Comparative Prognostic Utility of Indexes of Microvascular Function Alone or in Combination in Patients With an Acute ST-Segment–Elevation Myocardial Infarction

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STEMI patients
Screening, enrolment, and data collection were prospectively performed by cardiologists in the cardiac catheterization laboratories of the Golden Jubilee National Hospital, Glasgow, United Kingdom. This hospital is a regional referral center for primary and rescue percutaneous coronary intervention (PCI). The hospital provides clinical services for a population of 2.2 million. A screening log was recorded, including patients who did not participate in the cohort study. Patients were invited to undergo cardiac magnetic resonance imaging (CMR) 2 days and 6 months after hospital admission (1) (2).

Coronary angiogram acquisition
Coronary angiograms were acquired during usual care with cardiac catheter laboratory X-ray (Innova) and IT equipment (Centricity) made by GE Healthcare.

Percutaneous coronary intervention
Consecutive admissions with acute STEMI referred for emergency percutaneous coronary intervention (PCI) were screened for the inclusion and exclusion criteria. During ambulance transfer to the hospital, the patients received 300 mg of aspirin, 600 mg of clopidogrel and 5000 IU of unfractionated heparin (3,4). The initial primary PCI procedure was performed using radial artery access. A conventional approach to primary PCI was adopted in line with usual care in our hospital (3,4). Conventional bare metal and drug eluting stents were used in line with guideline recommendations and clinical judgement. The standard transcatheter approach for reperfusion involves minimal intervention with aspiration thrombectomy only or minimal balloon angioplasty (e.g. a compliant balloon sized according to the reference vessel diameter and inflated at 4-6 atmospheres 1-2 times). During PCI, glycoprotein IIbIIIa inhibitor therapy was initiated with high dose tirofiban (25 g/kg/bolus) followed by an intravenous infusion of 0.15 g/kg/min for 12 hours, according to clinical judgement and indications for bail-out therapy (3,4). No reflow was treated according to contemporary standards of care with intra-coronary nitrate (i.e. 200 g) and adenosine (i.e. 30 -60 g) (3,4), as clinically appropriate. In patients with multivessel coronary disease, multivessel PCI was not recommended, in line with clinical guidelines (3,4). The subsequent management of these patients was symptom-guided.

Measurement of IMR and CFR at the end of PCI
We adopted a thermodilution technique rather than Doppler, in order to implement a method that is potentially transferable to routine clinical practice. The Doppler measurements are more time-consuming, require considerable experience, may be less reproducible (5), and the guidewire is typically more expensive. The Doppler method less transferrable to every-day practice than the thermodilution method.
A coronary pressure-and temperature-sensitive guidewire (St Jude Medical, Uppsala, Sweden) was used to measure coronary flow reserve (CFR) and the index of microvascular resistance (IMR) in the culprit coronary artery at the end of primary or rescue PCI. The guidewire was calibrated outside the body, equalized with aortic pressure at the ostium of the guide catheter and then advanced to the distal third of the culprit artery. Coronary flow reserve is defined as the mean transit time at rest divided by the mean transit time during hyperemia.
IMR is defined as the distal coronary pressure multiplied by the mean transit time of a 3 ml bolus of saline at room temperature during maximal coronary hyperemia, measured simultaneously (mmHg x s, or units) (6)(7)(8).
Hyperemia was induced by 140 /kg/min of intravenous adenosine preceded by a 2 ml intracoronary bolus of 200 µg of nitrate. The mean aortic and distal coronary pressures were recorded during maximal hyperemia. We have previously assessed the repeatability of IMR using duplicate measurements 5 minutes apart in a subset of 12 consecutive patients (8).

Coronary artery anatomy
The coronary anatomy and disease characteristics of the study participants were described based on the clinical reports of the attending cardiologist. Coronary dominance were assigned as left, right or balanced according to the origin of the posterior descending coronary artery.

Coronary artery disease severity
Quantitative coronary analysis (QCA) of the culprit vessel was performed by two trained observers (J.C., V.Y.T.M) using standard methods (Centricity, GE Healthcare, Pollards Wood, UK). All coronary angiograms were analysed on a single image analysis software platform using de-identified images. Automatic edge detection algorithms were used to determine the vessel contours by assessing brightness along scan lines perpendicular to the vessel center. Image analysis was performed by two experienced observers supervised by an expert physician, all of whom were blinded to the other study data. End-diastolic frames were used to assess disease severity using angulations reveal the stenosis at its most severe degree with minimal foreshortening and branch overlap. The coronary artery segments in the culprit artery included all those with a reference diameter 1.5 mm.

TIMI flow grade
Coronary blood flow can be described based on the visual assessment of coronary blood flow revealed by contrast injection into the coronary arteries (3,4,9).

Myocardial perfusion
Angiographic evidence of myocardial perfusion will be evaluated using the TIMI myocardial perfusion grade (TMP) at the end of the PCI procedure (10). Good blush with slow clearing of myocardial contrast (present at end of cine but gone at beginning of next) 3 Good blush and normal clearing (ie. gone by end of cine)

Assessment by corrected Thrombolysis In Myocardial Infarction Frame Count
Corrected TIMI frame count (cTFC) was calculated as the number of frames for dye to reach a standardised distal landmark in each angiographic territory. The first frame taken for the measurement was the frame in which dye touched both borders of the coronary artery in question and moved forward with at least 70% of the vessel lumen opacified. The standardised distal landmarks were taken as the first branch of the postero-lateral artery for the right coronary artery, the most distal branch of the obtuse marginal for the circumflex, and the distal bifurcation of the left anterior descending (LAD) coronary artery. The number of frames from the first frame to the last frame when the dye entered the standardised distal landmark was counted. A standard image acquisition speed of 30 frames per second was used. The correction factor used to account for the increased length of the LAD compared to the right and circumflex arteries was 1.7 thereby giving a "corrected TIMI frame count".

CMR acquisition and analyses
We used CMR to provide reference data on LV function, pathology and surrogate outcomes, independent of the invasive tests.
In order to assess early microvascular obstruction, early gadolinium enhancement imaging

MR image analyses
The images were analysed on a Siemens work-station by observers with at least 3 years CMR experience (N.A., D.C., I.M). All of the images were reviewed by experienced CMR cardiologists (C.B., N.T.). LV dimensions, volumes and ejection fraction were quantified using computer assisted planimetry (syngo MR®, Siemens Healthcare, Erlangen, Germany).
All scan acquisitions were spatially co-registered. contrast enhanced T1-weighted, T2-weighted and cine images, respectively). The infarct zone region-of-interest was defined as myocardium with pixel values (T2) >2 SD from remote myocardium on T2-weighted CMR (11,12). The infarct core was defined as an area in the center of the infarct territory having a mean T2/ T2* value of at least 2 standard deviations (SDs) below the T2/ T2* value of the periphery of the area-at-risk.
In healthy volunteers, the mid-ventricular T2/T2* map was segmented into 6 equal segments, using the anterior right ventricular-LV insertion point as the reference point (2). T2/T2* was measured in each of these segments, and regions-of-interest were planimetered distinct and separate from blood-pool and tissue interfaces. These segmental values were also averaged to provide one value per subject. Results are presented as average values for segments and slices.

Infarct definition and size
The presence of acute infarction was established based on abnormalities in cine wall motion, rest first-pass myocardial perfusion, and delayed-enhancement imaging. In addition, supporting changes on the ECG and coronary angiogram were also required. Acute infarction was considered present only if late gadolinium enhancement was confirmed on both the axial and long axis acquisitions. The myocardial mass of late gadolinium (grams) was quantified using computer assisted planimetry and the territory of infarction was delineated using a signal intensity threshold of >5 standard deviations above a remote reference region and expressed as a percentage of total LV mass (15). Infarct regions with evidence of microvascular obstruction were included within the infarct area and the area of microvascular obstruction was assessed separately and also expressed as a percentage of total LV mass. The measurements of infarct size were performed by I.M. and N.A.

Microvascular obstruction
Microvascular obstruction was defined as a dark zone on EGE imaging 1, 3, 5 and 7 minutes post-contrast injection that remained present within an area of late gadolinium enhancement at 15 minutes. Identification of microvascular obstruction was performed independently by I.M. and N.A.

Myocardial hemorrhage
Myocardial hemorrhage was scored visually. On the T2* maps, a region of reduced signal intensity within the infarcted area, with a T2* value of <20 ms (16)(17)(18)(19), was considered to confirm the presence of myocardial hemorrhage.

Myocardial salvage
Myocardial salvage was calculated by subtraction of percent infarct size from percent area-atrisk (8,20,23). The myocardial salvage index was calculated by dividing the myocardial salvage area by the initial area-at-risk.

Reference ranges
Reference ranges used in the laboratory were 105 -215 g for LV mass in men, 70 -170 g for LV mass in women, 77 -195 ml for LV end-diastolic volume in men, 52 -141 ml for LV end-diastolic volume in women, 19 -72 ml for LV end-systolic volume in men and 13 -51 ml for LV end-systolic volume in women. Organization.

Electrocardiogram
The extent of ST-segment resolution on the ECG assessed 60 minutes after reperfusion compared to the baseline ECG before reperfusion (3) was expressed as complete (70%), incomplete (30% to < 70%) or none (30%). ECG evidence of reperfusion injury was taken as persistence of ST segment elevation resolution post-procedure, and specifically 30% STsegment resolution post-PCI.

Biochemical and hematologic measurements
Blood samples were obtained immediately after reperfusion in the cardiac catheterization laboratory, and subsequently between 0600 -0700 hrs each day during the initial in-patient stay in the Coronary Care Unit.

Biochemical assessment of infarct size
Troponin T was measured (Elecsys Troponin T, Roche) as a biochemical measure of infarct size. The high sensitive assay reaches a level of detection of 5 pg/ml and achieves less than 10% variation at 14 pg/ml corresponding to the 99th percentile of a reference population. The peak troponin T value for each patient was recorded in the study database.

Biochemical assessment of inflammation
C-reactive protein (CRP) was measured in an NHS hospital biochemistry laboratory using a particle enhanced immunoturbimetric assay method (Cobras C501, Roche) and the manufacturers calibrators and quality control material, as a biochemical measure of inflammation. The high sensitive assay CRP measuring range is 0.1-250 mg/L. The expected CRP values in a healthy adult are < 5 mg/L, and the reference range in our hospital is 0 -10 mg/L.

Hematological measurement of inflammation
Leucocyte count and leucocyte sub-populations were measured as a hematologic measure of inflammation using sheath flow technology incorporating semi-conductor laser beam, forward and side scattered light (Sysmex XT200i and XT1800i for white blood cell and differential white blood cell counts, respectively). The linearity ranges for white blood cells

Pre-specified health outcomes
We pre-specified adverse health outcomes that are pathophysiologically linked with STEMI (26,27). The primary composite outcome was (1) all-cause death or first heart failure event following the initial hospitalization (Supplementary Methods).
Research staff screened for events from enrolment by checking the medical records and by contacting patients and their primary and secondary care physicians, as appropriate with no loss to follow-up ( Figure 2 Differences in categorical variables between groups were assessed using a Fisher's test. Two raters assessed the angiograms of 30 subjects randomly selected from the whole cohort. Inter-rater reliability for angiographic parameters was assessed using weighted Cohen's kappa and the intra-class correlation coefficient (ICC) with random effects models (Supplementary Results).
Univariable and multivariable associations are assessed using binary logistic regression or linear regression where appropriate. Binary logistic models are compared using Harrel's C-statistic. Linear regression models are compared using the Bayesian Information Criterion (BIC). The likelihood ratio test was used to compare the binary logistic and linear regression models with IMR, or an IMR>40 and CFR≤2.0. A p-value <0.05 favors including the variable in the model.
Logistic regression (odds ratio, 95% confidence interval) was used to identify potential clinical predictors of all-cause death/heart failure events, including patient characteristics, CMR findings, and IMR and CFR.
All p-values are 2-sided and a p-value > 0.05 indicates the absence of a statistically significant effect. Statistical analyses were performed using R version 2.15.1 or SAS v 9.3, or higher versions of these programs.

Trial Management
The study was conducted in line with Guidelines for Good Clinical Practice (GCP) in Clinical Trials. http://www.mrc.ac.uk/documents/pdf/good-clinical-practice-in-clinical-trials/

Trial management included a Trial Management Group, and an independent Clinical Trials
Unit. Day to day study activity was coordinated by the Trial Management Group who was responsible to the Sponsor which was responsible for overall governance and that the trial was conducted according to GCP standards.
Clinical events were assessed and validated by an independent cardiologist (A.M.) who had access to relevant source clinical data. This cardiologist followed an agreed charter and he was blinded to all of the other clinical data.