An observational prospective study was conducted with patients over 18 years of age who had been admitted for evaluation of suspected CAD in the tertiary hospital, NEIGRIHMS, Shillong, Meghalaya, India, in the period between November, 2014 and November, 2016. This study had been approved by Institute Medical Ethical Committee. Written consents were taken from all the patients.

This study consist of three different group of population on the basis of coronary angiography findings, in which group A, B and C consist of isolated CAE patients, obstructive coronary artery disease patients and normal coronaries i.e., control group respectively. Group A, B and C had 56, 58 and 65 patients respectively. The estimated sample size was calculated using the formula,

where, p (prevalence) =10%; q=1-p, d (standard error=5%) which yielded a required sample of 71. However, due to constraints of time the total number of patients in each group was 56, 58 and 65 respectively.

Patients with active infection, liver disease, renal failure, alcoholism, leukaemia, lymphoma, haemolytic anaemia, receiving chemotherapy and radiation treatment, severe valvular heart disease, non O-CAD and O-CAD with ectasia on angiogram were excluded from study.

The patient clinical characteristics including age, sex, smoking status, diabetes mellitus, and hypertension were recorded. All the routine blood investigation including differential leukocyte count was done followed by echocardiography and coronary angiogram. Indication for CAG was either the presence of typical angina or positive result of treadmill test for myocardial ischaemia. These groups were compared for clinical characteristics and N/L ratio. Peripheral venous blood samples were drawn after overnight fasting. Total and differential leukocyte counts were measured using an automated haematology analyser.

Selective CAG was performed predominantly by radial route in multiple projections without the use of nitroglycerin or any other coronary epicardial dilator like adenosine and calcium channel blocker. CAGs were analysed by three experienced angiographers who were blinded to patient clinical and haemotological profile. The vessel diameter was calculated quantitatively in case of conflicts about CAE. The severity of isolated CAE was determined according to the Markis classification [1]. In decreasing order of severity, it classifies Type I as diffuse ectasia in at least two vessels, Type II as diffuse ectasia in one vessel and discrete ectasia in another vessel, Type III as diffuse ectasia in only one vessel without any evidence of ectasia in other vessels and Type IV as only discrete ectasia involving vessel. CAD was defined as stenosis of more than 50% of the diameter in one or more major epicardial artery.

SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Continuous variables were expressed as Mean±Standard Deviation (SD). Categorical variables were expressed as percentages. Group means for continuous variables were compared with ANOVA Categorical variables were compared with the chi square test. F ratio was measured which is a statistical term named after scientist Ronald A Fischer. A p-value of ≤0.05 was considered statistically significant.

The study population consisted of 179 patients. The mean age of the patients in Group A, B and C was 53.72±4.9, 56.15±7.3 and 53.25±8.4 years respectively. The patients with isolated CAE were relatively younger as compared to O-CAD. Male gender constituted 106 (59.2%) of the total patients. We found that isolated CAE were most likely to occur in right coronary artery 36 (64.2%) closely followed by 34 (60.7%) in Left Anterior Descending (LAD) coronary artery, less commonly Left Circumflex Artery (LCX) was involved 26 (46.4%) and mostly involved single vessel 25 (44.6%). According to the Markis classification, Type 4 (32.1%) was the most common types of isolated CAE [Table/Fig-1].

It was found that there were no significant differences between the groups with reference to hypertension, Type 2 diabetes mellitus, dyslipidemia, and smoking status (p>0.05). The LVEF however was lower in Group B and was statistically significantly different from the other two groups (p<0.01).

The mean WBC count and N/L ratio was found to be higher in both the CAE as well as the CAD group in comparison to the control group. Patients with CAE had a higher mean WBC count and N/L ratio than patients with CAD [Table/Fig-1].

On comparing the groups using ANOVA, statistically significant difference was found in WBC count between the CAE, CAD and control groups with F ratio of 22.16 and a p-value of <0.001 [Table/Fig-2]. Similarly statistically significant difference was found in N/L ratio between the CAE, CAD and control groups with an F ratio of 79.79 and a p-value of <0.001 [Table/Fig-3].

In this study, we found that there were statistically significant increased mean levels of total leukocyte count as well as ratio of N/L in patients with coronary ectasia and O-CAD as compared to normal coronaries (p<0.01). There was also statistically significant increased WBC count and N/L ratio in CAE as compared to O-CAD (p<0.01).

Balta S et al., in their study, found a higher N/L ratio in the CAE and O-CAD groups compared to the control group [9], They reported no difference between CAE and O-CAD groups however, there was statistically significant difference in CAE and CAD as compared to normal coronaries patients. However, Kalaycıoğlu E et al., reported significant difference between CAE and O-CAD [10]. CAE group had higher WBC count and N/L ratio as compared to O-CAD and NCA, similar to findings of present study. Thus in the present study, N/L ratio was associated with the increased likelihood of isolated CAE. Therefore, CAE may be related to more severe inflammation when compared to O-CAD and control groups.

The medial layer of the vascular wall contains a well arranged layer of smooth muscle with extracellular matrix proteins like elastin and collagen, which forms a structure that maintains vascular wall integrity [11]. The extensive destruction of this important medial layer of the vessel wall in the ectatic segment has been reported in postmortem histopathologic studies. Infiltration of the media layer by inflammatory cells is significant finding that can be seen in ectatic segments [12]. Markis JE et al., stated that the destruction of the vascular media as the principal cause of ectasia [1].

Previous studies have reported that Neutrophil Elastase (NE), a serine proteinase, may play a crucial role in the aetiopathogenesis of CAE [12]. NE is predominantly present in neutrophils and can digest vascular medial layer content namely elastin, collagen and proteoglycans. Akyel A et al., found that higher Neutrophil Gelatinase-Associated Protein (NGAL) levels were detected in patients with CAE compared to those with normal coronaries [13]. NGAL prevents degradation of MMP-9 which has a role in the degradation of collagen. Therefore, NE or NGAL may explain the relationship between N/L ratio and CAE.

The association between inflammation and CAE was evaluated based on the findings of previous postmortem studies. Higher levels of Interleukin-6 (IL-6) [14], Matrix Metalloproteinase-3 (MMP-3) [15], high sensitivity (hs-CRP) [16] have been seen in patients with isolated CAE, compared to patients with O-CAD. Additionally, Kocaman SA et al., also reported that patients with isolated CAE had significantly higher leukocyte and neutrophil levels than patients with non O-CAD and normal coronaries [17]. Yilmaz H et al., reported that patients with isolated CAE have raised levels of plasma soluble Intercellular Adhesion Molecule-1 (ICAM-1) [18], E-selectin and Vascular Cell Adhesion Molecule-1 (VCAM-1) in comparison to patients with O-CAD and normal coronaries.

Studies in the recent past have reported that elevated levels of inflammatory indicators are markers of atherosclerotic disease activity and also indicate an increased risk of the progression of atherosclerosis [19]. Although the underlying mechanism of abnormal luminal dilatation is not well known, yet the histopathological characteristics of CAE are similar to those of coronary atherosclerosis. Leukocyte subtype and N/L ratio are also indicators of systemic inflammation [6,7]. These markers have prognostic value in cardiovascular disease. Zazula AD et al., found N/L ratio was significantly higher in patients in acute coronary syndrome patients compared to patients diagnosed with non cardiac chest pain [20]. The N/L ratio levels give information about CAD severity in patients with acute myocardial infarction [21]. Because of all of these findings from previous studies, aetiology of the relationship between N/L ratio and CAE may be inflammation and atherosclerosis. N/L ratio may appear additive to conventional risk factors and commonly used biomarkers. In addition, interestingly, the N/L ratio has remained as a predictor of all-cause mortality in patients with normal WBC counts [22].

CAE can be identified by more sensitive and specific cardiovascular imaging tools. However, these tools are expensive and with ill effects such as exposure to radiation. Therefore, N/L ratio, which is low cost and readily done blood test, can be used as an initial filter criteria, and will help in determining the need for further imaging modalities in the assessment of CEA.

Major limitation of the study was small number of patients which may limit the generalisability of our findings. Secondly, it didn’t study the correlation of N/L ratio with short and long-term events. Thirdly, it did not assess the predictive value of other inflammatory markers such as CRP, TNF-α and IL-6. Lastly, the pathological role of elevated WBC and N/L ratio in patients of CAE has not been shown, so association may not prove causality. These issues should be addressed by large scale studies in future.

This study shows that a more severe inflammatory process may be involved in the development of CAE as compared to O-CAD. This severe involvement leads to abnormal dilatation of coronary artery by damaging its medial layer rather than causing stenotic lesion. To conclude, N/L ratio may be turned into a valuable parameter for the preliminary approach of patients with suspicion of CAD to rule out CAE.