Analysis and Comparative Evaluation of Aortic Calcium by Computed Tomography and Histopathology in Patients With Aortic Stenosis

1. Title Analysis and comparative evaluation of aortic calcium by computed tomography and histopathology in patients with aortic stenosis.
2. Background. Calcified aortic valve disease (CAVD) was initially considered to be a passive degeneration of the extracellular matrix (EM). Recent studies have shown that within the development of the damage mechanism there is complexity and it is an active (1) and degenerative process where genetic, environmental and epigenetic phases overlap and interact leading to hemodynamic changes in blood flow, endothelial dysfunction and metabolic disorders that lead to low-density lipoprotein cholesterol deposits in the ME where at the same time there is an underlying infiltration of macrophages, oxidative stress, and release of pro-inflammatory cytokines. (2) However, although there are several theories of damage, and some of them are attributed to a diversity of origin such as genetic, congenital, infectious, and degenerative. Finally, the dynamic and functional impact of the heart affects equally. It leads to organic deterioration when the AD reaches clinical and echocardiographic criteria of severity, and the only therapeutic option is the change or replacement of the aortic valve. The participation of imaging methods such as computerized tomography about to the concerning the aortic valve through the measurement of the aortic calcium score (AVC-CT) as a method to determine the severity of aortic stenosis (AS) in those cases where echocardiographic measurements are conflicting, allows us to see the interest in defining this problem through different angles of investigation (7). In AD, progressive calcification is the trigger factor that also predominantly leads to aortic valve obstruction. (3) Currently, such calcification can be easily quantified using the same acquisition protocol and Agatston Scoring Method as used for a coronary artery calcium scan and is particularly attractive because it provides an assessment of the severity of AD, which is independent of the geometry and ventricular ejection fraction. Advances in the field of CT CVA measurement have been demonstrated in the last decade and are closely associated with hemodynamic measurements of moderate to severe AD in Doppler echocardiography, however, until recently, significant over-positioning of calcium from the aortic valve area among patients with hemodynamically severe AD and those with non-severe AD made it challenging to implement in clinical practice. This development has generated significant interest in the use of CT-CVA as an alternative and complementary assessment of the severity of AD, especially in patients with discordant echocardiographic findings. (11) In fact: 1) CVA is strongly but non-linearly associated with hemodynamic measures of severity in moderate to severe AD (12). Thus, the determination of CVA may help discriminate severe AD from non-severe AD in patients with discordant findings measured by echocardiography, and 3) in patients with moderate to severe AD, CVA is also likely associated with faster progression of AD and with survival. However, it should be mentioned that the analysis of that series did not make adjustments to the calcium measured in the patients' coronary artery. Although the CVA score has been recommended in the European Society of Cardiology Guidelines to confirm the severity of AD in the following conditions 1) patients with low left ventricular ejection fraction (LVEF), gradient and low flow, in which dobutamine stress echocardiography is inconclusive and 2) patients with low gradient AD and preserved LVEF, and this is not yet part of the American Heart Association (AHA) and American College of Cardiology (ACC) guidelines (13). So it has been suggested that clinical applicability should be established, so what are the next steps? While the reproducibility of coronary calcium quantification by multidetector CT has been tested for various workstation platforms before, (14,15) the same has not been done so far for CVA quantification through variability with standard Gold through the histopathological study and determine the variability of the CVA score measured by CT by determining its relationship through the flow and gradient status of patients with AD and define what implications can be suggested for the patient's treatment(13,16)
3. Problem statement. There is a high prevalence of aortic valve calcification in patients who are subjected to valve change regardless of the cause (genetic, congenital, or degenerative structural malformation). Early identification of calcification in these patients is critical since they will receive a valve change that is likely to return to dysfunction if there is no history of the type of damage with or without preceding valve calcification. Currently, there is controversy about the ideal time to initiate valve therapy, as it has not been possible to identify those patients who will develop significant valve calcification. However, little is known about the relationship between the new imaging methods that are promising for measuring it and its correlation with standard gold, that is, what is quantified at the histopathological level. 3.1. Justification. Magnitude: The global prevalence of aortic stenosis is 0.3-0.5% and 2-7% in >65 years and of valvular calcification. According to its definition and criteria to classify valve stenosis, up to 30% will require aortic valve change due to valve insufficiency. Significance: In the last two decades, the incidence of aortic valve insufficiency has increased exponentially and has been associated with unfavorable outcomes. Vulnerability: Currently, the cornerstone of aortic stenosis management includes prevention and early support management. The identification of calcification has not been entirely clear either through laboratory parameters or about to with concerning risk factors, which are inadequate and late. Early detection and intervention of calcification during the disease can improve the prognosis if it is recognized early. Feasibility: The study will be carried out in the two departments of Immunology through Dr. Soto who will coordinate the research) (Cardiovascular Medicine through Dr. Israel Pérez Torres who will evaluate calcification and histological changes in the valve tissue) (Pathology through Dr. Aranda who will manage and treat the valve samples) and the Image Department coordinated by Dr. Sergio Críales Vera who will quantify the valve calcium). All departments are recognized for their extensive participation in research. The study of the pathogenesis of persistent inflammatory damage in aortic stenosis in relation to the type of prosthesis will lead in the future to evaluate if it is a factor involved in the recurrent damage in stenosis, calcification, and deterioration. In the last decades in Mexico, the demographic transition increased the number of people over 65 years old, the population in which the most significant complication of valve dysfunction has been seen. However, regardless of age and etiology, this is currently a population group with significant expansion and is expected to triple by 2030. This inflammatory damage will increase the prevalence of aortic stenosis, and therefore more patients will require valve replacement surgery. Our Institute is a national reference hospital that attends a significant number of patients with aortic stenosis, among others. Of the valves that are implanted, a quarter is manufactured at the Institute. Therefore, evaluating processes highly related to valve dysfunction such as calcification is a priority and is considered a health problem that requires prevention. 3.2. Research question. In patients who have undergone aortic valve surgery, will the quantification of calcium using the 4D computerized tomography method have a reasonable correlation with that measured by histopathology, and will it also allow it to be proposed as an early predictor of dysfunction?
4. Hypothesis. Working hypothesis (H1): The quantifiable assessment of valve calcium by CT is similar to that measured by histopathology and predicts early valve dysfunction. Null hypothesis (H0): The quantifiable assessment of calcium by CT scan is different from that measured by histopathology and does not predict early valve dysfunction.
5. Methodology. 5.1. Study design. Cohort, observational, comparative, and prospective study. 5.2.5. Elimination criteria.
6. Not having an adequate sample or staining of the valve tissue. 5.3. Sample size. For the calculation of the sample size, a determination of the sample size (two tails) shall be made, taking into account the means and standard deviation (SD) of the items corresponding to each one. All patients who meet the inclusion criteria described above shall be considered for evaluation during the period 2017-2020. 5.4. Data collection technique Data collection will be divided into three stages: Pre-surgical: Once the study population has been identified, a personal interview will be conducted, the objective of the study will be explained to the patient, and informed consent will be obtained. Various echocardiographic parameters will be collected for each patient (pre-surgical echocardiogram). The study of the valve, which is requested before surgery, will be analyzed in a tomography scan, and this is the one that will be used to measure the calcium. Post-surgery Employing the sample of the valve tissue obtained from the surgery, the appropriate cuts and stains for the measurement of the calcium will be requested. Echocardiographic study. The echocardiographic study performed will be re-evaluated by two expert echocardiographers following current recommendations.16 The Philips iE33 xMATRIX (Philips, Andover Massachusetts) ultrasound system with X5-1 transducer. The dimensions of the left ventricle will be quantified in the long parasternal axis; the volumes of the left atrium and left ventricle will be calculated with the Simpson method. The aortic valve area will be estimated with the continuity equation. The mass of the LV was estimated with the Devereaux method.17 The mean and maximum transvalvular aortic gradients will be acquired in the apical projection of three or five chambers, where a suitable continuous Doppler spectrum and the highest transaortic velocity were obtained. The pulmonary artery systolic pressure will be estimated by adding the right atrial pressure to the tricuspid insufficiency gradient. Computed tomography evaluation. The study was performed with a multidetector with double energy source of 256 cuts definition Flash Medical Systems Forcheim Germany (128 x 2), performing prospective additive in the best diastolic phase (65-70 %), with 3 mm thick reconstructions and 3mm spacing with Kernel B 35f filter and visual field adjusted only to the heart. The images were processed on a workstation (SmartScore by Syngo via, Waukeesha WI Electronics for Medicine. The results were expressed in Agatston units. The study was carried out without a contrast medium and with a radiation equivalent to 1.0 mSv. Tissue evaluation Histological technique. A 5-micron segment of the aortic valve, obtained by cardiothoracic surgery, was washed in 0.9% NaCl for 30 seconds immediately after the tissues were fixed by immersion in phosphate buffer with 10% formalin (pH 7.4) for 24 hours. Histological sections of the aortic valve were processed according to conventional histological procedures using Von Kossa staining. The technique is based on the reaction of silver nitrate in the presence of a reducing agent, usually, hydroquinone, resulting in insoluble silver phosphate that can be seen under the microscope in black. Histological sections were analyzed using a Carl Zeiss light microscope (Carl Zeiss, West Germany, Germany) (model 63,300) equipped with a Tucsen digital camera (9 megapixels) with TSview 7.1 software (Tucsen Imaging Technology Co., Ltd. Chuo, Japan, at 25x magnification. The photomicrographs were analyzed by densitometry using Sigma Scan Pro 5 Systat Software Inc. San Jose, California, CA, USA. Density values are expressed as pixel units.
7. Statistical analysis plan.

Categorical variables were expressed as proportions, continuous variables as means with standard deviation or median with interquartile range according to the distribution. Comparisons will be made for categorical variables with Chi-square or Fisher exact test and dimensional variables with Student t or Mann-Whitney U. Correlations with Pearson's r for quantitative variables or Spearman's rho for qualitative variables. Related samples were analyzed with ANOVA for repeated measurements and Friedman's test according to distribution with post-hoc analysis to identify differences between groups. The diagnostic test study will include the ROC curve to analyze cut-off points. The p-value of statistical significance was established to be less than 0.05 at two-tailed. Statistical analysis was performed with SPSS version 22 software (SPSS Inc. Chicago, Illinois).