Association of Neutrophil-to-Lymphocyte Ratio with the Severity and Morphology of Coronary Atherosclerotic Plaques Detected by Multidetector Computerized Tomography

Abstract

Background

Studies have demonstrated a consistent relationship between white blood cell (WBC) counts and coronary artery disease (CAD). The neutrophil/lymphocyte ratio (NLR) has been considered as a potential marker for identifying individuals under risk of CAD and associated events. In this study, we aimed to evaluate whether NLR was associated with the severity and morphology of coronary atherosclerotic plaques shown by multidetector computed tomography (MDCT).

Methods

Our study population consisted of 684 patients who underwent dual-source 64 slice MDCT for the assessment of CAD. Coronary arteries were evaluated on a 16-segment basis and critical coronary plaque was described as luminal narrowing > 50%, whereas plaque morphology was assessed on a per segment basis. Total WBC, neutrophil and lymphocyte counts were determined using commercially available assay kits.

Results

WBC count [7700 (6400-8800) vs. 6800 (5700-7900), p < 0.05] and NLR [2.40 (1.98-3.07) vs. 1.86 (1.50-2.38), p < 0.001] were found to be higher in patients with critical stenosis than in those without. In the binary logistic regression analysis, NLR was a predictor of critical stenosis (odds ratio, 1.68; 95% confidence interval, 1.39-2.03, p < 0.001). NLR levels differed among plaque morphology subtypes (p < 0.05) and was significantly higher in non-calcified plaque (NCP) compared to mixed plaque (MP) and calcified plaque (CP) (p < 0.05). In the multinomial logistic regression analysis, NLR was found to be an independent predictor of NCP, MP and CP (p < 0.001).

Conclusions

These data show that NLR is associated with both the severity and morphology of coronary atherosclerotic disease.

INTRODUCTION

Coronary artery disease (CAD), the leading cause of mortality worldwide, places a serious economic burden on healthcare systems. CAD is mainly due to atherosclerosis, an inflammatory process that is based on the interaction between immune mechanisms¹ and metabolic risk factors.² White blood cell count has been shown to be an independent indicator for adverse cardiovascular events and all-cause mortality.³ Neutrophil-to-lymphocyte ratio (NLR), calculated as the ratio of absolute neutrophil count and absolute lymphocyte count, has recently been considered as a potential marker for identifying individuals with a risk of cardiovascular disease (CVD) and associated events.^4,5 Patients with suspected but no prior diagnosis of CAD can be accurately evaluated with multidetector coronary tomography (MDCT), which is an established, quantitative and objective non-invasive method of assessing the presence, extent and severity of coronary atherosclerosis. This modality also generates additional information regarding plaque morphology and composition.^6,7 In this study, we aimed to demonstrate the relationship between NLR and the severity and morphology of coronary atherosclerotic disease detected by MDCT.

METHODS

Study population

This retrospective study was performed on a group of patients with stable angina who were admitted to our outpatient clinics, in whom coronary MDCT was performed following a detailed clinical and laboratory evaluation. Patients with a history of CAD, renal dysfunction (serum creatinine ≥ 1.5 mg/dL), hepatic disorders, malignancy, inflammatory disease, infection, and surgery or trauma within the previous six months were excluded. The study was approved by the local ethics committee and informed consent was received from each participant (HEK 08/181).

Baseline definitions and risk factor assessment

The recorded clinical characteristics of the patients included age, gender, anthropometric measurements, risk factors for CAD, comorbidities and medications. The presence of risk factors for atherosclerosis was derived from the patient’s chart and/or direct assessment. Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg or currently receiving antihypertensive treatment. Dyslipidemia was defined as total cholesterol ≥ 200 mg/dL, low-density lipoprotein (LDL) cholesterol ≥ 130 mg/dL, high-density lipoprotein (HDL) cholesterol < 30 mg/dL or currently receiving lipid modifying agents. Diabetes mellitus was defined as fasting glucose ≥ 126 mg/dL or currently receiving hypoglycemic treatment. Smoking was classified as current smoking if the patient smoked or quit in the last 30 days, or not smoking if the patient never smoked or smoked in the remote past. A family history of premature CAD was considered present if a first degree relative suffered an acute coronary syndrome (ACS) or was subjected to coronary revascularization prior to age 55 for men and age 65 for women.

Biochemical and hematologic measurements

Samples of peripheral venous blood were drawn from the antecubital vein for routine laboratory analysis following overnight fasting, including complete blood count and serum biochemistry tests (liver enzymes, kidney function tests and lipid profile). Total and differential leukocyte counts (neutrophils, eosinophils, basophils, lymphocytes and monocytes) and serum biochemistry tests were determined using a Beckman Coulter® High Wycombe, UK Gen-S automated analyzer and an assay kit (Hitachi® P800, Holliston, Massachusetts, USA), respectively.

Computed tomography (CT) imaging protocol

All scans were performed on a dual-source 64-slice CT scanner [Leonardo (Siemens Inc, Erlangen, Germany)],⁸ and breath holding was utilized to minimize motion artifact. Computed tomographic angiography (CTA) was performed according to the following protocol: detector collimation, 32 mm × 0.6 mm; slice acquisition, 64 mm × 0.6 mm; gantry rotation time, 33 s; temporal resolution, 83 ms; pitch, 0.2-0.47 adapted to the heart rate; tube current, 390 mAs per rotation; and tube potential, 100-140 kV depending on the patient’s weight. Beta-blockers were not given before scanning due to the enhanced temporal resolution of the scanner. Before injection of iodinated contrast for the performance of coronary CTA, sublingual nitrate [5 mg of isosorbide dinitrate, (Isordil^®, Fako, İstanbul, Turkey)] was administered to dilate the coronary arteries. The coronary angiographic scan was obtained following the injection of 80 mL of nonionic contrast medium (350 mg I/mL Iomeprol, Iomeron^®, Bracco, Milan, Italy) (adjusted for body weight 1.25 cc/kg) at a flow rate of 5 mL/sec followed by 50 mL of saline solution with the same injection rate to wash out the contrast material from the right ventricle. Contrast administration was controlled with bolus tracking, and scanning time was approximately 5.7-8.4 sec, depending on the cardiac dimensions and pitch. Reconstructed stacked short-axis images were obtained via filtered-back projection, spanning from the bronchial carina to the diaphragm. Electrocardiography (ECG) tube-current modulation (ECG gating) for radiation dose reduction was used for all patients. Retrospective gating technique was used to synchronize data reconstruction with the ECG signal. Axial images were reconstructed with 0.75 mm slice thickness and a reconstruction increment of 0.5 mm. The reconstruction interval with the fewest motion artifacts was chosen and utilized for further analysis.

CT evaluation

All images were interpreted immediately after scanning by an experienced radiologist who was blinded to clinical variables. Normal coronary arteries were defined as coronary arteries with no stenosis causing lesions seen in MDCT. Coronary plaque was defined as any clearly discernible structure attributable to the coronary artery wall in at least two independent image planes. Non-significant coronary plaque was defined as lesions causing ≤ 50% luminal narrowing, and significant coronary plaque was defined as lesions causing > 50% luminal narrowing. For categorization of the coronary plaques, the coronary system was divided into 16 separate segments based on a modified American Heart Association classification.⁹ Original axial images of thin slice (5 mm) and maximal intensity projections and cross-sectional reconstructions orthogonal to the long axis of each coronary segment (0.75 mm thickness) were acquired. Each segment was categorized associated with the morphology of the atherosclerotic plaque, namely: 1) none, 2) calcified (defined as lesion density > 130 Hounsfield units), 3) non-calcified (defined as lesion density < 130 Hounsfield units), 4) and mixed (having both calcified and non-calcified components)¹⁰ (Figure 1). Regarding the predominant plaque morphology in all segments, the patient was said to have non-calcified plaques (NCP), mixed plaques (MP) or calcified plaques (CP).

Figure 1.

Contrast-enhanced multidetector computed tomographic image of (A) significant (> 50% luminal stenosis), non-calcified plaque at proximal left anterior descending artery (LAD) (arrow); (B) nonsignificant, mixed plaque at the middle segment of intermediate artery (arrow); (C) significant diffuse calcified plaque at the proximal segment, non-significant calcified plaque at the middle LAD and nonsignificant calcified plaque at proximal right coronary artery (arrows).

Statistical analysis

Normally distributed parameters were presented as mean ± standard deviation and skewed parameters are expressed as median (interquartile range). Descriptive data are presented as frequencies and compared using chi-square test. Univariate analyses were performed on continuous variables with the use of the independent Student’s t-test for normally distributed variables, and the Mann-Whitney U test for non-normally distributed data. The ANOVA and Kruskal-Wallis tests were used for comparing more than two groups on continuous variable with normal and non-normal distribution, respectively. To determine independent predictors of severity and morphology of CAD, binary and multinomial logistic regression analyses were performed, respectively. Receiver operating characteristic curve analysis was used to determine the optimum cut-off levels of NLR value in predicting the presence of significant coronary atherosclerotic plaque using MDCT. Statistical analyses were performed using SPSS statistical software (version 20.0; SPSS Inc., Chicago, Illinois, USA). A two tailed p < 0.05 was considered statistically significant.

RESULTS

Our study population consisted of 684 subjects, with a mean age 56.12 ± 11.47 years, and 47.4% men. Baseline demographic and clinical characteristics of subjects are shown in Table 1. Among the patients that were analyzed, 62.57% had hypertension, 18.13% had diabetes mellitus, 28% were smokers, and 12.43% had a history of premature CAD in their family.

Table 1. Baseline demographic and clinical characteristics of patients regarding the severity of coronary atherosclerotic disease (n = 684).

	Non-critical stenosis (n = 479)	Critical stenosis (n = 205)	p value
Clinical parameters
Age (years)	54.65 ± 11.67	59.54 ± 10.22	< 0.001
Male gender (%)	41.54	60.98	< 0.001
BMI (kg/m2)	27.30 ± 4.97	27.03 ± 4.66	NS
Hypertension (%)	61.38	65.37	NS
Diabetes mellitus (%)	14.20	27.32	< 0.001
Dyslipidemia (%)	52.82	62.44	NS
History of smoking (%)	24.63	36.27	< 0.05
History of premature CAD (%)	10.44	17.07	< 0.05
Laboratory parameters
Total cholesterol (mg/dL)	207.15 ± 43.82	208.47 ± 45.98	NS
Triglyceride (mg/dL)	132 (96-175)	150 (101-200)	< 0.05
HDL cholesterol (mg/dL)	50.40 (42-62)	49 (41-57.5)	NS
LDL cholesterol (mg/dL)	131 (105-156)	130.50 (107.25-157.75)	NS
Serum creatinine (mg/dL)	0.83 (0.71-0.96)	0.85 (0.74-1.00)	NS
WBC count (/μL)	6800 (5700-7900)	7700 (6400-8800)	< 0.05
NLR	1.86 (1.50-2.38)	2.40 (1.98-3.07)	< 0.001
Medications
Acetylsalicylic acid (%)	49.89	54.90	NS
Statin (%)	33.54	34.31	NS
Beta blocker (%)	24.63	33.82	< 0.05
Calcium channel blocker (%)	11.79	14.71	NS
ACE inhibitor (%)	14.32	16.67	NS
Angiotensin receptor blocker (%)	27.58	25.98	NS

ACE, angiotensin-converting enzyme; BMI, body mass index; CAD, coronary artery disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NLR, neutrophil-to-lymphocyte ratio; NS, non-significant; WBC, white blood cell.

Critical coronary plaques were detected in 205/684 (30%) of subjects. The baseline characteristics of the two groups classified according to the severity of coronary atherosclerotic lesions detected by MDCT are shown in Table 1. BMI, total cholesterol, HDL cholesterol, LDL cholesterol and serum creatinine levels were similar between the two groups. No significant differences were detected between the groups regarding history of hypertension or dyslipidemia. Subjects with critical stenosis were older (59.54 ± 10.22 vs. 54.65 ± 11.67, p < 0.001) and predominantly male (60.98% vs. 41.54, p < 0.001). Diabetes mellitus (27.32% vs. 14.20%, p < 0.001), history of premature CAD (17.07% vs. 10.44%, p < 0.05) and history of smoking (36.27% vs. 24.63%, p < 0.05) were more prevalent in subjects with critical stenosis. White blood cell (WBC) count and NLR were found to be higher in patients with critical stenosis than in those without [7700 (6400-8800) vs. 6800 (5700-7900), p < 0.05 and 2.40 (1.98-3.07) vs. 1.86 (1.50-2.38), p < 0.001, respectively].

We also analyzed the association between NLR and the morphology of the atherosclerotic plaque. 431 (63.01%) patients having atherosclerotic plaques in their coronary arteries were stratified by the morphology of the plaque as having primarily (defined as > 70% of all segments) NCP, MP or CP. The percentages of patients having primarily NCP, MP and CP were 49.88%, 22.74% and 27.38%, respectively (Table 2). NLR was significantly higher in patients with NCP, MP and CP, irrespective of the plaque morphology, compared to those with normal coronary arteries [2.45 (1.95-3.12), 2.19 (1.71-2.73), 2.01 (1.58-2.42) and 1.72 (1.32-2.08) respectively, p < 0.001].

Table 2. Baseline characteristics of patients regarding the morphology of atherosclerotic disease.

	Normal (n = 253)	Non-calcified plaque (n = 215)	Mixed plaque (n = 98)	Calcified plaque (n = 118)	p value
Age (years)	51.47 ± 10.98	58.70 ± 9.67	57.22 ± 11.29	60.43 ± 12.36	< 0.001
Gender (male) (%)	42.69	58.14	37.76	45.76	< 0.050
BMI (kg/m2)	26.98 ± 5.33	27.65 ± 4.42	27.02 ± 4.98	27.09 ± 4.57	NS
Hypertension (%)	53.36	65.58	66.33	73.73	0.001
Diabetes mellitus (%)	11.07	22.79	23.47	20.34	< 0.05
Dyslipidemia (%)	48.22	60.00	56.12	63.56	< 0.05
History of smoking (%)	26.88	33.64	27.55	21.19	NS
History of premature CAD (%)	11.07	12.09	14.29	14.41	NS
Total cholesterol (mg/dL)	205.02 ± 40.34	207.59 ± 46.70	205.96 ± 38.70	214.50 ± 52.60	NS
Triglyceride (mg/dL)	133 (101-176)	140 (100-190)	133 (100-175)	125 (91-177)	NS
HDL cholesterol (mg/dL)	50 (42-62)	47.50 (41-57)	52 (43-62)0	53.50 (42-62)	< 0.05
LDL cholesterol (mg/dL)	130 (107-153)	131 (104-159)	131 (103-151)	133 (108-164)	NS
Serum creatinine (mg/dL)	0.84 ± 0.19	0.88 ± 0.18	0.82 ± 0.16	0.89 ± 0.52	NS
WBC count (/μL)	6800 (5600-7800)	7400 (6300-8700)	7300 (6100-8700)	7100 (5900-8425)	< 0.05
NLR	1.72 (1.32-2.08)	2.45 (1.95-3.12)	2.19 (1.71-2.73)	2.01 (1.58-2.42)	< 0.001
Medications
Acetylsalicilic acid (%)	41.50	55.87	60.82	56.90	0.001
Statin (%)	25.79	41.04	34.02	37.61	< 0.05
Beta blocker (%)	22.13	28.17	31.96	33.62	NS
Calcium channel blocker (%)	7.91	16.43	11.34	17.24	< 0.05
ACE inhibitor (%)	14.62	15.96	14.43	14.66	NS
Angiotensin receptor blocker (%)	24.11	29.58	25.77	30.12	NS

ACE, angiotensin-converting enzyme; BMI, body mass index; CAD, coronary artery disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NLR, neutrophil-to-lymphocyte ratio; NS, non-significant; WBC, white blood cell.

NLR levels differed among atherosclerotic plaque morphology subtypes (p < 0.05). NLR was found to be significantly higher in NCP compared to other subtypes (p < 0.05 for NCP vs. MP and p < 0.001 for NCP vs. CP) (Figure 2).

Figure 2.

Comparison of neutrophil-to-lymphocyte ratios of patients according to coronary plaque morphology.

In the binary logistic regression analysis, NLR was found to be an independent predictor of severity of CAD detected by MDCT [odds ratio (OR): 1.68; 95% confidence interval (CI) 1.39-2.03, p < 0.001] (Table 3). Apart from NLR, age (OR: 1.04; 95% CI 1.03-1.06, p < 0.001), male gender (OR: 2.23; 95% CI, 1.49-3.33, p < 0.001), history of diabetes mellitus (OR: 1.74; 95% CI 1.09-2.77, p < 0.05), history of smoking (OR: 1.59; 95% CI 1.06-2.39, p < 0.05) and history of premature CAD (OR: 1.90; 95% CI 1.09-3.32, p < 0.05) remained significant predictors of the severity of CAD following adjustment of other cardiovascular risk factors (Table 3).

Table 3. Binary logistic regression analysis demonstrating the association between cardiovascular risk factors and critical stenosis detected by MDCT.

Variable	OR (95% CI)	p value
Age (years)	1.04 (1.03-1.06)	< 0.001
Male gender	2.23 (1.49-3.33)	< 0.001
Hyperlipidemia	1.35 (0.90-2.00)	NS
History of smoking	1.59 (1.06-2.39)	< 0.05
Diabetes mellitus	1.74 (1.09-2.77)	< 0.05
History of premature CAD	1.90 (1.09-3.32)	< 0.05
Triglyceride (mg/dL)	1.00 (1.00-1.01)	NS
WBC count (/μL)	1.00 (1.00-1.00)	NS
NLR	1.68 (1.39-2.03)	< 0.001

CAD, coronary artery disease; NLR, neutrophil-to-lymphocyte ratio; WBC, white blood cell.

In the multinomial regression analysis, NLR was found to be an independent predictor of NCP, MP and CP (OR: 2.70; 95% CI, 2.04-3.59, p < 0.001; OR: 2.05, 95% CI, 1.49-2.83, p < 0.001; OR: 1.59, 95% CI, 1.14-2.22, p < 0.001, respectively) (Table 4). On receiver-operating characteristics curve analysis, a NLR value of > 1.95 had 76.6% sensitivity and 58% specificity (area under the curve 0.70, 95% CI 0.664-0.734, p < 0.001) in predicting the presence of significant coronary atherosclerotic plaque using MDCT (Figure 3).

Table 4. Multinomial logistic regression analysis demonstrating the association between cardiovascular risk factors and the morphology of CAD.

Variable	Non-calcified plaque		Mixed plaque		Calcified plaque
	OR (95% CI)	p value	OR (95% CI)	p value	OR (95% CI)	p value
Age (years)	1.06 (1.04-1.08)	< 0.001	1.04 (1.01-1.06)	< 0.05	1.06 (1.04-1.08)	< 0.001
Male gender	2.16 (1.39-3.37)	0.001	1.13 (0.66-1.92)	NS	1.62 (0.97-2.70)	NS
Dyslipidemia	1.74 (1.10-2.73)	< 0.05	1.33 (0.78-2.82)	NS	1.78 (1.05-2.99)	< 0.05
HDL (mg/dL)	0.99 (0.97-1.00)	NS	1.01 (0.99-1.03)	NS	1.00 (0.98-1.02)	NS
Hypertension	1.21 (0.76-1.93)	NS	1.19 (0.69-2.07)	NS	1.62 (0.93-2.84)	NS
Diabetes mellitus	1.36 (0.76-2.44)	NS	1.72 (0.87-3.40)	NS	1.46 (0.75-2.82)	NS
WBC count (/μL)	1.00 (1.00-1.00)	NS	1.00 (1.00-1.00)	NS	1.00 (1.00-1.00)	NS
NLR	2.70 (2.04-3.59)	< 0.001	2.05 (1.49-2.83)	< 0.001	1.59 (1.14-2.22)	< 0.001

HDL, high-density lipoprotein cholesterol; NLR, neutrophil-to-lymphocyte ratio; WBC, white blood cell.

Figure 3.

Receiver-operating characteristics curve analysis showing that the NLR value of > 1.95 had 76.6% sensitivity and 58% specificity (area under the curve 0.70, 95% CI 0.664-0.734, p < 0.001) in predicting the presence of significant coronary atherosclerotic plaque using MDCT.

DISCUSSION

NLR has raised interest as a potential biomarker for identifying patients at high cardiovascular risk. Our study found that higher NLR levels were independently associated with the severity and non-calcified morphology of coronary atherosclerotic plaques detected by MDCT apart from other well-known cardiovascular risk factors, and that this relationship persisted after adjustment for multiple cardiovascular risk factors.

Some evidence has indicated that there was a consistent association of WBC count with the cardiovascular disease and adverse cardiovascular outcomes.^3,11,12 In a previous study, it has been shown that total WBC count was associated with the presence, severity and extent of coronary atherosclerosis detected by MDCT.¹³ It has been concluded that WBC count is an independent predictor of CVD-associated and all-cause mortality, and can determine high-risk patients who are not identified by established CVD risk factors.³ Dragu et al. have shown that among all WBC subtypes (neutrophils, monocytes, lymphocytes), elevated neutrophil count best correlates with mortality in patients with acute myocardial infarction (AMI).^14,15

Neutrophil/lymphocyte ratio (NLR), calculated as the ratio of absolute neutrophil count and absolute lymphocyte count, has recently been considered as a potential marker for identifying individuals with a risk of CVD and associated events. It is reported that NLR is better at predicting CVD-associated events than WBC count.¹⁶ The work of Arbel et al. demonstrated for the first time that NLR was independently associated with CAD severity in patients undergoing conventional coronary angiography.¹⁷ In patients with clinically suspected CAD and no history of myocardial infarction, who were evaluated with conventional coronary angiography and followed up for a definite time course, it was shown that higher NLR levels independently predicted mortality and future myocardial infarction risk.^18-20 NLR has also been associated with in-hospital major adverse cardiovascular events (MACE) and long-term mortality in patients with ST-segment elevated myocardial infarction undergoing primary percutaneous coronary intervention (PCI).^17,21

However, the inflammatory mechanisms that link CAD to leukocyte counts are poorly understood. Activated neutrophils release a variety of proteolytic enzymes such as myeloperoxidase, which are responsible for tissue injury.²² On the other hand, for patients diagnosed with ACS, complications have been associated with lower lymphocyte count,²³ which has been explained by elevated cortisol levels that induce apoptosis.²⁴

In our study, NLR levels were found to be higher in patients with critical stenosis than without. This result is consistent with other studies that have shown the relationship between NLR levels and CAD severity. NLR levels were also significantly higher in NCP compared to MP and CP. Since the non-calcified type of plaque morphology is known to be susceptible to rupture in nature, and cause coronary artery thrombosis, the association found between NCP and NLR may explain the relationship between higher NLR levels and worse outcomes reported in previous studies.

Study limitations

There were several limitations of this study. First of all, because of the cross-sectional study design, the possible cause and effect relation between NLR levels and severity and morphology of coronary atherosclerotic disease remains unknown. Additionally, due to its retrospective design, this study lacks the capacity to demonstrate the role of NLR levels on cardiovascular outcomes. Another major limitation is that values of commonly used biomarkers for atherosclerosis, such as hs-CRP, have not been included in our study. Previously, Arbel et al. have demonstrated that NLR provided supplementary information on CAD severity and CVD outcome after controlling for hs-CRP.¹⁷

CONCLUSIONS

In the coming years, CAD is expected to be the main cause of mortality globally, as it has been in the last decades. Therefore, new strategies should be developed for the prediction, prevention and treatment of CAD. NLR is a simple and inexpensive marker that can help identifying individuals with severe CAD or those at risk of developing ACS. Ultimately, NLR may prove to be a novel component of an important risk stratification scoring system, which could positively impact the medical community’s ability to diagnose and treat CAD around the world.

CONFLICT OF INTEREST

The author(s) received no financial support for the research, authorship and/or publication of this article. The authors declare that there is no conflict of interest.

FUNDING

None declared.