Liver transplantation (LT) for patients with end-stage liver disease and hepatocellular carcinoma has seen considerable advancements in recent years. Nonetheless, biliary complications (BCs) continue to be a significant cause of morbidity following both deceased donor liver transplantation (DDLT) and living donor liver transplantation (LDLT) [1]. Biliary strictures are most commonly observed at the anastomosis site [2,3]. The incidence of biliary anastomotic strictures ranges from 8% to 31% after LDLT and from 5% to 15% following DDLT, with 70% to 87% of cases being diagnosed within the first postoperative year [4-6]. The incidence of biliary leaks varies from 2% to 25%, while the incidence of diffuse cholangitis ranges from 2% to 20% [7]. In LDLT, the bile duct anastomosis is a technically demanding procedure, frequently leading to post-LT BCs such as leaks and strictures. Li et al. [8] suggested that indocyanine green (ICG) fluorescence imaging can be valuable in hepatobiliary surgery for liver mapping, cholangiography, tumor visualization, and the evaluation of vascular flow during partial liver graft procedures. The utility of ICG is expanding into the field of LT, where it aids in predicting outcomes. This study explores the near-infrared (NIR) fluorescence intensity (FI) of ICG in the common bile duct (CBD) and its correlation with BCs within three months post-LDLT.

The clinical data and ICG fluorescence imaging data of nine patients who underwent LDLT from May to October 2022 were prospectively collected. Patients eligible for the study were 19 years or older and under 80 years, who underwent LDLT. Patients who had undergone re-transplantation or received other multi-organ transplants were excluded from the study.

The study protocol was approved by the Institutional Review Board of Seoul National University Hospital (No. 2302-132-1408). All patient records were anonymized and de-identified after data collection to ensure privacy and confidentiality.

A 0.05 mg/kg dose of ICG was intravenously administered to the recipient immediately before the bile duct anastomosis following the reconstruction of the hepatic vein, portal vein, and hepatic artery (HA) [9]. ICG FI curves typically showcase a transitional area, which acts as an optimal zone for analyzing fluorescence patterns [10,11]. CBD perfusion imaging was recorded for three minutes using an ICG fluorescence camera (Stryker), positioned 5 cm from the CBD. To minimize signal interference, the liver parenchyma was covered with gauzes, and the operating room lights were turned off during the imaging.

Fluorescence images were analyzed at 10-second intervals using ImageJ software (National Institutes of Health [NIH]) (Fig. 1, 2). FI was measured in a 1 cm × 1 cm area as close as possible to the recipient's proximal CBD margin.

Fig 1. Changes in ICG fluorescence intensity over time. After administering 0.05 mg/kg of ICG, the recipient CBD (arrow) perfusion images were captured using an ICG fluorescence camera. (A) After 10 seconds. (B) After 20 seconds. (C) After 30 seconds. (D) After 40 seconds. ICG, indocyanine green; CBD, common bile duct.

Fig 2. The ICG angiography images were analyzed at 10-second intervals using the Image J software program. The fluorescence intensity was measured in a 1 × 1 cm area (square), positioned as close as possible to the recipient`s CBD (dotted line) proximal margin. ICG, indocyanine green; CBD, common bile duct.

Parameters including F max, F_1/2 max, T_1/2 max, and slope were evaluated. In the time-fluorescence curve of ICG angiography (Fig. 3), F max denotes the peak FI, F_1/2 max represents the midpoint of the FI, T_1/2 max indicates the time to reach F_1/2 max, and the slope (F max/T max) quantifies the rate of change in FI. These metrics provide a quantitative assessment of the fluorescence response [12,13].

Fig 3. Time–fluorescence curve of ICG angiography. F max, peak fluorescence intensity; F_1/2 max, fluorescence intensity halfway between the baseline and F max; T_1/2 max, the time at which the fluorescence intensity reaches F_1/2 max; Slope, F max/T max.

Statistical analysis

Given the limited sample size (n = 9), non-parametric methods were employed for analysis. Continuous variables were analyzed using the Mann-Whitney U test, whereas categorical variables were compared using Fisher’s exact test. A p-value of < 0.05 was deemed statistically significant. All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 29 (IBM Corp.).

Biliary complications

Data on BCs such as bile leaks, bile duct strictures, and cholangitis were prospectively collected, and complications were graded using the Clavien-Dindo classification [14]. Cholangitis was diagnosed based on clinical symptoms, such as fever and abdominal pain, and abnormal liver function tests. A bile leak was defined as a bilirubin concentration in the Jackson-Pratt drainage fluid that was at least three times higher than the serum bilirubin level or when radiological evidence required intervention. Biliary strictures were diagnosed through imaging and clinical or biochemical markers of cholestasis, including elevated levels of alkaline phosphatase and gamma-glutamyl transferase that exceeded twice the upper limit of normal. The date of post-LT complications and subsequent treatments were analyzed.

Table 1 displays the clinical characteristics and surgical findings of the patients. Among the nine patients, five were male, predominantly in their 60s. The majority had ABO-compatible (ABOc) blood types (n = 8). Three patients had HBV-related liver cirrhosis (LC), and three had non-B, non-C (NBNC) LC. Six patients underwent HA anastomosis using the recipient's left HA, while the remaining three underwent the procedure using the recipient's right HA. Single bile duct anastomosis was performed in six patients, and double anastomosis in three. The table also provides comprehensive details on the surgical type, operation name, duration of surgery, cold and warm ischemic times, graft-to-recipient weight ratio, and donor information.

Table 1 . Clinical characteristics and operation findings

No.	Sex	Age (yr)	BMI (kg/m2)	ABO	Underlying liver disease	Operation type/name	Operation time (min)	Cold/warm ischemic time (min)	GRWR (%)	HA anastomosis	Donor’s BD opening	Donor information (sex/age [yr], BMI [kg/m2])
1	F	58	24.0	ABOc	NBNC LC	Open/LDLT	430	115/39	0.70	LHA	2	M/27, 21.0
2	M	61	22.5	ABOi	HBV LC, PSE	Open/LDLT	530	107/34	1.21	LHA	1	F/32, 25.6
3	F	64	24.8	ABOc	HCV LC	Open/LDLT	305	207/37	2.02	RHA	1	F/33, 25.7
4	F	64	20.8	ABOc	NBNC LC	Open/LDLT	375	55/36	1.09	LHA	2	M/39, 27.6
5	M	59	23.2	ABOc	Alcoholic LC	Open/LDLT	355	97/25	1.36	LHA	1	M/28, 30.8
6	M	54	27.9	ABOc	HBV LC, HCC	Open/LDLT	495	109/39	0.95	LHA	2	F/21, 26.1
7	M	63	21.4	ABOc	NBNC LC	Open/LDLT	325	102/19	0.92	RHA	1	F/59, 22.0
8	M	62	25.3	ABOc	HBV LC	Open/LDLT	365	125/33	1.00	LHA	1	M/24, 20.9
9	F	66	20.3	ABOc	Biliary cirrhosis LC	Open/LDLT	310	136/31	2.69	RHA	1	M/39, 26.9

F, female; M, male; BMI, body mass index; ABOc, ABO compatible; ABOi, ABO incompatible; NBNC, non-B non-C; LC, liver cirrhosis; HBV, hepatitis B virus; PSE, portosystemic encephalopathy; HCV, hepatitis C virus; HCC, hepatocellular carcinoma; LDLT, living donor liver transplantation; GRWR, graft-to-recipient weight ratio; RHA, right hepatic artery; LHA, left hepatic artery; BD, bile duct.

Both patients with postoperative BCs exhibited the longest T_1/2 max values and lower slopes, suggesting a correlation between prolonged T_1/2 max and BCs (Table 2).

Table 2 . ICG parameters and BD complications

No.	Sex	Age (yr)	F max	TTP	Slope	F1/2 max	T1/2 max	BD complication
1	F	58	46.4	80	0.6	23.2	38	Not occurred
2	M	61	24.3	60	0.4	12.2	36	Not occurred
3	F	64	33.7	60	0.6	16.9	27	Not occurred
4	F	64	67.1	50	1.3	33.6	36	Not occurred
5	M	59	48.6	50	1.0	24.3	31	Not occurred
6	M	54	54.9	60	0.9	27.5	17	Not occurred
7	M	63	28.9	40	0.7	14.5	43	Occurred
8	M	62	69.5	40	1.7	34.8	17	Not occurred
9	F	66	28.9	50	0.6	14.5	47	Occurred

ICG, indocyanine green; BD, bile duct; F, female; M, male; F max, maximum fluorescence; TTP, time to peak; Slope, F max/T max; F_1/2 max, the midpoint of the peak fluorescence intensity; T_1/2 max, time to half-maximum fluorescence.

Fig. 4 shows the FI of ICG at 10-second intervals. Two distinct patterns emerged: one group, represented by patients 6 and 8, showed a rapid FI peak and high F max, while the other group, including patients 7, 8, and 9, exhibited delayed peaks and lower F max values.

Fig 4. ICG fluorescence intensity at 10-second intervals over time. It illustrates two ICG fluorescence patterns: one with a rapid peak FI and high F max, and another with a delayed peak FI and lower F max. Post-LDLT BCs occurred in the latter group, with patients no. 7 and no. 9 exhibiting lower slopes and prolonged T_1/2 max, indicating delayed CBD perfusion. ICG, indocyanine green; FI, fluorescence intensity; LDLT, living donor liver transplantation; BC, biliary complication; CBD, common bile duct.

BCs, specifically biliary strictures, occurred in the latter group. Patient 7, a 63-year-old male with NBNC LC, developed a biliary stricture a month post-LDLT and required endoscopic retrograde biliary drainage (ERBD). Patient 9, a 66-year-old female diagnosed with biliary cirrhosis LC, developed a stricture two months postoperatively and underwent multiple ERBD procedures (Table 3).

Table 3 . Postoperative complications including bile ducts and interventions

No.	Sex	Age (yr)	Operation date	Postoperative complication
7	Male	63	2022/08	POD 28, biliary stricture ERBD (RA) POD 2 mon, 4 mon, 8 mon, and 9 mon, ERBD revision (RA, RP) POD 1 mon cholangitis, antibiotics treatment
9	Female	66	2022/10	POD 1 wk, infection, antibiotics treatment POD 1 wk, CMV infection, Ganciclovir POD 2 mon, biliary stricture, ERBD (RA, RP) POD 4 mon, CMV retinitis POD 7 mon, ERBD revision (RA, RP) POD 10 mon, ERBD removal POD 13 mon, biliary stricture, ERBD (RA) POD 13 mon, PTBD (RP)

POD, postoperative day; ERBD, endoscopic retrograde biliary drainage; RA, right anterior; RP, right posterior; CMV, cytomegalovirus; PTBD, percutaneous transhepatic biliary drainage.

ICG is a water-soluble dye that predominantly undergoes hepatic metabolism through binding to plasma proteins [15]. The rate of ICG metabolism in the liver is determined by hepatic blood flow, hepatocellular function, and bile excretion [15]. Therefore, ICG metabolism quantification is sometimes conducted preoperatively in patients scheduled for liver resection to determine the extent of the surgical resection. On the other hand, ICG fluorescence imaging during surgery can predict liver graft function and identify perfusion defects [16].

ICG fluorescence imaging in LDLT has primarily been studied for visualizing donor-recipient biliary anastomoses and assessing donor liver quality. In 2010, Mizuno et al. [17] injected ICG through the cystic duct during open left hemihepatectomy to guide real-time visualization of the bile ducts under fluorescence. In 2017, Hong et al. [9] administered 0.05 mg/kg ICG intravenously 30 to 60 minutes prior to laparoscopic liver resection, enabling precise bile duct incisions by clearly delineating the bile duct structures. Unlike traditional angiography, ICG fluorescence offers a multi-angle perspective, enhancing understanding of the spatial relationships between the bile duct and hepatic hilar structures for more accurate incision planning.

Kawaguchi et al. [16] described a case in which ICG was injected during LDLT to detect perfusion defects and identify associated portal vein thrombosis. Dousse et al. [18] performed an analysis involving 76 patients who had undergone DDLT; ICG was administered at a dose of 0.01 mg/kg body weight, and Fluobeam was used to evaluate the correlation between FI and the three-month survival rate, as well as its association with re-transplantation.

There are very few studies that evaluate bile duct perfusion using ICG fluorescence imaging in LT. Coubeau et al. [19] utilized ICG fluorescence angiography to assess the vascularization of the bile duct by performing intraoperative fluoroscopy after administering ICG (0.25 mg/kg) via a central catheter. This technique facilitated the identification and removal of ischemic areas. Follow-up percutaneous cholangiography, conducted six months post-operation, revealed no evidence of stenosis or leakage. This study is analogous to ours in terms of observing bile duct perfusion and identifying ischemic areas with ICG fluorescence angiography. However, our study is more sophisticated: using ICG angiography, we acquired CBD perfusion images before bile duct anastomosis following HA anastomosis in engraftment, analyzed the ICG FI graph patterns, and demonstrated that these patterns vary between patients who developed BCs and those who did not. This study suggests a correlation between BCs and changes in ICG FI over time in the bile duct.

In LT and hepatobiliary surgery, most existing studies using ICG angiography have primarily focused on visualizing bile duct anatomy [20-22]. Unlike these, our study represents a novel approach as it employed ICG angiography to obtain CBD perfusion images of the recipient during LDLT, analyzing the ICG FI graph over time and identifying two distinct patterns. A significant finding from this research is the correlation between extended T_1/2 max durations, lower slope, and the occurrence of BCs in patients undergoing LDLT.

Son et al. [12] demonstrated that quantitative analysis of ICG perfusion patterns using T_1/2 max, slope, and TR (T_1/2 max/T max) can accurately identify poorly perfused segments. While our study aligns with these findings in associating a lower slope and a prolonged T_1/2 max with poor perfusion and anastomosis complications, it differs in that it applied ICG angiography to bowel anastomosis in colorectal surgery and examined the correlation by measuring TR.

Study limitations include the interference from previous ICG injections in the donor, causing the graft bile duct to appear excessively bright and preventing accurate measurement of ICG FI. Furthermore, the study's small sample size of only nine LDLT recipients limits our ability to draw definitive conclusions from the data. The research methodology employs an ICG fluorescence camera and uses Image J (NIH) for measuring NIR FI, which, while sophisticated and accurate, restricts a comprehensive analysis of factors associated with BCs due to the small participant pool.

Despite such limitations, the study offers initial insights that may inform the development of new tools for predicting BCs. Verification through larger, more diverse studies is necessary to strengthen the correlations established and to enhance the predictive accuracy of ICG fluorescence imaging parameters for BCs.

In conclusion, ICG fluorescence imaging is a promising tool for assessing bile duct perfusion and predicting BCs in LDLT patients. Further studies are necessary, but the preliminary findings suggest that ICG imaging could significantly improve postoperative management and patient outcomes.

This study was funded by the Research Program of the Korean Association for the Hepato-Biliary-Pancreatic Surgery for 2023 (KAHBPS-23-02).

No potential conflict of interest relevant to this article was reported.

Conceptualization: YRC, NJY, SKH, KWL. Data curation: JL, JYK, SYH, JML. Methodology: JL, YRC, NJY. Visualization: JL, YRC, NJY, KSS. Writing - original draft: JL. Writing - review & editing: YRC, NJY, KSS.