Application of C4d Immunohistochemistry in ABO-Compatible and ABO-Incompatible Liver Transplantations

271 words) Antibody-mediated rejection (AMR) is difficult to diagnose after ABO-compatible or ABO-identical (ABO-C) liver transplantation. To confirm whether C4d immunostaining is useful for diagnosing AMR, we compared the results of C4d immunohistochemistry in allograft biopsies with assays for anti-donor antibodies performed at the time of biopsies. A total of 114 patients with ABO-C grafts and 29 patients with ABO-incompatible (ABO-I) grafts were included. Linear C4d endothelial staining identifiable by x4 objective lens or staining seen in >50% of portal tracts was considered positive. Five of 114 (4%) patients with ABO-C and 15 of 29 (52%) patients with ABO-I showed C4d positivity. In ABO-C cases, C4d positivity was associated with ≥stage 2 fibrosis (METAVIR score) and presence of donor-specific anti-HLA DR antibodies (HLA-DR DSA) with more than 5000 mean fluorescence intensity (MFI) by the Luminex single antigen bead assay in late (≥30 days posttransplantation) biopsies (p=0.01 and 0.04, respectively). Conversely, presence of HLA-DR DSA was associated with presence of ≥stage 2 fibrosis, acute cellular rejection, and C4d positivity. During two-year follow-up, neither C4d positivity nor HLA-DR DSA was related to graft loss. In ABO-I, C4d positivity was not associated with allograft dysfunction or fibrosis. Only three of 15 (20%) C4d-positive patients showed periportal hemorrhagic edema, which could be a histological sign of AMR in ABO-I, and were the only cases associated with elevations in anti-donor A/B antibody titers. In conclusion, C4d endothelial positivity in ABO-C was an uncommon event that could be associated with chronic graft damage with or without clinical AMR. C4d positivity is common in ABO-I grafts, and may not be associated with allograft dysfunction if alloantibody titers are not elevated. Salah. C4d immunohistochemistry in liver allografts 4 Antibody-mediated rejection (AMR) in liver allografts is recognized as a possible cause of early and late allograft injury, and poor prognosis (1-8). However, unlike acute cellular or chronic rejection, the diagnosis of AMR in liver allografts is often difficult to establish. One of the main reasons for this is due to the difficulty in interpreting C4d deposition, which is the most widely used marker of clinical AMR in renal, cardiac, and pancreatic transplantations (8-11). The specificity of C4d staining in liver allografts is controversial. Ali S et al. (3) and Lunz J et al. (4) correlated diffuse portal tract vascular endothelial C4d deposition with AMR. However, C4d positivity was also reported in other medical conditions, such as acute cellular rejection (ACR) (1,3-5,12,13), chronic rejection (CR) (3,5,12,13), ischemic injury (1,3,12), hepatitis (1,3,4,14) and cholangitis (1,3,4). Unfortunately, most of these previous studies performed C4d staining on non-consecutive biopsies from unstable liver grafts (15). A more comprehensive study is required to understand the significance of C4d and its utility in AMR, in combination with tests for alloantibodies. In addition, sites of C4d deposition differ between and within these reports, including portal vessels (1-6,12,14), portal stroma (1,2,5), and sinusoids (3,4,5,12). The lack of agreement in staining patterns may also be related to the low specificity of C4d for AMR and may prevent clinicians and pathologists from using C4d in the routine histological diagnosis of liver allografts. Kozlowski et al. (7) recently suggested that strong linear staining in the sinusoid, rather than the portal tract, was a better marker for AMR and recommended the use of immunofluorescence on frozen sections. As they pointed out, immunoperoxidase staining is insensitive and frozen sections may be a better tool to demonstrate C4d deposition. However, frozen sections are not suitable for conventional histological evaluations, and formalin-fixed paraffin embedded tissue is additionally required. Considering the rarity Salah. C4d immunohistochemistry in liver allografts 5 of clinical AMR in liver transplantation (LT), we suggest that establishing a method to evaluate C4d by immunoperoxidase alone may be practical. Here, we designed a non-selective prospective study in which we performed C4d staining on all liver allograft biopsies obtained over four consecutive months, and every clinically indicated biopsy was included in this study. The presence of anti-blood group (Anti-A/B) antibodies or anti-human leukocyte antigen (anti-HLA) antibodies was evaluated during the same period. All patients were followed up for 2 years to clarify the significance of C4d in liver allografts. We adopted endothelial staining for this study although we previously reported the stromal deposition of C4d as an ominous sign of ABO-I LT (2). The main reason to exclude stromal staining in this study was that only endothelial staining has been used as the standard in other solid organ transplantations (16). The second reason is that stromal staining alone is often difficult to differentiate from the non-specific staining seen in elastic fibers or necrotic tissue (1, 17). When we picked up every portal stromal or endothelial staining, C4d staining was seen in various types of liver allograft injuries and did not show clinical significance (1). Since extensive C4d staining covers the endothelia of portal, sinusoidal, and perivenular areas (1, 2), we now assume that endothelial staining alone is adequate for evaluating C4d. Materials and methods Study population and biopsies In a prospective and non-selective manner, regardless of indication, we studied all liver allograft biopsies obtained between July and October 2011 at Kyoto University Hospital. Patients who underwent LT outside Kyoto University hospital were not included. Liver allograft biopsies were performed to determine allograft dysfunction or evaluate graft fibrosis when immunosuppression weaning was intended. If a patient underwent multiple biopsies during this period, the first biopsy that showed C4d positivity was Salah. C4d immunohistochemistry in liver allografts 6 selected for analysis. When all biopsies were negative for C4d, the first biopsy was selected. In each case, the biopsy specimen for analysis was classified as early (taken within 30 days after transplantation) or late (taken 30 days or more after transplantation). All patients were followed up until July 2013. Clinical and serological data were obtained from electronic patient charts. The Institutional Review Board of Kyoto University approved this study. Immunosuppression The baseline immunosuppression protocol consisted of tacrolimus and oral prednisolone in both ABO-C and ABO-I patients. The lower limit of the target for whole blood tacrolimus levels was 10 to 15 ng/mL during the first 2 weeks, 10 ng/mL thereafter, and 5 to 8 ng/mL from the second month on. Acute rejection was treated by a 3-day course of intravenous methylprednisolone bolus therapy (10 mg/kg). Mycophenolate mophetil was administered to patients who underwent refractory rejection or plasma cell hepatitis simulating autoimmune hepatitis. Immunosuppression was weaned in selected pediatric patients, according to the previously described protocol (18). All ABO-I patients underwent preoperative plasmapheresis or blood exchange in order to reduce antidonor A/B antibodies to 1:8 or lower. In addition, patients who underwent ABO-I transplantation after 2006 received rituximab (anti-CD 20 monoclonal antibody) approximately two weeks before transplantation (19). Adult patients were given prostaglandin E1 and methylprednisolone via a portal vein or hepatic artery. Clinical AMR, consisted of an elevation in postoperative anti-donor A/B antibody titers and graft dysfunction, was treated for about 5 days by plasmapheresis or intravenous immunoglobulin, with steroid bolus therapy. Salah. C4d immunohistochemistry in liver allografts 7 Histopathology Liver allograft biopsies were processed for routine light microscopy. Biopsy specimens were fixed in 10% buffered formalin, sliced 3 μm thick, and stained with hematoxylin and eosin (H&E), Masson Trichrome, and Cytokeratin 7 (clone OV-TL 12/30, Dako, Glostrup, Denmark; dilution 1:200). ACR and chronic rejection were diagnosed according to Banff criteria (20, 21). AMR was diagnosed according to the criteria used in other solid organ transplantations; i) clinical evidence of graft dysfunction, ii) histologic evidence of graft injury, iii) immunopathologic evidence of antibody action (C4d deposition), and iv) serologic evidence of anti-HLA or anti-donor antibodies at time of the biopsy (22). A combination of periportal edema, hemorrhage, and neutrophilic infiltration was regarded as an indicator of AMR in ABO-I patients (8,23). Allograft fibrosis was staged according to the METAVIR scoring system (24). C4d immunohistochemical staining A rabbit polyclonal anti-human C4d antibody (BIOMEDIA, Bl-RC4D, 1:50 dilution) was used to detect C4d. Staining was performed on an autostainer machine (Ventana Benchmark ULTRA). Sections were treated with protease (Ventana, 0.5 U/mL) at 37 °C for 20 minutes for antigen retrieval. C4d immunostaining using formalin-fixed, paraffin embedded tissue was available in our laboratory since August 2003, but it was applied only to selected cases and was not used routinely before this study. C4d interpretation Staining was recorded as diffuse when linear C4d deposition in the portal tract vascular endothelium was seen in 50% or more of portal tracts. Staining of fewer than 50% of portal tracts was considered focal. We also evaluated the intensity of staining, which was Salah. C4d immunohistochemistry in liver allografts 8 recorded as strong when linear C4d deposition was seen on low power magnification (x4 objective lens), and weak when staining was confirmed only on higher magnification. Completely negative (Score 0) or focally weak (Score 1) staining was considered negative and equivocal, respectively. Diffus