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Histological Evaluation of Arterial Seals Produced by a New Bipolar Radio Frequency (RF) Current System R.D. Tucker University of Iowa Hospitals and Clinics, Iowa city, IA ABSTRACT Background: Histology of arterial seals produced by a new RF bipolar system (LTC) was examined. The system consists of instruments and a generator that automatically delivers energy with a unique waveform, which provides collagen fusion for vessel and duct closure, lung resection and bowel anastomosis. The technology has demonstrated pressure seals in arteries well above physiologic levels. Methods: Various diameter porcine arteries were fused in vivo. Arteries were removed and pressure tested. Next the vessels were placed in formalin, cut longitudinally, embedded in paraffin and sections were stained with H&E. The thermal damage from the patent end of the vessel was measured via calibrated ocular. Results: Tissue under the electrode jaws exhibited loss of normal architecture, hyperchromic cytoplasm, loss of cellular nuclei and hyperchromic pyknotic nuclei. The fused vessel plug, extending laterally from the necrotic tissue under the electrodes, averaged 0.72 mm. The preserved vessel demonstrated an area with denatured collagen but normal cytoplasm and cellular nuclei; the distance this extended from the patent vessel plug toward the electrode edge averaged 0.33 mm. Conclusions: Histology demonstrated that arterial seals produced necrotic tissue under the electrodes but did not produce denatured collagen or abnormal cells that extend beyond the thickness of the vessel plug.

INTRODUCTION The use of histology to quantify the damage caused by radio frequency electrosurgery was started during the initial commercialization of the technology in the 1920’s. We similarly employed histology to examine arterial seals made by a new vessel sealing system produced by Living Tissue Connect (LTC) and compare these results with a commercially available system from Gyrus, Inc. The system consists of bipolar forceps and a generator. The generator automatically provides energy with a unique waveform that fuses tissue and produces little collateral damage. The produced seals have three distinct areas of characteristic histology: 1) the area under the forceps jaws, 2) the area adjacent to the jaws where the side walls of the vessel are fused together forming a tissue plug at the end of the vessel and 3) the un-fused vessel wall extending away from the seal. Typically pathologists would measure the damage extending away from the jaw and term this lateral thermal spread or damage. However, this damage does not characterize the damage which truly affects an adequate seal. The area where the vessel walls are fused forming a tissue plug at the end of the vessel is of variable thickness. The tissue damage to the intact vessel walls extending away from this plug should have little to no damage; damage to these walls could cause necrosis leading to perforation and a delayed bleed. As measuring the lateral thermal spread from the jaw does not take into account the variable thickness of the tissue plug and does not measure the amount of thermal damage to the remaining intact vessel wall, we developed a new method for histological analysis to more adequately characterize an adequate electrosurgical vessel seal. The new analysis method was applied to both the LTC system and the commercially available Gyrus system.

MATERIALS and METHODS Sus Scrofia pigs were given IM ketamine at 14.7 mg/kg and IV acepromazine at 1.5 mg/kg as a preanesthetic. Surgical plane was maintained by inhalation of isoflurane, 3% solution. All animals were connected to heart rate monitor, oxygen monitor and ventilator. Standard cut downs were performed on the external femoral, femoral, left iliac, brachial and carotid arteries. After the fascia was removed from the vessel, the electrode was cross clamped on the artery and the RF current was applied until the vessel was sealed. In the case of the LTC system the generator is preprogrammed to pass current until a seal is obtained; the Gyrus system was energized until a generator sound change indicated that the seal was complete, and at this point, the current was manually stopped. The vessel was next placed in 10% buffered formalin for at least 24 hours. The vessels were then cut longitudinally and placed in paraffin blocks. The blocks were sectioned and 3 micron cuts were stained with hematoxylin and eosin. All samples were then examined blindly by a single pathologist. The thermal damage was measured by a calibrated ocular using standard light microscopy and polarized light microscopy. The amount of vessel damaged was measured as in Figure 1. The area under the vessel jaw is indicated as J. The variable thickness of the vessel plug or fused vessel wall is marked as the distance T. The beginning of the plug or the tissue plug farthest from the forceps jaw is considered as the “0 point” from which measurements are made. It is at this point that the vessel wall should show little or no damage in order to preserve the vessel. The distance, D, extending from the “0 point” toward the jaw is considered as a positive distance or good outcome; while the distance extending up the vessel wall away form the “0 point” is considered a negative distance or a negative outcome.

RESULTS Figure 1 shows a representative H&E of a vessel cross section for a seal performed with the LTC system. The forceps jaw edge is indicated by J. The thickness of the vessel plug is shown as the distance T, while the distance the thermal damage extends from the beginning of the vessel plug is indicated by D which in this case is a positive number on both sides of the cross section.

Figure 5 is a high magnification of the vessel plug where the opposing walls have been fused at low temperature. The area has mostly normal nuclei with some area of thermal damage indicated by hyperchromic and pyknotic nuclei. The cytoplasm is normal in color indicating little cellular damage.

The tissue under the jaw, J, exhibited the loss of normal architecture, hyperchromic cytoplasm, the loss of most of the cellular nuclei with the remaining nuclei appearing pyknotic and hyperchromic; a high magnification of this area in shown in Figure 2. In this area the macro molecules of the cell are irreversibly denatured with the long chains of collagen unraveled and intertwined with other collagen molecules and other long chains such as proteoglycans; these molecules are probably also chemically cross linked in unintended ways. There is no charring and therefore the large molecules have not been reduced to small constituents. The thermal necrosis extends for a small distance into the base of the remaining vessel. Figure 5: Fused area or plug, normal area=N The summary of the measured data is indicated in Table 1; distances are measured in mm. The number of vessels analyzed for the LTC and Gyrus systems were 26 and 25, respectively.

LTC G y rus

Figure 2: high mag of jaw area, J Figure 3 is a high magnification of the vessel wall thermal damage. This is a transition zone from necrotic tissue to normal tissue; the necrotic area, which shows little typical architecture, is show by Nec while normal is indicated by N. The boundary of the necrotic/normal tissue is seen by the line of hyperchromic cytomplasm indicated by, B. The seal failure is also seen and is shown as SF.

M ea su r ed Va lu es ( in mm) T (Avg ± SE) D (Avg ± SE) 0.72 ± 0.04 0.33 ± 0.05 0.85 ± 0.07 0.11 ± 0.06

No. of Negative of D ’ s N 4 26 15 25

The average thickness, T, produced by both systems is similar and the averages are not statistically different. The average distance, D, from the end of the vessel closure for both systems is positive, indicating that the collagen in the intact vessel is not thermally damaged and remained normal past the seal in the vessel. However, the average distance for the seals performed with the LTC system is significantly greater than the average of the Gyrus seals at p = 0.025. The number of negative D values for the Gyrus system is approximately 4 times that of the LTC system. These data may suggest that the LTC seals give added protection against delayed bleeds by preserving more of the vessel beyond the seal. While this data demonstrates that there is a potential for vessel wall weakness from thermal damage that could erode the wall in an area where it is not sealed, it must be remembered that the wall is also strengthened by the clot formed from blood stasis and initiation of the clotting cascade from endothelia damage to the vessel.


Figure 1: diagram of vessel wall with variable dimensions - J, T, 0 point, + and – distances, D

• We have developed a new method of histologically examining

At the conclusion of the experiments the pigs were euthanized by an IV injection of pentobarbital, 90 mg/kg, into an ear vein. Figure 3: high mag of wall damage-transition zone of N to Nec The damage to the vessel wall can also be demonstrated by polarized light. Normal collagen is isotropic and, therefore, birefringent and appears as a bright area while denatured anisotropic collagen appears dark. This is shown in Figure 4. In this case the distance D is positive, indicating a good seal.

vessel sealing by RF electrosurgery. The method involves measuring thermal spread not from the forceps jaw but from the vessel wall seal or plug up the normal remaining vessel wall; this measurement is believed to be more physiologically important as a possible indicator of delayed bleeding.

• The LTC system produced significantly fewer numbers of negative measurements or negative outcomes, where the damage to the vessel wall did not extend beyond the beginning of the point of fusion, than the Gyrus system.

• The LTC system also provides a greater margin of safety by preserving more artery past the beginning of the fusion point toward the jaw.

Figure 4: vessel wall damage by polarized light, +D

LTC Study Histological Data Poster3