|Year : 2021 | Volume
| Issue : 3 | Page : 82-90
Estimation of postmortem interval by postmortem myocardial computed tomography value
Zhiyuan An1, Hongxia He1, Qing Niu1, Haibiao Zhu1, Yucong Wang1, Ran Liu1, Weiliang Hou1, Peng Tang2, Tiantong Yang1, Dong Zhao1
1 Key Laboratory of Evidence Law and Forensic Science, Ministry of Education (China University of Political Science and Law), Beijing, China
2 Judicial Authentication Institute of PLA, Beijing, China
|Date of Submission||23-Jun-2021|
|Date of Decision||09-Aug-2021|
|Date of Acceptance||09-Aug-2021|
|Date of Web Publication||27-Sep-2021|
No. 25 Xitucheng Road, Haidian District, Beijing 100088
Source of Support: None, Conflict of Interest: None
Background: The estimation of postmortem interval (PMI) is one of the most important topics in forensic medicine research. We speculate that with an increased PMI, the computed tomography (CT) values of different tissues may show regular changes. Purpose: To use postmortem computed tomography (PMCT) to measure the myocardial CT value (unit: Hounsfield Unit, HU) of the heart to explore its pattern in postmortem change, and to discuss whether it can serve as a new parameter for PMI estimation. Methods: A total of 10 healthy adult New Zealand rabbits were selected and then put into a 20°C incubator after being sacrificed. Within 0–156 h after death, CT scans were performed every 12 h to detect changes in the myocardial CT value of the heart over time. Regression analysis was used to determine the relationship between the myocardial CT value of the heart and PMI. At the same time, HE and Masson were used to stain the cardiac tissue sections detected by PMCT at 0h, 48h and 156h, respectively. Results: During 0–156 h, the overall myocardial CT value showed a trend of first rising and then decreasing with the increase of PMI. The fitting regression equation was y = −2873.193 + 143.866x − 1.728x2 (x: myocardial CT value, unit: Hu; y: PMI, unit: h, R2 = 0.466, P < 0.05). During 48–156 h, the overall myocardial CT value decreased gradually with the increase of PMI. The fitting regression equation was y = −93.038 + 18.700x − 0.321x2 (x: myocardial CT value, unit: Hu; y: PMI, unit: h, R2 = 0.963, P < 0.001). The results of the morphological changes of the myocardial tissue structure after death showed that the myocardial cell structure was relatively complete at 0−48 hours after death; and the myocardial cell structure disappeared at 156 hours after death. Conclusions: Our results revealed evident postmortem changes in the myocardial CT value of the heart. Accordingly, measuring the myocardial CT value through PMCT shows promise for being used as a parameter for PMI estimation in forensic medicine and is worthy of further studies. The morphological changes of the myocardial tissue structure after death provide morphological basis for postmortem changes of tissue density, and further prove the reasons for the changes of CT value.
Keywords: Computed tomography, forensic pathology, myocardium, postmortem change, postmortem interval
|How to cite this article:|
An Z, He H, Niu Q, Zhu H, Wang Y, Liu R, Hou W, Tang P, Yang T, Zhao D. Estimation of postmortem interval by postmortem myocardial computed tomography value. J Forensic Sci Med 2021;7:82-90
|How to cite this URL:|
An Z, He H, Niu Q, Zhu H, Wang Y, Liu R, Hou W, Tang P, Yang T, Zhao D. Estimation of postmortem interval by postmortem myocardial computed tomography value. J Forensic Sci Med [serial online] 2021 [cited 2022 Dec 2];7:82-90. Available from: https://www.jfsmonline.com/text.asp?2021/7/3/82/326803
| Introduction|| |
Postmortem interval (PMI) is defined as the time between death and the discovery of the corpse. Estimation of PMI is very important for judging the nature of cases and restricting the scope of investigation and suspect, which is always an emphasis and difficulty for forensic pathology. Because of the importance of PMI estimation, scholars have mainly been focusing on PMI estimation methods. The most common research directions involve morphology and biochemistry,,, as well as studies in entomology and microbiology.,, However, there is still a lack of estimation methods for practical application. One of the relevant research directions for PMI estimation is the use of new technologies for the detection of substances or parameters that demonstrate a certain pattern in postmortem change.
Postmortem computed tomography (PMCT) is one of the new methods applied to PMI estimation in forensic medicine. Its essence is to discover density and structural changes or substance losses since death. The computed tomography (CT) value is a measurement unit used to determine the density of a local tissue or organ in the human body. As a result, the CT value can be used to reflect the postmortem change trends in the density of a substance. From the perspective of changes in the substance density after death, CT technology is expected to become one of the most powerful tools for PMI estimation.
The heart is located in the chest, protected by the thorax and surrounding organs, and is therefore in the most central position of the human body. Because it is relatively isolated from the outside environment, the heart is one of the most useful specimens for PMI estimation.
Currently, there are some PMI studies based on heart data. However, most focus on the analysis of nucleic acid and protein components in the heart, such as beta-actin mRNA and oxidant/antioxidant parameters with PMI., Some scholars have studied PMI estimation according to the morphological and histological changes in the heart. Yet, there are few studies on the application of CT technology in the postmortem changes of the cardiac muscle or the myocardium.
In view of the current situation of PMI estimation, taking the heart as the research sample, PMCT was used to detect the changes of myocardial CT value after death, and the statistical analysis was carried out. Finally, the myocardial tissues at 0 h, 48 h, and 156 h after death were sliced, stained, and observed to find the pathomorphological basis for the changes of myocardial CT value after death. This study will provide new research methods and ideas for forensic PMI estimation.
| Materials and Methods|| |
Preparation before experiment
A total of 10 New Zealand rabbits, male or female, were provided by the Beijing Keyu Animal Breeding Center and used in this study. Rabbit weight ranged between 1.5 kg and 2.0 kg. Before the experiment, all rabbits had free access to food and water at room temperature and under the conditions of natural lighting and adaptive feeding for 72 h. Afterward, the rabbits were sacrificed by auricular vein gas embolism. CT examination was performed immediately after their death, which was used as the data at 0 h. Next, all the animals were placed in an incubator (MJ-70-I, the temperature control range was 0°C–60°C, the temperature control precision was ± 0.5°C, Shanghai Bluepard) stored at 20°C ± 0.5°C. Myocardial CT scans were performed at 12-h intervals up to 156 h. The study was approved by the Ethics Committee of the Institute of Evidence Law and Forensic Science of China University of Political Science and Law (CUPL). The experiment was conducted in the lab of the Institute of Evidence Law and Forensic Science of CUPL. All animal studies were done in compliance with the regulations and guidelines of the Institute of Evidence Law and Forensic Science of CUPL and conducted according to the AAALAC and the IACUC guidelines. Ethics committee approval was obtained from the Institutional Review Board of the China Political Science and Law(#202005) on May 23/2020.
Computed tomography scan and analysis
Before the CT scan, the rabbit was placed in a supine position, with both forelimbs placed on both sides of the chest, with the head straight. The CT equipment used was the German Siemens SOMATOM Scope 16, and the scanning conditions were as follows: tube voltage 130 kV, tube current 40 mA, pitch 0.6 mm, acquisition layer thickness 16 mm × 0.6 mm, acquisition field of view 500 mm, and air calibration of the CT before each scan. The reconstruction range is the scanning range, the slice thickness is 1.0 mm, the slice distance is 1.0 mm, and the reconstruction field of view is 500 mm. Bone algorithm and soft-tissue algorithm are used for reconstruction, respectively.
The image workstation (Anythink CT , V4.2; CREALIFE Medical Technology Company, Beijing, China) is used for multiplanar and three-dimensional reconstruction of data. The volume analysis function is used to draw the region of interest of the heart. The volume analysis function is used to map the region of interest of the heart, measure the CT value of the myocardium [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6] and calculate the average CT value of the myocardium. All reconstructions and measurements are performed by a forensic doctor under the supervision of another senior forensic doctor (with at least 5 years of work experience). Only when two medical examiners reach a consensus, reconstruction and measurement are considered valid.
|Figure 2: Measuring the volume of region of interest of scanned image at 0 h since death. The region of interests of heart (shown in green) was collected and the computed tomography value of the heart was measured|
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|Figure 4: Measuring the volume of region of interest of scanned image at 48 h since death. The region of interests of heart (shown in green) was collected and the computed tomography value of the heart was measured|
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|Figure 6: Measuring the volume of region of interest of scanned image at 156 h since death. The region of interests of heart (shown in green) was collected and the computed tomography value of the heart was measured. The organs rotted and underwent autolytic changes, a large amount of putrefactive gas could be seen, and the normal heart structure was destroyed|
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The IBM SPSS statistical software (version 22.0, IBM Corp, Armonk, NY, USA) was used to perform statistical analysis on the collected data. The Shapiro–Wilk test and Levene's test were used for the normality test and the homogeneity test for variance, respectively. Under the premise that the data are normally distributed, and the variances are homogeneous, the CT value of each PMI time point (including 0 h) is compared by analysis of variance. When the data were not normally distributed and/or the variance was heterogeneous, intragroup differences were tested with the nonparametric method. The Friedman test was used to test the data for intragroup differences. P ≤ 0.05 was considered as statistical significance; P ≤ 0.01 was considered as dramatic statistical significance; P ≤ 0.001 was considered as extremely significant statistical significance. The SPSS software was used to draw the error line graph and fit the regression equation.
Paraffin embedding and sectioning
- Draw materials: take heart tissue at 0 h, 48 h, and 156 h after death, and cut into tissue pieces about 3–4 mm in one direction with a blade, no more than 5 mm
- Fixation: put the cut tissue block into 4% paraformaldehyde (PFA) and fix it. The volume ratio of the tissue block to 4% PFA is 1:7
- Wash off PFA: after fixing, rinse with running water 3 times, 5 min each time
- Dehydrated and transparent: dehydration: 30% ethanol → 50% ethanol → 70% ethanol (key steps: 2 h or overnight) → 95% ethanol 1 → 95% ethanol 2 → absolute ethanol; transparent: 50% ethanol + 50% xylene → xylene 1 → xylene 2 → xylene 3
- Wax dipping: melt the paraffin and keep the temperature at about 55°C. It takes three passes of wax
- Embedding: put the tissue block into the mold containing wax liquid, and the required tissue cut surface is parallel to the bottom. Because the wax liquid is easy to solidify in cold environment, this step should be as fast as possible
- Slicing, spreading, and baking: the myocardial tissue was cut into 4–6 um thick sections. The cut pieces were carefully transferred to the clear water in the developing table at 38°C–40°C with a brush, and the pieces with complete tissue shape and no knife marks were selected for picking up and developing. Then put it into a 60°C oven to bake the slices overnight, take it out, and store it at room temperature.
Hematoxylin and eosin staining
- The slices were dewaxed in xylene and gradient ethanol, and then washed with running water
- Hematoxylin semen was stained for 5 min and washed with running water for 5 min
- One percent hydrochloric acid ethanol solution for 30 s and water washing for 30 s
- Staining with 0.5% eosin solution for 2 min and washing with running water for 30 s
- Gradient ethanol dehydration, transparent xylene, and neutral gum sealing.
- Put the slices into Bouin solution and put them into 37°C incubator for 2 h for mordant dyeing and washing
- Dyeing with azure blue for 2–3 min and washing with water
- Hematoxylin semen was stained for 2–3 min, differentiated with 1% hydrochloric acid ethanol differentiation solution, and washed with water
- Dye with fuchsin solution for 10 min and wash with water
- One percent molybdophosphoric acid solution for 10 min
- Aniline blue dye for 5 min
- Treatment with weak acid solution for 2 min
- Gradient ethanol dehydration, transparent xylene, and neutral gum sealing.
| Results|| |
When the experiment was carried on for 168 h, the organs rotted and underwent autolytic changes, a large amount of putrefactive gas could be seen, and the normal heart structure was destroyed, so the CT value of the heart cannot be measured and the experiment was thus terminated. In total, 14 groups of CT value data at different PMI time points were obtained. The mean value, standard deviation, and 95% confidence interval for each group of data were obtained [Table 1]. The Shapiro–Wilk test and Levene's test were used for the normality test and the homogeneity test for the variance of the data, respectively. The Shapiro–Wilk test showed that the CT values in the PMI-0 h group did not conform to normal distribution; Levene test showed that P < 0.05, so it showed that there was heterogeneity in the data, and nonparametric test should be carried out for statistical analysis [Table 2] and [Table 3]. An intragroup difference analysis was performed with the Friedman test and P < 0.05, indicating that the intragroup differences were statistically significant [Table 4].
|Table 2: The experiment groups were tested in terms of normality by using the Shapiro-Wilk test|
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|Table 3: The experiment groups were tested in homogeneity of variance by using Levene's test|
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|Table 4: Computed tomography value of myocardium multiple comparisons of the groups with nonparametric tests|
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Denoting PMI as the independent variable, CT value as the dependent variable, and 95% of the average CT value as the confidence interval, the error line diagram shows that the average CT value increases with increasing PMI within 0–48 h, and decreases with increasing PMI within 48–156 h. From the error plot and the standard deviation of CT values in each group, it can be seen that the degree of data dispersion within each group increased from 96 h after death [Figure 7].
|Figure 7: Error line graph of computed tomography values of the myocardium in 14 groups at different postmortem intervals|
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Curve fitting of computed tomography value in 0–156 h
The regression equation between PMI (0–156 h) and myocardial CT value was established by a nonlinear fitting curve. The linear equation was y = 321.702 − 5.472x (R2 = 0.210, P = 0.099). The quadratic equation was y = −2873.193 + 143.866x − 1.728 × 2 (R2 = 0.466, P = 0.032). The results show that the quadratic equation (P < 0.05) has significant statistical difference.
The actual mean CT value of the myocardium in each group was, respectively, substituted into the linear and quadratic equations to calculate the estimated PMI. The actual PMI and estimated PMI were compared, and the absolute error of PMI in each group was calculated by subtracting the actual PMI from the estimated PMI to observe the effect of the fitting equation. The absolute error of PMI in the linear equation ranged from − 40.63 to 119.78 h; it ranged from − 68.81 to 82.44 h in the quadratic equation. The results suggest that the PMI obtained with the quadratic equation can better reflect the true value [Table 5].
|Table 5: Postmortem interval (0-156 h) obtained from regression equation|
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Curve fitting of computed tomography value in 48–156 h
The regression equation between PMI and CT value was established by using a nonlinear fitting curve. PMI was calculated from the CT value at 48 h, and the linear and quadratic equations were obtained as follows: Y = 510.351 = s. 232x (R2 = 0.946, P < 0.001) and y = −93.038 + 18.700x − 0.321 × 2 (R2 = 0.963, P < 0.001). The results show that the linear and quadratic equations have extremely significant statistical significance, and the quadratic equation has a better fitting effect.
The actual mean CT value of the myocardium in each group was, respectively, substituted into the linear and quadratic equations to calculate the estimated PMI. The actual PMI and estimated PMI were compared, and the absolute error of PMI in each group was calculated by subtracting the actual PMI from the estimated PMI to observe the effect of the fitting equation. The absolute error of PMI in the linear equation ranged from − 12.18 to 10.90 h; it ranged from − 7.79 to 14.58 h in the quadratic equation. The results suggest that the PMI obtained with the quadratic equation can better reflect the true value [Table 6].
|Table 6: Postmortem interval (48-156 h) obtained from regression equation|
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Pathological changes of hematoxylin and eosin staining of myocardial tissue since death
- PMI 0 h: myocardial cell structure was intact and cytoplasmic contrast was clear. Myocardial striations were clearly seen and the structure of the leap disc was clear. There was congestion in the myocardial interstitium. Local eosinophil enhancement occurred in myocardium, and individual myocardial fibers ruptured [Figure 8]
- PMI 48 h: the structure of myocardial cells was still intact, the contrast between nucleus and cytoplasm was clear, the myocardial striation was visible, the intercalated disc structure was clear, the myocardial interstitium was loose, some fibrous connective tissues disintegrated, the connection of fibrous cells was loose, and the cells were scattered [Figure 9]
- PMI 156 h: the nucleus of myocardial tissue disappeared, the cytoplasm changed homogeneously, the local fragmentation was fine granular, the interstitial was loose, and a large amount of homogeneous staining solution could be seen [Figure 10].
|Figure 8: Hematoxylin and eosin staining of myocardial tissue at 0 h since death|
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|Figure 9: Hematoxylin and eosin staining of myocardial tissue at 48 h since death|
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|Figure 10: Hematoxylin and eosin staining of myocardial tissue at 156 h since death|
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Pathological changes of Masson staining of myocardial tissue since death
- PMI 0 h: the myocardial fibers were red, the staining of fuchsin was clear, the myocardial structure was clear, the collagen fibers were sky blue, the structure was complete, and the contrast was clear [Figure 11]
- PMI 48 h: the myocardial fiber was stained with fuchsin, the myocardial structure was clear, the staining of azure was enhanced, the collagen fiber was stained with blue, the structure was complete, and the contrast with myocardial was not clear [Figure 12]
- PMI 156 h: myocardial fibers were stained red, and their structure was unclear and granular. Collagen fibers were stained blue, and their structures were disintegrated and discontinuous [Figure 13].
| Discussion|| |
Statistical results and reliability
The purpose of this study was to observe the changes of myocardial CT value with time after death in rabbits. We found that the CT value of the myocardium increased first, and then decreased with increasing PMI, peaked at 48 h after PMI, and then decreased gradually. The differences observed were statistically significant [Tables 4 and 5]. The CT values of 0–156 h and 48–156 h after death were fitted by statistical methods. The regression equation with the myocardial CT value as the independent variable and PMI as the dependent variable was established. The quadratic equation with the highest R2 value was selected as the best mathematical model [Figure 14] and [Figure 15].
Selection of test methods
In forensic medicine, the prevalent methods used for determining PMI are based on the physical parameters of detection such as rigor mortis, algor mortis, livor mortis, supravital reaction, and postmortem decomposition.,,, However, these methods do not give an exact PMI. Therefore, attention has now been shifted toward forensic pathology to determine a more precise time elapsed since death. Because of its many advantages, such as non-/minimally invasive examination, long-term storage, and repeated use of data, PMCT has been widely used in the field of virtual anatomy in recent years. In terms of pathomorphology, PMCT has shown important findings and can be used to determine the direct cause of death. With the gradual development of PMCT technology, previous research reports stated that some CT values in the corpse were found to show regular changes with increasing PMI. Therefore, in recent years, there has been a PMI estimation study using PMCT technology to observe the generation of putrefaction gas and the change of tissue density after death.,
In this study, to not damage the heart structure, PMCT was used to continuously measure the changes of myocardial CT value within 156 h after death. The CT value was positively correlated with PMI within 0–48 h and negatively correlated within 48–156 h. The linear and quadratic regression equations with the 0–156-h CT value and 48–156-h CT value as the independent variable and PMI as the dependent variable were, respectively, established. The fitting effect of the quadratic regression equation was good. This method of measuring organ density by PMCT can be used as one of the methods of PMI estimation. It is especially worthy of further study in the application of limited human test materials.
Because of the unavailability of human samples and the difficulty in precisely controlling external environmental factors, especially the impact of temperature on PMI estimation, New Zealand rabbits were selected as the experimental subjects in the present study. The sample size was increased, the postmortem observation time was lengthened, the method of measuring the myocardial CT mean value was adopted for research, and regression equations were established. It is important to note that human samples are required for verification at a later stage to make this method truly applicable to human PMI estimation.
Analysis of the causes for changing myocardial computed tomography values
The heart is located in the center of the body and protected by the thorax, which is not easily damaged and contaminated, making it one of the best samples for inferring PMI. The results showed that with increasing PMI, the value of myocardial CT first increased, and then decreased.
Within 48 h after death, with the prolongation of PMI, the myocardial CT value gradually increased. This observation may be related to the abnormal metabolism leading to rigor mortis. Because of the gradual stoppage of oxidative phosphorylation in vivo, adenosine triphosphate (ATP) is no longer synthesized after death. The existing ATP in muscle is sharply reduced from hydrolysis, which leads to the opening of calcium ion ATPase pumps on the cell membrane, increased intracellular calcium concentrations, and condensation of muscle fibers into actin, resulting in loss of muscle elastic contracture., As we can see on the pathological slices: (1) under hematoxylin and eosin (HE) staining, from 0 h to 48 h after death, the myocardial cell structure is relatively complete, the nucleoplasmic contrast is clear, myocardial stripes are clearly visible, and the intercalary disc structure is clear. (2) Under Masson staining, from 0 h to 48 h after death, the myocardial fiber was red, the myocardial structure was clear, the collagen fiber was sky blue, the structure was complete, and the contrast was clear. These pathological results indicate that the cardiomyocytes may still be in a living state.
At 48–156 h after death, with the prolongation of PMI, the myocardial CT value gradually decreased, which may be related to autolysis. After death, the lysosome in the cytoplasm of cardiac myocytes breaks down and releases various hydrolases, such as histone hydrolase. These hydrolases gradually degrade the macromolecular compounds, such as tissue proteins, nucleic acids, glycoproteins, glycolipids, and other complexes, and destroy the morphology of tissue cells until they are completely dissolved and liquefied., As we can see on the pathological slices: (1) under HE staining, from 48 h to 156 h after death, the structure of myocardial cells gradually disintegrated into fragments and became fine particles. The horizontal stripes and intercalary disc structure gradually disappeared. (2) Under Masson staining, from 48 h to 156 h after death, the red-stained myocardial fiber structure gradually disintegrated into fragments and granular, and the blue-stained collagen fiber structure gradually disintegrated and became discontinuous. These morphological changes all suggest that the tissue density becomes smaller, which is in line with the law of gradual decrease of myocardial CT value.
This study has certain limitations. First of all, considering that the change after death is closely related to the ambient temperature, this study controlled the ambient temperature. However, in practical applications, the changes in corpses encountered in forensic medicine are affected by many internal and external factors such as age, gender, cause of death, and external environment. In addition, the heart structure of New Zealand rabbits is slightly different from that of human hearts, so the results of this study cannot be directly applied to the estimation of human PMI. Therefore, in future research, the accuracy of the fitting equation can be improved by improving temperature conditions, shortening the sampling interval, and controlling different postmortem environments.
| Conclusion|| |
This study found that the myocardial CT value after death showed that the myocardial CT value increased first and then decreased with the increase of PMI within 0–156 h and established a PMI estimation mathematical model based on the myocardial CT value. In addition, the myocardial tissues of PMI 0 h, 48 h, and 156 h were stained and observed to confirm the pathomorphological reasons for the changes in the myocardial CT value.
This study initially realized the feasibility of using myocardial CT value changes to estimate PMI, indicating that myocardial CT value can be used as one of the evaluation indicators for PMI estimation. However, it should be pointed out that the regression equation established in this study can only reflect the change of CT value at 20°C, and cannot be used to estimate PMI at other temperatures. In order to further improve the accuracy of PMI estimation, future research should include more temperature conditions, postmortem environment, time points, and animal sample selection. This is to detect the change of myocardial CT value after death and obtain more comprehensive and practical experimental results. In future, human samples should be studied to explore its application value in human PMI estimation.
We thank J. Iacona, Ph.D., from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
Financial support and sponsorship
This work was supported by the General Program of National Natural Science Foundation of China (grant number 81971796), Beijing Natural Science Foundation (grant number 7192121), Chinese Academy of Engineering Advisory Project (grant number 2019-XZ-31), and Production-Study-Research Project for graduate students of China University of Political Science and Law (grant number CXY2012).
Conflicts of interest
Dr. Dong Zhao is the editorial board member of Journal of Forensic Science and Medicine.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]