Study setting {9}
This study is a prospective, observational, single-center study for acute myocardial infarction. Data would be collected in China-Japan Union Hospital.
Eligibility criteria {10}
Admission criteria:
Patients with acute ST-segment elevation myocardial infarction will be admitted according to the diagnostic criteria recommended in 2017 by the European Society of Cardiology (ESC) guidelines for ST-segment elevation myocardial infarction12:
(1) Clinical symptoms of myocardial ischemia; (2) The electrocardiogram showed new myocardial ischemia changes and new ST-segment elevation changes of the left bundle branch block. (3) Pathological Q wave appeared in Electrocardiograph (ECG).
Exclusion criteria:
(1) Refusal to participate in the study; (2) Refusal to cooperate with the collection of information, or unwilling to follow up; (3) Age over 90 years old; (4) Thyroid dysfunction (thyrotropin < the lower limit of the normal value, or > 1.5 times the upper limit of the normal value, free thyroxine T4 level is outside the normal range); (5) Severe renal insufficiency (estimated glomerular rate filter <20 ml/min/1.73 m2); (6) Severe liver insufficiency (aspartate aminotransferase or alanine aminotransferase >3 times the normal value); (7) Transplant of any major organ; (8) Pregnancy or lactation; (9) Suffering from mental illness; the presence of any infectious or systemic inflammatory disease; (10) Hematological disease; (11) Tumor; (12) Metabolic disease.
Who will take informed consent? {26a}
Professional clinicians from China-Japan Union hospital will make preliminary diagnoses and evaluate emergency patients. Those patients who meet the criteria must sign an informed consent form before the collection of any data, including medical records, contact information, emergency contacts and other information.
Additional consent provisions for collection and use of participant data and biological specimens {26b}
This informed consent is to solicit your opinions on preserving and using your hospital diagnosis and treatment information and the remaining blood and body fluids during the diagnosis and treatment process. Specific instructions are as follows:
About patients:
Allow researchers to view, collect, and use information about your illness. The collection of samples is carried out after the bedside blood collection and other medical treatment, which does not harm your body, does not affect or damage your definitive diagnosis and treatment and does not harm your health. You will not have to pay any additional fees and will not benefit financially in any way from this study. To protect personal information and privacy, no other person is allowed to know the information related. Patients may voluntarily provide samples and may cancel and withdraw the consent at any time; Even if the patients refuse to give the sample, it will not affect all your standard diagnosis and treatment process.
About researchers:
Researchers will keep these samples and clinical information to use in this study in the future.
Even if the patients refuse to provide the sample, we will still provide him/her with the same medical services as the sample provider. All samples will be used up anonymously. If they need to be discarded, the relevant personal information will be destroyed at the same time. Researchers will publish the results of our research for the benefit of medical development without disclosing the personal data of the sample providers. Intellectual property derived from research results belongs to the research unit and the researcher, not to the sample provider.
Interventions
Explanation for the choice of comparators {6b}
Not applicable. This study is an observational cohort study. Grouping is determined by whether the subjects had a primary or secondary endpoint event during the follow-up period after admission. No randomization procedure or interventions would be used.
Intervention description {11a}
Not applicable.
Criteria for discontinuing or modifying allocated interventions {11b}
Not applicable.
Strategies to improve adherence to interventions {11c}
Not applicable.
Relevant concomitant care permitted or prohibited during the trial {11d}
Not applicable.
Provisions for post-trial care {30}
Not applicable.
Outcomes {12}
Primary endpoint
The main endpoint of this study will be the compound endpoint of cardiovascular events within 12 months of onset, including the cumulative incidence of all-cause death, heart failure events, or coronary revascularization events.
Definition of all-cause death: death within 12 months after onset, regardless of cause. If death occurs before discharge, it will be subdivided into cardiac death and non-cardiac death according to physical and chemical examination, clinical diagnosis, and other information. After discharge, death found by follow-up is difficult to determine and so will be classified as all-cause death.
Definition of heart failure: the occurrence of heart failure within 12 months after the onset of the disease. Clinicians will assess in-hospital heart failure events based on new heart failure symptoms, new objective evidence of heart failure, and initial intensive treatment specifically for heart failure. Heart failure found by the follow-up after discharge will be defined as the patient having new symptoms of heart failure, has new objective evidence of heart failure, or is treated with anti-heart failure drugs. The criteria for judging heart failure are the need to meet at least two symptoms and at least one laboratory standard or objective index.
Symptoms of heart failure include (1) Dyspnea (dyspnea, dyspnea in expiratory phase, paroxysmal nocturnal dyspnea, shortness of breath in supine position), (2) Decreased exercise tolerance (due to symptoms of dyspnea or fatigue), or reduced ability to perform large-scale skeletal muscle dynamic exercise, and (3) Severe end-organ failure (brain, kidney, liver, abdomen, or deterioration of symptoms, such as fever, dizziness, syncope, confusion, restlessness, cognitive decline, nausea, abdominal distension, abdominal discomfort, abdominal tenderness, chills relief, limb discoloration, extreme reduction of jaundice in urine). The New York heart function classification was III and IV. Class III is defined as a marked limitation of physical activity; comfortable at rest, but less than ordinary activity causes fatigue, palpitations, or dyspnea. Class IV is defined as being unable to carry out any physical activity without discomfort; symptoms of cardiac insufficiency at rest.
The signs of heart failure are as follows: (1) Peripheral edema (edema of lower extremities, compression of ankles, legs, thighs, abdomen, upper limbs, and facial edema), (2) Abdominal distension or increased ascites (in the absence of primary liver disease), (3) Pulmonary rales, pulmonary edema, pleural effusion, (4) Elevated jugular pressure and hepatic-jugular reflux, and (5) Heart auscultation gallop rhythm.
Objective evidence standard: (1) Cardiac color ultrasound: ejection fraction is less than 45%, (2) The concentration of plasma BNP or NT-proBNP increased, BNP > 500 pg/mL or NT-proBNP > 2000 pg/mL, (3) The walking distance of fewer than 150 meters in 6 minutes indicates severe cardiopulmonary insufficiency. The walking distance of 150-450 meters shows moderate cardiopulmonary insufficiency, and the walking distance of more than 450 meters shows mild cardiopulmonary insufficiency.
The definition of coronary artery revascularization: coronary revascularization therapy is performed again because of coronary artery stenosis in the hospital or after discharge. If vascular lesions are evaluated for the first time in the hospital, the lesions that need to be treated in the hospital are not recorded. The site of coronary artery stenosis can include original stenosis and new or progressive stenosis of non-target diseases. Revascularization therapy includes balloon dilatation, thrombus aspiration, stent implantation, and rotational grinding of coronary plaques.
Secondary endpoints
Secondary endpoints include all-cause death, non-fatal myocardial infarction, non-fatal stroke, malignant arrhythmia, heart failure, unstable angina pectoris, and target vessel revascularization. The above endpoints are defined according to 2014 American College of Cardiology (ACC) and American Heart Association (AHA) key data elements and definitions for cardiovascular endpoints in clinical trials11.
Participant timeline {13}
Figure 1 shows the timeline of our study.
Sample size {14}
Sample size calculation is based on the main endpoint of this study: the compound endpoint of cardiovascular events within 12 months of onset, including the cumulative incidence of all-cause death, heart failure, and coronary revascularization events. We will use an overall Class I error of 0.05, and the risk ratio of the composite research endpoint is assumed to be 0.8. According to the results of OMEGA Study13 and CHINA PEACE14 studies, the incidence of all-cause death in patients with ST-segment elevation myocardial infarction was 2.7% and 3.7% within one year, and the incidence of compound events was about 18.2%. It is estimated that the total incidence of events at the main endpoint of this study will be approximately 19.9%. There are many kinds of free fatty acids, and different free fatty acids have different effects on the prognosis of AMI. We will calculate the sample size of this study according to the literature data of total FFA, EPA, and ALA. Shrieks et al.,14 found that a total FFA level of 7 was a predictor of poor prognosis in ACS, with an Hazard Ratio (HR) of 1.12. EPA and ALA are protective factors for the prognosis of AMI. It was shown that the HR of EPA to MACE events in patients with AMI was 0.76 8. The HR value of ALA for all-cause death was 0.65. However, DHA seems to show different results in different studies. The relationship between other free fatty acids and the prognosis of AMI is not clear and consequently the sample size is estimated based on the above literature data, and the lost follow-up rate is estimated to be 20%. Power and Sample Size software will be used for calculation, and the estimation is based on the literature15. The sample size estimated by total FFA, EPA, and ALA is 747, 23, and 609, respectively. The process is shown in Table 1, and the final estimated sample size is 747.
Recruitment {15}
This study was conducted in the Department of Cardiovascular Medicine, China-Japan Union Hospital of Jilin University. Suppose the cardiovascular physician encounters a suitable candidate during the diagnosis and treatment of acute chest pain in the Emergency Room (ER). In that case, the study physician will be notified to conduct further screening and initiate informed consent. The number of emergency patients in the China-Japan Union Hospital of Jilin University is large enough to guarantee adequate participant enrolment.
Assignment of interventions: allocation
Sequence generation {16a}
Not applicable.
Concealment mechanism {16b}
Not applicable.
Implementation {16c}
Not applicable.
Assignment of interventions: Blinding
Who will be blinded {17a}
Not applicable.
Procedure for unblinding if needed {17b}
Not applicable.
Data collection and management
Plans for assessment and collection of outcomes {18a}
Professional clinicians will make preliminary diagnoses and evaluate emergency patients. Those patients who meet the criteria must sign an informed consent form before the collection of any data, including medical records, contact information, emergency contacts, and other information, to prevent loss of follow-up. At the same time as patients undergo standardized treatment, blood samples will be taken for free fatty acid spectrum determination by high-performance liquid chromatography-mass spectrometry. Measurements will be collected for total free fatty acids, totalΩ3, total Ω6, total monounsaturated fatty acids, total polyunsaturated fatty acids, triene/tetraene, Ω3/Ω66, lignocerric acid, nervonic acid, behenic acid, erucic acid, docosatetraenoic acid (DTA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), arachidic acid, eicosanoic acid, mead acid, eicosadienoic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), stearic acid, oleic acid, linoleic acid, linolenic acid, palmitic acid, palmitoleic acid, myristic acid, myristoleic acid, laurric acid, and decanoric acid. On the second day, the baseline questionnaire data will be collected by trained personnel through face-to-face interviews, to maximize the data collection, implementation, and quality. The content of the face-to-face interview is shown in supplementary material. When the patient is discharged from the hospital, the researchers will take the medical records for filing. The information includes all medical records, results of biochemical blood tests, images of coronary angiography and surgical records, blood-free fatty acid test results, heart ultrasound results, ECG results, data structured processing and registration by the researchers if the patient has related clinical events during hospitalization. Trained doctors will use the standard definition of clinical events to collect and judge medical records.
Plans to promote participant retention and complete follow-up {18b}
Out-of-hospital follow-up will be conducted in 2, 6, and 12 months through centralized telephone interviews. Participants will be asked to report on quality of life, drug compliance, medical records, and descriptions of related events. A professional clinician will assess the definition of the events after the description.
Data management {19}
The trained researchers are responsible for recording the baseline information and medical records. The follow-up information would be recorded in the process during the follow-up telephone. After data entry is complete, Data monitoring committee (DMC) will randomly audit 20% of the medical records and monitor the summary's quality to ensure that the overall variable accuracy is more than 98%.
Confidentiality {27}
When a participant takes informed consent, all data records will be replaced by a participants’ unique number to protect their privacy. When the trial is over, the published data set will not contain their names, contact details, and other private information. Data entry, review, and proofreading will be based on the participants’ number.
Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}
Use of blood samples:
For each enrolled participant, the test of 31 free fatty acids is described in Plans for Assessment and Collection of Outcomes and any tests necessary to ensure participants' safety. Including virology testing, testing for COVID-19, etc. This testing aims to ensure that analytical methods produce valid data throughout the study. It may also include, but is not limited to, investigations with unexpected results, in-process analysis of samples, and method transfer and comparability analysis. Our research team may conduct additional tests on the remaining samples to study and better understand free fatty acid products or markers of cardiovascular disease and molecular characterization. The results of these analyses will be recorded and maintained but may not be reported as part of the study.
Laboratory evaluation
After the informed consent form, the blood samples were collected before the operation, and the plasma-free fatty acids were tested. The rest of the blood sample will be sent to the cardiovascular laboratory in an icebox immediately. In the laboratory, the blood was centrifuged at 3000 rpm at 4℃ for 10 mins. After centrifugation, the plasma and white cell layers were separated into tubes. The patient information and tube location information were electronically recorded and stored in the -80℃ refrigerator.
Biomarkers and genetic studies
Biomarkers are objectively measured to evaluate biological and pathophysiologic processes. Research techniques will be further developed in the future, so it is difficult to determine which biomarker should be tested at this stage. If markers can be correlated with the data in this study, they may provide prognostic indicators for patients with acute ST-segment elevation myocardial infarction in the future. Genetic studies can better reveal the distribution of genes in a population, disease susceptibility, and other related issues. The serum and leukocyte layers stored in this study may be tested for biomarkers and genetic material in the future.
Storage and destruction of samples
The serum and white blood cells from the remaining blood samples in the study will be preserved until 10 years after the end of the study, which will be used to study scientific problems related to cardiovascular disease, metabolic disease, inflammation and so on. By contacting and authorizing the key researchers, the subjects retained the right to destroy the sample material at any time. The researcher is the exclusive owner of the derivative of any data, discovery, or sample material source. The researchers destroyed the samples centrally when the storage period arrived.
Statistical methods
Statistical methods for primary and secondary outcomes {20a}
Participants will be divided into ‘event’ group and ‘non-event’ group according to whether the main endpoint event occurred. The baseline levels of all hospitalization data between the two groups will be compared. The continuous data of normal distribution is expressed by mean ± standard deviation (SD). In contrast, the skewed distribution data is expressed by median [quartile], and the classified data is expressed by n (%). The students' test will be used to compare the difference of continuous data of normal distribution between the two groups. The Mann-Whitney test will be used to compare the variables of skewed distribution, and the chi-square test used to compare the difference of rates between the two groups. Pearson or Spearman correlation coefficients determine the correlation between the two variables. The levels of serum-free fatty acids in patients with ‘non-events’ and ‘events’ will be compared, and various free fatty acids analyzed by cluster analysis. After adjusting for age, gender, history of hypertension, diabetes, cerebrovascular disease, heart failure, myocardial infarction, glomerular filtration rate, triglyceride, total cholesterol, left ventricular ejection fraction, and other confounding factors, multivariate analysis will be carried out. The predictive role of each FFA in the prognosis of AMI will be determined by Cox survival regression. The results will be expressed as Hazzard ratio (HR) and the corresponding 95% confidence interval (CI). SPSS software (SPSS 25.0, Chicago, Illinois) will be used for statistical analysis.
Interim analyses {21b}
In this study, there are no planned interim analysis or termination rules. This study is observational. All participants receive standard treatment. There are no interventions in this study, generally do not terminate the trial in advance.
Methods for additional analyses (e.g. subgroup analyses) {20b}
According to the data collected and the relationship between plasma FFA and the prognosis in patients with AMI, researchers may conduct gender subgroup analyses to see whether the plasma FFA predicts the cardiovascular event differently in gender. Meanwhile, researchers would explore the influence factor of plasma FFA.
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}
The definition of withdrawal:
Participants signed the informed consent form and selected the cases required to withdraw during the follow-up after selecting qualified participants for the study.
The definition of elimination:
Participants signed an informed consent form to enter the study. However, during the study period, if participants were found that they meet the excluded criteria. They would be excluded from the study.
The data of participants who were withdrawal or eliminated would be delete in the study.
Plans to give access to the full protocol, participant level-data and statistical code {31c}
The data/figures used and/or analyzed in this study are available from the corresponding author upon reasonable request.
Oversight and monitoring
Composition of the coordinating centre and trial steering committee {5d}
The Ethics Committee approved the study at the China-Japan Friendship Hospital of Jilin University, on October 30th 2019, approval number: (2019) Review No. (2019103004). The scientific design and implementation of the study will be carried out under the supervision and guidance of the Ethics Committee of the China-Japanese Union Hospital of Jilin University. The Ethics Committee will oversee the security and ethical aspects of the data. Authorized research doctors will collect data collection, and research assistants will assist in data collation and entry. All researchers involved in the project will receive unified questionnaire training to reduce data bias.
Research and data Collection Monitoring Group are responsible for contacting the researchers in order to inspect the facility and, as required, check various records of the clinical studies (for example, eCRF and other relevant data), provided that the confidentiality of the subjects is respected. During the study, inspectors are responsible for regular verification of eCRFs to verify compliance, completeness, accuracy and data consistency of the program, as well as compliance with relevant local clinical research regulations. The inspector should have the right to obtain the subject's medical records and other research-related records that need to be verified as entered in the eCRF. The researchers agreed to work with monitors to ensure that any problems found during these monitoring visits, including delays in the completion of the eCRF, were resolved.
Composition of the data monitoring committee, its role and reporting structure {21a}
Data monitoring committee (DMC) is composed of 2 professors and 2 attendance doctors. DMC will randomly audit 20% of the medical records and monitor the summary's quality to ensure that the overall variable accuracy is more than 98%.
Adverse event reporting and harms {22}
Not applicatble.
Frequency and plans for auditing trial conduct {23}
Not applicatble.
Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}
Any protocol modifications such as eligibility criteria, outcomes, analyses, et al. will be Submitted to Chinese Clinical Trail Registry (https://www.chictr.org.cn/), the Ethics Committee of Jilin University and the Journal for written approval. If any one above is not approved, no modification will be made.
Dissemination plans {31a}
The result of this study will be submitted to an appropriate journal. All the data would upload to Chinese Clinical Trail Registry. The data/figures used and/or analyzed in this study are available from the corresponding author upon reasonable request.