This was an observational, prospective, single-center study. The study was conducted in accordance with the Helsinki declaration and was approved by the Fundação Universitária de Cardiologia, Research Ethics Committee. All participants signed an informed consent form.

Using the fourth universal definition of myocardial infarction,⁸ we evaluated patients undergoing cardiac catheterization at the catheterization laboratory of Instituto de Cardiologia do Rio Grande do Sul, in Porto Alegre, from April 2016 to April 2017. We consecutively recruited patients with a diagnosis of acute ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (NSTEMI). The control group consisted of patients with no obstructive lesions on coronary angiography. Controls and cases were matched 2:1 by age and sex.

(a) Adults > 18 years old with an (b) episode of ACS in the last 24 hours and (c) established coronary artery disease who (d) consented to participate in the study.

Patients with (a) severe conditions and a life expectancy < 5 years, (b) a family history of blood dyscrasias, (c) severe anemia, (d) cancer, active infection, or inflammatory diseases, (e) renal failure on dialysis, (f) previous splenectomy, or (g) bipolar or mental disorders; patients who underwent (h) surgery in the last 7 days; patients (i) on immunosuppressants; and any patient (j) from whom we were not able to collect blood samples.

Patient demographics and clinical characteristics were collected, as well as a blood sample for identifying monocyte subsets through flow cytometry. Additional blood samples were collected from patients in the STEMI group on days 3, 7, and 30. Peripheral venous blood was collected and processed by flow cytometry in 60 minutes. Plasma was stored at 70°C for analysis.

Flow cytometry analyses were performed at the Laboratory of Cellular and Molecular Cardiology of Fundação Universitária de Cardiologia using anti-CD14 (fluorescein isothiocyanate and phycoerythrin) and anti-CD16 (PE-CyTM5) antibodies. The protocol was performed according to Imanishi et al.⁹

From the total blood volume, 500 μl were incubated with 5 mL of lytic solution for 15 minutes, and 20 mL of FCS buffer was later added to the sample. The sample underwent centrifugation at 1,500 pm for 5 minutes. After centrifugation, an additional 2 mL of buffer was added to the solution, which was then separated into 10 200-μL tubes and 5 500-μL frozen vials. The samples were subsequently marked with 3 μL of CD14/CD16, and 400 μL of FCS was added to the solutions, which were incubated for 60 minutes. After incubation, the samples were washed with 1 mL of buffer and divided into 200-μL tubes. Finally, flow cytometry (FACSCanto™, BDBiosciences) was conducted using Cell Quest and FACS Diva software, version 6.1.3 (BD Biosciences). Flow cytometry and biomarker analysis were performed at the Molecular Biology Laboratory of Instituto de Cardiologia.

Sample size calculation: with a power of 80% and a significance level of p < 0.05, 28 patients would be required to detect a difference of 30 cells/μL in CD14⁺⁺CD16⁺ monocytes with a standard deviation (SD) of 39 cells/μL. To detect a difference of 250 cells/μL in CD14⁺CD16^- monocytes with a SD of 250 cells/μL, the sample would need to include 17 patients. The Shapiro-Wilk test was used to assess the normality of continuous variables. Variables with normal distribution were described as means and SDs, whereas variables without normal distribution were described as median and interquartile range. Categorical variables were expressed as absolute numbers and percentages. The nonparametric Kruskal-Wallis test was used to assess differences between groups, and Dunn's post hoc test was used to identify which groups were different. Monocyte measurements and groups were obtained on days 1, 3, 7, and 30 in STEMI patients. Friedman's test was used to evaluate repeated measures. Cuzick's test for ordered group trends was used to assess falling or rising trends.¹⁰ Statistical analyses were performed using Stata software, version 13.0. The significance level was set at 5%.

We consecutively recruited 47 patients with ACS, of whom 34 had STEMI and 13 had NSTEMI. Mean patient age (mean ± SD) was 9.9 ± 7.3 years in the STEMI group and 60.2 ± 5.2 years in NSTEMI group. Men accounted for 71% of participants in the STEMI group and 58% in the NSTEMI group. The control group consisted of 19 patients, 37% of whom were men, with a mean age of 59.7 ± 6.4 years ( Table 1 ).

The monocyte profile of each group was analyzed by flow cytometry. There was an average of 2,620 events per microliter in the control group, 2,539 events per microliter in the NSTEMI group, and 5,571 events per microliter in the STEMI group ( Graph 1 ). The difference between groups was statistically significant, and the control group was different from the STEMI D1 group.

There was a difference between groups (Kruskal-Wallis, p = 0.00301) regarding classic monocyte count ( Graph 2 ). Dunn's post hoc test showed that the control group was different from the STEMI D1 group (p = 0.0058). There was a significant rising trend between groups according to Cuzick's test (p = 0.00112). There was no difference between STEMI and NSTEMI groups according to Friedman's test.

There were no differences between groups when comparing intermediate monocytes (Kruskal-Wallis, p = 0.3119; and Friedman, p = 0.1761).

In nonclassical monocyte analysis, there was no difference between groups (Kruskal-Wallis, p = 0.3658) ( Graph 03 ). In patients with STEMI, there was an increase in nonclassical monocyte count from the first collection to the seventh day, but the difference was not statistically significant according to Friedman's test (p = 0.1519).

This was an observational, single-center study with a limited number of participants. In addition, there was a predominance of male participants and a non-negligible heterogeneity regarding the use of statin and dual antiplatelet therapies between groups. This study is intended to raise hypotheses and should be complemented with new evidence in this clinical and laboratory setting.