Junki Ren,Cho Yun,Tong Zhng,b,c,*

aDepartment of Hepatic Surgery and Liver Transplantation Center,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou,China

bLiver Transplantation Center of Sun Yat-sen University,Guangzhou,China

cOrgan Transplantation Institute of Guangdong Province,Guangzhou,China

ABSTRACT

Indocyanine green(ICG)is a fluorescent dye that is widely used in hepatobiliary surgery for fluorescence contrast.ICG is selectively absorbed by the liver after intravenous injection and then secreted into the bile.ICG's unique catabolism and fluorescence characteristics allow for multiple applications in liver transplantation.We have divided the applications of ICG fluorescence imaging in liver transplantation into:(i)cholangiography,(ii)evaluation of liver transplantation vessels,(iii)liver mapping,and(iv)evaluation of donor liver quality.In this review,we summarize the current status of applications of ICG fluorescence imaging in liver transplantation.

Keywords:

Indocyanine green(ICG)

Fluorescence imaging

Liver transplantation

Liver

Surgery

1.Introduction

The identification of liver segments and the anatomical visualization of specific components during liver transplantation are the keys to reducing postoperative morbidity and mortality.Real-time liver visualization can help surgeons perform hepatectomy in a relatively safe condition,thereby reducing postoperative complications such as bile leakage caused by bile duct damage.1An intravenous injection of indocyanine green(ICG)is non-toxic and avoids the shortcoming of conventional radiography that may cause liver and kidney damage.2ICG fluorescence imaging can detect biliary leakage on the liver section after partial liver transplantation,as well as extrahepatic biliary leakage after liver transplantation.It can also be used to evaluate vascular reconstruction and out flow after liver transplantation and to evaluate donor liver quality.This study aimed to review the different applications of ICG fluorescence imaging in liver transplantation.

2.Characteristics of ICG

ICG is a water-soluble anionic tricarbocyanine dye that was approved by the US Food and Drug Administration(FDA)for clinical application in 1959.3Originally,ICG was used in the field of heart disease to do things like measuring the cardiac output in valve or septal defects.The potential applications in liver diseases have been attracted much attention since its early application in that ICG is mainly metabolized by the liver.4These studies measured the levels of ICG in the blood only.In the 1970s,researchers discovered that ICG fluoresces when exposed to near-infrared (NIR)light(750-810 nm).5With the progress of technology,ICG can also be utilized to measure tissue perfusion,6tumor detection,7and treatment.8It is widely used in heart surgery,neurosurgery,liver surgery,and eye surgery.4The ICG dye has a molecular weight of 776 Daltons.3After intravenous injection,ICG first binds to albumin but does not change the structure of the protein until it is removed from the blood and absorbed by liver cells.2-4Depending on the vascularization and function of the liver,the dye is almost completely excreted from the bile within approximately 8 min after injection.3ICG becomes fluorescent after using NIR light excitation.When the ICG protein complex is excited by a light source with a wavelength between 750 nm and 810 nm,it emits fluorescence at a wavelength of approximately 830 nm.9This type of fluorescence is invisible to the naked eye and requires the aid of specific imaging equipment.ICG can be seen through 5-10 mm thick tissues;so,deep tissues are invisible with this technique.1,8The LD50(median lethal dose)of ICG is 50-80 mg/kg and the standard dose is less than 2 mg/kg,which is far below the toxic dose;so,ICG is actually non-toxic as long as the patient is not allergic to iodine.4,10

3.The applications of ICG fluorescence imaging in liver transplantation

ICG's premium catabolic characteristics and unique fluorescence features make it particularly suitable for visualizing the anatomical and pathological structure of the liver.So,it is suitable for liver transplantation,especially with its fluorescence characteristics.We divided the applications of ICG fluorescence imaging in liver transplantation into the following four categories(Table 1):(i)cholangiography,(ii)evaluation of liver transplantation vessels,(iii)liver mapping,and(iv)evaluation of donor liver quality.

Table 1The applications of indocyanine green(ICG) fluorescence imaging in liver transplantation.

3.1.Intraoperative fluorescence cholangiography

Surgeons often face anatomical difficulties with the bile duct when performing living donor liver transplantation(LDLT)or split liver transplantation(SLT).Unlike blood vessels,the biliary tract is located in the Glissonian sheath and buried in the surrounding connective tissue,making it difficult to observe and separate during surgery.11ICG has fluorescence characteristics,and using a NIR camera to detect fluorescence can effectively observe the biliary system.4The ICG fluorescence imaging is currently mostly used for extrahepatic biliary tract imaging and intraoperative inspection for biliary leakage.The human bile contains lipoprotein and albumin.12Fluorescent images of the biliary tract can be acquired by injection into the biliary tract.ICG was diluted to 0.025 mg/mL preoperatively and entered the bile duct through a catheter before the hilar bile duct division.Before ICG injection,a small amount of bile(less than 1 mL)was inhaled into the syringe to promote the binding of bile proteins to ICG.Intravenous injections can also obtain fluorescent images of the biliary tract because ICG is discharged through the liver into the biliary tract after entering the blood.ICG was diluted in 1 mL of a solution by taking 2.5 mg of ICG before surgery and injecting through a central venous catheter or a peripheral vein.Although bile excretion of ICG starts within a few minutes after its intravenous injection,ICG should be injected at least 15 min before imaging for better signal-background contrast.1

3.1.1.Extrahepatic cholangiography

Biliary tract complications remain the Achilles’heel of liver transplantation.13These complications can be avoided by determining the optimal bile duct segmentation point.14Intraoperative cholangiography(IOC)and preoperative magnetic resonance cholangiopancreatography(MRCP)or anatomic and cystic duct exploration around the bile duct are helpful in determining the optimal segmentation point.14IOC is considered the gold standard in most centers for determining the optimal segmentation point of the bile duct.14Mitsuhashi et al.15first applied ICG fluorescence imaging in cholangiography.It neither involves radiation nor requires a large C-arm fluoroscope,which reduces the radiation exposure for medical personnel.In a study that enrolled 82 patients,16all patients underwent near-infrared fluorescence cholangiography(NIRFC)and IOC.NIRFC was significantly faster than IOC and the visibility of the gallbladder,common bile duct,and common hepatic duct using NIRFC was better than that using IOC.In addition,NIRFC successfully identified biliary structures in 80% of the 20 patients who were unable to perform IOC.Hong et al.14reported ten cases of pure 3D laparoscopic living-donor hepatectomy using fluorescent imaging systems.In all ten donors,the biliary system around the hepatic portal plate,including any abnormal hepatic duct,could be delineated.Real-time inspection facilitated the determination of the dividing point of the bile duct after marking and the degree of patency after the bile duct was truncated.All patients were discharged from the hospital on the 6th to 9th days after surgery without complications.ICG cholangiography is a convenient and efficient technology that determines the best segmentation point.The surgeon can be more confident in the determination of the separation point and increase the efficiency of the operation.However,ICG fluorescence imaging is not effective in overweight and obese patients(body mass index(BMI)＞26.35 kg/m2).2Because patients with a high BMI are more likely to develop hilar fat(which requires additional dissection to see the bile ducts clearly),this technique may not be suitable for patients with a high BMI.14

3.1.2.Intrahepatic cholangiography

Bile leakage is one of the most common biliary complications of liver transplantation,with an incidence of 2-25%.17,18Bile leakage can lead to abdominal bleeding,sepsis,immune disorders,and liver dysfunction,19and ultimately reduce graft and patient survival.Most bile leaks occur in the first 30 days after liver transplantation.20The study by Fang et al.17has shown that the one-year survival differs little between the biliary fistula group and the nonbiliary fistula group but one-year and two-year graft survivals and the two-year survival are significantly reduced in patients with biliary fistula.However,if diagnosed early and treated immediately,bile leakage can almost return to normal.21Since ICG's fluorescence penetration ability is relatively weak,the intrahepatic bile duct cannot be clearly seen;however,the bile leakage that is ignored by routine examination can be detected.2Sakaguchi et al.22first proposed to detect intraoperative biliary leakage with ICG compared with the traditional normal saline solution,with 27 patients who underwent hepatectomy undergoing the ICG leakage test.Biliary leakage was found and repaired in eight patients,and there was no biliary leakage after the operation.In contrast,two of 32 patients who underwent the standard bile leak test using saline solution had postoperative biliary fistulas.The detection of biliary leakage by ICG fluorescence imaging can effectively prevent postoperative biliary leakage.Similar results were obtained by Kaibori et al.23who divided 102 patients without biliary tract reconstruction after hepatectomy into two groups.In the control group(50 cases),bile leakage was detected by ICG staining alone,while in the experimental group(52 cases),both ICG staining and fluorescein angiography were performed.There were five cases of bile leakage in the control group,and no case of bile leakage in the experimental group.Since SLT and LDLT are similar to liver resection,the method of obtaining the donor's liver is also prone to postoperative bile leakage.Although there is currently no report of ICG fluorescence intrahepatic cholangiography in liver transplantation,we believe that this technology can be applied in SLT and LDLT in the future.

3.2.Evaluation of blood vessels for liver transplantation

Vascular complications of liver transplantation include stenosis,thrombosis,and pseudoaneurysms.These are further classified as affecting the hepatic artery,portal vein,inferior vena cava,and hepatic vein.24Very few vascular complications occur after liver transplantation according to a single-center study.25The probability of vascular complications occurring during dead donor liver transplantation is close to 7%,while the probability of these complications occurring during LDLT is close to 13%.Because the clinical manifestations of vascular injury are nonspecific,early imaging examination plays an important role in early diagnosis and determination of the optimal treatment regimen,which can rescue the donor.26Doppler ultrasound plays a critical role in the detection of these diseases.24,26,27Fluorescence imaging clearly shows the reconstructed hepatic artery and portal vein,and the combination of Doppler ultrasound and ICG fluorescence imaging can ensure the patency of reconstructed blood vessels and reduce the occurrence of vascular complications caused by incomplete technology.28

3.2.1.In flow evaluation

Kubota et al.28were the first to perform ICG in the visualization of reconstructed blood vessels.Three patients who underwent LDLT received intravenous injections after portal vein and hepatic artery reconstruction.A solution of 25 mg ICG in 10 mL of saline was prepared,and 1.5 mL of it(3.75 mg of ICG)was injected intravenously and then flushed with 20 mL of saline.Fluorescent images were recorded using the SPY system,which was designed to visualize grafts in coronary artery bypass grafting.29,3010 s later,the first photographs were recorded over a 30 s period.The above steps are repeated to obtain the images after 2 min(the second photographic record).Finally,after 40 min,images were performed without additional ICG injection(the third photographic record).The first photographic recordings clearly showed a reconstructed hepatic artery blood flow(without bending or narrowing)in all three patients.The second photographic record showed the flow of the portal vein without narrowing,kinking,or stagnation.The third images showed the excretion of ICG into the bile duct after 40 min,which confirms the production of bile in the transplanted graft.After surgery,color Doppler ultrasonography evaluated sufficient blood flow in the hepatic artery and portal vein in all three patients who returned to normal.No postoperative vascular complications occurred.During liver transplantation,ICG fluorescence imaging can display real-time areas of damaged perfusion caused by portal vein thrombosis(PVT),as well as liver blood flow from reconstructed vessels,to assess areas of venous occlusion.Kawaguchi et al.6reported a 60-year-old woman with end-stage liver disease associated with chronic hepatitis C who underwent LDLT with a left liver graft.After all hepatic vascular reconstructions, fluorescence imaging with ICG was administered to assess the reconstructions of the hepatic artery and portal vein anastomosis and venous congestion associated with venous reconstructions within the graft.The fluorescence intensity(FI)of the left surface of liver transplantation gradually increased,while the FI of the surface of section 4 was lower than that of the left lateral sector.Previously,the same research team31,32proved that the FI in the venous occlusion area was lower than that in the non-venous occlusion area.When ICG fluorescence imaging showed that the FI on the liver surface is uneven,the liver graft may suffer hypo-perfusion due to thrombosis,stenosis,or portal vein occlusion.Intraoperative Doppler ultrasonography detected no abnormalities in the blood flow of the 4th venous branch but showed PVT in the same branch.Washing with heparin saline successfully ablated the thrombus.The patient did not develop PVT postoperatively.One of the disadvantages of this technique is that it cannot be utilized repeatedly to evaluate hepatic perfusion of the graft in principle because the fluorescence on the liver surface lasts a few hours.6

3.2.2.Out flow evaluation

Hepatic venous out flow tract obstruction(HVOO)is a rare but serious complication of liver transplantation.HVOO can lead to decreased hepatic sinusoid perfusion,hepatic sinusoid congestion,and hepatocyte apoptosis,causing liver dysfunction such as venous occlusive disease,Budd-Chiari syndrome,and congestive liver disease associated with heart disease.Since the donor liver for pediatric liver transplantation is almost exclusively derived from adults,this liver needs to be trimmed,and the trimmed donor liver generally does not include an inferior vena cava;so,out flow tract obstruction is more common in children.33This complication is often diagnosed late due to the lack of clinical symptoms and the sensitivity and specificity of noninvasive radiological examination.However,this complication requires early treatment because it is the only way to save the graft.33ICG fluorescence imaging can accurately estimate the local liver volume of postoperative venous occlusion.34,35The method for the intraoperative identification of these areas has not been established.Hashimoto et al.36quantitatively assessed the local microcirculation of the residual liver by ICG.Compared with non-venous occluded areas,hepatic ICG uptake was reduced in venous occluded areas.Kawaguchi31reconstructed the residual roots of segment 5,segment 8,or both in a right liver graft with no middle liver vein in order to prevent postoperative liver dysfunction due to venous occlusion.After reconstructing segments 5 and 8,ICG(1.65 mg or 2.5μg/mL of graft liver volume)was administered intravenously to determine the area of venous occlusion.After 300 s,a clear boundary appeared between the venous occlusion area and the non-venous occlusion area.With the ICG fluorescence imaging technique,intraoperative differences in local venous occlusion can be assessed immediately after venous reconstruction as a basis for guiding physicians to determine whether further venous reconstruction is needed.One of the drawbacks of this method is that it cannot be applied to evaluate postoperative venous obstruction without laparotomy due to the need to directly observe the surface of the liver.The ICG fluorescent image combined with the vein clamping technique can better distinguish the non-occluded area,vein-occluded area,and ischemic area.35However,it cannot be utilized to identify the degree of occlusion.

3.2.3.Evaluate bile duct vascularization

A well-vascularized bile duct is a prerequisite for successful bile duct reconstruction.Vascular assessment is usually performed by assessing the macroscopic aspects of the recipient and donor bile duct stump.Coubeau et al.13used ICG fluorescence angiography to evaluate bile duct vascularization.Intraoperative fluoroscopy was performed after injecting ICG(0.25 mg/kg)through a central catheter.Fluorescence was seen in the vascularized liver graft 28 s after intravenously administering ICG and approximately 45 s later in the biliary tract stump.After ICG evaluation,the ischemic area was found and removed.Postoperative pathology revealed the loss of bile duct epithelial cells and separation of the epithelium from subepithelial connective tissue,both of which are typical signs of ischemia.Routine percutaneous cholangiography showed no stenosis or leakage six months after the operation,and follow-up showed no abnormality 10 months later.This is the first reported case of bile duct vascularization evaluated with ICG fluorescence imaging.The application of ICG fluorescence imaging in the assessment of the blood supply of the bile duct in liver transplantation needs further demonstration.

3.3.Liver mapping

Due to the scarcity of donor livers,SLT and LDLT have become the first choice in many regions.The donor liver is obtained in a similar way to liver resection.The surgeon cannot fully see the structure in the parenchyma.The liver cross-section margin is usually determined by ischemia or intraoperative ultrasound.ICG fluorescence imaging technology is developing rapidly in the field of hepatobiliary surgery.Under the guidance of real-time fusion of ICG fluorescent images,surgeons do not have to rely on guessing the intersegmental plane;neither do they frequently attempt to track landmark blood vessels.Additionally,this technology can protect the donor's residual liver to the greatest extent.Nishino et al.37used ICG fluorescence imaging for liver resection.Twentythree patients underwent hepatectomy under the guidance of ICG fluorescence imaging,and ICG fluorescence clearly revealed the boundary in 21 of them.However,the boundary was not found in two patients.In recent decades,advances in the number and technique of laparoscopic hepatectomy have led to its expansion into transplantation.Because it is difficult to perform bile duct intubation and angiography under laparoscopy,and the failure to recognize the anatomy of the bile duct may lead to conversion to laparotomy.ICG fluorescence imaging has become a better way of solving the problem.Meng et al.38reported the first case of ICG fluorescence image-guided total laparoscopic living-donor right hepatectomy in China's mainland.Two milliliters of ICG(0.5 mg/mL)were injected directly into the right portal branch through a scalp needle.Through the fusion image installed on the laparoscopic system,the fluorescence area of the right lobe could be clearly seen.The parenchyma was divided along the fluorescence boundary line of the right lobe.Soft-tissue dissection was carried out with a Cavitron Ultrasonic Surgical Aspirator(CUSA).Bipolar coagulation and clamps are applied to stop the bleeding.The donor liver was successfully removed,and the patient was discharged from the hospital on the seventh postoperative day.ICG fluorescence imaging technology is feasible and suitable for laparoscopic donor liver acquisition.ICG fluorescence imaging has the potential to simplify procedures and improve safety and accuracy.However,more practice and technical improvement are needed.

3.4.Evaluation of donor liver quality

Primary graft dysfunction(PGD)is the leading cause of graft loss after liver transplantation.As the rate of transplants from marginal donors(such as deceased heart donors)increases,so does the risk of complications after transplantation.39There are two main forms of PGD:initial functional poor(IPF),which is reversible in most cases,and initial primary non-function(PNF),which is an aggravating form of reperfusion injury,and the only treatment is immediate re-transplantation.Currently,there is no reliable method of predicting the occurrence of this complication intraoperatively.Figueroa et al.40first reported the intraoperative use of ICG as a predictor of graft function in liver transplantation.The study included 72 patients who received intraoperative ICG injections after undergoing graft vascularization(venous and arterial anastomoses),and ICG(25 mg vials)was re-suspended in 10 mL of sterile water for injection to obtain 2.5 mg/mL of the stock solution.2 mL or 4 mL,equivalent to 5 mg or 10 mg,are given intravenously.After 5 min of dye injection, fluorescence imaging recordings were performed to systematically capture images of all anterior liver surfaces for approximately 2 min.According to the new classification of implant perfusion fluorescence imaging,the entire population was divided into three groups based on the parenchymal uptake of ICG(the “perfusion area”)and whether this uptake was uniform.Type I:homogeneous fluorescence.The entire graft ingests ICG in a similar manner,with NIR light showing the uniform surface of the liver.Type II:non-perfused area.ICG uptake was uniform except for certain regions.NIR light shows some dark areas with uniform fluorescence around them.Type III:non-uniform fluorescence.The ICG uptake was irregular and the fluorescence was patchy.NIR light shows some fluorescent spots on a dark background.The overall incidence of PGD was 29%(21 cases):IPF was present in 19 cases(26%)and PNF in two cases(3%).IPF occurred in eight patients(17%)and three patients(30%)in the type II fluorescence group.In the type III group,PGD was present in ten cases(59%),including two cases of PNF.Univariate logistic regression analysis identified an abnormal ICG fluorescence pattern as a risk factor for PGD after liver transplantation (odds ratio(OR)=2.92,95% confidence interval(CI)1.56-5.44,P＜0.001).Almost 60% of patients with type III developed PGD,and there were only two cases of PNF in the cohort corresponding to this type.For type I and type II,the incidence of PGD was 17% and 30%,respectively.For type II,this result may be due to venous out flow problems,creating a congestive region that produces a non-perfused fluorescent region.Type III may be impaired in hepatocyte-level function,leading to impaired ICG uptake after cell reperfusion.The same research team quantified ICG during liver transplantation in real-time to predict its three-month survival rate.41ICG was injected intravenously,and the NIR camera captured the fluorescence of the graft and quantified its gray-scale postoperatively.Then,a multi-parameter model of the real-time FI curve was established to predict the survival of the graft,and the prediction model was tested.A total of 76 liver transplantations were enrolled from July 2017 to May 2019,and six of them underwent repeat transplantations.The time course parameter a150of FI increased significantly in the repeat liver transplantation group.This parameter was the only independent predictor of graft survival at three months(OR=2.4,95%CI 1.05-5.50,P=0.04).41The best cutoff value of the parameter a150predicts the survival of the graft at three months with a sensitivity of 83% and a specificity of 79%(area under the curve(AUC)=0.82(0.67-0.98),P=0.01).41Whether or not ICG can be utilized to assess donor liver quality requires more confirmatory prospective studies.

4.Conclusions

ICG is a safe and simple fluorescence imaging method.It is suitable for liver transplantation.Although some applications in this article have not been reported for liver transplantation,we believe that they will be welcomed in the future.The shortcomings of ICG fluorescence are also obvious and are mainly manifested as poor tissue penetration.Further research is needed to evaluate the application of ICG fluorescence imaging in liver transplantation.

Authors’contributions

J.Ren and C.Yuan wrote the manuscript.T.Zhang wrote and revised the manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the Natural Science Foundation of Guangdong Province of China(No.2019A1515011850).