Study population
In this retrospective study, eligible aortic dissection patients were selected from two Affiliated Hospital of China Medical University (Shenyang, China). In brief, 131 consecutive AAD patients with corresponding peripheral blood samples and clinical data were collected from the Department of Vascular Surgery of the First Hospital of China Medical University (CMU) immediately after admission between May 2014 and February 2019. In addition, another 93 AAD patients were collected from the Department of Clinical Laboratory, Shengjing Hospital of CMU between January 2016 and January 2018. Among these 93 patients, 50 and 43 patients were diagnosed with TAAD and TBAD, respectively, during hospitalization (Fig. 1). The diagnosis of AD was based on imaging outcomes (CT, MRI and echocardiography). The AD was categorized in line with Stanford classification [18].
Definitions
AD could be divided into acute as well as chronic phases. AAD is diagnosed if a patient is admitted to hospitalization within 14 days of symptom onset, otherwise, chronic AD is considered [5]. Moreover, acute AD patients admitted to the department of emergency of our two clinical centers were clearly diagnosed after careful assessment within 24 h following symptom onset. The diagnostic criteria of hypertension included a clinical record of systolic blood pressure (BP) ≥140 mmHg and/or diastolic BP ≥90 mmHg, and the administration of anti-hypertensive drugs. The definition of diabetes mellitus (DM) included fasting glucose level ≥ 7.0 mmol/L, glycosylated haemoglobin A1c ≥ 6.5% and the administration of oral hypoglycaemic drugs or insulin. In addition, the smoking status was judged in accordance with self-report current smokers.
The definitions of terms utilized in this research were listed in the following. The definition of a communicating false lumen in AAD is the opacification of at least partial false lumen with contrast media except ulcer-like projection [19]. On the contrary, a non-communicating false lumen in AAD is defined by the completely occlusive false lumen by a thrombus, as well as ulcer-like projection. Moreover, the definition of upper and lower strata SAA levels were set in line with cut-off value of receiver operating curve (ROC), which was 0.427 mg/L as cut-off value in the present study.
Exclusion criteria
The AD patients with the following disorders were eliminated: chronic aortic dissection, malignancy, autoimmune disorders, severe aortic stenosis which was defined by an aortic valve area less than 1.0cm2 or less than 0.6cm2/m2 if indexed to body surface area [20], hematological disorders, infectious diseases, coronary artery disease which was defined as the presence of at least one≥50% stenosis in a coronary artery≥2.0 mm in diameter based on either coronary computed tomography angiography or invasive coronary angiography examination [21], severe organ failure, congenital heart disorders, previous aortic operation, Marfan syndrome, Ehlers-Danlos syndrome, other types of combined connective tissue or vascular disorders, and those receiving non-steroidal anti-inflammatory drugs or steroids. These disorders were ruled out using angiographic diagnosis, imaging examinations, laboratory tests, echocardiography, and other medical examinations based on the clinical presentations and medical history of patients. Eventually, there were 55 from First Hospital and 28 from Shengjing Hospital incongruent patients eliminated in this study (Fig. 1).
Aortic dissection blood sample biobank
After excluding incongruent participates, on February 2019, eight and twelve patients were unwillingly to provide their inpatient record for publication, including their clinical data or blood samples, respectively (Fig. 1). Finally, aortic dissection blood samples from 68 patients were eligible for further analyses as Biobank, which registered in the First Hospital of CMU Aortic Dissection Blood sample Biobank (1H-ADBB/CMU) were selected to identity AAD. As a result, a total of 53 blood samples from AAD patients registered in Shengjing Hospital of CMU Aortic Dissection Blood sample Biobank (SJH-ADBB/CMU) were eligible and further enrolled in this study.
Venipuncture was conducted on eligible patients after their admissions, followed by sample collection of in EDTA plastic tubes (BD Vacutainer® lavender, 5.0 mL) and anticoagulant and silica/gel plastic tubes (SST BD Vacutainer® gold, 5.0 mL). Moreover, blood sample was centrifuged to collect plasma, which was reserved at − 80 °C for further test (up to 1 year). Peripheral blood mononuclear cells (PBMCs) of these AD patients were extracted by Ficoll-sodium diatrizoate density gradient centrifugation as described previously [22].
The present AD Biobank research was conducted in line with the Guidelines of the World Medical Association Declaration of Helsinki, and was approved by the Ethics Committee of Shengjing Hospital (Ethics Approval No. 2016PS085K) of China Medical University. All 121 subjects signed written informed consent.
Serum measurements
ELISA kits (SAA: Wuhan Boster Biotechnology Company, China) were purchased to determine the SAA levels according to the manufacturer’s protocol.
Laboratory examinations
Lipid panel
The plasma levels of low-density lipid cholesterol (LDL-C) and high-density lipid cholesterol (HDL-C) were directly detected using selective solubilization method (LDL-C test Kit or Determiner L HDL, Kyowa Medex, Tokyo). Additionally, levels of total cholesterol (TC) as well as TG were measured by enzymatic methods. Automatic biochemistry analyzer (ARCHIRECT ci16200, Abbott Laboratories, USA) was utilized to produce lipid profiles.
Additional biochemistry
Alanine aminotransferase (ALT) and glutamic oxalacetic transaminase (AST) were determined utilizing International Federation of Clinical Chemistry approach (Abbott Laboratories, USA). The plasma concentration of total protein (TP) was determined using biuret method (FUJIFILM Wako Pure Chemical industries Ltd., Japan). The fasting plasma glucose (FPG) levels were determined by urease GLDH and glucose oxidase methods (DiaSys Diagnostic Systems GmbH, Germany).
D-Dimer and CRP
Immunoturbidimetry was used to assess plasma D-dimer levels (Diagnostica Stago, France, normal limit ≤0.5 μg/mL). High-sensitivity assay with BN II nephelometer (Dade Behring, Germany) was used to detect CRP levels (normal limit ≤0.17 mg/L).
All blood analyses were carried out by Department of Clinical Laboratory at CMU Shengjing Hospital and Department of Clinical Laboratory of First Hospital of China Medical University for SJH-AADBB/CMU Biobank and 1H-AADBB/CMU Biobank, respectively. Follow by clinical data collection, blood Biobank establishment, and laboratory examinations, total of 87 eligible participates (TAAD = 37 and TBAD = 50) were finally identified to meet the inclusion criteria. A summary of the flow of participants’ selection and inclusion process is illustrated in Fig. 1.
Control group (ctrl)
The blood sample from 87 patients with AAD were available from the venipuncture together with the blood samples from 63 matched controls. Shengjing Hospital of China Medical University Hospital medical examination database was used to identify healthy control by thoroughly searching all patients admitted to the emergency department diagnosed with trauma or motor vehicle accident during the 2014–2018 period (with imaging examinations, CT and/or MRI, at admission). All participants that were hemodynamicly stable and had non-typical symptoms, were considered as candidates of the control group. The control group was selected because these patients would not be expected to present any inherent bias favoring AAD. We also selected 20 matched patients diagnosed with stable angina as another control group. Stable angina was defined to chest discomfort that is classically retrosternal, triggered by exertion, and relieved by rest or nitrates within minutes [23]. Subjects diagnosed with vascular or connective tissue disorders using imaging examinations, CT and/or MRI, at admission were excluded from the control group. Other exclusion criteria included malignancy, infection, drug history, or any other immune-related disorders. Eventually, 63 participants in the control group and 20 patients in the angina group were included in this study. And all 83 subjects as control also signed written informed consent and conducted in line with the Guidelines of the World Medical Association Declaration of Helsinki.
Statistical analysis
SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was utilized for statistical analysis. Data were shown as medians with upper or lower quartiles for continuous variables due to the non-Gaussian data distribution. The difference between two groups was measured by non-parametric Mann-Whitney test, respectively. Comparisons among three groups were performed by one-way ANOVA, followed by Tukey’s post-hoc test, or nonparametric tests, followed by Kruskal-Wallis 1-way ANOVA test, according to the normality of the values. Categorical variables were shown as numbers with percentages, and the differences between two groups (for both biochemical and clinical parameters) was determined by Chi-square test. Correlations between continuous variables were analyzed through partial correlation analysis accounting for age, gender, and smoking. Furthermore, multiple logistic regression analysis was conducted to evaluate the correlation of serum SAA and D-Dimer or CRP with AAD risk following the adjustment of possible confounding factors. For instance, demographic characteristics and comorbidities were adjusted as confounding factors in all multivariable logistic regression models. Receiver operator characteristic (ROC) curves with area under the curve (AUC) along with logistic models were employed to determine the corresponding cut-off points and to assess the diagnostic performance of serum SAA and D-Dimer or CRP individually, and combined for AAD detection. P values < 0.05 were considered as statistical significance.