Monocyte/lymphocyte ratio is associated with carotid stenosis in ischemic stroke: A retrospective analysis

Abstract Background Carotid artery stenosis, mainly caused by carotid atherosclerosis, is related to ischemic stroke. This study was to investigate whether monocyte/lymphocyte ratio (MLR) was associated with increased severity of carotid stenosis in patients with ischemic stroke. Methods A total of 395 participants with ischemic stroke were retrospectively analyzed. The severity of carotid stenosis was evaluated by ultrasound examination. Patients were divided into two groups: nonsevere stenosis group and severe stenosis group. Multivariate logistic analysis was used to evaluate risk factors. Results A significant correlation was found between MLR and the severity of carotid stenosis in patients with ischemic stroke. MLR was the independent risk factor of carotid stenosis (OR: 9.74, 95% CI: 1.16–81.54). In the ROC curves analysis, a cutoff value of 0.20 for MLR predicted the severity of carotid stenosis with a sensitivity of 80.40% and specificity of 26.40% (ROC area under the curve: 0.598, 95% CI: 0.53–0.67, p = .004). Conclusion Monocyte/lymphocyte ratio plays important roles in carotid stenosis in patients with ischemic stroke and is an independent risk factor of the severity of carotid stenosis. Therefore, MLR might be considered a potential index in the diagnosis of carotid stenosis in patients with ischemic stroke.

diseases associated with a long-term disability. Carotid artery stenosis is closely related to ischemic stroke (Eckstein et al., 2013;Gupta et al., 2012;Lu & Wang, 2016) and is mainly caused by carotid atherosclerosis (Adams, Bojara, & Schunk, 2018;Liu et al., 2017) because of embolic sequelae as a consequence of underlying atherosclerosis which can incur/ cause secondary hemodynamic disturbances. The role of inflammation in the pathogenesis of carotid atherosclerosis has been well established (Borne et al., 2017;Casiglia & Tikhonoff, 2007), and the role of various inflammatory cells and mediators involved in carotid disease and stroke should be well investigated in order to better predict/ identify at-risk patients (Ammirati, Moroni, Norata, Magnoni, & Camici, 2015;Casiglia & Tikhonoff, 2007;Horne et al., 2005;Woollard & Geissmann, 2010).
The increasing number of leukocyte subtypes, such as eosinophils, neutrophils, and monocytes, is associated with adverse cardiovascular and cerebrovascular events and carotid stenosis (Gijsberts et al., 2017;Hyun et al., 2015;Nunez et al., 2009;Vakili et al., 2017). Monocyte/ lymphocyte ratio (MLR, monocyte count/lymphocyte count), neutrophil/lymphocyte ratio (NLR, neutrophil count/lymphocyte count), and platelet/lymphocyte ratio (PLR, platelet count/lymphocyte count) are indicators reflecting the degree of inflammation (Gijsberts et al., 2017;Hyun et al., 2015;Vakili et al., 2017). These parameters or indices are derived from calculations formulated from results obtained from a routine blood analysis; the predictive value of these results obtained from laboratory testing provides a relatively noninvasive and accessible means of obtaining information and profiling individual patients in a time-sensitive manner in the workup for patient with stroke. In 2015, Hyun et al. (2015) found that NLR may be a clinically significant predictor of the degree of carotid stenosis in patients with ischemic stroke.
Inflammation plays critical role in the pathogenesis of carotid atherosclerosis in the pathogenesis of carotid atherosclerosis, and the increase in monocyte number in blood routine is positively related to carotid stenosis (Tanaka et al., 2016). A lymphocyte count decrease was found in cardiovascular and cerebrovascular events, suggesting a poor prognosis (Nunez et al., 2009). Therefore, MLR, closely related to monocytes number and lymphocytes number, as an indicator reflecting the degree of inflammation, its role in carotid artery stenosis in patients with ischemic stroke should be studied at present. Thus, we hypothesized that MLR might be related to the severity of carotid artery stenosis and could be a potential index in the diagnosis of carotid artery stenosis in patients with ischemic stroke. This study mainly aimed at investigating whether MLR was related to the severity of carotid artery stenosis in patients with ischemic stroke. We also investigated the relationship between PLR/NLR and carotid artery stenosis and compared the role of the aforementioned parameters in patients with carotid artery stenosis and ischemic stroke.

| Study population
A total number of 395 participants with ischemic stroke who were treated in the neurology department of Peking University People's Hospital from January 2013 to December 2015 were retrospectively analyzed. Ischemic stroke was diagnosed according to the diagnostic criteria of the ischemic stroke revised by the Fourth National Conference on cerebrovascular diseases (Neurology Branch of Chinese Medicine Association, 1996). Exclusion criteria were the following: 3 days after the onset of disease, chronic inflammatory disease, or any systemic infection, severe hepatic and renal insufficiency, tumors, or immunosuppressive disease; patients under immunosuppressants and hormones therapy; and diseases of the blood, digestive or endocrine system. For patients screened for these conditions above, we selected based on medical history, related blood tests, and routine examinations, such as abdominal ultrasound, chest X-ray, or chest CT. This study was approved by the ethics committee of Peking University People's Hospital, and written informed consent was obtained from all patients.

| Data collection
After admission, 10 ml of venous blood were collected after fasting for 10 hr and analyzed for leukocyte count and basic biochemical clinical data, including fasting plasma glucose and uric acid using an automatic blood analyzer. Patients' demographic data, such as medical history, personal history, and family history, were acquired, while the diagnosis of hypertension and diabetes was determined accord-

| Carotid ultrasonography
Carotid ultrasonography was carried out by qualified ultrasound technologists, and all protocols were performed according to the Chinese stroke vascular ultrasound examination guidelines. Each common carotid artery, internal carotid artery, external carotid artery, and bulb were examined and recorded for the presence of atherosclerotic plaques in the longitudinal and transverse planes. The severity of carotid artery stenosis was assessed according to the criteria recommended by a multidisciplinary panel of the Society of Radiologists in Ultrasound (Grant et al., 2003). Chinese guidelines for the management of carotid artery stenosis (Vascular Surgery Group, 2017) suggest to re-examine and monitor the development of carotid artery stenosis in patients with carotid artery stenosis was not <50%. According to this suggestion, thus, stenosis severity was classified into nonsevere stenosis (stenosis < 50%) and severe stenosis (stenosis ≥ 50%) and patients were divided into two groups: nonsevere stenosis group (n = 298) and severe stenosis group (n = 97) according to this classification.

| Statistical analysis
SPSS16.0 software was used for statistical analysis. Continuous variables were expressed as mean ± SD or median (interquartile range) when appropriate. Discrete variables were presented as percentages; the intergroup differences were analyzed using variance test for normally distributed measurement data, while rank sum test was used for non-normally distributed data; the intergroup differences were analyzed using chi-square test or Fisher's exact test. Univariate logistic regression analysis was performed in all clinical variables, and results with p < .05 were considered statistically significant. Variables included in logistic regression analysis were determined based on the results above and professional knowledge with p < .05 as statistical significance to comprehensively analyze stenosis risk factors of carotid arteries. ROC curve of MLR was developed to calculate the area under the curve and the 95% confidence interval. Since MLR was mainly used to screen patients with severe carotid stenosis, optimal operating point (OOP) was determined as the cut point to obtain a sensitivity of at least 80% and maximize specificity (Critschristoph, Muenz, & Tu, 2003). Two-sided difference with p < .05 was considered statistically significant.  Table S1). Table S2 shows the correlation between MLR and other inflammatory indexes from blood routine examination, which indicated that MLR could reflect the inflammation degree and should be further studied (see details in Table S2).

| RE SULTS
The population was divided into two groups according to the MLR median (0.25) in Table 1

| D ISCUSS I ON
Monocyte/lymphocyte ratio, calculated from a blood routine analysis, is inexpensive, simple, safe, and noninvasive. Our study confirmed its potential as a good inflammatory index and its strong correlation with white blood cells, neutrophils, and NLR. Therefore, the clinical utility of these indices could prove informative in expanding our knowledge on the biochemical profiles of patients with TIA or stroke. High MLR values indicate a poor prognosis of multiple malignancies. Indeed, MLR was initially applied as a predictor of lymphoma, nasopharyngeal carcinoma, and bladder, lung, and breast cancer (Li et al., 2012;Ni et al., 2014;Temraz et al., 2014;Teng, Zhang, Zhang, Zhang, & Li, 2016). In addition, MLR was valuable in the diagnosis of infectious diseases such as malaria, AIDS, and tuberculosis (Naranbhai, Hill, et al., 2014;Naranbhai, Kim, et al., 2014;Warimwe et al., 2013). Therefore, we focused our attention on MLR to evaluate its potential as a diagnostic marker. Li et al found that MLR was closely related to the degree of coronary plaque lesions in patients with non-ST-elevated myocardial infarction (Ji et al., 2017), suggesting that MLR may be an indicator of atherosclerosis.
To the best of our knowledge, no reports are available regarding the relationship between MLR and the degree of carotid stenosis.
Therefore, this study mainly focused on and demonstrated the relationship between MLR and carotid artery stenosis in ischemic stroke patients. Our study found that MLR had a positive correlation with the degree of carotid artery stenosis in ischemic stroke patients, and it was an independent factor, suggesting that MLR might be a potential diagnostic marker for the severity of carotid artery stenosis.
Carotid artery stenosis development and occurrence is due to a long-term chronic inflammatory process. Lymphocytes and mononu-   (Casiglia & Tikhonoff, 2007;Incalcaterra et al., 2013). The lymphocyte levels represent the level of cellular and humoral immunity in vivo, which is inversely proportional to the progress of atherosclerosis. In particular, regulatory T lymphocytes have significant anti-inflammatory effects. High monocyte counts and low lymphocyte counts were independent risk factors of cerebrovascular disease (Nunez et al., 2009;Tanaka et al., 2016). Therefore, MLR, such as the integration of monocytes and lymphocytes into a single index, might be a potential indicator of the severity of the atherosclerotic plaque. The results of this study confirmed that MLR was an independent risk factor for carotid artery stenosis, which was consistent with our expectations. Compared with NLR, MLR may more significantly influence on carotid artery stenosis. Horne et al. (2005) found that NLR was significantly and positively associated with the maximum carotid intima-media thickening value in men in a retrospective study with 252 patients. NLR may be a predictor of the severity of carotid stenosis in patients with ischemic stroke, which indicates that NLR, as well as MLR, is really worthy of studying, as other biomarkers assessing carotid stenosis are useful, but costly and complicated. This study showed that as well as NLR, MLR could be considered a potential clinical index. Except the reason above that MLR might reflect well on the progress of atherosclerosis and the severity of the atherosclerotic plaque, the difference on sample size, race of study population, and the variables included may also lead to the difference of the two studies.
The pathological mechanism might be related to the following processes: (a) the monocytes in the patients with high MLR level might produce a large number of inflammatory mediators to accelerate the inflammatory response, thereby promoting lipid deposition and plaque formation in the vascular walls. (b) Inflammatory cytokines released from inflammatory cells in patients with high MLR level might promote excessive proliferation of smooth muscle cells, thus promoting atherosclerosis (Gupta et al., 2012;Li et al., 2012). In this study, we found that MLR was the independent risk factor of the severity of carotid artery stenosis, while NLR was not. As we aforementioned, lymphocyte and mononuclear cells are the main inflammatory cells in the formation of carotid atherosclerotic plaque and carotid artery stenosis. Therefore, MLR, combined with lymphocyte count and monocyte count, might reflect the inflammatory state of carotid stenosis. MLR could be a potential index to predict the progression of carotid stenosis in patient with ischemic stroke.
Our study has some limitations. First, only MLR baseline measurements were collected, and MLR values over time were not obtained. More information should be collected in future to get more reliable conclusion. Second, this study is a retrospective single-center study, a prospective, and multi-center cross-sectional research should be designed in future. Third, the relationship between MLR and the incidence of MACCE (major adverse cardiac and cerebral vascular events) and death was not explored in this study. Thus, further study is needed to be performed in the future.
In conclusion, our study found that MLR was significantly correlated with the severity of carotid artery stenosis and was an independent risk factor. In addition, the combination of blood routine TA B L E 4 Potential interactions between risk factors on severity of carotid stenosis examination and carotid artery ultrasound examination has a potential clinical value in the diagnosis of carotid artery stenosis and being potentially useful to screen carotid stenosis in patients with carotid artery stenosis.

ACK N OWLED G M ENTS
We thank sincerely all patients and researchers contributing to this study.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.