atherosclerotic lesions are the leading cause of ischemic stroke worldwide.1 ) the primary factor in the pathogenesis of atherosclerosis is inflammation .
development and progression of an atherosclerotic plaque is mainly regulated by inflammatory and immune mechanisms.2 ) the risk of rupture of atherosclerotic plaques is more serious than the severity of stenosis caused by plaques.3 ) detection of early neutrophil infiltration in the atherosclerotic plaques allows prediction of the risk of plaque rupture.4 ) histopathological examinations of carotid artery plaques demonstrated that the rupture - prone lesions are associated with higher neutrophil count.5 ) in a study investigating the carotid endarterectomy ( cea ) specimens , coexistence of neutrophil infiltration and hemorrhage into the plaque was demonstrated particularly in the culprit zone of the carotid plaque.6 ) previous studies have shown that lymphopenia is one of the early markers that develop secondary to stress - related cortisol release in patients who have experienced a myocardial infarction.7 ) there is a negative correlation between lymphocyte count and cardiovascular prognosis.8)9 ) currently , neutrophil / lymphocyte ratio ( nlr ) is considered to be a good marker that simultaneously shows the negative effects of neutrophil elevation as an indicator of acute inflammation and lymphocyte depletion as an indicator of physiological stress , as well as it helps prediction of mortality and prognosis of stroke patients.10)11 ) plaque morphology is an independent risk factor for prognosis in coronary artery disease patients .
after 78-months of follow - up , mortality rate among patients with calcified coronary plaques , mixed plaques and non - calcified plaques was shown to be 1.4% , 3.3% and 9.6% , respectively.12)13 ) coronary computed tomography angiography can provide reliable information on plaque morphology.14 ) embolism and stroke risks associated with calcified and non - calcified plaques in the carotid artery are different .
histopathological examinations performed after cea have demonstrated that calcified plaques are more stable , and they carry a lower risk of rupture and thromboembolism.15 ) on the other hand , non - calcified and ulcerous plaques are more fragile and they carry a higher risk of rupture ; therefore they are associated with increased risk of embolism and stroke.16 ) severity of a carotid artery lesion is an important parameter that affects the risk of stroke .
the risk of stroke increases with the severity of carotid artery lesion.17 ) in order to perform carotid artery intervention , the level of stenosis should angiographically be at least 50% in symptomatic patients and at least 70% in asymptomatic patients.18 ) in symptomatic patients , a plaque that causes 50% stenosis in the carotid artery may pose a risk of recurrent stroke and requires surgical intervention or placement of a stent .
there is currently no test that can be used to predict the risk of stroke in asymptomatic patients with intermediate ( 50 - 70% ) carotid artery stenosis .
we believe that non - calcified plaques that cause asymptomatic intermediate carotid artery stenosis are more active in terms of inflammation and subsequent atherothrombosis ; therefore they may be associated with a higher risk of stroke than calcified plaques .
the present study compares calcified and non - calcified plaques that cause asymptomatic intermediate carotid artery stenosis with respect to total neutrophil count , lymphocyte count and nlr .
twenty - five patients were excluded from the study due to various reasons and the analyses were performed on the data obtained from 139 patients ( fig .
the patients were divided into two groups , as those having calcified ( 73 patients ) and non - calcified ( 66 patients ) plaques .
patients with 50 - 70% stenosis of the carotid artery were included in the study . initially carotid doppler ultrasound ( cdus ) and then computed tomography angiography ( cta ) were performed in all patients . the study cohort consisted of patients who had been assessed by a team that included a neurologist , a cardiologist , a radiologist and a cardiovascular surgeon and who had been confirmed to be asymptomatic with respect to the carotid artery lesion .
symptomatic patients were defined as those who experienced an ischemic cerebrovascular event with or without a sequel , a transient ischemic attack or amaurosis fugax within the last 6 months , and they were excluded from the study .
patients with diabetes mellitus were examined by an endocrinologist and they were included in the study after their blood glucose levels had been regulated .
patients were included in the study at least two months after initiation of antiaggregant , antihyperlipidemic and antihypertensive therapies .
laboratory parameters that have been analyzed in this study represent the mean value of two measurements taken at different time points by using the same device .
patients with a history of ischemic or non - ischemic stroke , systemic inflammatory disease , cancer , acute coronary syndrome , previous myocardial infarction , heart failure , significant valvular disease , chronic obstructive pulmonary disease , renal or liver failure , an hematological disease ; patients whose body temperature was 37 ; patients who had an active infection , white blood cell count ( wbc ) > 12.000 per l ; and patients who were using anti - inflammatory drugs or antibiotics were excluded from the study .
fasting blood samples were drawn from a large antecubital vein of each patient for determination of biochemical and haemostatic parameters .
total cholesterol , low - density lipoprotein , triglyceride and high - density lipoprotein levels were measured by colorimetric method ( abbott laboratories , abbott park , il , usa ) .
hypertension was defined as a systolic / diastolic blood pressure of  140/90 mmhg ; or patients using antihypertensive medications .
diabetes mellitus was defined as a fasting plasma glucose level of 126 mg / dl or patients actively using oral antidiabetics and/or insulin .
coronary artery disease was defined angiographically as the presence of a plaque resulting in 50% or more stenosis in a major coronary artery .
body mass index ( bmi ) was calculated by dividing the body weight ( kg ) to the square of the height ( m ) .
cdus examination was performed using esaote s.p.a mylabclass c ( florence - italy ) device and a linear arrayed probe that allows selection of frequencies between 3 - 11 mhz .
cta was performed using a ct device with the philips brilliance 64 detector ( holland ) .
after venous access was established through the antecubital vein and 80 ml non - ionic contrast agent was administered at a rate of 4.5 ml / sec , axial - plane ct images of the carotid and cerebral arteries were obtained using the tracking method . acquired slices were transferred to the work - station ( philips intellispace portal ) and multi - plane images , maximum intensity projection and volume rendering 3-dimensional images were developed by post - processing the original slices via appropriate software ( ava ) .
the stenosis caused by plaques as observed using cdus was assessed according to the criteria developed by the internal carotid artery stenosis criteria consensus committee .
internal carotid artery plaque characteristics were evaluated by a 64 slice ct and plaque burden was evaluated according to the thickness of the plaque .
the measurement of the stenotic arterial lumen was performed where it was the narrowest and in the thicker region of the plaque .
the level of stenosis based on cta was evaluated according to the nascet ( north american symptomatic carotid endarterectomy trial ) criteria .
based on the cdus and cta examinations , plaques that were fully soft and calcified less than 50% were categorized as non - calcified plaques whereas those that were fully hard and calcified more than 50% were categorized as calcified plaques . in other words , hyperdense plaques ( containing more than 50% calcific component and a measured plaque density of more than 120 hounsfield units [ hu ] ) were regarded as calcified plaques , and hypodense plaques ( containing less than 50% calcific component and a measured plaque density of less than 120 hu ) were considered as non - calcified plaques.19 ) data were analyzed by the spss software version 15.0 for windows ( spss inc . ,
categorical variables were presented as frequency and percentage . the  test and fisher 's exact test
student 's t - test was used for variables with normal distribution and the values were presented as meansd .
continuous variables without normal distribution were analyzed using mann - whitney u test and obtained values were presented as median ( 50 ) values and interquartile ranges ( 25 and 75 ) .
receiver - operating characteristic ( roc ) curves were drawn with sensitivity and specificity analyses .
multivariate logistic regression analysis was used to evaluate the independent associates of the risk of non - calcified plaque .
the odds ratios ( or ) and 95% confidence intervals ( ci ) were calculated .
twenty - five patients were excluded from the study due to various reasons and the analyses were performed on the data obtained from 139 patients ( fig .
the patients were divided into two groups , as those having calcified ( 73 patients ) and non - calcified ( 66 patients ) plaques .
patients with 50 - 70% stenosis of the carotid artery were included in the study . initially carotid doppler ultrasound ( cdus ) and then computed tomography angiography ( cta ) were performed in all patients . the study cohort consisted of patients who had been assessed by a team that included a neurologist , a cardiologist , a radiologist and a cardiovascular surgeon and who had been confirmed to be asymptomatic with respect to the carotid artery lesion .
symptomatic patients were defined as those who experienced an ischemic cerebrovascular event with or without a sequel , a transient ischemic attack or amaurosis fugax within the last 6 months , and they were excluded from the study .
patients with diabetes mellitus were examined by an endocrinologist and they were included in the study after their blood glucose levels had been regulated .
patients were included in the study at least two months after initiation of antiaggregant , antihyperlipidemic and antihypertensive therapies .
laboratory parameters that have been analyzed in this study represent the mean value of two measurements taken at different time points by using the same device .
patients with a history of ischemic or non - ischemic stroke , systemic inflammatory disease , cancer , acute coronary syndrome , previous myocardial infarction , heart failure , significant valvular disease , chronic obstructive pulmonary disease , renal or liver failure , an hematological disease ; patients whose body temperature was 37 ; patients who had an active infection , white blood cell count ( wbc ) > 12.000 per l ; and patients who were using anti - inflammatory drugs or antibiotics were excluded from the study .
fasting blood samples were drawn from a large antecubital vein of each patient for determination of biochemical and haemostatic parameters .
total cholesterol , low - density lipoprotein , triglyceride and high - density lipoprotein levels were measured by colorimetric method ( abbott laboratories , abbott park , il , usa ) .
hypertension was defined as a systolic / diastolic blood pressure of  140/90 mmhg ; or patients using antihypertensive medications .
diabetes mellitus was defined as a fasting plasma glucose level of 126 mg / dl or patients actively using oral antidiabetics and/or insulin .
coronary artery disease was defined angiographically as the presence of a plaque resulting in 50% or more stenosis in a major coronary artery .
body mass index ( bmi ) was calculated by dividing the body weight ( kg ) to the square of the height ( m ) .
cdus examination was performed using esaote s.p.a mylabclass c ( florence - italy ) device and a linear arrayed probe that allows selection of frequencies between 3 - 11 mhz .
cta was performed using a ct device with the philips brilliance 64 detector ( holland ) .
after venous access was established through the antecubital vein and 80 ml non - ionic contrast agent was administered at a rate of 4.5 ml / sec , axial - plane ct images of the carotid and cerebral arteries were obtained using the tracking method .
acquired slices were transferred to the work - station ( philips intellispace portal ) and multi - plane images , maximum intensity projection and volume rendering 3-dimensional images were developed by post - processing the original slices via appropriate software ( ava ) .
the stenosis caused by plaques as observed using cdus was assessed according to the criteria developed by the internal carotid artery stenosis criteria consensus committee .
internal carotid artery plaque characteristics were evaluated by a 64 slice ct and plaque burden was evaluated according to the thickness of the plaque .
the measurement of the stenotic arterial lumen was performed where it was the narrowest and in the thicker region of the plaque .
the level of stenosis based on cta was evaluated according to the nascet ( north american symptomatic carotid endarterectomy trial ) criteria . based on the cdus and cta examinations , plaques that were fully soft and calcified less than 50% were categorized as non - calcified plaques whereas those that were fully hard and calcified more than 50% were categorized as calcified plaques . in other words , hyperdense plaques ( containing more than 50% calcific component and a measured plaque density of more than 120 hounsfield units [ hu ] ) were regarded as calcified plaques , and hypodense plaques ( containing less than 50% calcific component and a measured plaque density of less than 120 hu ) were considered as non - calcified plaques.19 )
data were analyzed by the spss software version 15.0 for windows ( spss inc . , chicago , il , usa ) .
student 's t - test was used for variables with normal distribution and the values were presented as meansd .
continuous variables without normal distribution were analyzed using mann - whitney u test and obtained values were presented as median ( 50 ) values and interquartile ranges ( 25 and 75 ) .
receiver - operating characteristic ( roc ) curves were drawn with sensitivity and specificity analyses .
multivariate logistic regression analysis was used to evaluate the independent associates of the risk of non - calcified plaque .
the odds ratios ( or ) and 95% confidence intervals ( ci ) were calculated . a two - tailed p value of < 0.05 was considered statistically significant .
table 1 shows the demographic and clinical characteristics in addition to laboratory results of 139 patients ( 73 with calcified plaques , 66 with non - calcified plaques ) who were included in the study .
no significant difference was noted between the two groups in terms of the baseline parameters .
mean hemoglobin level was 13.51.5 g / dl in the calcified plaque group and 12.81.4 g / dl in the non - calcified plaque group .
wbc count was 8.01.6 10/mm in the calcified plaque group and 7.61.8 10/mm in the non - calcified plaque group ; the difference between wbc counts in the two groups was not statistically significant ( p=0.667 ) . mean total neutrophil count in the calcified and non - calcified plaque groups was 4.8 10/mm and 5.2 10/mm , respectively .
the increase in total neutrophil count in the non - calcified plaque group did not reach the level of statistical significance ( p=0.227 ) .
mean total lymphocyte count in the calcified and non - calcified plaque groups was 2.3 10/mm and 2.1 10/mm , respectively ; and the difference between the two groups was statistically significant ( p=0.002 ) .
nlr was 2.1 in the calcified plaque group and 2.6 in the non - calcified plaque group , and comparison of nlr between the two groups demonstrated that it was statistically significantly higher in the non - calcified plaque group ( p<0.001 ) ( fig .
an nlr cut - off value of > 2.54 was found for calcified - non calcified plaque discrimination among carotid arteries plaques which caused intermediate stenosis ( area under curve [ auc ] : 0.699 , ci 95% 0.611 - 0.787 , p<0.001 ) .
nlr of > 2.54 , had 53% sensitivity and 84% specificity in calcified - non calcified plaque discrimination ( fig .
the multivariate logistic regression analysis including age , hypertension , diabetes mellitus , coronary artery disease , hyperlipidemia , statin use , bmi , acetylsalicylic acid ( asa ) use and nlr showed that the nlr was independently associated with non - calcified carotid plaques ( or 5.686 , 95% ci 2.498 - 12.944 , p<0.001 ) ( table 3 ) .
this is the first study comparing calcified and non - calcified plaques that cause asymptomatic intermediate carotid artery stenosis with respect to inflammatory parameters . in the present study ,
stenosis and plaques in the carotid artery were assessed by cdus and cta ; and the calcified plaque group was compared with the non - calcified plaque group in terms of total neutrophil count , lymphocyte count and the nlr .
the findings of this study indicate that among asymptomatic plaques that cause intermediate carotid artery stenosis , non - calcified plaques are associated with a lower total lymphocyte count and a nlr that is significantly higher than those observed in the presence of calcified plaques .
atherosclerosis and cardiovascular diseases are the most common causes of mortality worldwide.20 ) inflammation plays a role in every stage of atherosclerosis , including its development , progression and rupture of the atherosclerotic plaque.21 ) neutrophils have certain effects on plaque rupture , reperfusion damage and remodeling.6 ) nasr et al.22 ) have previously demonstrated an association between neutrophil count and cerebral microembolization in symptomatic patients with carotid artery stenosis .
ionita et al.23 ) have noted a correlation between the severity of carotid atherosclerotic lesion and basal neutrophil count . in their study , the number of macrophages was increased and neutrophil count was higher in the presence of rupture - prone atherosclerotic plaques that are characterized by low collagen content and smooth muscle cells .
previous studies have established that the activated neutrophil count in the circulation is higher in patients with coronary and carotid atherosclerotic plaques , and neutrophils play crucial roles in the development of atherosclerotic plaques as well as rupture of a pre - existing plaque.23 ) lymphopenia is the most common inflammatory marker that develops in response to increased corticosteroid level secondary to stress24 ) and it occurs due to the increased apoptosis of lymphocytes during inflammatory process.25 ) lymphopenia has been associated with mortality in patients who have experienced st - elevation myocardial infarction.26 ) nlr is considered to be a marker that simultaneously shows the negative effects of neutrophil elevation as an indicator of acute inflammation and lymphocyte depletion as an indicator of physiological stress.11 ) nlr can be easily calculated based on the white blood cell count , and it is also used as a marker of mortality and prognosis in patients who have experienced an ischemic stroke.10 ) since nlr reflects both the neutrophil and lymphocyte counts , we believe that it is a more reliable marker than neutrophil or lymphocyte count alone to assess the inflammatory status of the carotid plaques that cause intermediate stenosis .
our study group consisted of asymptomatic patients who had intermediate stenosis of the carotid artery .
this patient group is currently being followed up by medical means in the light of available information .
it is difficult to predict which of these patients will become symptomatic during follow - up .
studies performed on coronary artery plaques showed that rupture - prone plaques are more active in terms of inflammation.27)28 ) non - calcified carotid plaques are more unstable than calcified plaques , and therefore , they are associated with a higher risk of rupture , thromboembolism and stroke.16 ) van lammeren et al.16 ) showed that patients with truly asymptomatic carotid atherosclerotic plaques have relatively more stable plaque characteristics with a higher plaque smooth muscle cell content , a higher proportion of heavily calcified plaques , and less frequent intraplaque haemorrhages compared with patients who had ipsilateral cerebrovascular events . in the present study , nlr was higher in the non - calcified plaque group compared to the calcified plaque group ; confirming that non - calcified carotid artery plaques are more active in terms of inflammation .
based on this finding , it may be argued that among the non - calcified plaques that cause asymptomatic intermediate carotid artery stenosis , those that are associated with increased nlr ( nlr cut - off value of > 2.54 was found in our study ) may reflect a higher risk of stroke and such cases should be closely monitored .
hemoglobin level was significantly lower in the non - calcified plaque group compared to the calcified plaque group .
the reason for such a difference between the two groups with respect to hemoglobin levels is not clear .
we believe this may be due to the higher rate of asa use in the non - calcified plaque group and the associated non - clinical gastrointestinal bleeding events .
nlr can be affected by coexisting comorbidities and concomitant medical therapies , particularly by statins . in the present study
, blood samples were obtained from all the analyzed patients after their comorbidities had been stabilized and they had regularly used medical therapy for at least 2 months .
mean values of the parameters measured in the blood samples drawn at two different time points were analyzed .
arrangements have been made to ensure that the hematological markers that are evaluated as the endpoints of this study reflect the actual values .
an important factor that distinguishes the present study from previous studies is that in this study , carotid stenosis and plaques were assessed by both cdus and cta .
cta is a more convenient method than magnetic resonance angiography and doppler ultrasonography for assessing the severity of carotid artery stenosis.29 ) occasionally , the degree of stenosis as demonstrated by cdus and by cta may be different .
cta is a particularly reliable method to evaluate the nature of a plaque and extent of plaque calcification in carotid arteries.30 ) one of the limitations of this study is the lack of comparison between neutrophil and lymphocyte counts and the levels of other inflammatory markers .
all of the comorbidities and environmental factors that might have affected the inflammatory markers were not taken into account .
also , our findings may not reflect the outcomes in female patients since the majority of the study cohort was male .
additional limitations of this study may be listed as it being a single - center study and its relatively small sample size .
nlr is increased in the presence of non - calcified carotid artery plaques that cause asymptomatic intermediate stenosis .
nlr may be used as a marker of the level of inflammation in carotid artery plaques , and consequently as a predictor of stroke risk .
one of the limitations of this study is the lack of comparison between neutrophil and lymphocyte counts and the levels of other inflammatory markers .
all of the comorbidities and environmental factors that might have affected the inflammatory markers were not taken into account .
also , our findings may not reflect the outcomes in female patients since the majority of the study cohort was male .
additional limitations of this study may be listed as it being a single - center study and its relatively small sample size .
nlr is increased in the presence of non - calcified carotid artery plaques that cause asymptomatic intermediate stenosis .
nlr may be used as a marker of the level of inflammation in carotid artery plaques , and consequently as a predictor of stroke risk .
 background and objectivesnon - calcified carotid plaques are more unstable than calcified plaques , and they are associated with a higher risk of rupture , thromboembolism , and consequently , stroke . 
 the purpose of the present study is to compare calcified and non - calcified plaques that cause intermediate carotid artery stenosis with respect to neutrophil / lymphocyte ratio ( nlr).subjects and methodsa total number of 139 asymptomatic patients with 50 - 70% stenosis of the carotid artery were included in this study . 
 carotid doppler ultrasound imaging and computed tomography angiography were performed to divide the carotid artery plaques into two groups as calcified and non - calcified . 
 patients included in the calcified ( n=73 ) and non - calcified ( n=66 ) plaque groups were compared with respect to total neutrophil count , lymphocyte count and nlr.resultstotal lymphocyte count was statistically significantly lower in the non - calcified plaque group compared to the calcified plaque group ( total lymphocyte count in non - calcified / calcified plaque groups [ 103/mm3 ] : 2.1/2.3 , respectively ) ( p=0.002 ) . 
 nlr was statistically significantly higher in the non - calcified plaque group compared to the calcified plaque group ( nlr in non - calcified / calcified plaque groups : 2.6/2.1 , respectively ) ( p<0.001 ) . 
 the cut - off value for nlr was found to be > 2.54 . 
 multivariate regression analysis showed that nlr was independently associated with non - calcified carotid artery plaques ( odds ratio 5.686 , 95% ci 2.498 - 12.944 , p<0.001).conclusionsnlr is increased in the presence of non - calcified carotid artery plaques that cause asymptomatic intermediate stenosis . 
 increased nlr can be used as a marker to assess the risk of rupture of non - calcified carotid artery plaques . 
Introduction
Subjects and Methods
Study population
Blood samples
Definitions
Doppler ultrasonography and computed tomography angiography assessments
Statistical analyses
Results
Discussion
Limitations of the study
Conclusion