This study was a retrospective, single-center study. From March 2006 to March 2010, a total of 399 CAD patients with 471 coronary lesions who underwent pre-intervention VH-IVUS at Chonnam National University Hospital were enrolled in this study. The presence of stable angina was determined by typical effort-induced chest pain which was relieved by resting. The presence of unstable angina was determined by chest pain within the preceding 72 hr with or without ST-T wave changes of positive cardiac biochemical markers. The presence of ST-segment elevation myocardial infarction was determined by >30 min of continuous chest pain, a new ST-segment elevation ≥2 mm on at least two contiguous electrocardiographic leads, and creatine kinase-myocardial band (MB) >3 times normal. The presence of non-ST-segment elevation myocardial infarction was diagnosed by chest pain and a positive cardiac biochemical marker without new ST-segment elevation. We excluded patients with subacute or late stent thrombosis, restenosis after stenting, coronary artery bypass graft failure, factors associated with increased risk of bleeding, severe heart failure or cardiogenic shock, important systemic disease, or creatinine ≥2.5 mg/dL, and patients in whom adequate IVUS images could not be obtained. The NLR was calculated as the ratio of neutrophil count to lymphocyte count. Based on the previously published article (11), we decided the cut-off value of NLR as 2.73, and we classified the patients into two groups according to the NLR on admission {low NLR group (NLR≤2.73 [370 lesions in 315 patients]) vs. high NLR group (NLR>2.73 [101 lesions in 84 patients])}.

The blood samples were centrifuged, and serum was collected and stored at -70℃ until the assay was performed. Absolute creatine kinase-myocardial band levels were determined by radioimmunoassay (Dade Behring Inc., Miami, FL, USA). Cardiac-specific troponin I levels were measured by a paramagnetic particle, chemiluminescent immunoenzymatic assay (Beckman, Coulter Inc., Fullerton, CA, USA). Serum levels of total cholesterol, triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were measured by standard enzymatic methods. High-sensitivity C-reactive protein was analyzed turbidimetrically with sheep antibodies against human C-reactive protein; this has been validated against the Dade-Behring method (12).

Coronary angiogram was analyzed with validated QCA system (Phillips H5000 or Allura DCI program, Philips Medical Systems, Eindhoven, the Netherlands) (13). With the outer diameter of the contrast-filled catheter as the calibration standard, the minimal lumen diameter and reference diameter were measured in diastolic frames from orthogonal projections.

All IVUS examinations were performed a 20-MHz, 2.9F IVUS imaging catheter (Eagle Eye, Volcano Corp, Rancho Cordova, CA, USA) was advanced >10 mm beyond the lesion; and automated pullback was performed to a point >10 mm proximal to the lesion at a speed of 0.5 mm/sec.

Grey-scale IVUS and VH-IVUS data were analyzed by 2 independent observers. The levels of reproducibility for external elastic membrane, lumen, and plaque plus media cross-sectional areas using the Spearman rank-order correlation coefficients were 0.95, 0.97, and 0.97, respectively. Similarly, for plaque components by VH-IVUS, reproducibility for the fibrous, fibro-fatty, dense calcium, and necrotic core volume measurements using the Spearman rank-order correlation coefficients were 0.95, 0.92, 0.93, and 0.93, respectively.

Quantitative volumetric grey-scale and VH-IVUS analyses were performed across the entire lesion segment, and cross-sectional analysis was performed at the minimum lumen area sites and at the largest nectoric core sites. Conventional quantitative volumetric grey-scale IVUS analysis was performed according to the American College of Cardiology Clinical Expert Consensus Document on Standards for Acquisition, Measurement and Reporting of Intravascular Ultrasound Studies (14). Measurements were made by every 1-mm interval for the region of interest, which was defined as the segment between distal to proximal reference sites that were the most normal looking within 5 mm proximal and distal to the lesion. Volumetric data were generated by the software using Simpson's method. External elastic membrane and lumen cross-sectional areas were measured. Plaque plus media cross-sectional area was calculated as external elastic membrane minus lumen cross-sectional area; and plaque burden was calculated as plaque plus media divided by external elastic membrane minus lumen cross-sectional area. Total atheroma volume (TAV) was calculated by summation of atheroma area from each measured image as: TAV=Σ(external elastic membrane area-lumen area). The percent atheroma volume (PAV) was determined using the formula: PAV=100×(Σ[external elastic membrane area-lumen area]/Σ[external elastic membrane area]). VH-IVUS analysis classified the color-coded tissue into four major components: green (fibrous), yellow-green (fibro-fatty), white (dense calcium), and red (necrotic core) (15). VH-IVUS analysis was reported in absolute amounts and as a percentage of plaque area or volume. We defined thin-cap fibroatheroma (TCFA) as necrotic core ≥10% of plaque area in at least 3 consecutive frames without overlying fibrous tissue in the presence of ≥40% least 3 plaque burden (16).

The statistical Package for Social Sciences (SPSS) for Windows, version 19.0 (Chicago, IL, USA) was used for all analyses. Continuous variables were presented as the mean value±1SD; comparisons were conducted by Student's t-test, Discrete variables were presented as percentages and frequencies; comparisons were conducted by chi-square test, where appropriate. Multivariate analysis was performed to determine the independent predictor of TCFA. A P value<0.05 was considered statistically significant.

This study protocol was reviewed and approved by the institutional review board of Chonnam National University Hospital, Gwangju, Korea (CNUH-2013-054). Informed consent was waived by the board.

The baseline characteristics are summarized in Table 1. High NLR group had more acute coronary syndrome (ACS) compared with low NLR group. High NLR group had higher white blood cell counts and high-sensitivity C-reactive protein, and lower ejection fraction compared with low NLR group. The creatine kinase-myocardial band and troponin-I, and N-terminal pro-B type natriuretic peptide level were significantly higher in high NLR group.

Coronary angiographic findings are summarized in Table 2. The minimal luminal diameter was significantly smaller and percent diameter stenosis was significantly greater in high NLR group.

Grey-scale IVUS findings are summarized in Table 3. At the proximal reference, lumen cross-sectional area was significantly greater in high NLR group. At the minimum lumen area site, external elastic membrane cross-sectional area and plaque plus media cross-sectional area and plaque burden were significantly greater, and IVUS lesion was significantly longer in high NLR group. At the largest necrotic core site external elastic membrane cross-sectional area and plaque plus media cross-sectional area and plaque burden were significantly greater in high NLR group. By volumetric analysis, external elastic membrane volume and lumen volume and total atheroma volume were significantly greater in high NLR group.

VH-IVUS findings are summarized in figures. At the minimum lumen area site, absolute fibrous and necrotic core areas were significantly greater in high NLR group (Fig. 1A). At the largest necrotic core site, absolute and relative necrotic core areas were significantly greater in high NLR group (Fig. 2). By volumetric analysis, absolute fibrous and fibro-fatty and dense calcium and necrotic core volumes were significantly greater in high NLR group (Fig. 3A). Also TCFA was observed more frequently in high NLR group compared with low NLR group (Fig. 4).

We performed separate analysis in the stable angina and ACS patients. In a subgroup analysis, ACS patients had greater total atheroma volume and absolute necrotic core volume compared with stable angina patients (216.2±141.9 µL vs. 177.2±110.7 µL, P=0.004, and 26.9±23.2 µL vs. 20.0±15.8 µL, P<0.001, respectively), and TCFA was observed more frequently in ACS patients compared with stable angina patients (24% vs. 13%, P=0.009).

We performed analysis in patients with ACS and stable angina using reference value of NLR (NLR: 2.7), respectively. We classified the lesions into two groups according to the NLR on admission in patients with ACS {low NLR group (NLR<2.7 [n=242]) vs. high NLR group (NLR≥2.7 [n=92]) and stable angina {low NLR group (NLR<2.7 [n=126]) vs. high NLR group (NLR≥2.7 [n=11]), respectively}. In ACS patients, at the minimum lumen area site and at the largest necrotic core site, high NLR group had greater absolute nectoric core area compared with low NLR group (1.58±1.04 mm² vs. 2.02±1.38 mm², P=0.002, and 2.16±1.11 mm² vs. 2.72±1.50 mm², P<0.001, respectively), and high NLR group had greater total atheroma volume and absolute necrotic core volume compared with low NLR group (201.7±134.6 µL vs. 254.3±153.8 µL, P=0.002, and 24.3±20.8 µL vs. 33.7±27.7 µL, P=0.001, respectively), and TCFA was observed more frequently in high NLR group compared with low NLR group (35% vs. 20%, P=0.007). However, there were no significant differences in VH-IVUS parameters between high NLR group and low NLR group in stable angina patients using reference value (NLR: 2.7).

Independent predictors of TCFA are summarized in Table 4. The following variables were tested to determine the independent predictors of TCFA (variables with P<0.2 in univariate analysis): ST segment elevation myocardial infarction, NLR>2.73, total atheroma volume, high-sensitivity C-reactive protein, plaque burden at the minimum lumen area site, plaque burden at the largest necrotic core site, hypertension, creatine kinase-myocardial band, ejection fraction. ST segment elevation myocardial infarction (odds ratio [OR], 2.159; 95% CI, 1.000-4.660, P=0.050) and NLR>2.73 (OR, 1.848; 95% CI, 1.016-3.360, P=0.044) and total atheroma volume (OR, 1.003; 95% CI, 1.001-1.004, P=0.004) were independent predictors of TCFA.