利用靛氰綠螢光進行非侵入性的肝功能檢測

Abstract

根據衛生署2014年調查的結果，癌症為台灣十大死因之首，而肝癌又是癌症中致死率第二高的，每年台灣都有超過一萬人死於肝病、肝硬化以及肝癌，因此提高肝癌治療的成功率是很重要的。肝癌治療的方式包括肝切除術、移植、化療或放療等等，其中，肝切除與移植能根治肝癌的機率較高。然而，並非所有的病患都適合做切除手術，若是術後的肝臟無法負擔全身的代謝量，會導致致命的肝衰竭現象，因此術前的肝功能評估極為重要。 目前主流的評估方法為靛氰綠測試，靛氰綠是一種只由肝臟代謝的染劑，藉由量測靛氰綠在血液中的滯留率來判斷肝功能好壞，以決定肝切除術的可行性及肝臟切除比例。一般而言，健康的肝臟能夠在注射靛氰綠15分鐘內，代謝掉90％的靛氰綠，但受損嚴重的肝在15分鐘內可能連60％的靛氰綠都無法代謝掉。傳統的測量方式使用具侵入性的抽血，人為影響大，且抽血只能取得單一時間點資料，無法連續的量測，較不準確。 本研究使用了光學方法，從體外量測皮膚表層血管中的靛氰綠螢光訊號，再推算出實際靛氰綠滯留量，以達到低背景訊號、連續監測及非侵入性的效果。此實驗分成兩階段確效，在第一階段中，我們同時監控雙光子螢光及三倍頻影像，使其能夠準確定位血管位置。在此階段中我們確認靛氰綠不會擴散至血管外，因此只收集血管內的螢光訊號是可行的，而動物實驗中測量出的靛氰綠滯留曲線也與傳統方法的結果一致：控制組中，15分鐘後的靛氰綠螢光強度不到初始強度的10％；而肝癌組中，15分鐘後的靛氰綠螢光強度還有初始強度的40％。在第二階段，我們致力於使用更低的靛氰綠劑量做出相同的結論，因此使用單光子雷射，並且移除了顯微鏡與掃描鏡系統，只量測單光子螢光訊號。由於此系統有良好的訊號收集效率，使得所注射的靛氰綠劑量能夠降低至1/10倍的醫院用劑量。 靛氰綠劑量的降低有三大優點：從技術層面上來看，低濃度的靛氰綠，其螢光強度與濃度呈線性關係，使得準確率提高；從實際層面上來看，節省價格昂貴的靛氰綠的用量，能夠使檢測成本降低；從安全層面上來看，低濃度的靛氰綠對人體的毒性較小，提升檢驗的安全性。此為本研究所致力追求的貢獻。

According to the investigation of the Ministry of Health and Welfare in 2014, cancer is the most common cause of death in Taiwan, and hepatic cancer is the second leading cause of cancer-related death. Over 10 thousands of people die of hepatic diseases, so it is essential to raise the survival rate in treatments to hepatic cancer. Methods of treatment to hepatic cancer include hepatectomy, liver transplant, chemotherapy, radiotherapy, etc. Hepatectomy and liver transplant are the most feasible methods to eradicate hepatic cancer. However, it is impossible to conduct hepatectomy on every patient. If the postoperative liver cannot afford the metabolism of the whole body, it will lead to liver failure which is immortal. Therefore, the preoperative assessment of liver function is of importance. Currently, the standard assessment method is Indocyanine Green (ICG) clearance test, and ICG is a kind of dye which would be metabolized almost by the liver only. By measuring the retention rate of ICG in blood, doctors can evaluate liver functions in order to determine the feasibility of hepatectomy and the ratio of the liver to be resected. Generally, after 15-minute administration, the retention of ICG of healthy people will below 10%, but the retention of ICG of patients with severe hepatic diseases could be over 40%. To quantify ICG concentration, the traditional way is to draw blood after administration at 5, 10, 15 minutes and measure the ICG concentration in the blood samples by spectrophotometry. This method is invasive, discontinuous, and with human-caused errors, so it is more incorrect. Developing a fluorescence-based detector system excited by a single photon laser, we are able to detect the ICG fluorescence noninvasively, and then calculate the retention time of ICG in blood. The advantages of this approach are noninvasively and continuously monitoring the ICG retention rate with high contrast. In the first step, we acquired the THG images and the ICG two-photon fluorescence images at the same time so that the imaging plane of blood vessels could be fixed. We ensured that ICG would not diffuse out of vessels, so it is feasible to collect the ICG fluorescence by large area excitation. The result showed that, in the control group, the ICG fluorescence intensity after 15-minute administration is lower than 10% of the initial intensity; in the HCC group, the intensity after 15-minute administration is over 40% of the initial intensity, which is consistent with the traditional method. In the second step, we transfer the design to single-photon excitation scheme. We used a single-photon laser as the light source and removed the telescope and the scanner parts. The only signal we detected came from the ICG fluorescence. Moreover, the efficiency of collecting signals of this system is high. As a result, the required dose of ICG can be lower to 1/10 times of the dose usually used in hospitals. There are three major advantages of the reduction of the ICG dose. First, in terms of technique, the ICG fluorescence intensity is linearly dependent on the ICG concentration when the dose is as low as 0.116 mg/dl, and that improves the accuracy of assessment. Second, lower dose brings lower cost, saving the usage of expensive ICG is equivalent to reducing the cost. Third, lower ICG dose is less toxic to human body, therefore the assessment can be more safe. We anticipate this method in the future can be further applied not only to the hepatic assessment before liver resection surgery, but also to the postoperative evaluation of the effectiveness of liver transplant or resection surgery.