We reviewed 740 consecutive patients with ACS who underwent grey-scale and VH-IVUS between January 2007 and September 2010. Anemia was defined according to criteria of the World Health Organization (i.e., hemoglobin levels < 13 g/dL in men and < 12 g/dL in women) (9). We compared baseline characteristics, coronary angiographic findings, and grey-scale and VH-IVUS findings between anemia group (171 patients, 260 lesions) and non-anemia group (569 patients, 881 lesions). The presence of unstable angina was determined by chest pain within the preceding 72 hr with or without ST-T-wave changes or positive cardiac biochemical markers (creatine kinase-myocardial band or cardiac specific troponin-I). 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 2 contiguous electrocardiographic leads, and creatine kinase-myocardial band (MB) or cardiac-specific troponin I above 2 times of normal. The presence of non-ST-segment elevation myocardial infarction was diagnosed by chest pain and positive cardiac biochemical markers (creatine kinase-myocardial band or cardiac-specific troponin I) without new ST-segment elevation. Hypercholesterolemia was diagnosed in patients who received lipid-lowering medication or had a history of a low-density lipoprotein level > 130 mg/dL. Heart failure was defined as the presence of previous clinical signs and symptoms of pulmonary venous congestion or previous evidence of systolic dysfunction (left ventricular ejection fraction < 50%) on echocardiogram. We excluded patients with subacute or late stent thrombosis, totally occluded lesions, restenosis after stenting, coronary artery bypass graft failure, severe heart failure or cardiogenic shock, important systemic disease, or serum creatinine > 2.5 mg/dL, and patients in whom adequate IVUS images could not be obtained. Hospital records of all patients were reviewed to obtain clinical demographics and medical history.

Peripheral blood samples were obtained before coronary angiography using direct venipuncture. The blood samples were centrifuged, and serum was removed and stored at -70℃ until the assay could be performed. Absolute creatine kinase-MB 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). The 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 (10). A random morning urine specimen was obtained. Urine creatinine and albumin were measured on a Kodak Ektachem 700 Analyzer and Beckman Array 360 CE protein Analyzer (Beckman Instruments, Fullerton, CA, USA), respectively. Patients with albumin levels of 30 to 300 mg/g of creatinine were defined as having microalbuminuria (11).

Coronary angiogram was analyzed with validated QCA system (Phillips H5000 or Allura DCI program, Philips Medical Systems, the Netherlands). With the outer diameter of the contrast-filled catheter as the calibration standard, the reference diameter and minimal lumen diameter were measured in diastolic frames from orthogonal projections. Perfusion was evaluated according to thrombolysis in myocardial infarction (TIMI) criteria (12).

All IVUS examinations were performed after intracoronary administration of 300 µg nitroglycerin using a commercially available IVUS system (Volcano Corp, Rancho Cordova, CA, USA). The IVUS catheter was advanced distal to the target lesion, and imaging was performed retrograde to the aorto-ostial junction at an automatic pullback speed of 0.5 mm/sec. In case of total occlusion, we opened the occluded artery by thrombus aspiration or balloon angioplasty using small balloon.

Gray-scale IVUS and VH-IVUS data were analyzed by 2 independent observers. The levels of reproducibility for external elastic membrane (EEM), lumen, and plaque plus media (P&M) cross-sectional areas (CSAs) using the Spearman rank-order correlation coefficients were 0.96, 0.97, and 0.97, respectively. Similarly, for plaque components by VH-IVUS, reproducibility for the fibrous (FT), fibro-fatty (FF), dense calcium (DC), and necrotic core (NC) volume measurements using the Spearman rank-order correlation coefficients were 0.93, 0.94, 0.93, and 0.93, respectively.

Quantitative volumetric gray-scale and VH-IVUS analyses were performed across the entire lesion segment, and cross-sectional analyses were performed at the minimum lumen sites and at the largest NC sites. Conventional quantitative volumetric gray-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 (13). 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. References were the single slices with the largest lumen and smallest plaque CSAs within 10 mm proximally and distally. Hypoechoic plaque was less bright than the adventitia, hyperechoic noncalcified plaque was as bright as or brighter than the adventitia without acoustic shadowing, and hyperechoic calcified plaque was brighter than the adventitia with acoustic shadowing. When there was no dominant plaque composition, the plaque was classified as mixed.

Volumetric data were generated by the software using Simpson's method. EEM and lumen CSAs were measured. P&M CSA was calculated as EEM minus lumen CSA; and plaque burden was calculated as P&M divided by EEM CSA. VH-IVUS analysis classified the color-coded tissue into four major components: green (FT); yellow-green (FF); white (DC); and red (NC) (14-17). VH-IVUS analysis was reported as a percentage of plaque area or volume. We defined thin-cap fibroatheroma (TCFA) as a NC ≥ 10% of plaque area in at least 3 consecutive frames without overlying fibrous tissue in the presence of ≥ 40% plaque burden (15).

The statistical Package for Social Sciences (SPSS) for Windows, version 15.0 (Chicago, IL, USA) was used for all analyses. Continuous variables were presented as the mean ± SD; comparisons were conducted by Student's t-test or nonparametric Wilcoxon test if normality assumption was violated. Discrete variables were presented as percentages and relative frequencies; comparisons were conducted by chi-square statistics or Fisher's exact test as appropriate. Pearson's correlation coefficient was used to evaluate the associations between baseline hemoglobin level and each plaque components. Multivariate analysis was performed to determine the independent predictors of TCFA. A P value < 0.05 was considered statistically significant.

The study protocol was reviewed and approved by the institutional review board of Chonnam National University Hospital (1-2009-07-082). Informed consent was waived by the board.

The baseline characteristics are summarized in Table 1. Patients with anemia were significantly older, were more females, and were more diabetics and were less smokers, and had more history of previous coronary intervention compared with those without anemia. Glucose level, high-sensitivity C-reactive protein, and urinary albumin-to-creatinine ratio were significantly higher in patients with anemia compared with those without anemia. Anemia group had significantly lower low-density lipoprotein cholesterol and apolipoprotein B levels compared with non-anemia group.

Angiographic findings are summarized in Table 2. Type B₂/C lesions and multi-vessel diseases were observed more frequently and lesion length was significantly longer in patients with anemia compared with those without anemia. There were no significant differences in the target vessel, TIMI flow grade, minimal lumen diameter, and percent diameter stenosis between both groups.

Gray-scale IVUS results are summarized in Table 3. P&M CSAs were significantly greater at the proximal and distal references and at the minimum lumen site and at the largest NC site in patients with anemia compared with those without anemia. EEM CSAs were significantly greater at the distal references and at the largest NC site in patients with anemia compared with those without anemia. By volumetric analysis, anemia group had greater plaque volume and smaller lumen volume compared with non-anemia group.

At the minimum lumen site, absolute NC and DC areas were significantly greater and %NC area was significantly greater in patients with anemia compared with those without anemia (Fig. 1).

At the largest NC site, absolute NC, DC, and FT areas were significantly greater, and %NC and DC areas were significantly greater in patients with anemia compared with those without anemia; conversely %FT area was significantly smaller in patients with anemia compared with those without anemia (Fig. 2).

Absolute NC and DC volumes were significantly greater, and absolute FT and FF volumes were significantly smaller in patients with anemia compared with those without anemia; and %NC and DC volumes were significantly greater; and %FT and FF volumes were significantly smaller in patients with anemia compared with those without anemia (Fig. 3). TCFA was observed more frequently in patients with anemia compared with those without anemia (47% vs 35%, P < 0.001).

Hemoglobin level correlated negatively with absolute NC volume (r = -0.235, P < 0.001), %NC volume (r = -0.209, P < 0.001), absolute DC volume (r = -0.211, P < 0.001), %DC volume (r = -0.193, P < 0.001), and correlated positively with absolute FT volume (r = 0.205, P = 0.001), %FT volume (r = 0.164, P = 0.001), absolute FF volume (r = 0.187, P = 0.001), and %FF volume (r = 0.134, P < 0.001).

Multivariate analysis was performed to identify independent predictors of TCFA. The following variables were tested (all with P < 0.2 in univariate analysis) including microalbuminuria, age, male gender, diabetes mellitus, hypertension, smoking, ejection fraction, anemia, high-sensitivity C-reactive protein, and minimum lumen site plaque burden (all with P < 0.2 in univariable analysis). Independent predictors of TCFA included diabetes mellitus (odds ratio [OR], 2.213; 95% confidence interval [CI], 1.403-3.612, P = 0.006), high-sensitivity C-reactive protein (OR, 1.143; 95% CI, 1.058-1.304, P = 0.012), microalbuminuria (albumin levels of 30 to 300 mg/g of creatinine) (OR, 2.124; 95% CI, 1.041-3.214, P = 0.018), and anemia (OR, 2.112; 95% CI, 1.022-3.208, P = 0.028).