Prognostic Value of Admission Mean Corpuscular Volume for Major Adverse Cardiovascular Events following Stent Implantation in Nondiabetic and Diabetic Patients with Acute Coronary Syndrome

Background One of the key concerns of the clinician is to identify and manage risk factors for major adverse cardiovascular events (MACEs) in nondiabetic and diabetic patients with acute coronary syndrome (ACS) undergoing stent implantation. Mean corpuscular volume (MCV) is a marker of erythrocyte size and activity and is associated with prognosis of cardiovascular disease. However, the role of admission MCV in predicting MACEs following stent implantation in diabetes mellitus (DM), non-DM, or whole patients with ACS remains largely unknown. Methods and Results A total of 437 ACS patients undergoing stent implantation, including 294 non-DM (59.08 ± 10.24 years) and 143 DM (63.02 ± 9.92 years), were analyzed. Admission MCV was higher in non-DM than DM patients. During a median of 31.93 months follow-up, Kaplan-Meier curve demonstrated that higher admission MCV level was significantly associated with increased MACEs in whole and non-DM, but not in DM patients. In Cox regression analysis, the highest MCV tertile was associated with higher MACEs in whole ([HR] 1.870, 95% CI 1.113-3.144, P = 0.018), especially those non-DM ([HR] 2.089, 95% CI 1.077-4.501, P = 0.029) patients after adjustment of several cardiovascular risk factors. MCV did not predict MACEs in DM patients. During landmark analysis, admission MCV showed better predictive value for MACEs in the first 32 months of follow-up than in the subsequent period. Finally, the receiver operating characteristic (ROC) curve was conducted to confirmed the value of admission MCV within 32 months. Conclusion In patients with ACS, elevated admission MCV is an important and independent predictor for MACEs following stent implantation, especially amongst those without DM even after adjusting for lifestyle and clinical risk factors. However, as the follow-up period increased, the admission MCV lost its ability to predict MACEs.


Introduction
Coronary artery disease (CAD) is caused by atherosclerosis accompanied by stenosis or lumen obstruction, resulting in myocardial ischemia, hypoxia, and necrosis [1][2][3]. It has become an important threat to human life and health, especially in middle-and low-income countries [4,5]. Acute cor-onary syndrome (ACS) is a serious type of CAD that can lead to various adverse outcomes [6,7]. Percutaneous coronary intervention (PCI) is a common revascularization strategy in the management of ACS due to its safety profile, low complication rates, symptom relief, and survival benefits [8,9]. Although significant achievements have been made in the treatment, major adverse cardiovascular events (MACEs) remains a frequent and severe complication following stent implantation [10,11].
The incidence of diabetes mellitus (DM), a metabolic disorder characterized by hyperglycemia, is a major and important risk factor for cardiovascular events including ACS undergoing PCI [12,13]. Sustained hyperglycemia is shown to exacerbate cardiac damage through changes of erythrocyte [14]. Mean corpuscular volume (MCV) is the measure of the average size of erythrocytes, which is closely related to erythrocyte disorders [15,16]. It is reduced in diabetes, probably because iron deficiency anemia is common in people with diabetes [17,18]. Studies demonstrate that MCV is associated with the prognosis of several diseases, including peripheral arterial disease, CAD, and cerebral ischemic stroke [19][20][21][22][23]. The possible mechanism is that the increase of MCV impairs the antioxidant capacity of erythrocytes and decreases endothelial function, both of which promote the development of atherosclerosis [24]. In addition, high MCV would be related to a hindered flow of larger, less flexible erythrocytes through microcirculation, which might contribute to myocardial ischemia [25,26]. However, the role and difference of MCV in predict MACEs between nondiabetic and diabetic populations with ACS undergoing PCI still largely unknown. Therefore, exploring the relationship between MCV level and adverse outcomes following stent implantation in nondiabetic and diabetic patients with ACS is of great clinical value.
In this context, the present study was performed to explore the value of admission MCV for prognosis of MACEs in ACS patients, with or without DM in China.

Materials and Methods
2.1. Study Population. The present study was carried out in the First Affiliated Hospital of Medical College of Xi'an Jiaotong University from January 2013 to February 2014. The inclusion criteria were confirmed admission diagnosis of ACS including ST-elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI), and unstable angina pectoris (UA), successful treatment by stent implantation, without diabetic ketosis or nonketotic hyperosmolar coma. The exclusion criteria were patients with malignant tumors, severe renal and liver diseases, pregnant patients, patients with autoimmune diseases or blood disorders, severe cerebrovascular diseases, and prior history of surgical treatment within 2 weeks of this study, and data were not available for the MCV levels. Qualitative and quantitative coronary angiographic analyses were carried out according to standard methods. PCI was performed using standard techniques. All patients were given loading doses of aspirin (300 mg), clopidogrel (600 mg), and unfractionated heparin (100 U/kg) before coronary intervention. The treatment strategy, stenting techniques, and use of glycoprotein IIb/IIIa receptor inhibitors or intravascular ultrasound used at the discretion of the operator during the PCI procedures. Drug-eluting stents were used (Medtronic, Inc.). The success of PCI is defined as previously described [27,28]. Written informed consent was obtained from all study participants, approved by ethic committee approval at the First Affiliated Hospital of Xi'an Jiaotong University.
The estimation of sample size was performed using PASS software (version 11.0) [29]. A sample size of 311 achieves 90% power to detect an effect size of 0.202 using a 2 degrees of freedom chi-square test with a significance level of 0.050.   Disease Markers MI was based on a history of acute MI (AMI) or with signs of an infarction outside the area of the index infarction.

Biochemical Measurements.
Peripheral blood was sampled from patients within 24 h of admission prior to stent implantation. Triglycerides (TG), low-density lipoprotein cholesterol (LDL), high-sensitivity C-reactive protein (hsCRP), creatinine, Hemoglobin A1c (HbA1c), and pro-Btype natriuretic peptide (pro-BNP) were assayed using the Cobas Integra automated chemistry analyzer (Roche Cobas Integra 400 Plus, Roche Diagnostics, USA). High-sensitivity C-reactive protein was assessed using enzyme-linked immunosorbent assay according to the manufacturer's instructions (EIA-3954, DRG International Inc., Springfield Township, USA). Hemoglobin (HGB), MCV, and platelet counts were measured using the Sysmex KX 4000i haematology analyzer (Sysmex Corporation, Kobe, Japan) by standard biochemical techniques. The patients were further divided into 3 groups according to tertiles of MCV, and the reference MCV interval of our laboratory was 82-100 fL. Echocardiographs were performed on admission by experienced echo cardiologists, and systolic function was expressed as the ejection fraction (EF), which was calculated using Simpson's method.

Outcomes and Follow-Up.
MACEs were defined as composite endpoints, including all-cause death, nonfatal AMI, urgent coronary revascularization, UA, and stroke [31]. All patients were followed up by interview or telephone in our hospital, and the end of follow-up was the date of the first MACEs occurrence. Data are presented as mean ± SD, median (IQR) or number (%), "a" represents P value = 1. ACS: acute coronary syndrome; DM: diabetes mellitus; MI: myocardial infarction; PCI or CABG: past percutaneous coronary intervention or coronary artery bypass grafting; ACEI: angiotensin-converting enzyme inhibition; ARB: angiotensin receptor blocker.
3 Disease Markers 2.5. Statistical Analysis. The statistical analyses in this study were performed using SPSS for Windows 25.0 (SPSS Inc, Chicago, IL), R software (version 3.6.1), and EmpowerStats (http://www.empowerstats.com/). Continuous variables were expressed as mean ± SD or median (interquartile range (IQR)) according to different distributions, and in case of All probability values were two-tailed. P < 0:05 was considered statistically significant.

Clinical Characteristics of Patients with and without DM.
In the study, a total of 2153 patients were screened; 437 consecutive ACS patients who are undergoing stent implantation were analyzed, including 294 non-DM patients and 143 DM patients (Figure 1). During a median follow-up of 31.93

Comparison of Basic Characteristics Stratified by MCV
Tertiles. According to the above results, we found that the admission MCV level varied greatly between nondiabetic and diabetic patients. Therefore, all patients were segregated into three groups by tertiles of admission MCV level, and a comparison of the clinical data of these groups is shown in Table 2. The proportions of diabetes and HbA1c level were decreased as MCV level increased (all P < 0:05). Moreover, the incidence of MACEs in the MCV-H group (41.8%) was higher than that in the MCV-M (25.5%) and MCV-L group (28.8%, P = 0:007). No significant difference in other factors and medication use at discharge were observed amongst the three groups (all P > 0:05).
Moreover, the whole and non-DM population were subjected to landmark survival analysis with a landmark point of 32 months. The cumulative survivals of MACEs were statistically different in whole and non-DM patients within 32 months, but with no significant differences in the time period over 32nd month to the end of follow-up ( Figure 3). The results in Table 4 showed significantly higher MACEs in the MCV-H group than MCV-L group in the whole (HRs 2.979, 95% CI1.331-6.671, P = 0:020), especially non-DM population (HRs 4.054, 95% CI 1.192-13.789, P = 0:025) within 32 months of follow-up but not in the later period (over 32nd month to the end of follow-up). The corresponding graphs were shown in Figure 4.

Discussion
Previous studies showed that there was the high risk of allcause mortality in patients with ACS within one year after PCI [32]. Long-term glycometabolic disorder implied a high 6 Disease Markers risk for cardiovascular disease and adverse outcomes [33,34]. Consequently, it is urgent to identify and manage risk factors of nondiabetic and diabetic patients in order to ameliorate adverse cardiovascular events. In the current study, we noted that MCV levels were higher in nondiabetic than diabetic ACS patients following stent implantation. Intriguingly, Kaplan-Meier survival analysis and Cox regression analysis demonstrated that the incidence of MACEs following stent implantation was significantly higher in MCV-H group com-pared with MCV-L group of whole, especially those nondiabetic populations but not diabetic populations over a median of 31.93 months of follow-up. Moreover, landmark analysis indicated that, in comparison to the MCV-L group, the increased risk of MCV-H group within 32 months was lost after 32nd month to the end of follow-up. Finally, the prognostic value of admission MCV within 32 months was further confirmed by ROC curve analysis in whole and non-DM patients. To the best of our knowledge, this is the first  Chronic hyperglycemia induces ROS production, crosses the erythrocyte membrane, and oxidizes heme proteins, which have been shown to lead to the progressive loss of deformability of erythrocytes [35]. Diabetes can change hematological parameters that were related to hyperglycemia [14]. Previous studies observed that MCV was decreased in DM as compared to non-DM [17,36]. Macrocytosis increased mortality, as well as main adverse cardiovascular and cerebrovascular events, among consecutive patients  Disease Markers who underwent PCI [37]. Increased MCV is associated with a higher incidence of subsequent cardiovascular event or death in nonanemic AMI patients [25]. However, the level of admission MCV and its role in MACEs occurrence of diabetic and nondiabetic ACS patients undergoing stent implantation receives not much attention. In the present study, the admission MCV level of non-DM patients was higher than that of DM patients. In addition, patients with MCV-H values exhibited high risk of MACEs in whole and non-DM ACS patients but not DM patients. Our study not only confirms the relationship between MCV and DM but also further assesses the association of MCV with MACEs incidence following stent implantation in ACS patients with or without DM. However, the conflicting evidence regarding MCV level and cardiovascular outcomes is reported. Stable CAD patients undergoing PCI in the 1st quartile who had the lowest MCV values exhibited a high risk of restenosis [22]. Obviously, the target population of this study are participants with stable CAD, and their aim is to explore the association between MCV and incident restenosis; these details are very different from those of our study. High MCV reduces the antioxidant function of red blood cells and is an independent prognosticator of impaired endothelial function measured via flow-mediated dilatation [24]. The direct mechanism of high MCV effect may be related to the increase of red blood cells resistance through microcirculation, which may lead to myocardial ischemia [25,26]. These factors may also be involved in the high occurrence of MACEs of MCV-H group in ACS patients undergoing stent implantation in this study. It is noteworthy that the significant difference of MACEs only exists in whole and nondiabetic patients in the present study might be due to chronic hyperglycemia and high HbA1c leads to low MCV level in general in DM patients.
The important implication of the present study is that landmark analyses revealed a better predictive value of admission MCV for MACEs after stent implantation in the first 32 months than in the subsequent period, and ROC curve confirmed its predictive value within 32 months in whole, especially non-DM populations, suggesting that admission MCV level cannot accurately reflect patients' outcomes with prolonged follow-up. To identify high-risk patients, current guidelines recommend a standardized approach involving validated scoring systems such as the Global Registry of Acute Coronary Events (GRACE) score [38,39], which provides validated prognostic information for MACEs in ACS patients [40,41]. Therefore, combining admission MCV with the GRACE score may provide a more accurate risk estimation in ACS undergoing PCI which needs to be further explored.
The present study also has several limitations. Firstly, as an observational cohort study, there is inevitably some confounding bias, such as selection bias. Secondly, this research is a single-center study in a Chinese population, and the sample size is relatively small, especially in patients with DM; therefore, the comparisons of subgroups may lack power to detect significant differences for selected variables. Further multicenter and larger-scale surveys are required to confirm the results from this study and to elucidate the precise mechanisms. Moreover, the study only included ACS patients undergoing stent implantation, which suggests that the study results may not be extended to all ACS patients.

Conclusions
This study demonstrated that elevated admission MCV level was an important independent risk factor and had predictive value for MACEs following stent implantation in whole, especially those nondiabetic ACS patients. However, as the follow-up period increased, admission MCV lost its ability to predict MACEs. Measuring MCV, which is cheap and easy to obtain, might be helpful for a prognostic risk stratification