All 27 recipients were admitted to our institute 17 d before the day of scheduled operation (D-17) and receive a single dose of rituximab 170 mg/BSA (m²) on D-14. Preoperative PE was performed according to the serum isoagglutinin titer. The target serum isoagglutinin levels were the same or less than 1:16. PE was performed with 1.0 or 1.5 times the patient’s plasma volume, and the replacement fluid was composed of equal amounts of albumin and fresh frozen plasma (FFP) of blood type AB.

Lymphocyte subsets were routinely measured on the day of a patient’s admission (Lф-1), 4 d after rituximab administration (Lф-2), and 2 wk after ABO-I ALDLT (Lф-3). Immunophenotyping for lymphocyte subsets was performed by 4-color flow cytometry analysis with the tetraCXP system on fresh peripheral blood samples. Lymphocyte gating was performed on the basis of a primary CD45 antigen using the side scatter procedure. Among the CD45+ cells, CD3+ cells were counted as T-cells, CD3+CD4+ as Th-cells, CD3+CD8+ as Tc-cells, CD3-CD19+ as B-cells, CD3-CD56+ as natural killer (NK) cells.

Serum isoagglutinin levels were preoperatively measured on the day of the patient’s admission and after every PE. In the post-transplant period, isoagglutinins were measured daily during the first week, and then twice a week until discharge. The methods used to measure isoagglutinin titer were tube techniques including the immediate spin tube method for IgM and the tube with anti-human globulin method for IgG[7].

The recipient and donor operations were performed as previously described[8]. All 27 ABO-I ALDLTs were performed using right lobe grafts from 27 living donors. All recipients underwent duct-to-duct biliary reconstruction with a 4-french external biliary stent[9]. After biliary reconstruction, splenectomy was performed. The spleen was mobilized and divided with a vessel sealing system and stapling device[10]. Hepatic artery infusion therapy (HAIT) was performed in 6 patients. The HAIT regimen consisted of prostaglandin E1 (0.01 g/kg per minute), methylprednisolone (1 mg/kg per day), and gabexate mesylate (1 mg/kg per day), infused continuously for 21 postoperative days (POD).

Immunosuppression was started with intravenous infusion of methylprednisolone (1 g) during the anhepatic phase of the operation, followed by 200 mg/d given intravenously on POD 1. The dose was gradually tapered to 40 mg/d by POD 7, and then converted to oral prednisolone until it was discontinued after 3 mo. Tacrolimus was started immediately after the operation to reach a target serum trough level of 15 ng/mL until POD 7, then reduced to around 10 ng/mL; that level was maintained until discharge, at which point it was reduced to 5-8 ng/mL. Mycophenolate mofetil (MMF, 500 mg twice a day) was added to a drug combination with steroid and tacrolimus after POD 3. The dose of MMF was adjusted according to the level of tacrolimus in the blood and white blood cell (WBC) count. All recipients received 2 g/kg of intravenous immunoglobulin (IVIg) for the first 3 d after transplantation.

Isoagglutinin titers were measured every other day until POD 14, and twice a week thereafter until discharge. Lymphocyte subset was measured routinely on POD 14 (Lф -3). Post-transplant PE was performed when isoagglutinin titer increased to 1:64 or above or when AMR was diagnosed on a liver biopsy.

Liver function test (LFT) and complete blood cell count were measured daily until the date of discharge from the hospital. Surveillance cultures for bacteria and fungi from blood, urine, stool, sputum, and body fluid drainage were performed weekly until discharge. Also, cytomegalovirus (CMV) infection was assessed weekly with a CMV antigenemia assay. Preemptive anti-CMV therapy was performed using intravenous gancyclovir when the CMV infected WBC count was above 4/200000 WBCs.

Vascular flow in the graft was assessed daily using Doppler ultrasound for the first week and then twice a week until POD 28. Abdominal computed tomogram (CT) images were taken regularly once a week until POD 28. A direct cholangiography was performed routinely on POD 21 via the external biliary stent. A liver biopsy was performed only when the patient had signs of AMR or acute cellular rejection (ACR) and after excluding other causes for abnormal graft dysfunction. All liver biopsy procedures were performed with a trans-hepatic approach under ultrasound guidance.

ITBL was suspected when a recipient showed abnormal LFT with a cholestatic profile and/or dilatation of the intrahepatic bile duct on imaging studies during the post-transplant course. Then, a cholangiogram was taken via the external biliary stent in order to diagnose ITBL. In this study, ITBL after ABO-I ALDLT was defined when dilatation or stricture of the intrahepatic bile ducts with irregularity were observed on a cholangiogram after exclusion of hepatic artery thrombosis by Doppler ultrasound or angiogram.

Management of the ITBL was mainly composed of interventional and supportive treatment. Percutaneous transhepatic biliary drainage (PTBD) was performed to reduce biliary stasis and bile sludge. Supportive treatment consisted of ursodeoxycholic acid and antibiotics.

All liver biopsy specimens were pathologically evaluated for evidence of AMR, ACR, or other findings. Routine staining methods included H&E, Masson’s trichrome, and immunohistochemistry (IHC) with CD56 for NK cells and complement C4d. A pathologic finding of AMR was defined as periportal edema and necrosis or hemorrhage with neutrophil infiltration. Positive staining for C4d in the small vessels or periportal sinusoid supported the finding of AMR[11].

Spleen specimens were routinely stained with H&E and IHC for CD20 for B-cells and CD138 for plasma cell. Each spleen was reviewed by a pathologist, who measured the size of the B-cell follicles and counted the number of plasma cells. B-cell follicles in the spleen were semi-quantitatively scored according to the number and size of CD20+ follicles. Scoring ranged from 0 to 5, with 0 corresponding to an absence of B-cell follicles larger than 10 μm; 1, occasional staining of small follicles less than 100 μm; 2, frequent staining of small follicles less than 100 μm; 3, presence of medium follicles less than 300 μm; 4, presence of medium-to-large follicles less than 500 μm; 5, presence of large follicles more than 500 μm. The number of plasma cells is reported as the sum of CD138+ cells counted in 10 random fields under 400 × magnification using a light microscope. Six spleen specimens for control pathology were obtained from 6 adult recipients who received ABO-compatible liver transplants.

All data were analyzed using SPSS statistical software (Ver 22.0; SPSS Inc., Chicago, IL, United States). Data are expressed as mean ± SD or median values, ranges, and percentages. Univariate analysis was performed by the Mann-Whitney U test or Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and survival rates were compared using a log-rank test. Logistic regression analysis was performed to assess the risk factors of ITBL in terms of the odds ratio and 95%CI. P-values of less than 0.05 were considered statistically significant.

The characteristics of all 27 ABO-I ALDLT patients are listed in Table 1. The median age was 50 years old (range, 31-65), and 21 were male (77.8%). The underlying diseases were hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) (n = 17, 62.9%), HBV-related liver cirrhosis (n = 6, 22.2%), alcoholic liver cirrhosis (n = 2, 7.4%), hepatitis C virus-related HCC (n = 1, 3.7%), and Wilson’s disease (n = 1, 3.7%). The median value of the Model for End-Stage Liver Disease score was 11 (6-34).

The median age of donors was 24 (18-53) years, and 13 donors were male (48.1%). There were 20 donors who had blood type-A barrier to recipients and 7 with blood type-B barrier. Donors with A or AB blood type were placed in the A1 or A1B subgroup. The mean body mass index of the donors was 22.6 ± 3.1 (17.6-31.5). All donors recovered from right hepatectomy without serious complications and were discharged with normal LFT. The median length of hospital stay after donor hepatectomy was 9 d (7-11).

The mean weight of the 27 grafts was 667.9 ± 113.3 g (460-895), and the graft-to-recipient weight ratio was 0.97 ± 0.25 (0.60-1.55). The degree of macro-vesicular steatosis of the grafts was a median of 0% with a range of 0% to 5%. The cold and warm ischemic time of the grafts was 119.6 ± 51.6 min (25-290) and 46.2 ± 12.1 min (18-65), respectively. The total volume of RBC intraoperatively transfused to the recipients was 2.7 ± 3.7 units of RBC. The recipients’ operation took 658.1 ± 84.5 min (524-850).

The lymphocyte subset counts of the recipients at Lф-1 showed a B-cell count of 149.0 ± 108.7 per μL in blood, which was 16.4% ± 7.1% of total lymphocytes. B-cell depletion was successfully achieved in all recipients at Lф-2 and Lф-3. None of the recipients needed an additional dosage of rituximab before or after the operation.

The median titers of IgM and IgG isoagglutinin of the recipients at the time of admission were 1:32 (1:8-1:256) and 1:32 (1:2-1:1024), respectively. The median number of PEs was 2 (0-15) before the operation. All recipients achieved isoagglutinin titer ≤ 1:16 on the day of transplantation. The median peak titers of isoagglutinin IgM and IgG after transplantation were 1:4 (1:1-1:256) and 1:8 (1:0-1:256), respectively. Six recipients underwent PEs after transplantation.

The mean semi-quantitative score of the B-cell follicles in the spleen of ABO-I ALDLT recipients was 2.8 ± 1.1 (0-4). All control spleens had a score of 5. The scores of B-cell follicles were significantly decreased after B-cell depletion in the recipients compared to the control patients (P = 0.001). The plasma cell count was 76.3 ± 44.6 (10-168) in the ABO-I ALDLT recipients and 72.5 ± 30.3 (20-107) in the control patients (P = 0.846) (Figure 1).

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Figure 1 Immunohistochemistry of the spleen for CD20+ B-cell follicles and CD 138+ plasma cells. Reduced size of B-cell follicles after rituximab prophylaxis in the spleen (A) compared to a control (B) (× 40). Plasma cells in the spleen with rituximab prophylaxis (C) and without (D) (× 400).

The median follow-up time of the 27 recipients was 26 mo (8-84). The median length of hospital stay after ABO-I ALDLT was 28 d (20-75). Post-transplant laboratory tests showed that the peak levels of serum aspartate transaminase, alanine transaminase, and total bilirubin were 342.5 ± 268.9 IU/L, 342.5 ± 268.9 IU/L, and 4.2 ± 1.6 mg/dL, respectively. Graft function normalized between POD 12 and 21. All recipients showed satisfactory blood flows in the graft’s hepatic vein, portal vein, and hepatic artery without stenosis or obstruction on Doppler ultrasound and CT scan. None of 6 recipients with HAIT experienced infusion catheter-related complications. There were 4 recipients with intra-abdominal hemorrhage that developed between POD 2 - 16. The condition was were managed with angiographic embolization (n = 2) and abdominal exploration (n = 2). Post-transplant transfusion of packed RBC was performed in 17 recipients using 4.1 ± 4.4 units.

Eleven of the 27 recipients (40.7%) underwent a liver biopsy after ABO-I ALDLT. The total number of biopsy procedures was 13, because 2 patients underwent a second biopsy owing to repetitive graft dysfunction. Among the 13 biopsy results, 1 showed a finding compatible with AMR, 6 showed ACR, and 6 showed non-specific findings. One patient with non-specific inflammatory finding on the liver biopsy showed NK cells embedded in the liver tissue (Figure 2). Six of the 27 recipients (22.2%) experienced CMV infection after ABO-I ALDLT in our series, which was successfully treated with IV or oral gancyclovir. One patient developed MRSA pneumonia and ARDS followed by in-hospital mortality on POD 70. Five recipients passed away during the follow-up period from recurrent HCC (n = 3), post-transplant lymphoproliferative disease (n = 1), and recurrent cholangitis and liver abscess (n = 1). The 1-year, 3-year and 5-year survival rates of the 27 recipients after ABO-I ALDLT were 92.6%, 81.5%, and 73.0%, respectively.

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Figure 2 Liver biopsy pathology of case No. 12. Non-specific inflammation shown by H&E staining (A) (× 400). CD 56+ cells (black arrows) embedded in liver parenchyma (B) (× 400). The patient developed ITBL 23 d after the liver biopsy.

Among the recipients with ABO-identical or compatible ALDLT, 4 recipients (3.5%, 4 of 114) experienced in-hospital mortality after surgery due to bacterial pneumonia sepsis (n = 3), and pulmonary Aspergillosis (n = 1). Thirteen recipients died during the follow-up period after normal discharge due to HCC recurrence (n = 10), chronic alcohol abuse (n =1), and cerebrovascular infarction (n = 1). The 1-year, 3-year and 5-year survival rates of the ABO-identical or compatible ALDLT recipients were 93.9%, 84.8% and 82%, respectively. The survival rates of recipients with ABO-I ALDLT and ABO-identical/compatible ALDLT are compared in Figure 3 (P = 0.303).

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Figure 3 Overall survivals rates after adult living donor liver transplantation. 1-, 3-, and 5-yr survival rates are 93.9%, 84.8%, and 82% after ABO-identical/compatible ALDLT and 92.6%, 81.5%, and 73.0% after ABO-I ALDLT. There is no statistical difference between survival rates of the two groups (P = 0.303). ALDLT: Adult living donor liver transplantation.

No cases of ITBL developed among the 114 recipients with ABO-identical or -compatible ALDLT in this study. However, ITBL developed in 4 recipients (14.8%, 4 of 27) after ABO-I ALDLT during the period between POD 45 and POD 112 (Table 2). ITBL was confirmed by a cholangiogram via the external biliary stent (Figure 4). One of 4 recipients with ITBL had biopsy-proven AMR prior to development of ITBL; however, three had no evidence of AMR on biopsy. All patients with ITBL were treated with radiological intervention and supportive treatment. Among them, one died of recurrent cholangitis, liver abscess, and biliary sepsis 14 mo postoperatively. The remaining 3 patients underwent serial PTBD for more than 1 year. Two patients completed the interventional treatment, one with favorable liver function and the other with abnormal LFT due to diffuse intrahepatic portal vein thrombosis. One patient remains on PTBD treatment and has shown sustained hyperbilirubinemia for 20 mo.

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Figure 4 Ischemic-type biliary lesion diagnosed by the cholangiogram via external biliary stent. Diffuse dilatations of the intrahepatic bile ducts with irregularities are shown.

One recipient developed anastomosis stricture (AS) after ABO-I ALDLT in this series. The AS occurred on POD 135, and was successfully treated without sequelae by serial dilatation of the stricture site using a percutaneous transhepatic approach.

The clinicopathological characteristics of the 4 patients with ITBL were compared with those of the 23 recipients without ITBL. There was no difference in the perioperative characteristics of donors and recipients between the two groups (Table 3). The lymphocyte subsets analyses are compared between the recipients with and without ITBL in Table 4. The results at Lф-1, Lф-2, and Lф-3 showed no difference in terms of total lymphocyte count, B-cell count, T-cell count, and T4/T8 ratio between the recipients with ITBL and those without ITBL (P > 0.05). However, the NK cell counts were significantly higher in the recipients with ITBL than in those without ITBL at Lф-2 (P = 0.034) and Lф-3 (P = 0.027). ROC curve analysis of the NK cell counts showed optimal cut-off values of 150/μL at Lф-2 and 120/μL at Lф-3 for the development of ITBL. The sensitivity and specificity were 75% and 87% for the NK cell counts at Lф-2 and 75% and 83% for the counts at Lф-3, respectively. The relative risks of ITBL after ABO-I ALDLT according to NK cell count at Lф-2 and Lф-3 are given in Table 5.

Following the recent advancement in techniques for transplant surgery, the use of ABO-incompatible adult living donor liver transplantation (ABO-I ADLDT) has increased, with improved clinical outcomes. Antibody-mediated rejection (AMR), which is a major cause of the graft’s death after ABO-I ALDLT, seems to be overcome by B-cell depleting protocol using preoperative rituximab prophylaxis and plasma exchange with or without splenectomy. However, ischemic-type biliary lesion (ITBL) remains a problematic issue after ABO-I ALDLT.

There were previously no clearly identified risk factors or immunological mechanisms for the development of ITBL. This study was conducted to determine risk factors for the development of ITBL after ABO-I ALDLT. The authors extensively analyzed the clinico-pathological data from recipients with ABO-I ALDLT, including patient demographics, intraoperative outcomes, postoperative complications, and general serum and blood analysis in the perioperative period. Furthermore, perioperative serum agglutinin titer, blood lymphocyte subset count (B-cell, T-cell, and NK cell count), immunohistochemical staining for B-cells and plasma cells in the spleen and for NK cells in a liver biopsy were specially collected and analyzed for this study.

From our clinico-pathological analyses, the authors found that a perioperative high NK cell level in the blood was associated with the development of ITBL after ABO-I ALDLT. This novel finding could explain the clinical characteristics of ITBL after ABO-I ALDLT by microcirculatory disturbance following NK cell triggering antibody dependent cytotoxicity (ADCC).

The results of this study needs to be expanded through molecular research into NK cell activity in the development of ITBL via further clinical and experimental studies. Anti-NK cell treatment could be used as a preventive measure for the development of ITBL.

ABO-I ALDLT is a method of adult liver transplantation across the blood barrier of ABO-incompatibility using a partial liver graft from a living donor. AMR is a type of graft rejection triggered by an immunological reaction of donor-specific antibodies against graft antigens. ITBL is a radiological diagnosis, characterized by intrahepatic stricture and dilatation of the bile duct on a cholangiogram, in the absence of hepatic artery thrombosis. ADCC is the killing of an antibody coated target cell by a cytotoxic effector cell through a nonphagocytic process.

The authors have presented an interesting study of 27 patients who have undergone ABO incompatible liver transplantation. They present their own immunological protocol and the outcome of the 27 patients concentrating particularly on the incidence of ischaemic biliary injury. These data are very interesting and demonstrate that with careful attention to detail it is possible to achieve a satisfactory outcome in this group of patients.