Novel Ex Vivo Ablation Test Model for Monopolar Hot Biopsy Forceps. A thesis presented to. the faculty of. In partial fulfillment

Transcription

1 Novel Ex Vivo Ablation Test Model for Monopolar Hot Biopsy Forceps A thesis presented to the faculty of the Russ College of Engineering and Technology of Ohio University In partial fulfillment of the requirements for the degree Master of Science Liang Chen May Liang Chen. All Rights Reserved.

2 2 This thesis titled Novel Ex Vivo Ablation Test Model for Monopolar Hot Biopsy Forceps by LIANG CHEN has been approved for the Department of Chemical and Biomolecular Engineering and the Russ College of Engineering and Technology by JungHun Choi Assistant Professor of Mechanical Engineering Dennis Irwin Dean, Russ College of Engineering and Technology

3 Abstract 3 CHEN, LIANG, M.S., May 2014, Biomedical Engineering Novel Ex Vivo Ablation Test Model for Monopolar Hot Biopsy Forceps (106 pp.) Director of Thesis: JungHun Choi Previous studies on hot biopsy forceps for colorectal polypectomy indicated that simulated ablation tests could provide useful information on hot biopsy forceps design and ablation parameter optimization that would help reducing the rate of potential polypectomy complications such as perforation. In this thesis, a novel ex vivo ablation test model for colonoscopic monopolar hot biopsy forceps was designed and implemented; simulated ablation tests had also been performed on this ablation test model for (a) verifying the model design and implementation, and (b) finding the roles of the major ablation parameters in tissue injury during the ablation process. In the design of this novel ex vivo ablation test model, a body equivalent block was introduced to simulate the properties of the human body. Porcine colon was used as tissue source for the simulated ablation tests. Major ablation parameters that were tested on this model covered (a) output power level, (b) firing duration, (c) contact area at the electrode tip, and (d) electrical impedance. Simulated ablation tests that were performed on this ablation test model covered the main combinations of targeted ablation parameters. During simulated ablation tests various ablation sites were generated under different parameter combinations, which indicated that this ablation test model can be used for hot biopsy forceps ablation studies. Some ablation parameters such as output power level and firing duration had dominating effects on the resulting ablation site geometry and tissue injury. The simulated test results showed the potential applications for testing hot biopsy forceps and optimizing ablation parameters.

4 4 To my wife, Fei, and our parents.

5 Acknowledgments 5 First of all, I would like to thank my advisor, Dr. JungHun Choi, for his mentoring and support on my research project. Then I would like to thank all my graduate committee members, Dr. Bob Williams, Dr. John Cotton, Dr. Shiyong Wu, and Dr. David Drozek, for their time to review my proposal and final thesis, and their comments and suggestions. I also want to thank biomedical engineering program for providing me a chance to study at Ohio University. Special thanks to following people because they give me help during my study and research: graduate student Ye Yuan in Dr. Wu s lab, graduate students Randy Robert and Chen Tang in Dr. Choi s lab, Dr. Ramiro Malgor, Noriko Kantake, Dr. Gerardine Botte, Dr. Jenkinson, Dr. Yang Li, Randy Mulford, and Tom Riggs.

6 Table of Contents 6 Page Abstract Dedication Acknowledgments List of Tables List of Figures List of Acronyms Introduction Background Objectives of Study Methods Material Preparation Ablation Test System Experiment Design Post-Ablation Analysis Results Test Model Features Ablation Tests Results Discussion Ablation Test Model Ablation Test Results Comparison with Similar Studies Limitations and Future Plans Summary References Appendix A: Software Packages Appendix B: Source Code

7 List of Tables 7 Table Page 2.1 Gel block reagent list Detailed gel formula Electrical impedance measurement parameters Test parameter combinations Tissue dehydration procedure H&E staining procedure Variable names in R code ANOVA of contact area on site volume T-test on contact area settings T-test on impedance matching ANOVA of power level and duration on site volume ( Std gel) ANOVA of power level and duration on site volume ( HiR gel) P-values of t-test on mucosa layer ratio between Std and HiR gel P-values of t-test on submucosa layer ratio between Std and HiR gel P-values of t-test on total layer thickness ratio between Std and HiR gel Effect of power level and duration on mucosa layer ( Std gel)

8 List of Figures 8 Figure Page 1.1 Diagram of human digestive system Anatomy of colon Anatomical structure of colon Large intestine wall layers Comparison of in vivo and ex vivo experiments ex vivo test model overview Gel block mold Complete circuit diagram Biopsy forceps tip and RF connector Photograph of refined electrode Refined electrode holder Photograph of the supporting frame Gel block with tissue patch Histology analysis procedure Tissue cassette and spacer Measurements on tissue slice photograph Details of electrode tip and ablation site Diagram of spherical cap Std gel impedance-frequency curve Photographs of ablation site top views Photographs of ablation site cross-sectional views Effect of power level and duration on site diameter ( Std gel) Effect of power level and duration on site depth ( Std gel) Effect of power level and duration on site volume ( Std gel) Effect of power level and duration on site diameter ( HiR gel) Effect of power level and duration on site depth ( HiR gel Effect of power level and duration on site volume ( HiR gel) Mucosa layer on Std gel Submucosa layer on Std gel Muscularis layer on Std gel Mucosa & submucosa layers on Std gel Total thickness of layers on Std gel Mucosa layer on HiR gel Submucosa layer on HiR gel Muscularis layer on HiR gel Mucosa & submucosa layers on HiR gel Total thickness of layers on HiR gel

9 ESD ESU H&E EIT EEG ECG CT AC DC PAGE PAG PBS APS TEMED PMMA PVC RF SEM aka List of Acronyms electrosurgical device electrosurgical unit Haematoxylin and Eosin electrical impedance tomography electroencephalography electrocardiography computerize tomography alternating current direct current polyacrylamide gel electrophoresis polyacrylamide gel phosphate buffer solution ammonium persulfate tetramethylethylenediamine (N, N, N, N -tetramethylethylenediamine) polymethyl methacrylate polyvinyl chloride radio-frequency standard error of the mean also known as 9

10 1 Introduction Background Colorectal polyps are the abnormal growing tissue in large intestine, and the polyps may develop into colon cancer. Monopolar hot biopsy forceps are widely used in polypectomy for colorectal polyps removal. Minimizing the potential or unnecessary injury is highly considered during medical device design. Determining and monitoring the factors involved in the injury will be useful for injury prevention. Ex vivo simulation experiment is a better choice than in vivo experiment for repeatable, comparable tissue injury evaluation test: universal, comparable results; individual-independent and experience-independent; safety and ethical issues Colon and Colorectal Polyps Digestive system is the media for humans and high grade animals to acquire energy and nutrition from the environment. Human digestive system includes the secretion system and the gastrointestinal tract (Figure 1.1). The secretion system includes salivary glands, liver, gallbladder, pancreas. The gastrointestinal tract starts from the mouth, through the oral cavity, pharynx, esophagus, stomach, small intestine, large intestine, and ends at the anus. The large intestine is the part of gastrointestinal tract where feces are generated. It is also the important region for water and vitamins intake. The large intestine is made up by cecum, colon, and rectum. The whole colon includes ascending colon, transverse colon, descending colon and sigmoid colon. The relationship of different parts is shown in Figure 1.2. The anatomy structure of the colon is shown in Figure 1.3. The layers of the colon wall include mucosa, submucosa, and muscularis (internal/inner and external/outer layers) [1].

11 11 Figure 1.1: Diagram of human digestive system Colon polyps are the abnormal tissue which may develop into colorectal cancer[2]. They initiate from the epithelial and mucosal tissues in the colon wall and had no notable symptoms in feces[3]. Polyps have several different morphological types, such as stalked, round, and sessile. However, they can only be classified by histological evaluation on the biopsy specimens[4] Colonoscopy Screening and Polypectomy Without proper treatment malignant colorectal polyps may develop into colorectal cancer. Colorectal cancer is the third most common cancer in both men and women. In 2011 approximately 141,300 cases and 49,400 deaths of colorectal cancer were expected

12 12 Transverse colon Ascending colon Cecum Part of small bowel Sigmoid colon Descending colon Appendix Anus Rectum Figure 1.2: Anatomy of colon Tenia coli (longitudinal muscle) Appendix epiploica Circular muscle Mucosa Submucosa Figure 1.3: Anatomical structure of colon. in the US [5]. Although the post-surgery survival rate is relatively high, the population of expected colorectal cancer patients is still increasing [5].

13 13 Figure 1.4: Section of large intestine wall to show the layers. M, mucosa; SM, submucosa; MM, muscularis mucosae; ME, muscularis externa. (Reproduced with permission, from [1]) Early detection of colon polyps is the key for preventing colorectal cancer. Patients are suggested to take routine polyps screening every two or three years [4]. Many medical and clinical examination methods are applied for screening colon polyps such as X-ray, endoscopy and colonoscopy, computerize tomography (CT). Colonoscopy surgery is the practical and cost-efficient way to screen and detect polyps [6]. Colonoscopes are the medical devices for colorectal investigating and surgical monitoring[7]. The treatment sites of colon polyps are deep in patient s body, and the colorectal cavity is small, humid, dark, and hard to sterilization. These properties of the

14 14 colon determine the models and the ablation methods used in medical research and clinical practice. Because of the limited space of the colorectal cavity, the size of surgical instruments and devices is limited. The geometry of the colonoscope is usually small in diameter but long in length. The average diameter of the colonoscope is below three centimeters, but the average length of the colonoscope is over 90 centimeters[4]. The selection of the colonoscope geometry are based on the following aspects: average size of human anus, average length and diameter of human colon, the flexibility of the colonoscope required by physicians. Currently several different types of ablation methods are invited for polyps removing, such as argon coagulation, laser, electrothermal cauterization, and abdominal surgery. The hot biopsy forceps is commonly used instrument for polyps removal because it is suitable for both sampling and ablation. Polypectomy is the clinical practice of colorectal polyps removal. The method of removing colon polyps by using the colonoscope is called colonoscopic polypectomy. Different methods are applied to remove the polyps depending on the size and shape of the polyps. A previous study showed that colonoscopic polypectomy resulted in a lower-than-expected incidence of colorectal cancer [8]. However, some risks and complications are associated with colonoscopy and polypectomy. Common complications are bleeding, perforation, and post-polypectomy ablation syndrome [9]. The method of using thermal effect of electric current in polypectomy is called electrical ablation (electrocautery). Electrical ablation has a long clinical application history[10]. Although many different types of thermal-based polyp removal methods have been developed and applied in clinical practice, for example argon and laser, the electric current is still widely used. Electric current has many advantages over some other thermal

15 15 generating methods: simple, portable device; no special energy conversion step needed; easily controlled power output; and fast and direct thermal generation. The power generator for electrical ablation operations is named electrosurgical unit (ESU). All the surgical devices, including power generator, hot forceps, hot snare, are electrosurgical devices (ESDs). The electrosurgical device is one of the major instruments for the electrical ablation operations because it generates the current flows for cautery[11, 12]. Electrosurgical devices generate different types of waveform for cutting and coagulation; they have adjustable power output for different patient individuals. Although hot biopsy forceps are the most commonly used instrument in polypectomy, they still have some disadvantages such as incomplete removal, size limit, alternative site injury, and potential perforation risk [13]. Colorectal polyps surgical operations were helpful for preventing colorectal cancer, while the complications of polypectomy arise. Some complications such as perforations may result in death, therefore prevention of polypectomy complications is important. Studies on polypectomy[13 18] had shown that many factors may affect the resulting complications. Much effect had been made in the perforation prevention field, including redesigned surgical devices, enhanced physician training protocols, tissue injury evaluation, etc. Studies[19] on active electrode monitoring technique was a good example of finding potential ways of preventing unintentional thermal injury Tissue Injury: Evaluation and Prevention The mechanism of electrical ablation depends on the thermal effect of the electric current. Because the applied current passes through the body tissue, peripheral regions of the electrical ablation sites also receives the heat from the current or the passive distribution of the heat.

16 16 Some researchers have worked on modeling the theoretical heat and mass transfer functions of animal tissues[20]. Although the method of measurement of quantitative heat transfer was not mature, researchers often use duration time and output power of electrosurgical device for energy output estimation [21]. Some other researchers tested specially designed electrosurgical devices and compared them with conventional monopolar electrosurgical devices [22, 23]. Based on new devices, electrothermal injury could be well controlled and reduced, and the tissue healed well and fast [22]. Another study focused on the thermal management that could limit the heat effective field in the target tissue, and utilized a computer model for stimulated tests [23]. Finding optimal parameters for electrosugery is one hotspot for studies on conventional monopolar devices[24]. Many studies have attempted to find out methods to minimize or prevent tissue injury peripheral to the ablation sites[14]. In order to compare the tissue injury results with other studies, a grading system for scoring tissue injury is required. In previous studies[25, 26], a grading system for scoring the depth of tissue injury has been established based on the layers of the colon wall involved in tissue injury. A similar grading system was also used in colon polyps cautery evaluation[2]. In contrast, some other studies used the quantitative depth of the ablation sites to describe the degree of tissue injury that was caused by electrical incision[22]. Compared with the quantitative depth system, the tissue layer system is much better because it has greater clinical relevance [25] Tissue Impedance and ex vivo Simulation For ex vivo emulation of electrosurgical experiment, the proper simulation mechanisms should be applied for obtaining similar results in vivo (Figure 1.5). The most important parts are determining the electrical impedance of in vivo live tissue and building artificial materials for generating equivalent resistance.

17 17 (a) in vivo Colonoscope ESU Hot Biopsy Forceps Colon Wall Skin (b) ex vivo Colonoscope Hot Biopsy Forceps Colon patch ESU Equivalent Stack Figure 1.5: Comparison of in vivo and ex vivo experiments. Previous research[25] had measured tissue impedance during electrical ablation experiments, but the ex vivo simulations were just used for energy power consumption measurement. Precise measurement of tissue impedance is still unavailable or too hard to implement. As the individual differentiation also increases the difficulty off tissue impedance measuring Current Status and Future of ex vivo Model In vitro porcine training model for colorectal endoscopic dissection had been described by previous study[27], and their setup was simple. The simple setup connects

18 18 the gastrointestinal tract tissue with the electrical ablation circuit directly, for example, the GI tract may be placed directly on the returning pad, or placed in a metal plate with a returning pad attached to it. Some setup had saline in the metal plate and dipped part of the GI tract tissue in saline to emulate the normal physiological status in living animal body [28]. There are some studies of ex and in vivo test model and training models [29, 30], and some researchers admitted that variance exist between different species and organs [31]. The electrical properties (impedance, capacitance) and mechanical properties (hardness, tensile strength, etc) are important to build a good ex vivo emulation model. The current existing ex vivo models found in literature usually had simple design of the body environment emulation that only represented partial of the tissue properties. Another aspect of a good ex vivo model is the emulation of the normal or pathogenic in vivo physiological status of the human body tissue may provide advanced ablation test model. 1.2 Objectives of Study The purpose of this study is to build an ex vivo test model in order to determine the association between the electrical ablation environmental parameters and the electro-thermal ablation effects. The test parameters include the power output of electrical polypectomy operation, firing duration time, and the contact condition at the tip of forceps Establish An ex vivo Simulated Electrical Ablation Test Environment To better emulate the electrical ablation environment in the colon, an ex vivo simulated test environment is needed for this research. A previous study had used a similar method for energy measurement [25], but their study used an in vivo test model; the in vivo model involved many factors in their results. Similarly in vivo studies [14, 24] attempted to work on the electrical ablation parameters, but the results and conclusions were also limited by the test model. A universal and stable test model will help compare the different studies.

19 Post-ablation Morphological and Histological Analysis and Evaluation The new post-ablation evaluation included in this study will be based on the post-electroperation analysis that includes immunohistological analysis (in situ antibody labeling) and Haematoxylin and Eosin (H&E) staining. Immunohistological analysis of injured tissue is different from analysis of normal tissue because during the injury process there were many morphological, physical, chemical changes within the tissue, such as protein degeneration, dehydration, thermal effects. The H&E staining protocol for this research will be based on existing standards [32]. For data analysis and classification, a new grade evaluation system will be built based on the grade system from previous studies [25, 26, 33]. The results of this research will provide the methods and the model for finding the optimal parameters for treating colon cancer safely and effectively. It will also help to develop a new automatic computerized assistant colonoscopy movement control system.

20 2 Methods 20 The complete ex vivo ablation test model is the combination of material preparation, ablation system, ablation tests, and post-ablation analysis and evaluation. All the components of this ablation test model are explained with detailed information. Material preparation covers the tissue preparation and gel block preparation. Ablation system includes all the electrical circuits and supporting fixture. Post-ablation analysis and evaluation includes histology method analysis, ablation sites geometry measurement, and tissue injury evaluation. The overview of the testing procedure on this ex vivo test model is shown in Figure 2.1. Colon cutting Colon patch Tissue loading Ablation test Gel solution Gel formation Dehydrization Measurement Staining Slicing (cross dissection) Embedding Figure 2.1: Overview of the ex vivo test model. The complete ablation test procedure in this test model includes colon cutting, gel formation, tissue loading, ablation test, dehydration, embedding, slicing, staining, measurement, and data analysis (not shown in this figure).

21 Material Preparation Material preparation is the fundamental part of this ex vivo test model. It includes the colon tissue preparation and the gel block preparation. Both the tissue sample and the gel blocks should be well prepared before performing ablation tests. This section will describe the preparation procedures in details Colon Tissue Preparation In this test model the porcine colon was used as the source of the raw material. The fresh porcine colon was rinsed with tap water to remove feces. Then the porcine colon was cut longitudinally into strips and flattened. Flattened porcine colon strips were rinsed with PBS, then dried with paper towel. Finally the porcine colon strips were stored in -20 C freezer for temporary storage. Before the ablation test, the porcine colon strip was brought back to room temperature and rinsed with distilled water, then cut into 1 1 inch patches. The frozen porcine colon strips were used within one month since the frozen date, and any remaining frozen colon strips were discarded after one month since the frozen date Gel Block Preparation The gel block is an important part in this test model because one ablation parameter is related with it. Both the design and the preparation of the gel block are critical to this ablation test model Gel Block Design The gel block in this test model is required to have proper mechanical properties (size, shape, and stiffness) and electrical properties (impedance), and adjusting the properties should be easy. The gel blocks that were used in this test model were made from polyacrylamide gel (PAG) that is commonly used in biological sciences. Standard

22 22 polyacrylamide gel protocol[34] was followed with some modifications to fit the needs in this test model. The reagent list of standard polyacrylamide gel is shown in Table 2.1. The formula of polyacrylamide gel can affect the electrical properties and the stiffness of the final gel blocks; the geometry of the gel block can also affect the electrical properties and the stiffness: these features are useful to the gel block geometry design. Table 2.1: Gel block reagent list Reagent Acrylamide/Bis-Acrylamide Tris-HCl Buffer APS TEMED Description 40% solution 1.5M, ph 8.8 solution white to yellowish crystals solution Distilled degas water According to the gel protocol, the gel block should be made within a container (aka. gel block mold ) that holds the gel solution for a short period of time; and the shape of the gel block mold should not be very complex because it will cause problems when removing the final gel block out of the gel block mold. Therefore, the final design of the gel block was set as a straight cylinder. A simple gel block mold was designed in order to fit with the gel block. The final design of the gel block mold was a container with cylinder-shape internal space, and the bottom of the mold was removable. The gel block mold was made from a short segmentation of PMMA pipe and a piece of PMMA plate, and the two parts can be locked together (shown in Figure 2.2).

23 23 Figure 2.2: Photograph of the gel block mold. This gel block mold was made from a short segmentation of PMMA pipe and a piece of PMMA plate Preparation Procedure For gel block preparation, some procedures were needed. A typical preparation cycle for a gel block includes the following steps: gel block mold preparation and assembling, gel solution preparation, gel formation, transferring to storage Gel Mold Preparation For each gel block, all the components of the gel block mold must be carefully rinsed by distilled water, and then assembled together. When assembling the mold, vacuum gel

24 24 must be applied onto the contact surface of the mold components to prevent any potential leakage Gel Solution Preparation The formula for the gel solution is based on the standard 15% resolution PAG protocol[34], and the detailed gel formula (the quantity of gel reagents) was shown in Table 2.2. Standard polyacrylamide gel kit (Bio-Rad, USA) was used during solution preparation. The standard kit includes 40% acrylamide/bis-acrylamide solution, 1.5M Tris-HCl buffer solution Special Notes for Gel Solution Preparation Because APS solution is unstable it should be prepared at each time of gel preparation. The preparation of APS solution should be done as follows: proper amount of APS powder is weighted within dispensable plastic weighting dish; proper amount of deionized water is added into the plastic weighting dish, stir is needed until APS is fully dissolved in water; the fresh APS solution is dumped into the gel block mold (with acrylamide solution and Tris-HCl buffer already in it), and the weighting dish is washed by deionized water two or three times; all the water must be dumped into gel mold (making sure not exceed the total volume of the gel block), and the gel solution in the gel mold must be stirred Gel Formation The detailed gel formation procedure is described as follows: first, the acrylamide/bis-acrylamide solution and Tris-HCl buffer were mixed together in the gel block mold; second, the APS solution were added into the gel solution; the gel solution was carefully stirred; then the catalyst TEMED was added into the gel solution to start the consolidation procedure; about twenty minutes later the gel block was formed.

25 Transferring and Storage After the gel block became consolidated, contact surface of the gel block and gel mold should be cleaned by a nail pin. Then the gel mold was disassembled and the bottom blocker piece was removed. The final gel block was carefully removed out of the gel mold and transferred into cold distilled water for temporary storage before actual use. The polyacrylamide gel blocks should be kept in sealed containers with cold distilled water, and the gel containers should be placed in cool shady place, for example, in 4 C refrigerator. Table 2.2: Detailed gel formula Reagent Amount 60 ml gel 80 ml gel Acrylamide/Bis-Acrylamide 22.5 ml 30 ml Tris-HCl Buffer (ph 8.8) 15 ml 20 ml APS 60 mg 80 mg TEMED µl 80 µl Distilled degas water 22.5 ml 30 ml 2.2 Ablation Test System In this ex vivo ablation test model, all the simulated ablation tests were performed within the ablation test system. The ablation test system includes many components for simulating in vivo monopolar hot ablation operations. The components of this system includes the electrosurgical unit (ESU), the ablation electrode, ablation firing control system, body equivalent stack, and supporting frame.

26 26 All the components can be classified into two major categories: core parts and accessories. Core parts are those components that are directly involved in the ablation operations, such as ESU and body equivalent stack. Accessories are those components that are not directly involved in the ablation operations, such as supporting frame and connecting wires. The electrical circuit diagram of the ablation test system is shown in Figure 2.3. The two major parts within the electrical circuit are the energy generating circuit (Figure 2.3A), and the test zone (Figure 2.3B). The energy generating circuit includes the ESU and firing control system, and the test zone includes the electrode and tissue patch. Isolation switches (also shown in Figure 2.3) were used to isolate the high-voltage electrical current from the test zone for safety reasons. When the isolation switches are set to b, the electrode and returning pad are connected to the ground Energy Generating Circuit The firing part includes the power generator (ESU) and firing control circuit. The function of the firing part is generating and delivering electric power to the tissue sample with selected power level and duration. This part is shown in Figure 2.3A. When the isolation switches are set to a, the energy generating part is connected to the electrode tip and returning pad Power Generator (ESU) A clinical monopolar electrosurgical power generator, Olympus PSD-20 (Olympus, Japan) was used. No modifications were made to the electrosurgical power generator except for the foot pedal.

27 27 A ESU Isolation switch a B b Electrode Tip Firing Control a b Isolation switch Tissue Patch Gel Block Returning Pad (Grounding Patch) Figure 2.3: Complete circuit diagram. Both the energy generating part and the test zone were displayed. A, energy generating part (power generating and trigger control system); B, test zone. The isolation switches separate the energy generating part and testing part to protect the test operator from high voltage electric shock. When the isolation switches are set to a, the electrode and returning pad are connected to the energy generating part. When the isolation switches are set to b, the electrode and returning pad are connected to the ground Control Circuit An Arduino UNO microprocessor board was programmed and connected to the foot pedal of the ESU to trigger the ESU, and this circuit was used for precise ablation firing duration control. The firing duration is adjustable through the source code (source code is listed in Appendix B.1). The firing duration values are selected by three(3) push buttons on the control circuit.

28 Test Zone The test zone includes the electrode, returning pad, and the body equivalent stack (tissue-gel complex) Ablation Electrode and Holder In preliminary experiment[35] both the original hot biopsy forceps and radio-frequency cable connector (equivalent in size and shape) were used (photograph of forceps tip and connector is shown in Figure 2.4). The hot biopsy forceps was loaded in the colonoscope during ablation test. It provided the best emulation effects on the ablation test at the forceps tip, but there were some trade-offs: (a) the surface of the forceps tip is very complex and the exact contact status at the tip is unknown; (b) the cord of the biopsy forceps is flexible, which made it hard to maintain a stable contact at the tissue surface when the forceps clamp is closed; (c) mounting the colonoscope can not be very tight and stable; (d) no locking mechanisms were available to lock the forceps in fixed position. In order to reduce the systemic error induced by the colonoscope and the forceps, a short segmentation of RF cable was used to replace the colonoscope-forceps combination. A RF cable connector with similar diameter to the forceps tip was used to emulate the forceps tip (shown in Figure 2.4). The results of the preliminary experiment[35] showed no difference between real forceps and replaced electrode (RF connector). In addition, maintaining stable contact status (pushing force and contact area at the electrode tip) is the key element for performing comparisons among different ablation parameter combinations. In order to simplify the experiment design and maintain stable contact area at the electrode tip, a new fixture (aka. electrode holder ) for holding the electrode was designed and implemented. The new electrode holder was shown in Figure 2.5.

29 29 Figure 2.4: Biopsy forceps tip and RF connector. Forceps tip is on the left side, and the RF connector is on the right side. The detailed structure of the refined electrode holder is shown in Figure 2.6A. This new electrode holder will provide better control on the pushing force or pushing depth of the electrode tip against the colon patch surface than the real forceps tip or RF connector (shown in Figure 2.6C). The pushing depth of the tip is controlled by the length of the electrode tip and the total weight of the electrode and the holder Body Equivalent Stack Body equivalent stack (shown in Figure 2.3B) was made up by the tissue-gel complex (prepared gel block with porcine colon patch loaded on the top surface). The whole body equivalent stack was placed on the returning pad (the grounding patch of the ESU). The electrical impedance of the body equivalent stack can be changed in order to emulate the matching or dismatching effective electrical impedance of the patient s body during actual ablation.

30 30 Figure 2.5: Photograph showed the details of the refined electrode and the holder Supporting Frame The supporting frame is an assembled metal portal frame with the connecting wires and isolation switches mounted on it (shown in Figure 2.7). The test stack (electrode tip, tissue patch, gel block, and returning pad) was placed below the frame. ESU and firing control circuit were put away from the supporting frame to reduce potential electromagnetic interference.

31 31 A B PMMA plate PVC tube Copper electrode tip C Electrode holder Gel surface Figure 2.6: Refined electrode holder. A, cross sectional view of the electrode holder; B, aerial view of the electrode holder; C, cross sectional view of the electrode holder when placed on the gel block surface. 2.3 Experiment Design The overall experiment design for this ex vivo ablation test model includes (a) model design verification, and (b) simulated ablation test Model Design Verification The electrical impedance values of the prepared gel blocks under radio-frequency current were measured by Solartron (Solartron Analytical, USA) at Center of Electrochemical Engineering Research (CEER, Russ College of Engineering and Technology, Ohio University). Both the electrode tip and the returning pad (grounding patch) for ablation test were used in electrical impedance measurement, and the body equivalent stack was placed as it was used in simulated ablation test (Figure 2.8) to

32 32 Figure 2.7: Photograph of the supporting frame. The isolation switches were mounted on it. The measurement circuit was mounted on the rear side, not shown in this photo. The test stack was placed below the frame, not shown in this photo. minimize the possible differences. The test parameters for electrical impedance measurement were shown in Table Simulated Ablation Tests The experiment design of the simulated ablation tests was fully covered experiment: all the ablation parameter combinations were tested, and each combination had several repeats in order to eliminate random errors.

33 33 Figure 2.8: Gel block with tissue patch loaded. The refined electrode and the holder were also shown in this photo Parameter Selection The different combinations of ablation test parameters were shown in Table 2.4. All the values of ablation parameters were selected based on preliminary test results[35] Configuring Ablation Parameters in Test Model The values of output power level were manually input into ESU during ablation test. The values of firing duration were predefined in the source code of the firing control circuit (Appendix B.1) and complied and downloaded to the Arduino board; during ablation test, three different push buttons in the firing control circuit were used to switch among different duration values. Contact area was set when loading tissue patch and

34 34 Table 2.3: Solartron electrical impedance measurement parameters Parameter Experiment Type DC potential AC potential Frequency (lower bound) Frequency (upper bound) Frequency Sweep Type Frequency Step Value Frequency Sweep, Control Voltage 0 mv 2000 mv 200 Hz 300k Hz (device s maximal output freq.) Log Decade Frequency Interval 50 Connection scheme Working electrode Control electrode two electrode system electrode tip grounding patch Table 2.4: Test parameter combinations Parameter Unit Values Count power level watt 30, 60, 90 3 duration second 0.5, 1.5, 6 3 contact area small, middle 2 gel type Std, HiR 2 placing electrode holder on top surface of the gel block. Contact area between the colon patch and electrode tip was controlled by the pushing force of the electrode.

35 Repeated Trials For each individual ablation parameter combination, several repeats ( trials ) were needed. Due to the numbers of total trials, four(4) trials were performed for each combination Test Procedure For each combination of the test parameters, the test trial was performed with the following procedure: 1. All the components of the test model were well prepared, assembled together with wires connected. 2. The colon tissue patch was loaded on gel block surface, and the electrode holder was placed on top of the gel block. 3. Pre-selected ablation parameters were set, and pre-ablation measurement was performed. 4. Actual ablation was performed. (aka. firing ) 5. Post-ablation measurement was performed. 6. Tissue patch was unloaded from gel surface for histology analysis steps, and the other test model components were reset for the next test trial. 7. Repeat step 2 to 6 until all the test trials were performed, then clean up all the test model components. 2.4 Post-Ablation Analysis Post-ablation analysis is the key step to retrieve important data and information of the ablation sites. Post-ablation analysis includes performing histology analysis methods on the ablation sites, photographing and measurement under microscopy, and statistical data analysis and plotting.

36 Ablation Sites Photographing Photographing on ablation sites will be used for site geometry measurement. Because the histology analysis method is a non-reversible process, essential data, such as photographs of the ablation sites, must be collected before histology analysis Photographing before Histology Analysis Photographs of the top view of the ablation sites were taken immediately after the each ablation test. These photographs were used as the reference for indexing all the burned sites Photographing and Measuring under Microscopy after Histology Analysis After histology analysis, the cross sectional slices of the ablation sites were photographed under microscopy, and the measurements were performed on these photographs. The measurements were used in the ablation sites volume calculation and tissue injury evaluation Histology Analysis Histology analysis provides in-depth information about the tissue injury that was generated by electrical ablation, such as the tissue structure changes, cell and tissue functionality changes. Histology analysis utilizes many staining and labeling methods to enhance the ability and specificity of presenting micro-structure of the tissue specimens. In this ex vivo test model, a tissue staining method, Haematoxylin and Eosin ( H&E for short) staining, was used for presenting the layer structures within the porcine colon tissue specimen. H&E staining includes three general steps: specimen embedding, slicing, and staining. The ablated colon tissue patches were cut into small size specimens before embedding procedure. Photos of histology analysis procedure were shown in Figure 2.9.

37 37 Figure 2.9: Histology analysis procedure. Top left, embedded specimen blocks; top right, embedded block mounted on microtone for slicing; bottom left, specimen slices; bottom right, specimen slices mounted on glass slide Dehydration Protocol Specimens were manipulated with Ethanol gradient and Xylene for dehydration, then the patches were placed in melted paraffin to prepare for embedding. The detailed dehydration procedure was shown in Table Modifications for Dehydration Procedure In preliminary experiment[36] changes in the size and shape of the ablation sites were observed during the specimen dehydration procedure before embedding, therefore some modifications were made to the dehydration process. During dehydration procedure tissue specimen sometimes folded or rolled up, which resulted in changes in the size and

38 38 Table 2.5: Tissue specimen dehydration procedure. Step Reagent Time 1 70% Ethanol overnight 2 85% Ethanol 2 hour 3 95% Ethanol hour 4 100% Ethanol 30 min (repeat 3 times) 5 Xylene 20 min (repeat 3 times) 6 Paraffin I 1-2 hour 7 Paraffin II overnight 8 Paraffin III 30 min shape of the ablation sites. The modification is reducing the internal space of the tissue cassette in order to prevent tissue specimen from folding or rolling up. The method of implementation was: placing a small piece of Xylene-resistant plastic spacer in the cassette. The material was from the cassette cover. One third ( 1 3) in length of the cassette cover was cut off and the edges of the remaining part was polished. Finished cassette and several spacers are shown in Figure Embedding Dehydrated tissue specimens were submerged in melted paraffin before embedding. The embedding operations were performed on a Leica embedding station (Leica Embedding Center EG1160, Leica Biosystems). The specimen was put in the embedding cell and melted paraffin was added into the cell, then the embedding cell was cooled down to solidify the paraffin. After the paraffin solidification the tissue specimen was embedded in paraffin and ready for slicing.

39 39 Figure 2.10: Tissue cassette modifications. The spacers can prevent tissue specimen from folding or rolling up by reducing the internal space of the cassette. The spacers are made from cassette covers because the material of cassette covers is resistant to Xylene Slicing Dehydrated tissue specimens were embedded into paraffin blocks and then cross-dissected into 5-10µm thickness slides by a Leica microtome (Leica rotary microtome RM 2135, Leica Biosystems). Twelve(12) to twenty(20) slices per ablation site were collected at fixed interval for photographing under microscopy. All the collected slices were mounted on glass slides and dried for staining procedure Tissue H&E Staining Protocol The H&E staining protocol used in this test model was adapted from previous studies [32]. The general procedure of the H&E staining is: hydrating the specimen slices, then staining with Haematoxylin and Eosin, at last dehydrating again for fixation. Detailed procedure of H&E staining was shown in Table 2.6. When the staining was finished, the

40 40 slides were mounted permanently with permount mounting medium for photographing under microscopy Measurement on Photographs The measurements that were collected from the cross sectional photographs were used for tissue injury analysis. These measurements include the diameter and depth of each ablation site, the thickness of each layer at the central region of the ablation site and non-ablation regions. The detailed measurements were shown as the yellow bars in Figure For example, yellow bar A indicates the diameter of the ablation site, and yellow bar B indicates the depth of the ablation site. In order to simplify the microscopy measurement operation and the mathematical model of tissue injury, the layer structure of the tissue specimen (porcine colon) will be converted into a three-layer model: mucosa, submucosa, and muscle layer (muscularis). In Figure 2.11 yellow bars C, D, and E indicate the thickness of the layers at the central region of the ablation site, and yellow bars F, G, and H indicated the thickness of the layers at the non-ablation region. This simplified tri-layer model was also used for tissue injury evaluation Ablation Sites Volume Calculation Ablation sites were the place that tissue injury occurred during the ablation process. The lost volume of the ablation sites reflect the grade of the tissue injury that was caused by ablation operation. In this test model the terminology ablation site volume was used to refer the lost volume of the ablation site. Ablation sites were tiny and flat, therefore some assumptions were made to simplify the measurement and calculation process. Assumptions were made based on the components that were involved in the ablation process: The geometry of the electrode tip is the assembly of a cylinder and a hemisphere

41 41 Table 2.6: H&E staining procedure (including hydration and dehydration) Operation Time Hydration Submerge slides in Xylene I Submerge slides in Xylene II Submerge slides in Xylene III Submerge slides in 100% Ethanol I Submerge slides in 100% Ethanol II Submerge slides in 95% Ethanol Submerge slides in 70% Ethanol Wash slides in distilled water Submerge slides in Haematoxylin Wash slides in circulating bath with tap water Wash slides in distilled water 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 5 minutes 3 minutes 2 minutes 20 minutes 3 minutes Dehydration Submerge slides in 70% Ethanol Submerge slides in Eosin Submerge slides in 95% Ethanol Submerge slides in 100% Ethanol I Submerge slides in 100% Ethanol II Submerge slides in 100% Ethanol III Submerge slides in Xylene I Submerge slides in Xylene II Submerge slides in Xylene III 3 minutes 30 seconds 3 minutes 3 minutes 3 minutes 3 minutes 3 minutes 3 minutes 3 minutes

42 42 E C D B H A F G Figure 2.11: Measurements on tissue slice photograph. The structure of the tissue was simplified as a three-layer model: Mucosa, Submucosa, and Muscularis. The yellow bars in the figure indicate the measured length or thickness. The length of bar A is the diameter of the ablation site. The length of bar B is the depth of the ablation site. The length of bar C is the thickness of Mucosa layer at the central region of the ablation site. The length of bar D is the thickness of Submucosa layer at the central region of the ablation site. The length of bar E is the thickness of Muscularis layer at the central region of the ablation site. The length of bar F is the thickness of Mucosa layer at the non-ablation region. The length of bar G is the thickness of Submucosa layer at the non-ablation region. The length of bar H is the thickness of Muscularis layer at the non-ablation region. that the two objects have the same diameter. The electrode tip and tissue surface had close contact during the ablation trial. During the process of electrical ablation, the volume lost of the tissue occurred at the contact area between the tissue and electrode tip. Therefore the resulting sites was primarily shaped by the hemispherical electrode tip, and the shape of the ablation site volume should be a spherical cap (shown in Figure 2.12). The above assumptions were useful when a mathematical model was setup for calculating the ablation site volume during ablation process. The mathematical model (spherical cap) of the ablation site volume is shown in Figure In this figure, a is the radius of the base of the spherical cap, and h is the height of the spherical cap. Ablation site volume calculation was based on this spherical cap model. The base radius a of the

43 43 Electrode tip Ablation site Tissue sample Figure 2.12: Details of electrode tip and ablation site. During ablation test the resulting ablation site was primarily shaped by the hemispherical electrode tip. spherical cap was set to the radius of the ablation site, and the height h of the spherical cap was set to the depth of the ablation site. The volume of the spherical cap represents the ablation site volume. According to general geometry knowledge, the volume formula of the spherical cap is shown as Formula 2.1. V = πh 6 ( 3a 2 + h 2) (2.1) The variables in Formula 2.1 represent geometric parameters of the spherical cap: a represents the radius of the base of the spherical cap; h represents the height of the spherical cap; V represents the volume of the spherical cap. The R code of Formula 2.1 was listed in Appendix B.2.

44 44 h a (R-h) R Figure 2.13: Diagram of spherical cap. R is the radius of the sphere; a is the radius of the base of the spherical cap; h is the height of this cap; (R h) is the distance from the sphere center to the base of the spherical cap Tissue Injury Evaluation The preliminary experiment had shown that (a) layers of the colon wall were injured and compressed due to the electrical current and the pressure at the electrode tip, and (b) no perforations were observed. The thickness of all the layers of the colon wall can be measured under microscopy, which provided a possible method of evaluating tissue injury. The tissue injury evaluation was based on the measurement of the thickness of all the layers at the central point of the ablation sites and the peripheral non-ablation regions (as shown in Figure 2.11). Changes in the layers thickness between the central region of the ablation site and peripheral non-ablation region were calculated by the ratio of the thickness at ablation (central) and non-ablation (peripheral) regions (Formula 2.2). This numeric value of this ratio will be used as the index of the tissue injury.

45 45 Ratio = layer thickness at the central region layer thickness at the peripheral region Different layers were calculated respectively in order to determine the potential (2.2) distribution pattern of the tissue injury. Combinations of several layers thickness were also used for ratio calculation in order to determine the distribution of the tissue injury in different layers. The mapping between the variable names, tc1 to tc3 (central), and tp1 to tp3 (peripheral), of the R code in Appendix B.2 and the measured data (layer thickness that was measured as shown in Figure 2.11) was shown in Table 2.7. Table 2.7: Mapping between the R code variable name and measurement data variable name mapped data measurement tc1 thickness of mucosa layer at ablation region C tc2 thickness of submucosa layer at ablation region D tc3 thickness of muscularis layer at ablation region E tp1 thickness of mucosa layer at non-ablation region F tp2 thickness of submucosa layer at non-ablation region G tp3 thickness of muscularis layer at non-ablation region H ra1 ra2 ra3 ra12 ra123 ratio of mucosa layer ratio of submucosa layer ratio of muscularis layer ratio of thickness of mucosa + submucosa layers ratio of total thickness (mucosa + submucosa + muscularis)

46 Statistical Analysis and Plotting Statistical analysis including t-test and two-way ANOVA were performed on the data that collected from microscopy measurement to investigate the roles of all the parameters that affect the shape and geometry of the resulting ablation sites. In order to perform the above statistical analysis tests and plotting the analysis results some software packages were used: (a) GNU R with ggplot2 package [37, 38], and (b) Gnuplot. The volume of the ablation sites and the ratio of layer thickness at the ablation site to peripheral region were calculated by these software packages and analyzed. The source codes of GNU R and Gnuplot scripts that were used in analyzing and plotting were listed in Appendix (B.2, B.3).

47 3 Results 47 The experiment results and analysis results were presented in this chapter. First part contains the results that are related with test model, and the second part includes all the results of the ablation tests that were performed on this test model. Statistical analysis results were also presented along with the experiment data. 3.1 Test Model Features This ex vivo test model had some features that are different from the traditional in vivo test models. These features include the electrical properties of the gel block that provide better emulation to the real world human (patient) body. In this section the test results that were related with the features of ex vivo test model were presented Gel Electrical Impedance Result Gel block electrical impedance values represent the resistance of the gel block to external electric current. The impedance-frequency curve of the Std gel block was shown in Figure 3.1. In the gel block impedance-frequency curve (Figure 3.1), the gel block had high impedance values (about 750Ω) at the low frequency region (shown in the left part of the curve). As the frequency of external current went higher, the impedance value of the gel block remained the same until the frequency reached specific level (around 100kHz). At around 100kHz there was a shape jump (from 750Ω down to 350Ω) in the impedance-frequency curve, which is the unique feature of the gel block. At the high frequency region (frequency value is higher than 100kHz) the impedance value of the gel block was in the range of 280Ω 350Ω. The Std gel block had the impedance value of 300Ω at the frequency range of 250kHz to 300kHz, which is important for this test model.

48 z (Ω) e4 1.0e5 1.5e5 2.0e5 2.5e5 3.0e5 Frequency (Hz) Figure 3.1: Impedance-frequency curve of Std gel block. The x-axis represents the frequency of the external current, and the y-axis represents the magnitude of the electrical impedance that measured by Solartron. The range of test frequency was 200Hz to 300kHz. In this figure the low frequency was limited to 50kHz Ablation Tests Results Actual tissue ablation tests were performed on this ex vivo model, and the data was collected and analyzed. The data includes the photographs of top view and cross sectional view of the ablation sites, measurements of the features on the photographs, and calculation results Photographs of the Ablation Sites The top views and cross sectional photos of the ablation sites were shown in Figure 3.2 and Figure 3.3. In the top views of the ablation sites the edge The top views of the ablation sites provided little information about the power output settings or the size of the contact area unless the perforation occurred. In contrast, the cross sectional views provided more useful information about power settings and contact area. With higher

49 power output settings, the degree of the electrical injury became greater and the involved colon wall layers were more deeper. 49 Figure 3.2: Photographs of the top views of different ablation sites Effect of Ablation Parameters on Ablation Site Geometries In the design of this ex vivo test model, several ablation parameters were introduced, for example, power level, firing duration, and contact area. The contributions of each parameter to the electrical ablation were diverse. In order to determine the dominate ablation parameters, ablation site geometries (including diameter, depth, and volume) were used for determine the possible roles of the ablation parameters. And any parameters with minor effect on the electrical ablation should be ignored when in-depth analysis was

50 50 Figure 3.3: Photographs of the cross-sectional views of different ablation sites. performed. Detailed results about the effect of each ablation parameter on the ablation site geometries were analyzed respectively Contact Area at the Electrode Tip As in the experiment design, the contact area between the tissue sample surface and electrode tip may have effect on the shape and geometry of the resulting ablation sites. Tests with same power level (60 watts) and duration (1.5 seconds) but different levels of contact area ( small and middle ) were performed to investigate the effect of contact area on ablation sites. Table 3.1: ANOVA test of contact area s effect on ablation site volume Df Sum Sq Mean Sq F value Pr(>F) contac Residuals

51 51 ANOVA tests were performed on the data of different contact areas and the test result was shown in Table 3.1. In Table 3.1 the p-value was greater than 0.05, which indicated that no significant difference was generated by different levels of contact area. Table 3.2: P-values of t-tests on different levels of contact area settings t-test P-value diameter 0.74 depth 0.48 volume 0.58 mucosa 0.31 submucosa 0.19 muscularis 0.42 mucosa+submucosa 0.17 total (mucosa+submucosa+muscularis) 0.19 T-tests were also performed on ablation site geometry and tissue injury in order to determine whether different levels of contact area have significant effect on ablation results. The results of these T-tests were shown in Table 3.2. None of the P-values shown in Table 3.2 was less than 0.05 (p<0.05 is the common standard for significant difference in t-test), which indicated that contact area did not play an important and significant role in affecting the shape and geometry of the ablation sites. Because the results indicated that contact area at the electrode tip provided no significant effect on the electrical ablation, the following analysis will not include the contact area as an effective parameter.

52 Effect of Electrical Impedance Matching T-tests were performed against the impedance matching and dis-matching ( Std and HiR ) in the firing loop, and the P-values of t-tests were shown in Table 3.3. Table 3.3: P-values of t-tests on site volume between Std and HiR firing duration (sec) power level (watt) In Table 3.3, no significant differences were archived when either (a) firing duration was short and with relatively low power level, or (b) long firing duration with high power level. This result showed that insufficient duration inhabits the deep ablation, and prolonged ablation would cause deep damage no matter what power level it used. This result also provided information that different electrical impedance along the firing loop can change the distribution of the ablation energy that was delivered by ESU Effect of Output Power Level and Firing Duration On Std gel, the mean and standard error of the mean (SEM) of the diameter and depth of the ablation sites were shown in Figure 3.4 and Figure 3.5. The effect and role of both power level and firing duration on ablation site volume was evaluated by two-way ANOVA test, and ANOVA result was shown in Table 3.4. Figure 3.6 presented the site volume under different power level and firing duration combinations (tests performed on Std gel).

53 53 diameter, mean with SEM, on Std gel diameter duration power Figure 3.4: Effect of power level and duration on site diameter. Tests were performed on Std gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the site diameter. The length of each black bar represents the standard error of the site diameter. On HiR gel, the mean and standard error of the mean (SEM) of the diameter and depth of the ablation sites were shown in Figure 3.7, and 3.8. ANOVA analysis has shown that different power levels and different durations have significant effect on the ablation site volume. The combination of power level and

54 54 depth, mean with SEM, on Std gel depth duration power Figure 3.5: Effect of power level and duration on site depth. Tests were performed on Std gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the site depth. The length of each black bar represents the standard error of the site depth. duration did not show very significant effect on volume, this may due to the variance of the data and limited sample size. And the differences in volume may due to the calculation method (the mathematical function of volume).

55 55 Table 3.4: ANOVA test of power level and duration on site volume with Std gel Df Sum Sq Mean Sq F value Pr(>F) power dura power:dura Residuals Table 3.5: ANOVA test of power level and duration on site volume. ANOVA tests were performed on HiR gel. Df Sum Sq Mean Sq F value Pr(>F) power dura power:dura Residuals Effects of Ablation Parameters on Tissue Injury Detailed results about the effect of each ablation parameter on tissue injury were analyzed respectively. In the three-layer model, the ratio of the each layer (mucosa, submucosa, and muscularis) had been calculated respectively; the ratio of the layer combinations (for example, mucosa+submucosa, mucosa+submucosa+muscularis) was also calculated in order to find the potential role in tissue injury estimation. Because Table 3.2 showed that contact area at the electrode tip had no significant effect on tissue layer thickness ratio, no future analysis was done on contact area.

56 56 volume, mean with SEM, on Std gel 6 4 volume duration power Figure 3.6: Effect of power level and duration on site volume. Tests were performed on Std gel block. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the site volume. The length of each black bar represents the standard error of the site volume Gel Impedance Matching Table 3.6 showed the P-values of t-test between Std and HiR gels.

57 57 diameter, mean with SEM, on HiR gel diameter duration power Figure 3.7: Effect of power level and duration on site diameter. Tests were performed on HiR gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the site diameter. The length of each black bar represents the standard error of the site diameter Power Level and Firing Duration Power level had significant effect on the ablation site volume. As power level increased, the resulting ablation sites became larger in volume, and the compression ratio of the layers increased. Firing duration had significant effect on the ablation site volume.

58 58 depth, mean with SEM, on HiR gel depth duration power Figure 3.8: Effect of power level and duration on site depth. Tests were performed on HiR gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the site depth. The length of each black bar represents the standard error of the site depth. As duration increased, the resulting ablation sites became larger in volume, and the compression ratio of the layers increased. Table 3.9 showed the ANOVA test result of power level and duration on mucosa layer ratio.

59 59 volume, mean with SEM, on HiR gel 6 4 volume duration power Figure 3.9: Effect of power level and duration on site volumes. Tests were performed on HiR gel block. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the site volume. The length of each black bar represents the standard error of the site volume. Figure 3.10 showed that showed the Mucosa layer thickness ratio of the ablation tests that were performed on Std gel block. As the power level increased, the Mucosa layer thickness ratio decreased. As the firing duration increased, the Mucosa layer thickness ratio also decreased. Thickness ratio decreasing indicated that tissue layer were injured deeper during the ablation. Either at the same power level or the same firing

60 60 Table 3.6: P-values of t-test on mucosa layer ratio between Std and HiR gel. firing duration (sec) power level (watt) Table 3.7: P-values of t-test on submucosa layer ratio between Std and HiR gel. firing duration (sec) power level (watt) Table 3.8: P-values of t-test on total layer thickness ratio between Std and HiR gel. firing duration (sec) power level (watt) duration, the increases in values of the other parameter caused the decrease in ratio value.

61 Table 3.9: Effect of power level and duration on Mucosa layer thickness ratio. ANOVA tests were performed on Std gel. Df Sum Sq Mean Sq F value Pr(>F) power dura power:dura Residuals ra1, mean with SEM, on Std gel 0.75 ra duration power Figure 3.10: Mucosa layer ratio. Tests were performed on Std gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio.

62 For example, when power level was 30 watts, the Mucosa layer thickness ratio decreased while the firing duration increased ra2, mean with SEM, on Std gel 0.75 ra duration power Figure 3.11: Submucosa layer ratio. Tests were performed on Std gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. Figure 3.11 showed that showed the Submucosa layer thickness ratio of the ablation tests that were performed on Std gel block. The x-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level increased, the submucosa layer thickness ratio decreased. As the firing

63 63 duration increased, the submucosa layer thickness ratio also decreased. Thickness ratio decreasing indicated that tissue layer were injured deeper during the ablation. Either at the same power level or the same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the submucosa layer thickness ratio decreased while the firing duration increased ra3, mean with SEM, on Std gel 0.75 ra duration power Figure 3.12: Muscularis layer ratio. Tests were performed on Std gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. Figure 3.12 showed that showed the Muscularis layer thickness ratio of the ablation tests that were performed on Std gel block. The x-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing

64 64 duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level increased, the muscularis layer thickness ratio decreased. As the firing duration increased, the muscularis layer thickness ratio also decreased. Thickness ratio decreasing indicated that tissue layer were injured deeper during the ablation. Either at the same power level or the same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the muscularis layer thickness ratio decreased while the firing duration increased ra12, mean with SEM, on Std gel 0.75 ra duration power Figure 3.13: Mucosa & submucosa layers ratio. Tests were performed on Std gel. X- axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio.

65 65 Figure 3.13 showed that showed the Mucosa+Submucosa layers thickness ratio of the ablation tests that were performed on Std gel block. The x-axis of this figure represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level increased, the thickness ratio of two layers decreased. As the firing duration increased, the thickness ratio of two layers also decreased. Thickness ratio decreasing indicated that tissue layers were injured deeper during the ablation. Either at the same power level or the same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the thickness ratio of two layers decreased while the firing duration increased. Figure 3.14 showed the total layer thickness ratio of the ablation tests that were performed on Std gel block. The x-axis of this figure represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level increased, the total tissue thickness ratio decreased. As the firing duration increased, the total tissue thickness ratio also decreased. Thickness ratio decreasing indicated that tissue layers were injured deeper during the ablation. Either at the same power level or the same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the total thickness ratio decreased while the firing duration increased.

66 66 ra123, mean with SEM, on Std gel ra duration power Figure 3.14: Thickness ratio of total tissue layers on Std gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. On HiR gel, the general patterns of the effect of parameters are the same as on Std gel. But the grade of tissue injury that gained on HiR gel was relatively smaller than that on Std gel, as shown in the following figures. Figure 3.15 showed the Mucosa layer thickness ratio of the ablation tests that were performed on HiR gel block. The x-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level increased, the Mucosa layer thickness ratio decreased. As the firing duration increased, the Mucosa layer

67 67 ra1, mean with SEM, on HiR gel ra duration power Figure 3.15: Mucosa layer on HiR gel. Different colors of the bars represent the firing duration. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. thickness ratio also decreased. Thickness ratio decreasing indicated that tissue layer were injured deeper during the ablation. Either at the same power level or the same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the Mucosa layer thickness ratio decreased while the firing duration increased. Figure 3.16 showed the Submucosa layer thickness ratio of the ablation tests that were performed on HiR gel block. The x-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of

68 68 ra2, mean with SEM, on HiR gel ra duration power Figure 3.16: Submucosa layer on HiR gel. Different colors of the bars represent the firing duration. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level increased, the submucosa layer thickness ratio decreased. As the firing duration increased, the submucosa layer thickness ratio also decreased. Thickness ratio decreasing indicated that tissue layer were injured deeper during the ablation. Either at the same power level or the same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the submucosa layer thickness ratio decreased while the firing duration increased.

69 69 ra3, mean with SEM, on HiR gel ra duration power Figure 3.17: Muscularis layer on HiR gel. Different colors of the bars represent the firing duration. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. Figure 3.17 showed the Muscularis layer thickness ratio of the ablation tests that were performed on HiR gel block. The x-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level increased, the muscularis layer thickness ratio decreased. As the firing duration increased, the muscularis layer thickness ratio also decreased. Thickness ratio decreasing indicated that tissue layer were injured deeper during the ablation. Either at the same power level or the

70 70 same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the muscularis layer thickness ratio decreased while the firing duration increased ra12, mean with SEM, on HiR gel 0.75 ra duration power Figure 3.18: Mucosa & submucosa layers on HiR gel. Different colors of the bars represent the firing duration. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. Figure 3.18 showed the Mucosa+Submucosa layers thickness ratio of the ablation tests that were performed on HiR gel block. The x-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level

71 71 increased, the thickness ratio of two layers decreased. As the firing duration increased, the thickness ratio of two layers also decreased. Thickness ratio decreasing indicated that tissue layers were injured deeper during the ablation. Either at the same power level or the same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the thickness ratio of two layers decreased while the firing duration increased ra123, mean with SEM, on HiR gel 0.75 ra duration power Figure 3.19: Thickness ratio of total tissue layers on HiR gel. X-axis represents the power level of the ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. Figure 3.19 showed the total tissue thickness ratio of the ablation tests that were performed on HiR gel block. The x-axis of this figure represents the power level of the

72 72 ablation tests. Different colors of the rectangle bars represent the firing duration. The height of each rectangle bar represents the mean value of the thickness ratio. The length of each black bar represents the standard error of the thickness ratio. As the power level increased, the total tissue thickness ratio decreased. As the firing duration increased, the total tissue thickness ratio also decreased. Thickness ratio decreasing indicated that tissue layers were injured deeper during the ablation. Either at the same power level or the same firing duration, the increases in values of the other parameter caused the decrease in ratio value. For example, when power level was 30 watts, the total thickness ratio decreased while the firing duration increased. Compared with Figure 3.14, it showed that on HiR gel the total tissue thickness ratio was larger than that on Std gel, which indicated that less tissue injury was made on HiR gel than on Std gel, and high electrical impedance changed the energy distribution along the firing loop.

73 4 Discussion 73 Discussion on the experiment results, test model design, and suggestions for future study directions. 4.1 Ablation Test Model Colonoscopy with polypectomy is an efficient treatment method for pre-cancerous polyps in order to prevent colorectal cancer, while the complications of the colonoscopy operation with colorectal polypectomy are bleeding, perforation, and other post-polypectomy syndromes[9]. Previous studies[9, 39 46] had shown that (a) the greatest perforation risk area was sigmoid colon, (b) excessive tissue injury was the major reason causing perforation, and (c) many factors that can result in serious colorectal perforations including lacking of training or experience in colonoscopy operations. This ex vivo ablation test model was designed and built up in order to investigate the possible roles of all the major ablation parameters during the electrical ablation (polypectomy performed with hot biopsy forceps). The results of the simulated ablation tests that were performed on this test model provided detailed information about the ablation parameters. The important features of this ex vivo ablation test model are (a) the fresh tissue ablation and (b) electrical impedance matching with in vivo tests. Due to clinical and ethic issues, real tissue ablation tests can not be performed directly on human subjects.the practical ways of performing real tissue ablation tests are either (a) using living lab animals to emulate human body (in vivo), or (b) using the tissue samples that are usually obtained from lab animals (ex vivo), for example, porcine colon, sheep colon, dog colon, etc. These two different ways have their own advantages and drawbacks. Some studies[24 26, 31, 47 49] use in vivo ablation test models in order to maximize the similarity between animal test and patient surgery, but the costs and animal welfare issues need to be carefully considered because these in vivo test models are

74 74 usually non-survival experiments that the animals will be scarified in order to get the ablation sites for histology analysis. Ex vivo ablation test models can overcome some disadvantages of in vivo test models, some limitations still exists, for example, tissue samples are perishable and hard to keep the normal status as in vivo models. This ablation test model was designed for testing all the major parameters that involve in the ablation procedure. In order to simulate the working conditions of hot biopsy forceps in the colorectal cavity, raw fresh animal tissue is always the best choice. In this ex vivo ablation test model the fresh porcine colon tissue was used. Many existing ex vivo models[27 30, 50] are focusing on the training purpose, and their settings do not emulate the exact in vivo environment. Without matching the in vivo environment and the electric properties of human body, the results of the dissection operations can not be used as the evaluation of the performance and design of hot biopsy forceps. In order to maximize the similarity to in vivo settings, the design of this ex vivo ablation test model introduced the body equivalent stack.the body equivalent stack was used to emulate the electrical properties of human body, for example, the electrical impedance. Archiving proper electric impedance value was one of the major goals in the design of this ex vivo ablation test model that can compete other simple models. The electrical impedance is one of the electrical properties that human body has. During the electrical ablation procedure, radio-frequency current passed through human body, and the electrical impedance of the human body tissue converted the electric current energy into thermal energy that causes cells and tissue structures degeneration. Physicians usually adjust (a) the output power level of the ESU, and (b) ablation duration in order to prevent excessive ablation. In this ablation test model, the electric impedance feature was simplified so that the impedance value should match with in vivo tests. Selections of body equivalent materials that can generate proper electric impedance value with good mechanical

75 75 properties were difficult. Previous study [51] provided good hints on the selection of materials: chemical synthetic polymers. Polyacrylamide gel (PAG) is one kind of widely used chemical synthetic polymers. In biochemistry and molecular biology fields, polyacrylamide gel blocks are commonly used for separating/filtering/purifying bio-molecules by their weights or sizes. The formula (shown in Table 2.2) for the polyarcylamide gel blocks that were used in this ex vivo ablation test model was based on the standard 15% resolution PAG protocol. The gel block made for this ex vivo ablation test model was measured by electrochemistry instrument, Solartron, to verify the electrical impedance properties. Application of electrochemistry method and instrument in biomedical engineering fields is one of the novel parts in this ex vivo ablation test model. Previous studies [52 54] that were related with ion channels and skin permeability, also utilized electrical or electrochemical methods for measuring electric current, voltage, and resistance/impedance of biological tissue samples. As shown in Figure 3.1 the gel block impedance-frequency curve indicated that the gel block could archive 300Ω around 300kHz. Compared with previous study[25], the gel block archived similar electrical impedance value with the in vivo model, 4.2 Ablation Test Results Ablation test results of the parameter combinations provided useful information about the efficiency and safety of different designs of hot biopsy forceps tip. Analysis on the ablation effects were based on two major aspects: ablation site geometry and involved tissue layers. Ablation site geometry includes diameter, depth, and volume of the resulting site. The volume data of all the ablation sites were collected from cross sectional slices. Tissue injury evaluation was based on the changes of the colon wall layers, for example, changes in layer structure and layer thickness.

76 76 In the design of this ex vivo ablation test model, there were four(4) distinct ablation test parameters: gel block type (electric impedance of the stack), contact area between the electrode tip and the tissue sample surface, ESU output power level, and ESU firing duration. Some of the ablation test parameters may be ignored due to the fact that the test result of their effects was not significant and different values of these test parameters cause no significant differences in test result. The ablation test parameter that was ignored in in-depth analysis was the contact area at the electrode tip. In the initial design of this ablation test model, there were three(3) different contact area settings ( small, middle, and large ). The contact area was controlled by adjusting the pushing force at the electrode tip towards the gel block. The stronger pushing force the electrode tip has, the larger contact area it generates at the electrode tip. This is the idea behind the design of the refined electrode tip and its holder that were used in this ablation test model. Using large contact area which requires more pushing force at the electrode tip towards gel block than middle caused irreversible damage of the gel block surface (making cracks in the gel block), therefore only small and middle contact area settings were used in actual ablation tests. As shown in Table 3.1, no significant difference in ablation site volume was observed with different levels of contact area. Also differences in other ablation site geometries and tissue layer thickness under different contact area settings were observed (shown in Table 3.2), but no significant differences were reported by statistical analysis. The reason to this phenomenon may due to the method to gain different contact area by different push force at the tip of the electrode. Because the contact area at the electrode tip does not play an important and significant role in affecting the shape and geometry of the ablation sites, the following experiment data was collected under the same contact area setting ( middle ).

77 Site Geometry Three geometry values of the ablation sites were examined: diameter, depth, and volume. The diameter of the ablation site was the widest part of the site at the top surface of the colon tissue, and the size of the site diameter indicates the horizontal range of the tissue injury caused by electrical ablation. The depth of the ablation site was the distance from the deepest part at the central region of the ablation site to the top surface of the peripheral non-ablation region, and the size of the site depth indicates the vertical tissue injury caused by electrical ablation. The volume of the ablation site was calculated from the diameter and the depth of the site with the mathematical model, and the value of the site volume indicates the tissue volume loss caused by electrical ablation. The ablation tests performed in this study had gone through different output power levels and firing durations (the test parameter matrix) in order to cover the majority range in the ESU energy delivery space. The effects of output power level and firing duration on the diameter of the ablation sites were shown in Figure 3.4 (on Std gel) and Figure 3.7 (on HiR gel). The diameter of the ablation sites increased as the output power level increased or the firing duration increased, which indicates that higher energy output from ESU to the tissue would result in larger ablation region area. Therefore one possible method to limit the diameter of the ablation site is to limit the output power level or the firing duration. But the diameter of the ablation sites stopped increasing at high energy output, and the possible reason is that the size and the shape of the electrode tip limits the horizontal expansion of the tissue injury area. The effects of output power level and firing duration on the depth of the ablation sites were shown in Figure 3.5 (on Std gel) and Figure 3.8 (on HiR gel). The depth of the ablation sites increased as the output power level increased or the firing duration increased, which indicates that higher energy output from ESU to the tissue would result in deeper ablation sites. Therefore limiting the output power level or the firing duration

78 78 would also help limiting the depth of the resulting ablation sites. Compared with the results of the ablation site diameter, changes in ablation site depth were easier to be observed and measured than changes in ablation site diameter, and the increase of ablation site depth did not show any ceiling limit in the ablation test result. The effects of output power level and firing duration on the volume of the ablation sites were shown in Figure 3.6 (on Std gel) and Figure 3.9 (on HiR gel). Output power level had a dominate effect on the volume of the ablation site, and the site volume became larger as output power level increased. Firing duration also had positive effect on the ablation site volume, longer duration generally resulted in greater tissue injury at the ablation site. Therefore controlling output power level and firing duration would be a useful method for limiting tissue injury during electrical ablation. The effect of different electric impedance on the volume of the ablation sites were shown in Table 3.3. Significant differences in ablation site volume were on the medium energy level (the values on the secondary diagonal line were less than 0.05), while no significant differences in ablation site volume were on either (a) low output power level with short firing duration or (b) high output power level with long firing duration. The reason for this phenomenon may be that: (a) low energy level only generated small ablation sites, and the difference between the two gel types was too small; (b) high energy level generated deep and severe tissue injury in the colon wall, and the ablation sites were shaped by the electrode tip, therefore the difference between the two gel types was also small. The result indicated that electric impedance value differences can affect the energy distribution Tissue injury Generally all the ablation sites specimens had mucosa layer injured during the electrical ablation procedure, and various grades of tissue injury in submucosa layer and

79 79 muscle layer. In this ex vivo ablation test model the layer thickness ratio was used to evaluate the tissue injury caused by electrical ablation. Lower layer thickness ratio indicates higher compression ratio of the tissue layer, which means tissue injury at the ablation site. The effects of output power level and firing duration on mucosa layer thickness ratio were shown in Figure 3.10 (on Std gel) and Figure 3.15 (on HiR gel). At higher output power level and longer firing duration, the energy delivered by ESU caused severe tissue injury at the ablation site than that at lower output power levels or shorter firing durations. Therefore the output power level and firing duration are the dominant ablation parameters on tissue injury, and fine control on these parameters would help preventing excessive burning so that the perforations may be avoid. The effects of output power level and firing duration on the Submucosa layer thickness ratio were shown in Figure 3.11 (on Std gel) and Figure 3.16 (on HiR gel). At high output power level and long firing duration, the energy delivered by ESU caused the severe tissue injury at the ablation site than that at lower output power levels or shorter firing durations. Therefore the output power level and firing duration are the dominant ablation parameters on tissue injury, and fine control on these parameters would help preventing excessive burning so that the perforations may be avoid. The effects of output power level and firing duration on the total layers (colon wall) thickness ratio were shown in Figure 3.14 (on Std gel) and Figure 3.19 (on HiR gel). At high output power level and long firing duration, the energy delivered by ESU caused the severe tissue injury at the ablation site than that at lower output power levels or shorter firing durations. Therefore the output power level and firing duration are the dominant ablation parameters on tissue injury, and fine control on these parameters would help preventing excessive burning so that the perforations may be avoid.

80 80 Different gel blocks (electrical impedance matching) also, as shown in Table 3.6 (Mucosa layer), Table 3.7 (Submucosa layer), and Table 3.8 (total colon wall thickness). High electrical impedance reduced the tissue injury than low electrical impedance Summary The general pattern of the ablation test results showed that larger ablation sites and worse tissue injury resulted from(a) higher output power level, or (b) longer firing duration. This general pattern indicates that proper selection of output power level and firing duration may help preventing unnecessary tissue injury. 4.3 Comparison with Similar Studies The tissue injury estimation method that is used in this ex vivo ablation test model is different from other other studies [25]. Although anatomical structure-based injury grading system fits the clinical practice well, the alternative methods from engineers are more practical and easier to be implemented in automatic medical devices. Computational simulation models have been used in evaluation of hot biopsy forceps design and thermal energy management [23], but real tissue ablation tests are much more closer to the actual environment in patient body than computer simulations (in silico). Real tissue ablation tests can provide detailed information about the ablation sites and peripheral regions, cell and tissue structure changes within different layers, etc. Many ex vivo training models for clinic endoscopic surgery were built and used for training endoscopists and medical students[27 30, 49, 50]. These training models were designed for dissection training, and some models provide the capability of evaluating dissection performance, for example, canine model for submucosal dissection[31]. But all these training models require fresh tissue source, and this ex vivo ablation test model also relies on fresh tissue materials. The source of the fresh tissue material is the common

81 81 point of all the ex vivo models, and the quality of the fresh tissue sample may affect the results of the emulated ablation tests. The objective of this study is to investigate potential methods to help preventing perforations caused by electrical ablations. Perforation prevention is the common goal of many studies: the study[47] was focusing on the alternative technique for submucosal dissection that was taken on an animal model; the study[9] investigated the complications (for example, perforations) that are related with colonoscopy and polypectomy; The study[55] also pointed out that the post-colonoscopy perforations were usually caused by barotrauma; some techniques were introduced to close the perforation by using the endoscopy[48] instead of traditional methods such as primary repair and resection, while perforation prevention is still a better choice than repairing. All these studies tried to find good methods to prevent perforations, and fine control on the electrical ablation procedure is important. Although some studies[23] have looked deeply into the thermal management at the electrode tip and surrounding area, few of them had paid much attention on tiny surface details extraction and measurement methods on soft tissue and live tissue. This ex vivo ablation test model tried to make a step forward in this field, and its potential application include evaluating hot biopsy forceps design. 4.4 Limitations and Future Plans Some limitations were found during the model implementation and ablation tests. The limitations in the current implementation indicated the directions for future improvements of this ex vivo ablation test model Limitations The source of the raw porcine colon material is one potential bottleneck to this ex vivo model because only the fresh porcine colon may generate the best and reliable

82 82 results. The common methods for storing raw porcine colon material are not suitable for ablation test and post-ablation histology analysis: ethanol or formalin fixed tissue samples preserve the cell and tissue structure but they lose the capability of emulating the changes during the ablation procedure. Another problem is that the tissue source (raw porcine colon) does not contain polyps which is the main target for hot biopsy forceps. During the ablation tests no perforations were generated or observed. The reason of non-perforation may due to the operating procedure of this ablation test model an acute experiment method. The perforations usually happened after colonoscopic ablation surgery[9, 41, 46, 55] a chronic procedure that involves with the feces (food residuals), the contraction of the smooth muscles of the colon ( movement ), and deep tissue injury at the ablation sites ( excessive ablation ). This ex vivo ablation test model can not emulate this complicated procedure Future Plans Further work should be done to find out solutions to the above limitations and expand this model, for example, finding the proper tissue source that contains polyps would be a great opportunity for testing the real performance of this ex vivo ablation test model. Matching the electrical properties with in vivo models would help for better emulating the exact conditions in the living tissue. One study[56] suggested that ESU equipped with rapid electrical impedance measurement would help evaluating the tissue damage in situ. The electric capacitance also belongs to the electric properties of the tissue (or part of human body) which is part of the electric circuit during ablation. The detailed role of tissue capacitance value on ablation results still requires further investigation. Some studies archived useful results in dielectric properties of different types of fresh animal tissues[57], but for direct capacitance measurement with AC current under specific frequency range has not been done yet. One potential improvement of this

83 83 ex vivo ablation test model may be adding the capability of adjusting the electric capacitance of the body equivalent stack in this model. Although there are some methods of adjusting the surface capacitance values of the colon patch, matching both the electrical impedance and capacitance values with human body or lab animal body has not yet been implemented in this ex vivo ablation test model due to the complexity and stability of the current implementation methods. Also a well-designed and validate capacitance measurement method is required for investigating the potential role of the capacitance property of the tissue sample and body equivalent stack. This ablation test model may also be used for expanding the ex vivo training models to provide a comprehensive training model for physicians and medical students. Some training models[28, 49, 50] already use animal tissue as an important component, adding the ablation test modules would be beneficial to the users of the hybrid (colonoscopy operation and tissue ablation) model. 4.5 Summary This study established an ablation test model for simulating the electrical ablation procedure on colon tissue that is performed for polyps removing. This ablation test model built an ex vivo test environment with the real animal tissue (porcine colon) to investigate the roles of the ablation test parameters and the design of the hot biopsy forceps and ESUs. Actual ablation tests were performed with this ex vivo ablation test model and valuable test results were obtained. Test results provided useful information about the roles of the ablation parameters and the effects of the parameters on the ablation sites and tissue injury. This ex vivo ablation test model may be used for optimizing the electrical ablation parameters since it may provide useful information about the tissue ablation for both physicians and ESU designers. As the result presented that several different parameter

84 84 combinations may generate similar ablation effects, it may become a useful method to recommend the physicians the proper ablation parameter combinations to increase the safety and efficiency for polypectomy. This test model may also help the physicians for testing and training purposes, and the potential application of this model may be integrated with some colonoscopy training models to provide comprehensive training to physicians and medical students.

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89 [48] Zhang, X.L., Qu, J.H., Sun, G., Tang, P., and Yang, Y.S., 2012, Feasibility study of secure closure of gastric fundus perforation using over-the-scope clips in a dog model. J Gastroenterol Hepatol, 27(7), pp [49] Bok, G.H. and Cho, J.Y., 2012, ESD Hands-on Course Using Ex Vivo and In Vivo Models in South Korea. Clin Endosc, 45(4), pp [50] Sedlack, R.E., Baron, T.H., Downing, S.M., and Schwartz, A.J., 2007, Validation of a colonoscopy simulation model for skills assessment. Am J Gastroenterol, 102(1), pp [51] Holder, D.S. and Khan, A., 1994, Use of polyacrylamide gels in a saline-filled tank to determine the linearity of the Sheffield Mark 1 electrical impedance tomography (EIT) system in measuring impedance disturbances. Physiol Meas, 15 Suppl 2a, pp. A45 A50. [52] Kasting, G., Merritt, E., and Keister, J., 1988, An in vitro method for studying the iontophoretic enhancement of drug transport through skin, Journal of Membrane Science, 35(2), pp [53] Kasting, G.B. and Bowman, L.A., 1990, DC electrical properties of frozen, excised human skin. Pharm Res, 7(2), pp [54] Pliquett, F. and Pliquett, U., 1996, Passive electrical properties of human stratum corneum in vitro depending on time after separation. Biophys Chem, 58(1-2), pp [55] van der Sluis, F.J., Loffeld, R.J., and Engel, A.F., 2012, Outcome of surgery for colonoscopic perforation. Colorectal Dis, 14(4), pp. e187 e190. [56] Lewis, Jr, G.K., Lewis, Sr, G.K., and Olbricht, W., 2008, Cost-effective broad-band electrical impedance spectroscopy measurement circuit and signal analysis for piezo-materials and ultrasound transducers. Meas Sci Technol, 19(10), p [57] Gabriel, S., Lau, R.W., and Gabriel, C., 1996, The dielectric properties of biological tissues: II. Measurements in the frequency range 10 Hz to 20 GHz. Phys Med Biol, 41(11), pp

90 Appendix A: Software Packages 90 A.1 Micro-controller Programming Micro-controllers were used in this study to finish some simple but repeated tasks, for example, firing the ESU for specific time period. Some software and hardware development work was needed, such as coding in the programming language that the micro-controller uses, downloading the compiled firmware onto the micro-controller, and building the peripheral electrical circuit to connect with micro-controller. The standards that were used during the procedure of choosing the proper micro-controller for this study were: (a) availability and cost, (b) requirements on user s skills, (c) extensibility and peripheral hardware, (d) performance and durable, and (e) platform life-cycle and support. A.1.1 Overview of Arduino UNO Arduino is an open-source hardware project based on Atmel 8-bit AVR micro-processor. It is an electronics prototyping platform that can be used in many fields. As an open source project, it can be easily extended and re-designed, and many hardware add-ons ( shields ) and software modules ( libraries ) are available. Arduino UNO has an ATmega328 chip with 32kB flash memory, and a USB port for downloading and debugging. Advantages: (a) it is an open-source design, so anyone can make modifications or extensions; (b) it uses C++-like object-oriented programming language, which is easier for newbies than embedded C or assembly language. Disadvantages: (a) it has limited hardware power, so extension shields or peripheral circuits are required when Arduino is used for complex tasks; (b) it has fixed structure in the source code of user s programs, so coding and debugging complex programs are difficult.

91 91 Homepage: A.1.2 Arduino IDE Arduino IDE is a Java -base integrated development environment (IDE) for Arduino programming. It has a source code editor with graphical user interface (GUI), and it is also a frontend to the command-line tools such as compiler, firmware downloader, and bootloader flasher. Advantages: (a) it is a Java program, so it can run on all the major platforms with the same user interface; (b) it can call all the necessary command-line tools in the background, so it is easy to use for newbies. Disadvantages: (a) it is a Java program, therefore Java runtime environment (JRE) is required to run Arduino IDE; (b) it is less customizable than some general-purpose text editors and source code editors. Homepage: A.2 Scientific Analysis Tools Scientific analysis tools were used for data analysis and visualization. Proper selection of scientific analysis tools can simplify the analysis procedure and provide reliable results. A.2.1 ImageJ ImageJ is a Java -based image processing and analysis tool. It is developed at the National Institutes of Health (NIH), and it is an open-source program. Advantages: (a) it supports user developed plug-ins and user defined macros; (b) it has a board user community. Homepage:

92 92 A.2.2 Gnuplot Gnuplot is a command-line graphing tool. It has the ability to read from a text file ( script ) containing a batch of directive commands and generate graphs by following those commands. It can also output the graphs into various file formats such as PNG (Portable Network Graphics) and SVG (Scalable Vector Graphics). Advantages: (a) it supports script and batch processing, therefore it is easy for users to generate same plots on big datasets; (b) it can generate high quality vector graphs. Homepage: A.2.3 GNU R with ggplot2 R is an open source project for statistical computing and graphics. It has wide range of statistical functions and routines, and it can be easily extended with additional packages. Homepage: ggplot2 is a plotting system for R. Homepage: Advantages: (a) it is widely used, and well developed for statistical use; (b) it has a very large user community, so it is easy to find solutions for some problems.

93 Appendix B: Source Code 93 B.1 Arduino UNO Board Code for Ablation Firing Duration Control footpedalcontrol.ino 1 /* 2 * footpedalcontrol.ino 3 * Copyright (C) 2012, 2013, 2014, Liang Chen 4 ********************************************************** 5 * This program is free software; you can redistribute it 6 * and/or modify it under the terms of the GNU General 7 * Public License as published by the Free Software 8 * Foundation; either version 2 of the License, or (at 9 * your option) any later version. 10 * 11 * This program is distributed in the hope that it will be 12 * useful, but WITHOUT ANY WARRANTY; without even the 13 * implied warranty of MERCHANTABILITY or FITNESS FOR A 14 * PARTICULAR PURPOSE. See the GNU General Public License 15 * for more details. 16 * 17 * You should have received a copy of the GNU General Public 18 * License along with this program; if not, write to the 19 * Free Software Foundation, Inc., 51 Franklin Street, 20 * Fifth Floor, Boston, MA , USA 21 ********************************************************** 22 */ /* 25 * footpedalcontrol.ino 26 * Arduino UNO code for ESD foot pedal control 27 * 28 ********************************************************** 29 * In the footpedal of ESU there is an infrared light gate 30 * to detect whether the pedal is pressed down. When the 31 * pedal is pressed down, the infrared light is blocked and 32 * the voltage goes down to trigger the ESU. 33 */ // pins for input 36 int BTN_SHORT = 5; // short duration; 37 int BTN_MIDDLE = 6; // middle duration; 38 int BTN_LONG = 7; // long duration;

94 39 40 // pins for output 41 int LED_STATUS = 11; // LED indicator; 42 int EXT_TRIG = 12; // external trigger output; // duration, in milliseconds. 45 int DURA_SHORT = 500; 46 int DURA_MIDDLE = 1500; 47 int DURA_LONG = 6000; void setup() 50 { 51 pinmode(led_status, OUTPUT); 52 pinmode(ext_trig, OUTPUT); 53 //// 54 pinmode(btn_short, INPUT); 55 pinmode(btn_middle, INPUT); 56 pinmode(btn_long, INPUT); 57 //// 58 digitalwrite(ext_trig, HIGH); 59 } void feedback() 62 { 63 int cnt; 64 digitalwrite(led_status, HIGH); 65 delay (150); 66 digitalwrite(led_status, LOW); 67 delay (200); 68 //// 69 for(cnt =0; cnt <30; cnt ++){ 70 digitalwrite(led_status, HIGH); 71 delay(10); 72 digitalwrite(led_status, LOW); 73 delay(40); 74 } 75 //// 76 delay (200); 77 } void loop() 80 { 81 int btn_short_status = digitalread( BTN_SHORT); 94

95 82 int btn_middle_status = digitalread( BTN_MIDDLE); 83 int btn_long_status = digitalread( BTN_LONG); 84 //// 85 if( btn_short_status == HIGH) { 86 feedback(); 87 digitalwrite(ext_trig, LOW); digitalwrite(led_status, HIGH); 88 delay( DURA_SHORT); 89 digitalwrite(ext_trig, HIGH); digitalwrite(led_status, LOW);} 90 else if( btn_middle_status == HIGH) { 91 feedback(); 92 digitalwrite(ext_trig, LOW); digitalwrite(led_status, HIGH); 93 delay( DURA_MIDDLE); 94 digitalwrite(ext_trig, HIGH); digitalwrite(led_status, LOW);} 95 else if( btn_long_status == HIGH) { 96 feedback(); 97 digitalwrite(ext_trig, LOW); digitalwrite(led_status, HIGH); 98 delay( DURA_LONG); 99 digitalwrite(ext_trig, HIGH); digitalwrite(led_status, LOW);} 100 else { 101 digitalwrite(ext_trig, HIGH);} 102 //// 103 btn_short_status = LOW; 104 btn_middle_status = LOW; 105 btn_long_status = LOW; 106 digitalwrite(ext_trig, HIGH); 107 } 108 /*--eof --*/ 95

96 96 B.2 R Code for Data Analysis do3.r 1 # filename: do3.r 2 # Copyright (C) 2011, 2012, 2013, 2014, Liang Chen 3 ########################################################### 4 # This program is free software; you can redistribute it 5 # and/or modify it under the terms of the GNU General 6 # Public License as published by the Free Software 7 # Foundation; either version 2 of the License, or (at 8 # your option) any later version. 9 # 10 # This program is distributed in the hope that it will be 11 # useful, but WITHOUT ANY WARRANTY; without even the 12 # implied warranty of MERCHANTABILITY or FITNESS FOR A 13 # PARTICULAR PURPOSE. See the GNU General Public License 14 # for more details. 15 # 16 # You should have received a copy of the GNU General Public 17 # License along with this program; if not, write to the 18 # Free Software Foundation, Inc., 51 Franklin Street, 19 # Fifth Floor, Boston, MA , USA 20 ########################################################### 21 # Module: 22 # do3.r 23 # Purpose: 24 # statistical analysis and plotting for experiment data 25 # Parameters: 26 # None 27 # Pre - Conditions: 28 # None 29 # Post - Conditions: 30 # output to several TeX and PDF files 31 # Dependencies: 32 # xtable, plotrix, ggplot2 33 ########################################################### ############################## 36 RV <- as. numeric(r. version $major) 37 RVmin <- as. numeric(r. version $minor) USE_ GGPLOT <- ( (RV == 3) (RVmin > 14) ) 40 ##############################

97 41 # load pkgs 42 library( plotrix) 43 library( xtable) if( USE_ GGPLOT ) library(" ggplot2") 46 ############################## 47 ############################## 48 # common variables 49 ddcols <- c( "diam","depth","vol", "ra1","ra2","ra3","ra12","ra123" ) gels <- c("std", "hir") 52 pwrs <- c( 30, 60, 90 ) 53 dur <- c( 0.5, 1.5, 6 ) 54 ############################## 55 ############################## 56 # func:: volume 57 svol <- function( dia, height ){ 58 # parameters: dia(meter), height [unit:um] 59 # return: volume [unit:mm ˆ3] 60 #### 61 # convert from um(10e-6) to mm(10e-3) 62 r <- dia / h <- height / ## 65 res <- ( pi * height * ( 3 * r * r + h * h ) )/6 66 ## 67 return(res) 68 } 69 ############################## 70 ############################## 71 # func:: ratio 72 cratio <- function(atcenter, peripheral){ 73 res <- atcenter/ peripheral 74 return(res) 75 } 76 ############################## 77 ############################## 78 # func:: gtype 79 gtype <- function(a){ 80 if( a=="std" a =="Std" ){ 81 res < } 97

98 83 if( a=="hir" a=="hir" ){ 84 res < } 86 ## 87 if( a==1 ){ 88 res <- "std" 89 } 90 if( a==2 ){ 91 res <- "hir" 92 } 93 res; 94 } 95 ############################## 96 ############################## 97 get.data <- function( sfilename ){ 98 # read date from tsv file 99 Dat <- read. table(file=sfilename, sep="\t", header =TRUE, comment.char="#", row.names ="trial") 100 #### 101 # calculate volume 102 i_diam <- which( colnames(dat)=="diam") 103 i_ depth <- which( colnames(dat)=="depth") 104 Dat$vol <- apply(dat, 1, function(row) svol( row[ i_diam ], row[ i_depth ] )) 105 #### 106 # calculate layers 107 ic1 <- which( colnames(dat)=="tc1") 108 ic2 <- which( colnames(dat)=="tc2") 109 ic3 <- which( colnames(dat)=="tc3") 110 ip1 <- which( colnames(dat)=="tp1") 111 ip2 <- which( colnames(dat)=="tp2") 112 ip3 <- which( colnames(dat)=="tp3") Dat$ra1 <- apply( Dat, 1, function(row) cratio(row[ic1], row[ip1]) ) 115 Dat$ra2 <- apply( Dat, 1, function(row) cratio(row[ic2], row[ip2]) ) 116 Dat$ra3 <- apply( Dat, 1, function(row) cratio(row[ic3], row[ip3]) ) Dat$ra12 <- apply( Dat, 1, function(row) cratio(sum(row[ ic1],row[ic2]), sum(row[ip1],row[ip2])) ) 98

99 119 Dat$ ra123 <- apply( Dat, 1, function(row) cratio(sum(row[ ic1],row[ic2],row[ic3]), sum(row[ip1],row[ip2],row[ip3])) ) 120 #### 121 Dat; } 124 ############################## 125 ############################## 126 ca. anova <- function( Da, pa, t.p.val, t.d.val, bprint =FALSE ){ 127 message( sprintf(" contac.anova[one -way] on %s.",pa)) 128 #### 129 Td <- subset( Da, power ==t.p.val & dura ==t.d.val & gel ==1 ) 130 ## 131 Td$ contac <- factor(td$ contac) 132 Td$gel <- factor(td$gel) 133 tf <- formula( paste(pa," "," contac") ) 134 result <- aov(tf, data = Td) 135 ## 136 if( bprint ){ 137 FuncSaveFile <- sprintf("ca-anova -on -%s- stdgel.tex", pa) 138 message( sprintf("save TO %s", FuncSaveFile) ) 139 ## 140 sink( file= FuncSaveFile, type=" output" ) 141 print( xtable( result), floating =FALSE, booktabs =TRUE ) 142 sink() 143 } 144 #### 145 remove(td, tf, result) 146 } 147 ############################## 148 ############################## 149 ca.t.test <- function( Da, t.p.val, t.d.val, bprint =FALSE ){ 150 message( " contac.t-test on data, \t\twith stdgel." ) 151 #### 152 Td <- subset( Da, power ==t.p.val & dura ==t.d.val & gel ==1 ) 153 #### 154 mid <- Td[Td$ contac ==1 & Td$gel ==1, ddcols] 155 sml <- Td[Td$ contac ==0 & Td$gel ==1, ddcols] 156 #### 99

100 157 tdrowname <- c("diameter", "depth", "volume", "mucosa"," submucosa"," muscularis"," mucosa + submucosa","total ( mucosa + submucosa + muscularis)" ) 158 tddata <- c( t.test(mid$diam,sml$diam)$p.value, t.test(mid $depth,sml$ depth)$p.value, t.test(mid$vol, sml$vol)$p.value, t.test(mid$ra1, sml$ra1)$p.value, t.test(mid$ra2, sml$ra2) $p.value, t.test(mid$ra3, sml$ra3)$p.value, t.test(mid$ra12, sml$ra12)$p.value, t.test(mid$ra123, sml$ ra123)$p.value ) 159 ## 160 td <- data. frame(tdrowname, tddata) 161 colnames(td) <- c("t-test", "P-value") 162 #### 163 if( bprint ) { 164 FuncSaveFile <- "ca-t-test -on- stdgel.tex" 165 message( sprintf("save TO %s", FuncSaveFile) ) 166 ## 167 sink( file= FuncSaveFile, type=" output" ) 168 print( xtable(td), floating =FALSE, include. rownames = FALSE, booktabs =TRUE ) 169 sink() 170 } 171 #### 172 remove(td, mid, sml, td, tdrowname, tddata) 173 } 174 ############################## 175 ############################## 176 gel.t.test <- function( Da, pa, bprint =FALSE ){ 177 message( sprintf("gel.t-test on %s.", pa) ) 178 #### 179 dth <- c( " & \\ multicolumn {3}{c}{ firing duration (sec)} \\\\ \n power level (watt)", "0.5", "1.5", "6.0" ) 180 dt <- data. frame( power = integer(), dura. short= numeric(), dura. middle = numeric(), dura.long= numeric()) 181 ## 182 for( ii in c(30, 60, 90) ){ 183 newrow <- c( as. integer(ii) ) 184 for( jj in c(0.5, 1.5, 6) ){ 185 Td <- subset( Da, contac ==1 & power ==ii & dura ==jj ) 186 ## 187 stdgel <- Td[Td$gel ==1,] 188 hirgel <- Td[Td$gel ==2,] 189 ## 100

101 190 gel.t.test.pa. result <- t.test( stdgel[[pa]], hirgel[[ pa]] ) 191 ## 192 newrow <- c( newrow, gel.t.test.pa. result $p.value ) 193 } 194 ## 195 dt <- rbind(dt, newrow) 196 } 197 ## 198 colnames(dt) <- dth 199 #### 200 if( bprint ){ 201 FuncSaveFile <- sprintf("gel -t-test -%s.tex", pa) 202 message( sprintf("save TO %s", FuncSaveFile) ) 203 #### 204 sink( file= FuncSaveFile, type=" output" ) 205 print( xtable( dt ), floating =FALSE, booktabs =TRUE, include. rownames =FALSE, sanitize. colnames. function = identity ) 206 sink() 207 } 208 #### 209 remove(dth,dt,td,stdgel,hirgel) 210 } 211 ############################## 212 ############################## 213 pwr.dura. anova <- function( Da, pa, ga, bprint =FALSE ){ 214 message( sprintf("pwr.dura.anova[two -way] on %s, \twith %s gel.", pa, ga) ) 215 #### 216 gtt <- gtype(ga) 217 ## 218 Td <- subset(da, contac ==1 & gel == gtt) 219 ## 220 Td$ power <- factor( Td$ power ) 221 Td$dura <- factor( Td$dura ) 222 ## 223 tf <- as. formula( sprintf("%s power*dura", pa)) 224 tblres <- aov( tf, data=td ) 225 #### 226 if( bprint ) { 227 FuncSaveFile <- sprintf("pwrdura -aov -%s-on-% sgel.tex", pa, ga) 101

102 228 message( sprintf("save TO %s", FuncSaveFile) ) 229 ## 230 sink( file= FuncSaveFile, type=" output" ) 231 print( xtable( tblres ), floating =FALSE, booktabs =TRUE ) 232 sink() 233 } 234 #### 235 remove(gtt,td,tf,tblres) 236 } 237 ############################## 238 data.mean.sem <- function( Da, bprint =FALSE ){ 239 message( "--- mean and SEM ---" ) 240 #### 241 resth <- c("trial", 242 "gel", "power", "duration", 243 " diameter"," diameter.sem", 244 "depth","depth.sem", 245 "volume","volume.sem", 246 "tc1","tc1.sem", "tc2","tc2.sem", "tc3","tc3.sem ", 247 "tp1","tp1.sem", "tp2","tp2.sem", "tp3","tp3.sem ", 248 "ra1","ra1.sem", 249 "ra2","ra2.sem", 250 "ra3","ra3.sem", 251 "ra12","ra12.sem", 252 "ra123","ra123.sem" ) 253 DaCol <- c("diam", "depth", "vol", 254 "tc1", "tc2", "tc3", 255 "tp1", "tp2", "tp3", 256 "ra1", "ra2", "ra3", 257 "ra12", "ra123") 258 #### 259 res <- data. frame( row. names = 1: length( resth) ) 260 #### 261 lcnt < for( geltype in gels ){ 263 gt <- gtype( geltype) 264 for( pwrlevel in pwrs ){ 265 for( ftime in dur ){ 266 Td <- subset( Da, contac ==1 & gel ==gt & power == pwrlevel & dura == ftime ) 267 #### 102

103 268 lcnt <- lcnt newlin <- c( lcnt, gt, pwrlevel, ftime ) 270 ## 271 # calculate each data column and add result to newlin 272 for( ii in DaCol ){ 273 tmp.mean <- mean( Td[[ii]] ) 274 tmp.sem <- std.error( Td[[ii]] ) 275 #### 276 newlin <- c(newlin, tmp.mean, tmp.sem ) 277 } 278 #### 279 res <- rbind(res, newlin) 280 } 281 } 282 } 283 ## 284 colnames(res) <- resth 285 #### 286 if( bprint ){ 287 FuncSaveFile <- "all -mean -sem.tex" 288 message( sprintf("save TO %s", FuncSaveFile ) ) 289 #### 290 sink( file= FuncSaveFile, type=" output" ) 291 print( xtable( res ), floating =FALSE, booktabs =TRUE, include. rownames =FALSE ) 292 sink() 293 } 294 #### 295 res; 296 } 297 ############################## 298 ############################## 299 pwrdur.chart <- function(da, px, py, pcolr, yuplimit, ga){ 300 message( sprintf(" errbar chart %s,\ twith %s gel", py, ga) ) 301 #### 302 pye <- sprintf("%s.sem", py) 303 Dt <- subset( Da, gel == gtype(ga) ) 304 ## 305 Dt$ power <- factor(dt$ power) 306 Dt$ duration <- factor(dt$ duration) 307 #### 103

104 308 if( USE_ GGPLOT ){ 309 ggplot( Dt, aes_ string(x=px, y=py, fill=pcolr) ) geom_bar( position ="dodge", stat=" identity" ) geom_ errorbar( aes_ string( ymin= sprintf("%s-%s",py, pye), ymax= sprintf("%s+%s",py,pye) ), width =.2, position = position_dodge(.9) ) ggtitle( sprintf("%s, mean with SEM, on %s gel",py,ga) ) xlab( sprintf("%s", px) ) ylab( sprintf("%s",py) ) ylim(0, yuplimit) 316 }else{ 317 message("[ii] ggplot disabled.") 318 } 319 } 320 ############################## 321 ############################## #--eof --# 104

105 105 B.3 Gnuplot Script for Plotting b01.plot 1 # b01.plot 2 # Copyright (C) 2011, 2012, 2013, 2014, Liang Chen 3 ########################################################## 4 # This program is free software; you can redistribute it 5 # and/or modify it under the terms of the GNU General 6 # Public License as published by the Free Software 7 # Foundation; either version 2 of the License, or (at 8 # your option) any later version. 9 # 10 # This program is distributed in the hope that it will be 11 # useful, but WITHOUT ANY WARRANTY; without even the 12 # implied warranty of MERCHANTABILITY or FITNESS FOR A 13 # PARTICULAR PURPOSE. See the GNU General Public License 14 # for more details. 15 # 16 # You should have received a copy of the GNU General Public 17 # License along with this program; if not, write to the 18 # Free Software Foundation, Inc., 51 Franklin Street, 19 # Fifth Floor, Boston, MA , USA 20 ########################################################### # 23 # Nyquist plot and impedance - frequency curve # 24 # mydatafile = "../ gel -data/std+ sample.z" set key top left 29 set multiplot set style data linespoints 32 set size 0.5,1 33 set origin 0,0 34 set xlabel "z "; set ylabel "z " 35 set format x "%g" 36 set format y "-%g" 37 # unset yrange; set yrange [0:-600] 38 plot mydatafile using ($5):(-$6) every ::127 title "Std+ tissue" 39

106 40 set style data lines 41 set size 0.5, set origin 0.5, set logscale x 44 set xlabel "freq"; set ylabel " z " 45 set format x "10ˆ%L" 46 unset xrange; set xrange [50:300000] 47 # unset yrange; set yrange [0:+2500] 48 set format y "%g" 49 plot mydatafile using ($1):( sqrt(( $5)**2+( $6)**2)) every ::127 title "" set size 0.5, set origin 0.5,0 53 set logscale x 54 set xlabel "freq"; set ylabel "theta" 55 set format x "10ˆ%L" 56 unset yrange; set yrange [-6:6] 57 plot mydatafile using ($1):( atan2($6,$5)) every ::127 title "" set nomultiplot unset key 62 unset logscale 63 reset #--eof --# 106

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