration + cTnT concentration, and total cTnITC concentration + total complex concentration + cTnT concentration, all showed good diagnostic efficacy when used in combination to distinguish acute myocardial infarction (type 1) from chronic cardiac events. These results show that the combination of large-size cTnITC concentration and total cTnITC concentration, or at least one of them combined with other troponin fragment concentrations, has good performance in distinguishing acute myocardial infarction (type 1) from chronic cardiac events.

[0515] The diagnostic efficacy of a characteristic parameter derived from combining ratio parameters of multiple myocardial injury markers for distinguishing acute myocardial infarction (type 1) from chronic cardiac events in patients with troponin I values ​​between the 99th percentile and 5 times the 99th percentile was analyzed. Ratio parameters for myocardial injury markers included the ratios of large or total cTnITC concentration to total complex concentration, cTnT concentration, or cTnI concentration (i.e., large cTnITC concentration / total complex concentration, total cTnITC concentration / total complex concentration, cTnT concentration / total complex concentration, large cTnITC concentration / cTnT concentration, total cTnITC concentration / cTnT concentration, large cTnITC concentration / cTnI concentration, and total cTnITC concentration / cTnI concentration).

[0516] The large-size cTnITC concentration / total complex concentration, total cTnITC concentration / total complex concentration, and cTnT concentration / total complex concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters: Logit(P) = 6.880*(large-size cTnITC concentration / total complex concentration) + 13.985*(total cTnITC concentration / total complex concentration) - 0.619*(cTnT concentration / total complex concentration) - 1.201. The prediction probability (characteristic parameter 5) was calculated as: P = 1 / (1 + e-logit )*100%.

[0517] Large-size cTnITC concentration / cTnT concentration and total cTnITC concentration / cTn concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the formula for calculating the predicted value: Logit(P) = 30.283*(large-size cTnITC concentration / cTnT concentration) + 68.804*(total cTnITC concentration / cTnT concentration) - 4.519. The prediction probability (characteristic parameter 6) was calculated as: P = 1 / (1 + e -logit )*100%.

[0518] The AUCs for characteristic parameters 5 and 6 were 0.875 and 0.854, respectively, indicating good diagnostic efficacy (see Table 4-2-7). The optimal CUTOFF value was determined based on the maximum value of the Youden Index (see Table 4-2-8). Table 4-2-7: Diagnostic efficacy of combined analysis of multiple proportion parameters for differentiating acute myocardial infarction (type 1) from chronic cardiac events

[0519] Table 4-2-8: Sensitivity and specificity corresponding to the cutoff value of multi-proportional parameter joint analysis

[0520] In addition, other ratio parameter combinations including large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration, large-size cTnITC concentration / total complex concentration + cTnT concentration / total complex concentration, total cTnITC concentration / total complex concentration + cTnT concentration / total complex concentration, large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration + large-size cTnITC concentration / cTnT concentration, and large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration + total cTnITC concentration / cTnT concentration can be combined in the above manner to distinguish acute myocardial infarction (type 1) from chronic cardiac events, and all have good diagnostic efficacy. The results showed that the combination of the ratio of large-size cTnITC to other troponin fragments and the ratio of total cTnITC to other troponin fragments, or at least one of the two combined with the ratio of other troponin fragments, had better performance in distinguishing acute myocardial infarction (type 1) from chronic cardiac events.

[0521] These data suggest that large-size cTnITC concentration or total cTnITC concentration, particularly large-size cTnITC concentration, can be used alone to differentiate acute myocardial infarction (type 1) from chronic cardiac events in patients with troponin I values ​​between the 99th percentile and 5 times the 99th percentile. Furthermore, the ratio of large-size cTnITC concentration or total cTnITC concentration to total complex concentration, or the ratio of large-size cTnITC concentration or total cTnITC concentration to cTnT concentration, can also be used to differentiate acute myocardial infarction (type 1) from chronic cardiac events, with the ratio of large-size cTnITC concentration or total cTnITC concentration to cTnT concentration or the ratio of large-size cTnITC concentration to total complex concentration having superior diagnostic efficacy. The combined use of multiple parameters, such as large-size cTnITC concentration + total complex concentration, large-size cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total cTnITC concentration, or large-size cTnITC concentration + total cTnITC concentration + total complex concentration + cTnT concentration, or the combined use of large-size cTnITC concentration / total complex concentration, total cTnITC concentration / total complex concentration, cTnT concentration / total complex concentration, or large-size cTnITC concentration / cTnT concentration, total cTnITC concentration / cTnT concentration, can further improve the diagnostic efficacy.

[0522] Taking into account Examples 4-1 and 4-2, Example 4-2 is a secondary analysis conducted on patients with mild elevation of troponin among the patients enrolled in Example 4-1. In Example 4-2, the study found that for patients with mild elevation of troponin, the characteristic parameters obtained based on large-size cTnITC concentration and / or total cTnITC concentration had better diagnostic efficacy in acute myocardial infarction (type 1) and chronic cardiac events. Preferably, large-size cTnITC concentration or characteristic parameters obtained based thereon have better diagnostic and therapeutic performance. In addition, the ratio of total cTnITC concentration to cTnT concentration, or the characteristic parameters obtained based thereon, also has good diagnostic and therapeutic performance.

[0523] Example 5. Distinguishing between myocardial injury caused by invasive procedures and chronic cardiac events

[0524] 1. Patient enrollment

[0525] (1) Patients undergoing invasive procedures:

[0526] Patients aged 18 years or older who underwent invasive treatment were enrolled. Patients undergoing coronary artery bypass grafting (CABG) or heart valve replacement were included in the surgical group. Patients diagnosed with myocardial infarction and undergoing percutaneous coronary intervention (PCI) were included in the interventional group. Patients younger than 18 years were excluded; pregnant female patients were also excluded. Lithium heparin plasma samples were collected before invasive treatment and the first lithium heparin plasma sample after invasive treatment for analysis. Patients with elevated total complex levels postoperatively that were above the sex-specific 99th percentile upper reference limit (URL) were included in subsequent analyses. The patient enrollment process is shown in Figure 18.

[0527] (2) Chronic cardiac event patients group:

[0528] Patients aged 18 years or older were enrolled with an admission diagnosis and final diagnosis of chronic heart failure, cardiomyopathy, or pneumonia. Patients were excluded if the final diagnosis was not chronic heart failure, cardiomyopathy, or pneumonia. Patients were also excluded if they were also diagnosed with acute myocardial infarction. Patients younger than 18 years were excluded; pregnant women were excluded. Cardiomyopathy, chronic heart failure, and pneumonia were independently determined by hospital clinicians. Acute myocardial infarction was independently determined by hospital cardiologists based on the definition of acute myocardial infarction. The first lithium heparin plasma sample after admission was collected from these patients for analysis of troponin complex and fragment composition. The patient enrollment process is shown in Figure 19.

[0529] Patient information was recorded, including age, gender, medical history, hypertension, diabetes, smoking history, creatinine level, and glomerular filtration rate. Samples meeting the inclusion criteria were selected for relevant marker testing. A total of 82 patients undergoing invasive treatments were included, including 48 surgical procedures and 34 interventional procedures; 145 patients with chronic cardiac events were included. Patient information is provided in Table 5-1.

[0530] Table 5-1: Characteristics of enrolled patients

[0531] Note: Continuous variables are presented as medians (25th-75th percentiles); categorical variables are presented as numbers (percentages).

[0532] 2. Analysis of Troponin Fragments and Complexes in Myocardial Injury and Chronic Cardiac Events Induced by Invasive Procedures

[0533] After sample enrollment, samples were assayed for troponin markers, including total troponin complexes, large-sized cTnITC, total cTnITC, and cTnT, using a Mindray chemiluminescence analyzer and accompanying reagents. The differences in troponin composition between patients with invasive myocardial injury and those with chronic cardiac events were analyzed. The concentrations of total complexes, large-sized cTnITC, total cTnITC, cTnT, and cTnI in the patients' blood are shown in Table 5-2. The values ​​measured by the cTnI kit and the total complex kit were similar, indicating a certain degree of clinical equivalence between the two.

[0534] Table 5-2: Concentrations of total complex, large-sized cTnITC, total cTnITC, cTnT, and cTnI in patients with invasive myocardial injury and chronic cardiac events

[0535] Note: Values ​​are shown as median (25-75% interquartile range).

[0536] Figure 20 shows the relationship between the ratios of troponin complexes and fragments in patients with invasive myocardial injury and those with chronic cardiac events, including the ratio of large cTnITC concentration to total complex concentration, the ratio of total cTnITC concentration to total complex concentration, the ratio of cTnT concentration to total complex concentration, the ratio of large cTnITC concentration to cTnT concentration, and the ratio of total cTnITC concentration to cTnT concentration. The ratios of large cTnITC concentration to total complex concentration, total cTnITC concentration to total complex concentration, large cTnITC concentration to cTnT concentration, and total cTnITC concentration to cTnT concentration were higher in patients with invasive myocardial injury and lower in patients with chronic cardiac events. The ratio of cTnT concentration to total complex concentration was higher in patients with chronic cardiac events and lower in patients with invasive myocardial injury.

[0537] The concentrations of troponin complexes and fragments, as well as the ratio between them, are correlated with the type of injury. Specific detection of different troponin complexes can help differentiate and diagnose myocardial injury caused by chronic cardiac events and invasive treatments.

[0538] 3. Diagnostic efficacy of myocardial injury in distinguishing myocardial injury caused by invasive procedures from chronic cardiac events

[0539] Table 5-3 shows the diagnostic efficacy of the myocardial injury markers used. In this example, the patient underwent invasive treatment resulting in myocardial injury or suffered a chronic cardiac event. The chronic cardiac event patient suffered from cardiomyopathy, chronic heart failure, or pneumonia, and the concentration of the troponin complex in the patient's sample was higher than the 99th percentile reference upper limit.

[0540] The P values ​​for total complex concentration, large-size cTnITC concentration, total cTnITC concentration, cTnT concentration, cTnI concentration, the ratio of large-size cTnITC concentration to total complex concentration, the ratio of total cTnITC concentration to total complex concentration, the ratio of cTnT concentration to total complex concentration, the ratio of large-size cTnITC concentration to cTnT concentration, the ratio of total cTnITC concentration to cTnT concentration, and the ratio of large-size cTnITC concentration to cTnI concentration were all less than 0.05, indicating that the variables were significant. The area under the curve (AUC) for large-size cTnITC concentration, total cTnITC concentration, and the ratio of total cTnITC concentration to cTnT concentration were 0.952, 0.940, and 0.913, respectively, demonstrating good diagnostic efficacy. The results showed that compared with using the concentration of total complex, cTnT or cTnI alone, the characteristic parameters obtained based on the concentration of large-size cTnITC or total cTnITC can more effectively distinguish myocardial damage caused by invasive procedures from chronic cardiac events, among which the concentration of large-size cTnITC or total cTnITC has good diagnostic and therapeutic efficacy.

[0541] Table 5-3: Diagnostic efficacy of concentration and concentration ratio characteristic parameters for distinguishing invasive myocardial injury from chronic cardiac events

[0542] The Youden index was calculated using the sensitivity and specificity corresponding to different cutoff values ​​of the characteristic parameters. The optimal diagnostic cutoff value was determined based on the maximum value of the Youden index (see Table 5-4).

[0543] Table 5-4: Cutoff values ​​and corresponding sensitivity and specificity of concentration and concentration ratio characteristic parameters for distinguishing myocardial injury caused by invasive procedures from chronic cardiac events

[0544] 4. The diagnostic efficacy of combining multiple myocardial injury marker concentration parameters to distinguish between myocardial injury caused by invasive procedures and chronic cardiac events

[0545] Concentration parameters of myocardial injury markers include concentrations of troponin complexes and fragments, namely, total complex concentration, large-size cTnITC concentration, total cTnITC concentration, cTnT concentration, and cTnI concentration. In this example, the patient experienced invasive treatment leading to myocardial injury or suffered a chronic cardiac event. The chronic cardiac event patient suffered from cardiomyopathy, chronic heart failure, or pneumonia, and the concentration of troponin complexes in the patient's sample was higher than the 99th percentile upper reference limit.

[0546] Large-size cTnITC concentration and cTnT concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 0.0417*(large-size cTnITC concentration) - 0.0001*(cTnT concentration) - 1.346. The prediction probability (characteristic parameter 1) was calculated as: P = 1 / (1 + e -logit )*100%.

[0547] The large-size cTnITC concentration and the total complex concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 0.0522*(large-size cTnITC concentration) - 0.0009*(total complex concentration) - 1.2682. The prediction probability (characteristic parameter 2) was calculated as: P = 1 / (1 + e -logit )*100%.

[0548] Large-size cTnITC concentration and total cTnITC concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters, namely, Logit(P) = 0.0217*(large-size cTnITC concentration) + 0.0040*(total cTnITC concentration) - 1.3428. The prediction probability (characteristic parameter 3) was calculated as: P = 1 / (1 + e -logit )*100%.

[0549] Large-size cTnITC concentration and total cTnITC concentration, total complex concentration, and cTnT concentration were selected as characteristic variables for joint analysis. Characteristic parameters were constructed using a logistic regression algorithm. The coefficients for each characteristic variable were estimated by logistic regression, resulting in the following formula for calculating the characteristic parameter: Logit(P) = 0.0375*(large-size cTnITC concentration) + 0.0053*(total cTnITC concentration) - 0.0007*(total complex concentration) - 0.0001*(cTnT concentration) - 1.3256. The predicted probability (characteristic parameter 4) was calculated as: P = 1 / (1 + e-logit) * 100%.

[0550] The areas under the curve (AUCs) for characteristic parameters 1, 2, 3, and 4 were 0.940, 0.921, 0.955, and 0.937, respectively, demonstrating good diagnostic efficacy (see Table 5-5). The optimal cutoff values, determined based on the maximum Youden index, were 0.4163, 0.3081, 0.3169, and 0.2827, respectively (see Table 5-6).

[0551] Table 5-5: Diagnostic efficacy of combined analysis of multiple concentration parameters for distinguishing myocardial injury caused by invasive procedures from chronic cardiac events

[0552] Table 5-6: Sensitivity and specificity of the cutoff values ​​corresponding to the combined analysis of multiple concentration parameters

[0553] In addition, other concentration parameter combinations include: total cTnITC concentration + total complex concentration, total cTnITC concentration

[0554] The combined analysis of large-size cTnITC concentration, total cTnITC concentration, and total complex concentration, large-size cTnITC concentration, total cTnITC concentration, and cTnT concentration, large-size cTnITC concentration, total complex concentration, and cTnT concentration, and total cTnITC concentration, total complex concentration, and cTnT concentration all showed good diagnostic efficacy in distinguishing between myocardial injury caused by invasive procedures and chronic cardiac events. These results demonstrate that combined analysis of large-size cTnITC concentration or total cTnITC concentration with other troponin fragment concentrations has excellent performance in distinguishing between myocardial injury caused by invasive procedures and chronic cardiac events.

[0555] 5. The diagnostic efficacy of combining ratio parameters of multiple myocardial injury markers to distinguish between myocardial injury caused by invasive procedures and chronic cardiac events

[0556] Ratio parameters of myocardial injury markers include the ratio of large-size cTnITC concentration to total complexes, the ratio of total cTnITC concentration to total complexes, the ratio of cTnT concentration to total complexes, the ratio of large-size cTnITC concentration to cTnT concentration, the ratio of total cTnITC concentration to cTnT concentration, the ratio of large-size cTnITC concentration to cTnI concentration, and the ratio of total cTnITC concentration to cTnI concentration. In this embodiment, the patient undergoes invasive treatment resulting in myocardial injury or suffers from a chronic cardiac event. The chronic cardiac event patient suffers from cardiomyopathy, chronic heart failure, or pneumonia, and the concentration of troponin complexes in the patient's sample is higher than the 99th percentile reference upper limit.

[0557] The large-size cTnITC concentration / total complex concentration, total cTnITC concentration / total complex concentration, and cTnT concentration / total complex concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, resulting in the calculation formula for the characteristic parameters: Logit(P) = 4.775*(large-size cTnITC concentration / total complex concentration) + 10.015*(total cTnITC concentration / total complex concentration) - 0.413*(cTnT concentration / total complex concentration) - 0.251. The prediction probability (characteristic parameter 5) was calculated as: P = 1 / (1 + e -logit )*100%.

[0558] The large-size cTnITC concentration / cTnT concentration and total cTnITC concentration / cTnT concentration were selected as characteristic quantities for joint analysis. Characteristic parameters were constructed based on the logistic regression algorithm. The coefficients of each characteristic quantity were estimated by logistic regression, thereby obtaining the calculation formula for the characteristic parameters. Logit(P) = 9.933*(large-size cTnITC concentration / cTnT concentration) + 40.395*(total cTnITC concentration / cTnT concentration) - 2.094. The prediction probability (characteristic parameter 6) was calculated as: P = 1 / (1 + e -logit )*100%.

[0559] The areas under the curve (AUCs) for characteristic parameters 5 and 6 were 0.926 and 0.921, respectively, demonstrating good diagnostic efficacy (see Table 5-7). The optimal cutoff values, determined based on the maximum Youden index, were 0.6594 and 0.6053, respectively (see Table 5-8).

[0560] Table 5-7: Diagnostic efficacy of combined analysis of multiple proportion parameters for differentiating myocardial injury caused by invasive procedures from chronic cardiac events

[0561] Table 5-8: Sensitivity and specificity corresponding to cutoff values ​​in the combined analysis of multiple proportion parameters

[0562] In addition, other ratio parameter combinations, including large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration, large-size cTnITC concentration / total complex concentration + cTnT concentration / total complex concentration, total cTnITC concentration / total complex concentration + cTnT concentration / total complex concentration, large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration + large-size cTnITC concentration / cTnT concentration, and large-size cTnITC concentration / total complex concentration + total cTnITC concentration / total complex concentration + total cTnITC concentration / cTnT concentration, all showed good diagnostic efficacy when combined in the above manner to distinguish between myocardial injury caused by invasive procedures and chronic cardiac events. These results show that the combination of the ratio of large-size cTnITC to other troponin fragments and the ratio of total cTnITC to other troponin fragments, as well as the combination of the ratio between at least one of the two and other troponin fragments, has good performance in distinguishing between myocardial injury caused by invasive procedures and chronic cardiac events.

[0563] These data suggest that large-size or total cTnITC concentrations alone can be used to differentiate between myocardial injury caused by invasive procedures and chronic cardiac events. The ratios of large-size or total cTnITC concentrations to total complex concentrations, the ratios of large-size or total cTnITC concentrations to cTnT concentrations, and the ratios of large-size or total cTnITC concentrations to cTnI concentrations can also be used to differentiate between myocardial injury caused by invasive procedures and chronic cardiac events. The combined use of multiple parameters, such as large-size cTnITC concentration + total complex concentration, large-size cTnITC concentration + cTnT concentration, large-size cTnITC concentration + total cTnITC concentration, or large-size cTnITC concentration + total cTnITC concentration + total complex concentration + cTnT concentration, or large-size cTnITC concentration / total complex concentration, total cTnITC concentration / total complex concentration, cTnT concentration / total complex concentration, or large-size cTnITC concentration / cTnT concentration, or total cTnITC concentration / cTnT concentration, can also be used to distinguish between myocardial injury caused by invasive procedures and chronic cardiac events. Although not wishing to be bound by theory, it is possible that large-size cTnITC concentration or characteristic parameters derived therefrom are more suitable for distinguishing between myocardial injury caused by invasive procedures and chronic cardiac events.

[0564] Example 6 Assessment of the prognosis of acute myocardial injury

[0565] This embodiment is divided into two parts: the prognosis of acute myocardial injury in patients undergoing cardiac surgery and the prognosis of acute myocardial injury in patients with myocardial infarction. These two parts illustrate the application of the markers of this application in the prognosis of acute myocardial injury.

[0566] (1) Surgical operation

[0567] 1. Patient enrollment and follow-up methods

[0568] Patients aged 18 years or older who underwent cardiac surgery were enrolled. Cardiac surgery included (1) coronary artery bypass grafting (CABG) or (2) heart valve replacement. Patients younger than 18 years were excluded; pregnant women were excluded. Lithium heparin plasma samples were collected before and after invasive treatment (within 48 hours) for analysis. The patient enrollment process is shown in Figure 21.

[0569] Patient information was recorded, including age, gender, medical history, and glomerular filtration rate. Samples meeting the inclusion criteria were selected for relevant marker testing. This study included 311 patients undergoing cardiac surgery, including 133 patients undergoing coronary artery bypass grafting and 178 patients undergoing heart valve replacement. Patient information is provided in Table 6-1.

[0570] Table 6-1: Characteristics of enrolled patients

[0571] Continuous variables are presented as medians (25th-75th percentiles); categorical variables are presented as numbers (percentages).

[0572] Patients were followed for 3 months and 1 year, with information on clinical events recorded through office visits, telephone interviews, and / or electronic medical record collection. The primary clinical event was a composite of all-cause death, myocardial infarction, and unplanned coronary revascularization. Secondary clinical events included cardiovascular death, components of the primary clinical event, stroke, hospitalization for heart failure or emergency department observation for 24 hours or more, cardiac arrest or malignant arrhythmia, and other cardiovascular hospitalizations, as well as composite endpoints of these events.

[0573] 3. Evaluation of the predictive ability of troponin fragments and complexes for patient prognostic risk

[0574] A total of 311 patients undergoing cardiac surgery were enrolled in this study, of whom 133 (43%) underwent coronary artery bypass grafting. Within 3 months after surgery, the overall mortality rate was 0.6% (2 / 311 patients), and the composite endpoint of death and adverse cardiovascular events occurred in 5.1% (16 / 311 patients). Within 1 year after surgery, the overall mortality rate was 1.0% (3 / 311 patients), and the composite endpoint of death and adverse cardiovascular events occurred in 7.7% (24 / 311 patients).

[0575] Receiver operating characteristic (ROC) curve analysis was used to evaluate the efficacy of troponin complex and fragment markers in predicting patient prognosis (Table 6-2). The predicted outcome was a composite endpoint of death and adverse cardiovascular events within 1 year after surgery. Variables included troponin total complex concentrations, large-size cTnITC concentrations, total cTnITC concentrations, and cTnT concentrations within 24 hours after surgery.

[0576] The P values ​​for total complex concentration, large-size cTnITC concentration, total cTnITC concentration, and cTnT concentration were all less than 0.05, indicating significant differences. The area under the curve (AUC) for total complex concentration and absolute large-size cTnITC concentration was 0.765, and the area under the curve for absolute total cTnITC concentration was 0.771, indicating a certain ability to predict adverse cardiovascular events and mortality within one year. These results demonstrate that troponin total complex concentration, large-size cTnITC concentration, total cTnITC concentration, and cTnT concentration can assess patient prognostic risk.

[0577] Table 6-2: Evaluation of the ability of troponin complex and fragment markers to predict patient prognostic risk

[0578] The Youden Index was calculated using the sensitivity and specificity of different CUTOFF values ​​for the troponin total complex concentration, large-size cTnITC concentration, total cTnITC concentration, and cTnT concentration as markers to ensure high sensitivity and specificity. The optimal CUTOFF value was determined based on the maximum Youden Index (Table 6-3). A total complex concentration above 6848.6 ng / L had a predicted sensitivity of 66.7% and a specificity of 69.3%; a large-size cTnITC concentration above 1001.0 ng / L had a predicted sensitivity of 71.4% and a specificity of 78.7%; a total cTnITC concentration above 1897.1 ng / L had a predicted sensitivity of 81.0% and a specificity of 65.5%; and a cTnT concentration above 738.9 ng / L had a predicted sensitivity of 66.7% and a specificity of 76.0%.

[0579] Table 6-3: Sensitivity and specificity of troponin complex and fragment markers for predicting patient prognostic risk at cutoff values

[0580] In addition, the difference in troponin fragment and complex concentrations (postoperative concentration - preoperative concentration) and the fold change (postoperative concentration / preoperative concentration) between postoperative and preoperative levels were calculated to predict patient prognostic risk (Table 6-4). Variables included troponin total complex concentration (postoperative - preoperative), large-size cTnITC concentration (postoperative - preoperative), total cTnITC concentration (postoperative - preoperative), and cTnT concentration (postoperative - preoperative), as well as total complex (postoperative / preoperative), large-size cTnITC (postoperative / preoperative), total cTnITC (postoperative / preoperative), and cTnT (postoperative / preoperative).

[0581] The P values ​​for the difference between postoperative and preoperative cTnT concentrations, as well as the ratio of postoperative and preoperative cTnT concentrations, were all less than 0.05, indicating significance. The area under the curve (AUC) for large cTnITC (postoperative - preoperative) was 0.759, for total cTnITC (postoperative - preoperative), was 0.774, for large cTnITC (postoperative / preoperative), was 0.663, and for total cTnITC (postoperative / preoperative), was 0.660, demonstrating predictive power for adverse cardiovascular events and mortality within one year. The results showed that the difference or change multiple between the total complex, large-size cTnITC, total cTnITC, and cTnT before and after surgery can assess the patient's prognostic risk and predict the risk of myocardial injury after surgery in patients undergoing cardiac surgery.

[0582] Table 6-4: Evaluation of the ability of the postoperative and preoperative concentration differences and fold changes of troponin complexes and fragments to predict patient prognostic risk

[0583] Youden indices were calculated for sensitivity and specificity corresponding to different CUTOFF values ​​using the postoperative and preoperative differences and fold changes in total complex, large-size cTnITC, total cTnITC, and cTnT concentrations as parameters to ensure high sensitivity and specificity. The optimal CUTOFF value was determined based on the maximum Youden index (Table 6-5). A postoperative increase in large-size cTnITC concentration greater than 1008.0 ng / L compared with preoperative concentrations was associated with a predicted sensitivity of 68.4% and a specificity of 77.8%. A total cTnITC concentration increase greater than 1896.6 ng / L was associated with a predicted sensitivity of 84.2% and a specificity of 65.4%. A postoperative increase in large-size cTnITC concentration greater than 3320-fold compared with preoperative concentrations was associated with a predicted sensitivity of 63.2% and a specificity of 67.3%. A total cTnITC concentration increase greater than 970-fold was associated with a predicted sensitivity of 63.2% and a specificity of 55.3%.

[0584] Table 6-5: Sensitivity and specificity of the cutoff values ​​corresponding to the postoperative and preoperative differences and fold changes in concentrations of troponin complexes and fragments for predicting patient prognostic risk

[0585] 4. Evaluation of the predictive ability of combined analysis of troponin fragment concentrations for patient prognostic risk

[0586] Multi-parameter combined analysis of markers was used to predict the patient's prognostic risk. Parameters included the difference (postoperative concentration - preoperative concentration) and ratio (postoperative concentration / preoperative concentration) of troponin complex and fragment concentrations after surgery compared to before surgery.

[0587] Taking large-size cTnITC concentration and cTnT concentration as an example, a joint analysis was performed to construct a combined prediction parameter based on the logistic regression algorithm. The coefficients of each feature were estimated by logistic regression. The output prediction value calculation formula was: Logit(P) = 0.0002*(large-size cTnITC concentration) + 0.0002*(cTnT concentration) - 3.103. The prediction probability (prediction parameter 1) was calculated as: P = 1 / (1 + e -logit )*100%

[0588] ROC curve analysis showed that the P value for prediction parameter 1 was less than 0.05, indicating that the variable was significant. The area under the curve (AUC) was 0.789, indicating a slight improvement in diagnostic performance compared to using a single marker (Table 6-6).

[0589] Table 6-6: Multi-parameter joint analysis of absolute troponin concentration for predicting patient prognosis risk

[0590] In addition, other combinations, including large-size cTnITC + total complexes, large-size cTnITC + total cTnITC, total cTnITC + total complexes, total cTnITC + cTnT, large-size cTnITC + total cTnITC + total complexes, large-size cTnITC + total cTnITC + cTnT, large-size cTnITC + total complexes + cTnT, and total cTnITC + total complexes + cTnT concentrations, as well as the difference or ratio between postoperative and preoperative concentrations, were also used to assess patient prognostic risk. These results suggest that combined analysis of large-size cTnITC with other troponin fragments can be used to predict patient prognostic risk.

[0591] 5. Risk of composite endpoint events in different troponin fragment concentration groups

[0592] Taking the postoperative troponin concentration as an example, patients were divided into different subgroups according to the postoperative troponin concentration: Group 1 "< median concentration", Group 2 "median concentration - 75th percentile concentration", and Group 3 "> 75th percentile concentration". The corresponding subgroup concentrations of each marker are shown in Tables 6-7.

[0593] Table 6-7: Corresponding concentration ranges of different troponin fragment and complex concentration groups

[0594] Binary logistic regression analysis was used to analyze the risk of composite endpoint events in patients with different calcein fragment and complex concentration groups (Tables 6-8). Binary logistic regression analysis was used to analyze the correlation between marker parameters and the occurrence of primary endpoint events. A P value < 0.05 was considered significant.

[0595] The analysis found that the P values ​​of total complex group 2 and group 3 were less than 0.05, and the OR values ​​were 10.0 and 11.5, respectively; the P value of large-size cTnITC group 3 was less than 0.05, and the OR value was 11.6; the P value of total cTnITC group 2 and group 3 was less than 0.05, and the OR values ​​were 8.8 and 12.1, respectively; the P value of cTnT group 2 and group 3 was less than 0.05, and the OR values ​​were 4.2 and 8.9, respectively. The above results showed that the total complex concentration higher than 2964 ng / L, the large-size cTnITC concentration higher than 997 ng / L, the total cTnITC concentration higher than 956 ng / L, and the cTnT concentration higher than 354 ng / L were risk factors for patients experiencing composite endpoint events.

[0596] Table 6-8: Risk of composite endpoint events in different troponin fragment concentration groups

[0597] Cox regression was used to analyze the risk of composite endpoint events in patients with different calcineurin fragment and complex concentration groups (Tables 6-9), and survival curves for different patient groups were plotted (Figure 22). The Cox proportional hazards model was used to determine the relationship between marker parameters or their combinations and study subject outcomes. Hazard ratios (HRs) were calculated based on the Cox proportional hazards model to analyze the risk level of different risk groups with different marker parameter values, as a multiple of the risk of an endpoint event compared to the baseline group. A P value < 0.05 was considered significant.

[0598] The analysis found that the P values ​​of total complex group 2 (median concentration - 75th percentile concentration, 2964-9605 ng / L) and group 3 (>75th percentile concentration 9605 ng / L) were less than 0.05, and the HR values ​​were 8.0 and 9.63, respectively; the P value of large-size cTnITC group 3 (>75th percentile concentration 997 ng / L) was less than 0.05, and the HR value was 9.9; the P value of total cTnITC group 2 (median concentration - 75th percentile concentration, 956-4220 ng / L) and group 3 (>75th percentile concentration 4220 ng / L) was less than 0.05, and the HR values ​​were 7.8 and 10.1, respectively; the P value of cTnT group 3 (>75th percentile concentration 809 ng / L) was less than 0.05, and the HR value was 7.2. The above results show that a total complex concentration higher than 2964 ng / L, a large-size cTnITC concentration higher than 997 ng / L, a total cTnITC concentration higher than 956 ng / L, and a cTnT concentration higher than 809 ng / L are risk factors for patients experiencing composite endpoint events.

[0599] Table 6-9: Risk of composite endpoint events in different troponin fragment concentration groups (COX regression analysis)

[0600] The above data show that large-size cTnITC or total cTnITC, preferably large-size cTnITC, can be used to predict the prognosis of postoperative myocardial injury in patients undergoing cardiac surgery, and the grouping data also show that the higher the concentration of this marker, the higher the risk and the worse the prognosis.

[0601] (2) Myocardial infarction

[0602] 1. Patient enrollment and follow-up methods

[0603] Patients with acute myocardial infarction (type 1) were enrolled. Patients aged 18 years or older were selected for enrollment. Acute myocardial infarction was independently adjudicated by hospital cardiologists based on the definition of acute myocardial infarction. Adjudication of acute myocardial infarction was completed through clinical examinations, including physical examination, echocardiography, electrocardiogram (ECG), high-sensitivity troponin I (HSI) testing, high-sensitivity troponin T (HST) testing, and coronary angiography. Patients were excluded if the final diagnosis was not acute myocardial infarction or if the diagnostic information was incomplete. Patients under 18 years of age were excluded; pregnant women were also excluded. Initial lithium heparin plasma samples were collected from these patients at presentation and before interventional therapy for analysis of troponin complex and fragment composition. The patient enrollment process is shown in Figure 23.

[0604] Patient information was recorded, including age, gender, medical history, and glomerular filtration rate. Samples meeting the inclusion criteria were selected for relevant marker testing. A total of 324 patients with acute myocardial infarction were included. Patient information is shown in Tables 6-10.

[0605] Table 6-10: Characteristics of enrolled patients

[0606] Continuous variables are presented as medians (25th-75th percentiles); categorical variables are presented as numbers (percentages).

[0607] Patients were followed for 3 months and 1 year, with information on clinical events recorded through office visits, telephone interviews, and / or electronic medical record collection. The primary clinical event was a composite of all-cause mortality, myocardial infarction, and unplanned coronary revascularization. Secondary clinical events included cardiovascular death, components of the primary clinical event, stroke, hospitalization for heart failure or emergency department observation for 24 hours or more, cardiac arrest or malignant arrhythmia, and other cardiovascular hospitalizations, as well as composite endpoints of these events.

[0608] 2. Evaluation of the predictive ability of troponin fragments and complexes for patient prognostic risk

[0609] A total of 324 patients with acute myocardial infarction were enrolled in this study. Within 3 months of discharge, the overall mortality rate was 0.6% (2 / 324), and the composite endpoint of death and adverse cardiovascular events occurred in 6.5% (21 / 324). Within 1 year of discharge, the overall mortality rate was 1.2% (4 / 324), and the composite endpoint of death and adverse cardiovascular events occurred in 9.6% (31 / 324).

[0610] Patients were divided into different subgroups according to the median or 25th percentile of troponin concentration. The subgroup concentrations corresponding to each troponin fragment or complex are shown in Table 6-11.

[0611] Table 6-11: Corresponding concentration ranges of different troponin fragment and complex concentration groups

[0612] Cox regression was used to analyze the risk of composite endpoint events in patients with different calcineurin fragment and complex concentration groups (Tables 6-12), and survival curves for different patient groups were plotted (Figure 24). The analysis found that large cTnITC concentrations > the 25th percentile concentration of 1.1 ng / L had a P value less than 0.05 and a HR of 3.6; cTnT concentrations > the median concentration of 136.8 ng / L had a P value less than 0.05 and a HR of 2.2. These results indicate that large cTnITC and high cTnT concentrations are risk factors for composite endpoint events.

[0613] Table 6-12: Risk of composite endpoint events in different troponin fragment concentration groups (COX regression analysis)

[0614] The above data show that large-size cTnITC can be used to predict myocardial damage in patients with myocardial infarction, and the grouped data also show that the higher the concentration of this marker, the higher the risk and the worse the prognosis.

[0615] Example 7: Assessing the prognosis of patients with chronic heart damage

[0616] This example illustrates the application of the markers of the present application in the prognosis of myocardial injury in patients with chronic myocardial injury, such as chronic heart failure or cardiomyopathy.

[0617] 1. Patient enrollment and follow-up methods

[0618] Patients with chronic cardiac events were enrolled. Patients aged 18 years or older were selected for enrollment, with both an admission diagnosis and a final diagnosis of chronic heart failure or cardiomyopathy. Patients were excluded if the final diagnosis was not chronic heart failure or cardiomyopathy. Patients were also excluded if they were also diagnosed with acute myocardial infarction. Patients younger than 18 years were excluded; pregnant women were also excluded. Cardiomyopathy and chronic heart failure were independently adjudicated by hospital clinicians. Acute myocardial infarction was independently adjudicated by hospital cardiologists based on the definition of acute myocardial infarction. The first lithium heparin plasma sample after admission was collected from these patients for analysis of troponin complex and fragment composition. The patient enrollment process is shown in Figure 25.

[0619] Patient information was recorded, including age, gender, medical history, and glomerular filtration rate. Samples meeting the inclusion criteria were selected for relevant marker testing. A total of 179 patients with chronic cardiac events were included, including 29 diagnosed with chronic heart failure, 62 with cardiomyopathy, and 88 with both chronic heart failure and cardiomyopathy. See Table 7-1 for patient information.

[0620] Table 7-1: Characteristics of enrolled patients

[0621] Continuous variables are presented as medians (25th-75th percentiles); categorical variables are presented as numbers (percentages).

[0622] Patients were followed for 3 months and 1 year, with information on clinical events recorded through office visits, telephone interviews, and / or electronic medical record collection. The primary clinical event was a composite of all-cause death, myocardial infarction, and unplanned coronary revascularization. Secondary clinical events included cardiovascular death, components of the primary clinical event, stroke, hospitalization for heart failure or emergency department observation for 24 hours or more, cardiac arrest or malignant arrhythmia, and other cardiovascular hospitalizations, as well as composite endpoints of these events.

[0623] 2. Evaluation of the predictive ability of troponin fragments and complexes for patient prognostic risk

[0624] In this study, 179 patients with chronic cardiac events were enrolled, including 29 (16%) with chronic heart failure, 62 (35%) with cardiomyopathy, and 88 (49%) with both chronic heart failure and cardiomyopathy. Within 3 months of discharge, the overall mortality rate was 0.6% (1 / 179), and the composite endpoint of death and adverse cardiovascular events occurred in 12.8% (23 / 179). Within 1 year of discharge, the overall mortality rate was 1.1% (2 / 179), and the composite endpoint of death and adverse cardiovascular events occurred in 24.0% (43 / 179).

[0625] Receiver operating characteristic (ROC) curve analysis was used to evaluate the efficacy of troponin complex and fragment markers in predicting patient prognostic risk (Table 7-2). In this example, patients with chronic cardiac events suffered from cardiomyopathy or chronic heart failure. The predicted event was a composite endpoint of death and adverse cardiovascular events occurring within one year of discharge. Variables included troponin total complex concentration, large-size cTnITC concentration, total cTnITC concentration, and cTnT concentration.

[0626] The P values ​​for total complex concentration, large-size cTnITC concentration, and cTnT concentration were all less than 0.05, indicating significant differences. The area under the curve (AUC) for the absolute concentration of large-size cTnITC was 0.614, indicating a certain predictive ability for adverse cardiovascular events and mortality within one year. These results demonstrate that troponin total complex concentration, large-size cTnITC concentration, and cTnT concentration can assess patient prognostic risk.

[0627] Table 7-2: Evaluation of the ability of troponin complex and fragment markers to predict patient prognostic risk

[0628] The Youden Index was calculated using the sensitivity and specificity corresponding to different CUTOFF values ​​for the troponin total complex concentration, large-size cTnITC concentration, and cTnT concentration as markers. The optimal CUTOFF value was determined based on the maximum value of the Youden Index (Table 7-3). A total complex concentration higher than 11.7 ng / L predicted a sensitivity of 86% and a specificity of 42%. A large-size cTnITC concentration higher than 0.3 ng / L (approximately 2 times the LoD) predicted a sensitivity of 93% and a specificity of 30%. A cTnT concentration higher than 24.3 ng / L predicted a sensitivity of 83% and a specificity of 63%.

[0629] Table 7-3: Sensitivity and specificity of troponin complex and fragment markers for predicting patient prognostic risk at cutoff values

[0630] 3. Risk of composite endpoint events in different troponin fragment concentration groups

[0631] Patients were divided into different subgroups according to troponin concentrations, including group 1 "<25th percentile concentration", group 2 "25th percentile concentration - median concentration", and group 3 ">median concentration". The corresponding subgroup concentrations of each marker are shown in Table 7-4.

[0632] Table 7-4: Corresponding concentration ranges of different troponin fragment and complex concentration groups

[0633] Binary logistic regression analysis was used to analyze the risk of the composite endpoint in patients with different calcineurin fragment and complex concentration groups (Table 7-5). The analysis found that total complex concentrations in group 3 (>median concentration 19.5 ng / L) had a P value less than 0.05 and an odds ratio (OR) of 4.3; large cTnITC concentrations in group 2 (25th percentile concentration minus median concentration, 0.3-0.7 ng / L) and group 3 (>median concentration 0.7 ng / L) had P values ​​less than 0.05 and ORs of 4.0 and 3.1, respectively; and cTnT concentrations in group 3 (>median concentration 23.8 ng / L) had a P value less than 0.05 and an OR of 13.8. These results indicate that total complex concentrations above 19.5 ng / L, large cTnITC concentrations above 0.3 ng / L, and cTnT concentrations above 23.8 ng / L are risk factors for the composite endpoint.

[0634] Table 7-5: Risk of composite endpoint events in different troponin fragment concentration groups

[0635] Cox regression was used to analyze the risk of composite endpoint events in patients with different calcineurin fragment and complex concentration groups (Table 7-6), and survival curves for different patient groups were plotted (Figure 26). The analysis found that the total complex group 3 (> median concentration 19.5 ng / L) had a P value less than 0.05 and a HR value of 3.8; the large-size cTnITC group 2 (25th percentile concentration - median concentration, 0.3-0.7 ng / L) and group 3 (> median concentration 0.7 ng / L) had P values ​​less than 0.05 and HR values ​​of 3.4 and 2.8, respectively; the total cTnITC group 2 (25th percentile concentration - median concentration, 1.4-2.7 ng / L) had a P value less than 0.05 and a HR value of 2.8; and the cTnT group 3 (> median concentration 23.8 ng / L) had a P value less than 0.05 and a HR value of 9.9. The above results show that a total complex concentration higher than 19.5 ng / L, a large-size cTnITC concentration higher than 0.3 ng / L, a total cTnITC concentration between 1.4-2.7 ng / L, and a cTnT concentration higher than 23.8 ng / L are risk factors for patients experiencing composite endpoint events.

[0636] Table 7-6: Risk of composite endpoint events in different troponin fragment concentration groups (COX regression analysis)

[0637] The above data show that large-size cTnITC can be used to predict the prognosis of myocardial damage in patients with chronic cardiac events, such as chronic heart failure or cardiomyopathy, and the grouped data also show that the higher the concentration of this marker, the higher the risk and the worse the prognosis.

[0638] Example 8 Excluding chest pain subjects without myocardial injury events

[0639] 1. Patient enrollment and diagnosis

[0640] Consecutive patients with acute chest pain suspected of coronary syndrome who presented to the emergency department were enrolled to establish a threshold and process for the rapid exclusion of patients with non-ST-segment elevation myocardial infarction (NSTEMI) and to evaluate its safety and effectiveness. Inclusion criteria: (1) Chinese population aged 18 years and above, (2) patients presenting with symptoms or signs suspected of acute myocardial infarction (possible cardiac symptoms include: acute chest, upper abdominal, neck, jaw, or arm pain or discomfort or pressure), (3) patients undergoing blood sampling at the time of presentation; Exclusion criteria: patients with confirmed STEMI at presentation; pregnant female patients; patients who had undergone major surgery or trauma within four weeks and patients with chest pain clearly caused by non-cardiovascular reasons.

[0641] Acute myocardial infarction was independently adjudicated by hospital cardiologists based on the definition of acute myocardial infarction. Adjudication of acute myocardial infarction was accomplished through clinical evaluation, including physical examination, echocardiography, electrocardiogram (ECG), high-sensitivity troponin I (HSI) and high-sensitivity troponin T (HST) testing, and coronary angiography. Lithium heparinized plasma samples were obtained before treatment for analysis of troponin complex and fragment composition. The patient enrollment process is shown in Figure 27.

[0642] Patient information was recorded, including age, gender, medical history, hypertension, diabetes, smoking history, creatinine, and glomerular filtration rate. Samples meeting the inclusion criteria were selected for relevant marker testing. A total of 1,210 patients with suspected acute myocardial infarction were included, including 138 diagnosed with NSTEMI and 1,072 with non-myocardial infarction. Key patient information is shown in Table 8-1.

[0643] Table 8-1: Characteristics of enrolled patients

[0644] Continuous variables are presented as medians (25th-75th percentiles); categorical variables are presented as numbers (percentages).

[0645] 2. Evaluation of the efficacy of troponin fragments and complexes for ruling out NSTEMI

[0646] In this example, all patients with suspected acute coronary syndrome symptoms were selected for analysis, and a total of 1210 suspected NSTEMI patients were included, including 138 NSTEMI patients. Table 8-2 shows the diagnostic efficacy of the troponin complex and fragment markers used.

[0647] The P values ​​for the absolute concentrations of total complexes, large-size cTnITC, total cTnITC, cTnT, and cTnI were all less than 0.05, indicating significance. The area under the curve (AUC) for the absolute concentration of large-size cTnITC was 0.959, demonstrating good diagnostic efficacy. The values ​​measured by the cTnI kit and the total complexes were similar, demonstrating clinical equivalence. These results suggest that the absolute concentrations of large-size cTnITC or total cTnITC alone are more effective in predicting NSTEMI. Large-size cTnITC is preferred.

[0648] Table 8-2: Analysis of AUC values ​​of troponin complex and fragment markers for the diagnosis of NSTEMI

[0649] In patients suspected of myocardial infarction, using extremely low troponin thresholds allows for the early and safe exclusion of patients with non-MI conditions at the time of presentation. Exclusion thresholds for troponin complexes and fragments were established based on guideline-recommended criteria for myocardial infarction exclusion efficacy (requiring a diagnostic sensitivity of at least 99% and a negative predictive value (NPV) of at least 99.5%). Based on the exclusion thresholds, the sensitivity and NPV for excluding NSTEMI patients were calculated, along with the proportion of patients excluded. The calculation method is shown in Table 8-3. The established thresholds and diagnostic efficacy data are shown in Table 8-4. These data further demonstrate that both the absolute concentration of large-size cTnITC and total cTnITC can aid in the diagnosis of myocardial infarction and be used to exclude patients with non-MI conditions. Large-size cTnITC measurements can safely exclude patients with non-MI conditions at a higher rate, with efficacy comparable to that of total complexes and superior to that of cTnT.

[0650] Table 8-3: Analytical methods for diagnostic sensitivity and negative predictive value (NPV)

[0651] Sensitivity = a / (a+c)×100% NPV = d / (c+d)×100%

[0652] Table 8-4: Thresholds and diagnostic performance of troponin complex and fragment markers for ruling out NSTEMI

[0653] Note: Excluded patient ratio = number of excluded patients (marker values ​​lower than the set exclusion threshold) / total number of patients

[0654] 3. Evaluation of the efficacy of troponin fragments and complexes for ruling out NSTEMI (in patients with early chest pain)

[0655] In this example, patients with chest pain onset within 24 hours were selected as early-stage chest pain patients for analysis. A total of 764 suspected NSTEMI patients were included, including 88 NSTEMI patients. Table 8-5 shows the diagnostic efficacy of the troponin complex and fragment markers used. The P values ​​for the absolute concentration of total complex, the absolute concentration of large-size cTnITC, the absolute concentration of total cTnITC, and the absolute concentration of cTnT were all less than 0.05, indicating that the variables were significant. The area under the curve (AUC) for the absolute concentration of large-size cTnITC was 0.976, indicating good diagnostic efficacy.

[0656] Table 8-5: Analysis of AUC values ​​of troponin complex and fragment markers for the diagnosis of NSTEMI (patients with early chest pain, chest pain duration ≤ 24 hours)

[0657] In patients with early chest pain suspected of myocardial infarction (chest pain duration ≤ 24 hours), using very low troponin thresholds allows for early and safe exclusion of non-MI cases at the time of presentation. Rule-out thresholds for troponin complexes and fragments were established based on guideline-recommended criteria for rule-out of myocardial infarction (requiring a diagnostic sensitivity of at least 99% and a negative predictive value (NPV) of at least 99.5%). Based on these thresholds, the sensitivity and NPV for rule-out of NSTEMI patients were calculated, along with the proportion of patients excluded. The established thresholds and diagnostic efficacy data are shown in Table 8-6.

[0658] Table 8-6: Thresholds and diagnostic performance of troponin complex and fragment markers for ruling out NSTEMI (in patients with early chest pain, duration of chest pain ≤ 24 hours)

[0659] Note: Excluded patient ratio = number of excluded patients (marker values ​​lower than the set exclusion threshold) / total number of patients

[0660] In this example, patients with chest pain onset within 12 hours were selected as early-stage chest pain patients for analysis. A total of 617 suspected NSTEMI patients were included, including 59 NSTEMI patients. Table 8-7 shows the diagnostic efficacy of the troponin complex and fragment markers used. The P values ​​for the absolute concentration of total complex, the absolute concentration of large-size cTnITC, the absolute concentration of total cTnITC, and the absolute concentration of cTnT were all less than 0.05, indicating that the variables were significant. The area under the curve (AUC) for the absolute concentration of large-size cTnITC was 0.972, indicating good diagnostic efficacy.

[0661] Table 8-7: Analysis of AUC values ​​of troponin complex and fragment markers for the diagnosis of NSTEMI (patients with early chest pain, chest pain duration ≤ 12 hours)

[0662] In patients with early chest pain suspected of myocardial infarction (chest pain duration ≤12 hours), using very low troponin thresholds allows for early and safe exclusion of non-MI cases at the time of presentation. Rule-out thresholds for troponin complexes and fragments were established based on guideline-recommended criteria for rule-out of myocardial infarction (requiring a diagnostic sensitivity of at least 99% and a negative predictive value (NPV) of at least 99.5%). Based on these thresholds, the sensitivity and NPV for rule-out of NSTEMI patients were calculated, along with the proportion of patients excluded. The established thresholds and diagnostic efficacy data are shown in Table 8-8.

[0663] Table 8-8: Thresholds and diagnostic performance of troponin complex and fragment markers for ruling out NSTEMI (in patients with early chest pain, duration of chest pain ≤ 12 hours)

[0664] The above data show that large-size cTnITC complex of troponin and total cTnITC complex alone can be used to assist in the diagnosis of myocardial infarction in a cohort of patients with chest pain suspected of myocardial infarction, to exclude patients without myocardial injury, so that patients can leave the emergency room early, reduce the patient's observation time, and receive diagnosis and treatment of other diseases, and also speed up the turnover of the emergency room.

[0665] Furthermore, in patients with early chest pain (chest pain duration ≤ 12 or 24 hours), large-size cTnITC measurements can safely rule out non-MI patients at a higher rate. Compared with the total complex and cTnT, it has a higher proportion of excluded patients and superior diagnostic efficacy. Comparison of data from all patients with chest pain within 24 hours and within 12 hours showed that large-size cTnITC has a higher safe rule-out rate in patients with shorter chest pain duration, demonstrating its superiority over the total complex.

[0666] 3. Efficacy of combined troponin fragment detection for ruling out NSTEMI

[0667] Combined testing of multiple troponin fragment concentrations is used to quickly rule out NSTEMI.

[0668] The markers tested included total complex concentration, large-size cTnITC concentration, total cTnITC concentration, and cTnT concentration. Large-size cTnITC concentration and total complex concentration were selected as characteristic quantities for combined testing, and threshold values ​​were established for each. Patients whose large-size cTnITC and total complex concentrations were both below the threshold values ​​were excluded to maximize safety. The established threshold values ​​and diagnostic efficacy data are shown in Tables 8-9. These data demonstrate that combined testing of multiple troponin fragment concentrations can be used to rapidly rule out myocardial infarction and assist in the diagnosis of myocardial infarction. Compared with the use of a single marker, combined testing of multiple troponin fragments reduced the number of patients with missed myocardial infarction to zero, achieving 100% sensitivity and 100% negative predictive value.

[0669] Table 8-9: Thresholds and diagnostic performance of troponin complex and fragment markers for ruling out NSTEMI (combined use)

[0670] The data in Tables 8-9 show that a single marker cannot effectively solve the problem of missed diagnosis, but joint testing can do so, and the exclusion rate does not decrease much. It is the optimal solution to the contradiction between missed diagnosis and the proportion of excluded patients.

[0671] This example shows that the large-size cTnITC complex and total cTnITC complex of troponin can exclude patients without myocardial injury from patients clinically presenting with chest pain, with the large-size cTnITC complex being preferred as having better diagnostic and therapeutic effects than cTnT. In particular, the large-size cTnITC complex of troponin is more advantageous in patients with early-stage chest pain. Furthermore, its combination with the total complex can avoid missed detection while maintaining a good patient exclusion ratio, demonstrating excellent diagnostic efficacy in excluding myocardial injury events in subjects with chest pain, a diagnosis of myocardial injury.

[0672] In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for evaluating myocardial injury in a subject in vitro, comprising: detecting the level of one or more myocardial injury markers in a sample from the subject; Based on the content of the one or more myocardial injury markers, obtaining characteristic parameters for assessing myocardial injury; comparing the characteristic parameter with a reference value of the characteristic parameter; Based on the results of the comparison, assessing myocardial injury in the subject; Wherein, the one or more myocardial injury markers include large-sized cardiac troponin ternary complex and / or total cardiac troponin ternary complex; The large-size cardiac troponin ternary complex is a complex formed by the full-length protein or any amino acid fragment of troponin C, the full-length protein or any amino acid fragment of cardiac troponin I, one or more segments of amino acid residues 223-287 of cardiac troponin T, and one or more segments of amino acid residues 1-222 of cardiac troponin T; The total cardiac troponin ternary complex is a complex formed by the full-length protein or any amino acid fragment of troponin C, the full-length protein or any amino acid fragment of cardiac troponin I and one or more segments of amino acid residues 223-287 of cardiac troponin T, and optionally one or more segments of amino acid residues 1-222 of cardiac troponin T.

2. The method of claim 1, wherein: The myocardial injury markers further include one or more of the following: cTnI, including the full-length protein of cardiac troponin I or any amino acid fragment thereof; cTnT, including the full-length protein of cardiac troponin T or any amino acid fragment thereof; TnC, including the full-length protein of troponin C or any amino acid fragment thereof; A cardiac troponin binary complex, comprising a binary complex consisting of a full-length protein of troponin C or any amino acid fragment thereof and a full-length protein of cardiac troponin I or any amino acid fragment thereof; and Total cardiac troponin complex, including a binary complex consisting of the total cardiac troponin ternary complex, a full-length protein of troponin C or any amino acid fragment thereof, and a full-length protein of cardiac troponin I or any amino acid fragment thereof; Preferably, the one or more myocardial injury markers further include at least one of cTnI, cTnT and total cardiac troponin complex.

3. The method according to claim 1 or 2, wherein: Based on the content of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including: determining the content of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; or Characteristic parameters for assessing myocardial damage are obtained based on the content of the large-sized cardiac troponin ternary complex and the content of the total cardiac troponin ternary complex.

4. The method according to any one of claims 1 to 3, wherein: Based on the content of one or more myocardial injury markers, characteristic parameters for evaluating myocardial injury are obtained, including: inputting the content of the multiple myocardial injury markers into a preset function model to obtain the output of the preset function model as the characteristic parameter for evaluating myocardial injury.

5. The method according to any one of claims 1 to 4, wherein: Obtaining characteristic parameters for assessing myocardial injury based on the content of the one or more myocardial injury markers comprises: determining one of the following ratio parameters as the characteristic parameter for assessing myocardial injury, or obtaining the characteristic parameter for assessing myocardial injury based on at least two of the following ratio parameters, or obtaining the characteristic parameter for assessing myocardial injury based on at least one of the following ratio parameters and the ratio of the content of cTnT to the content of total cardiac troponin complex; The ratio parameters include: the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnI, the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnT, the ratio of the content of large-size cardiac troponin ternary complex to the content of total cardiac troponin complex, the ratio of the content of total cardiac troponin ternary complex to the content of cTnI, the ratio of the content of total cardiac troponin ternary complex to the content of cTnT, the ratio of the content of total cardiac troponin ternary complex to the content of total cardiac troponin complex, and the ratio of the content of large-size cardiac troponin ternary complex to the content of total cardiac troponin ternary complex; Preferably, the ratio parameter is the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnI, cTnT or total cardiac troponin complex; optionally, the ratio parameter further includes the ratio of the content of total cardiac troponin ternary complex to the content of cTnI, cTnT or total cardiac troponin complex, and / or the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complex.

6. The method according to any one of claims 1 to 5, wherein The method is used for diagnosing myocardial injury in a subject; preferably, characteristic parameters are obtained based on the large-size cardiac troponin ternary complex, and the characteristic parameters are used for diagnosing myocardial injury in a subject.

7. The method according to any one of claims 1 to 5, wherein The method is used to evaluate the prognosis of myocardial injury in a subject; preferably, characteristic parameters are obtained based on the large-size cardiac troponin ternary complex, and the characteristic parameters are used to evaluate the prognosis of myocardial injury in a subject.

8. The method according to any one of claims 1 to 6, wherein: Based on the results of the comparison, the myocardial injury of the subject is assessed, including: Based on the results of the comparison, the subjects are classified into myocardial infarction stages; Preferably, when the characteristic parameter is higher than a reference value of the characteristic parameter, the subject is judged to be in an early stage of acute myocardial infarction.

9. The method of claim 8, wherein: Based on the content of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including: determining the content of the large-sized cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; or Obtaining a characteristic parameter for assessing myocardial injury based on the content of the large-sized cardiac troponin ternary complex and the content of at least one of cTnI, cTnT, total cardiac troponin ternary complex and total cardiac troponin complex, preferably based on the content of the large-sized cardiac troponin ternary complex and the content of cTnT; or Characteristic parameters for assessing myocardial damage are obtained based on the ratio of the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex to the content of at least one of cTnI, cTnT and total cardiac troponin complexes, and optionally the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complexes.

10. The method according to any one of claims 1 to 6, wherein: Based on the results of the comparison, the myocardial injury of the subject is assessed, including: Based on the results of the comparison, determining whether the subject has a type I myocardial infarction or a type II myocardial infarction; Preferably, when the characteristic parameter is higher than a reference value of the characteristic parameter, it is judged that the subject has suffered type I myocardial infarction; or Preferably, a characteristic parameter for assessing myocardial damage is obtained based on the content of the large-sized cardiac troponin ternary complex or the ratio of the content of the total cardiac troponin ternary complex to the total cardiac troponin complexes.

11. The method according to any one of claims 1 to 6, wherein: Based on the results of the comparison, the myocardial injury of the subject is assessed, including: Based on the result of the comparison, determining whether the subject has a type I myocardial infarction or a chronic cardiac event; Preferably, when the characteristic parameter is higher than a reference value of the characteristic parameter, it is judged that the subject has suffered type I myocardial infarction.

12. The method of claim 11, wherein: Based on the content of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including: determining the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; or Obtaining characteristic parameters for assessing myocardial injury based on the content of the large-sized cardiac troponin ternary complex and the content of the total cardiac troponin ternary complex; or Obtaining characteristic parameters for assessing myocardial injury based on the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex and the content of cTnT or the total cardiac troponin complex; Preferably, characteristic parameters for assessing myocardial damage are obtained based on the ratio of the content of the large-size cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex to the content of at least one of cTnI, cTnT and total cardiac troponin complexes, and optionally the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complexes.

13. The method according to any one of claims 1 to 6, wherein: Based on the results of the comparison, the myocardial injury of the subject is assessed, including: Based on the comparison result, determining whether the subject has myocardial injury caused by invasive operation or chronic cardiac event; Preferably, when the characteristic parameter is higher than a reference value of the characteristic parameter, it is determined that the subject has suffered myocardial damage caused by invasive operation.

14. The method of claim 13, wherein: Based on the content of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including: determining the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; Preferably, based on the content of the large-size cardiac troponin ternary complex or the total cardiac troponin The ratio of the content of the ternary complex to the content of at least one of cTnI, cTnT and total cardiac troponin complex, and optionally the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complex, obtains characteristic parameters for assessing myocardial damage.

15. The method according to any one of claims 1 to 6, wherein based on the comparison result, subjects with chest pain who have not experienced a myocardial injury event are excluded; Preferably, when the characteristic parameter is lower than the reference value of the characteristic parameter, subjects with chest pain who have not experienced a myocardial injury event are excluded; Preferably, the myocardial injury event is myocardial infarction or NSTEMI.

16. The method of claim 15, wherein: Based on the content of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including: determining the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial damage; or A characteristic parameter for assessing myocardial injury is obtained based on the content of the large-sized cardiac troponin ternary complex or the content of the total cardiac troponin ternary complex and the content of the total cardiac troponin complex.

17. The method of claim 7, wherein the prognosis of myocardial injury in a subject with acute myocardial injury is evaluated based on the result of the comparison; preferably, the subject is a subject who has undergone cardiac surgery or has had myocardial infarction; Preferably, when the characteristic parameter is higher than a reference value of the characteristic parameter, the prognosis is judged to be poor.

18. The method of claim 17, wherein: Based on the content of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including: determining the content of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial injury; Preferably, the change in the content of large-sized cardiac troponin ternary complex or total cardiac troponin ternary complex in the plasma of the subject before and after receiving cardiac surgery is determined as a characteristic parameter for assessing the prognostic risk of receiving surgery.

19. The method of claim 7, wherein the prognosis of myocardial injury in a subject with chronic myocardial injury is assessed based on the result of the comparison; Preferably, the subject is a patient with cardiomyopathy, chronic heart failure, structural heart disease, infiltrative disease, stable Patients with type 2 coronary heart disease and persistent arrhythmia; Preferably, when the characteristic parameter is higher than a reference value of the characteristic parameter, the prognosis is judged to be poor.

20. The method of claim 19, wherein: Based on the content of the one or more myocardial injury markers, characteristic parameters for assessing myocardial injury are obtained, including: The content of the large-sized cardiac troponin ternary complex or the total cardiac troponin ternary complex is determined as a characteristic parameter for evaluating myocardial damage.

21. A device for obtaining characteristic parameters for evaluating myocardial damage in a subject, comprising: A data receiving module configured to receive the content of one or more myocardial injury markers obtained from a sample from a subject, wherein the one or more myocardial injury markers include a large-sized cardiac troponin ternary complex and / or a total cardiac troponin ternary complex; A data processing module is configured to process the content data of one or more myocardial injury markers received by the receiving module to obtain characteristic parameters for evaluating myocardial injury; and, an output module configured to output the characteristic parameters; The large-size cardiac troponin ternary complex is a complex formed by the full-length protein or any amino acid fragment of troponin C, the full-length protein or any amino acid fragment of cardiac troponin I, one or more segments of amino acid residues 223-287 of cardiac troponin T, and one or more segments of amino acid residues 1-222 of cardiac troponin T; The total cardiac troponin ternary complex is a complex formed by the full-length protein or any amino acid fragment of troponin C, the full-length protein or any amino acid fragment of cardiac troponin I and one or more segments of amino acid residues 223-287 of cardiac troponin T, and optionally one or more segments of amino acid residues 1-222 of cardiac troponin T.

22. The device of claim 21, wherein: The data processing module processes the content of the one or more myocardial injury markers to obtain characteristic parameters for evaluating myocardial injury, including: The data processing module determines the content of the cardiac troponin ternary complex or the total cardiac troponin ternary complex as a characteristic parameter for assessing myocardial injury; or The data processing module obtains characteristic parameters for assessing myocardial injury based on the content of the cardiac troponin ternary complex and the content of the total cardiac troponin ternary complex.

23. The device of claim 21, wherein: The myocardial injury markers further include one or more of the following: cTnI, including the full-length protein of cardiac troponin I or any amino acid fragment thereof; cTnT, including the full-length protein of cardiac troponin T or any amino acid fragment thereof; TnC, including the full-length protein of troponin C or any amino acid fragment thereof; A cardiac troponin binary complex, comprising a binary complex consisting of a full-length protein of troponin C or any amino acid fragment thereof and a full-length protein of cardiac troponin I or any amino acid fragment thereof; and Total cardiac troponin complex, including a binary complex consisting of the total cardiac troponin ternary complex, a full-length protein of troponin C or any amino acid fragment thereof, and a full-length protein of cardiac troponin I or any amino acid fragment thereof; Preferably, the one or more myocardial injury markers further include at least one of cTnI, cTnT and total cardiac troponin complex.

24. The device according to any one of claims 21 to 23, wherein: The data processing module processes the content of the one or more myocardial injury markers to obtain characteristic parameters for evaluating myocardial injury, including: the data processing module inputs the content of the multiple myocardial injury markers into a preset function model to obtain the output of the preset function model as the characteristic parameter for evaluating myocardial injury.

25. The device according to any one of claims 21 to 24, wherein: The data processing module processes the content of the one or more myocardial injury markers to obtain characteristic parameters for evaluating myocardial injury, including: the data processing module determines one of the following ratio parameters as the characteristic parameter for evaluating myocardial injury, or obtains the characteristic parameter for evaluating myocardial injury based on at least two of the following ratio parameters, or obtains the characteristic parameter for evaluating myocardial injury based on at least one of the following ratio parameters and the ratio of the content of cTnT to the content of total cardiac troponin complex; The ratio parameters include: the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnI, the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnT, the ratio of the content of large-size cardiac troponin ternary complex to the content of total cardiac troponin complex, the ratio of the content of total cardiac troponin ternary complex to the content of cTnI, the ratio of the content of total cardiac troponin ternary complex to the content of cTnT, the ratio of the content of total cardiac troponin ternary complex to the content of total cardiac troponin complex, and the ratio of the content of large-size cardiac troponin ternary complex to the content of total cardiac troponin ternary complex; Preferably, the ratio parameter is the ratio of the content of large-size cardiac troponin ternary complex to the content of cTnI, cTnT or total cardiac troponin complex; optionally, the ratio parameter further includes the ratio of the content of total cardiac troponin ternary complex to the content of cTnI, cTnT or total cardiac troponin complex, and / or the ratio of the content of cTnT to the content of cTnI or total cardiac troponin complex.

26. A sample analysis system, comprising: A sample carrying portion, used for carrying a container containing a sample of a subject; The sample dispensing part is used to draw the sample of the subject from the sample holding part and discharge it into the reaction cup to be loaded with the sample; A reagent carrying part, used for carrying detection reagents; The reagent dispensing part is used to absorb the detection reagent from the reagent carrying part and discharge it into the reaction cup to be added with the reagent; The reaction part is used to place the reaction cup so as to incubate the test solution obtained by the reaction of the subject's sample and the detection reagent in the reaction cup; a detection unit having a signal detector for detecting the signal of the test solution in the reaction cup to determine and output the content of one or more myocardial injury markers in the sample of the subject, wherein the one or more myocardial injury markers include a large-size cardiac troponin ternary complex and / or a total cardiac troponin ternary complex, wherein the large-size cardiac troponin ternary complex is a complex formed by the full-length protein or any amino acid fragment of troponin C, the full-length protein or any amino acid fragment of cardiac troponin I, one or more segments of amino acid residues 223-287 of cardiac troponin T, and one or more segments of amino acid residues 1-222 of cardiac troponin T; and the total cardiac troponin ternary complex is a complex formed by the full-length protein or any amino acid fragment of troponin C, the full-length protein or any amino acid fragment of cardiac troponin I, one or more segments of amino acid residues 223-287 of cardiac troponin T, and optionally one or more segments of amino acid residues 1-222 of cardiac troponin T; The data processing unit includes a processor and a computer-readable storage medium, wherein computer-readable instructions are stored on the computer-readable storage medium, wherein when the computer-readable instructions are executed by the processor, the processor is caused to perform the following steps: Receiving and processing the content of the one or more myocardial injury markers to obtain characteristic parameters for assessing myocardial injury; and The characteristic parameters are output.

27. Use of a reagent for quantitatively detecting a large-sized cardiac troponin ternary complex and / or a reagent for quantitatively detecting a total cardiac troponin ternary complex in a sample in the preparation of a kit, wherein the kit is used to assess myocardial damage in a subject, in, The large-size cardiac troponin ternary complex is a complex formed by the full-length protein or any amino acid fragment of troponin C, the full-length protein or any amino acid fragment of cardiac troponin I, one or more segments of amino acid residues 223-287 of cardiac troponin T, and one or more segments of amino acid residues 1-222 of cardiac troponin T; The total cardiac troponin ternary complex is a complex formed by the full-length protein or any amino acid fragment of troponin C, the full-length protein or any amino acid fragment of cardiac troponin I and one or more segments of amino acid residues 223-287 of cardiac troponin T, and optionally one or more segments of amino acid residues 1-222 of cardiac troponin T.

28. The use according to claim 27, wherein the kit further comprises a reagent for quantitative detection of total cardiac troponin complex, a reagent for quantitative detection of cTnI and / or a reagent for quantitative detection of cTnT, wherein: The cTnI includes the full-length protein of cardiac troponin I or any amino acid fragment thereof; The cTnT includes the full-length protein of cardiac troponin T or any amino acid fragment thereof; The total cardiac troponin complex comprises the total cardiac troponin ternary complex and a binary complex consisting of a full-length protein of troponin C or any amino acid fragment thereof and a full-length protein of cardiac troponin I or any amino acid fragment thereof.

29. The use according to claim 27 or 28, wherein the reagent for quantitative detection of large-sized cardiac troponin ternary complex in a sample comprises a first group of antibodies and a second group of antibodies, wherein: The first group of antibodies includes one or more antibodies 1-1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 67-222 of cTnT; The second group of antibodies includes one or more antibodies 1-2, each of which is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence; Optionally, the second group of antibodies further comprises one or more antibodies 1-3, each of the antibodies 1-3 is independently selected from an antibody that specifically binds to cTnIC; and / or, One or more antibodies 1-4, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 18-210 of cTnI; Preferably, the first group of antibodies does not include antibodies that specifically bind to any section of the amino acid sequence of cTnT at positions 223-287; Preferably, the first set of antibodies are capture antibodies and the second set of antibodies are detection antibodies.

30. The use according to any one of claims 27 to 29, wherein the reagent for quantitative detection of total cardiac troponin ternary complex in a sample comprises a first antibody group and a second antibody group, wherein: The first detection reagent comprises one or more antibodies 2-1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 223-287 of cTnT; The second detection reagent comprises one or more antibodies 2-2, each of the antibodies 2-2 being independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence; Optionally, the first detection reagent further comprises one or more antibodies 2-3, each of the antibodies 2-3 being independently selected from antibodies that specifically bind to any segment of the amino acid sequence at positions 67-222 of cTnT; Optionally, the second detection reagent further comprises: One or more antibodies 2-4, each of which is independently selected from an antibody that specifically binds to cTnIC; and / or One or more antibodies 2-5, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 18-210 of cTnI; Preferably, the first antibody group is capture antibodies and the second antibody group is detection antibodies.

31. The use according to any one of claims 27 to 30, wherein the kit is used for one or more of the following: 1) Myocardial infarction staging, especially determining whether the subject is in the early stage of myocardial infarction; 2) Differentiate between type I myocardial infarction and chronic cardiac events; 3) Excluding subjects with chest pain who did not experience myocardial injury events; 4) Differentiate between type I and type II myocardial infarction; 5) Differentiate between myocardial injury caused by invasive procedures and chronic cardiac events; 6) Evaluate the prognosis of chronic myocardial injury; 7) Evaluate the prognosis of acute myocardial injury.

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