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In ABO blood type-incompatible liver transplantation, acute humoral rejection triggered by antibodies against donor-type isoagglutinins is the most serious form of rejection and is often associated with graft loss. With the introduction of effective antihumoral rejection therapy such as the arterial/portal infusion of prostaglandin E1 and use of anti-CD20 monoclonal antibody, as well as preoperative plasma exchange, ABO-incompatible liver transplantation is becoming a choice to overcome the paucity of liver allografts from deceased donors in Japan.1–4 Acute humoral rejection, however, is still a major problem in patients with ABO blood type- incompatible grafts and the evaluation of humoral rejection is necessary.
We previously reported that periportal edema and necrosis (PEN) could be histological indications of the early phase of severe humoral rejection.5 In that report, all grafts with PEN resulted in massive parenchymal or biliary necrosis. Recent papers from other institutions, however, reported that histologically proven humoral rejection could be reversible.3, 6 Biopsies also demonstrated portal edema or hemorrhage, but no significant necrosis or endothelialitis has been documented. Portal hemorrhagic edema without significant necroinflammation may represent a milder degree of humoral rejection.6
To obtain the full histological spectrum of humoral rejection of ABO blood type-incompatible liver transplantation, we performed clinicopathological analysis with complement C4d immunostaining. C4d is a molecule that covalently binds to tissues after activation of the complement system, and immunostaining has been widely used to demonstrate humoral immunoreactivity or antibody-mediated rejection in other solid organ transplantations.7–10 The aim of this study was to clarify the clinicopathological features of acute humoral rejection caused by antidonor ABO blood group antigens using C4d immunostaining.
Between January 1999 and December 2004, 82 patients (35 children and 47 adults) received primary ABO blood type-incompatible living donor liver transplantation. Informed consent for ABO blood type-incompatible liver transplantation was obtained from the recipient and donor, and the Ethical Committee of the Medical School of Kyoto University approved the surgery and treatment procedures. Among them, 35 patients (13 children and 21 adults) underwent liver biopsy within the first 3 postoperative weeks. One adult patient was excluded from this study because the paraffin-embedded tissue was not available. The other 34 patients were enrolled in this study. As of April 2005, the clinical follow-up period for these patients ranged from 0.4 to 74 months (median, 16 months).
Antirejection Regimen and Clinical Monitoring
Baseline antihumoral rejection therapies included intravenous steroids and oral tacrolimus. Most adult patients underwent continuous prostaglandin E1 infusion via an intra-arterial or intravenous catheter for 2 to 3 weeks posttransplantation.1, 11
Splenectomy was performed for adult patients whose transplantation was performed between May 1999 and January 2003. To avoid splenectomy-related portal vein thrombosis, the administration of preoperative rituximab (anti-CD 20 monoclonal antibody) without splenectomy was introduced after April 2004.11 In addition, 2 adult patients were given postoperative rituximab as a therapy for humoral rejection.
Patients underwent preoperative plasmapheresis or blood exchange in order to reduce antidonor blood group A/B antibody (immunoglobulin M (IgM) and IgG) titers to ×8 or lower. A microhemagglutination assay was used to monitor the serum levels of antidonor ABO blood group antibodies at least 3 times per week during the first postoperative month. Patients were assigned to high-titer groups if the peak of the postoperative antidonor blood group IgM titers was ×64 or more, and to the low-titer group if it was less.5 In this study, there were 21 patients with a high titer and 13 with a low titer. Postoperative plasmapheresis was performed when severe humoral rejection was suggested clinically or histologically. Elevation of postoperative antidonor antibody titer not associated with liver dysfunction was treated by steroid bolus therapy or was managed with watchful observation alone.
Hepatic necrosis was diagnosed by enhanced computed tomography. The findings were diffusely spreading low-density lesions in the graft. Intrahepatic biliary complications were diagnosed by cholangiography, showing irregularity and beading throughout the intrahepatic biliary tree.
Pretransplant T-cell cross-match tests were performed as previously described.12 The test was interpreted as positive when 41% or more of donor lymphocytes were killed, weakly positive when 21 to 40% of donor lymphocytes were killed, and negative when no more than 20% of donor lymphocytes were killed. Cross-match tests were negative in 33 of 34 patients. One patient was interpreted as weakly positive.
Pathological diagnosis was made on a routine basis by 3 pathologists (H.H., T.S., and A.M.), with no information about immunostaining of immunoglobulins or complements, including C4d. The minimal quantitative requirement for the diagnosis of rejection was a biopsy containing at least 5 portal areas. Acute cellular rejection was diagnosed using Banff criteria.13 Histological acute humoral rejection was suspected when PEN or portal hemorrhagic edema was associated with the elevation of antidonor A/B antibody titers.5, 6 In each case of acute cellular rejection and acute humoral rejection, portal inflammation, bile duct damage, and venular endothelialitis were evaluated separately with the rejection activity index (RAI) of Banff criteria.13 To apply the RAI to humoral rejection, we regarded any leukocyte infiltration as portal inflammation and gave a score of 3 on periportal coagulative necrosis as part of portal inflammation.
C4d Immunostaining and Control Materials
Eighteen-gauge liver core tissue biopsies were placed in 10% buffered formalin from several hours to overnight, processed routinely, and sliced into 3-μm paraffin sections. Staining methods for routine histological evaluation included hematoxylin and eosin, Masson trichrome, and immunostaining of cytokeratin 7 (OV-TL 12/30, Dako, Glostrup, Denmark; dilution, 1:200). The polyclonal antibody against C4d complement (BI-RC4D, Biomedica, Vienna, Austria; 1:50) was used for immunostaining with an automated immunostainer (BENCHMARK XT, Ventana, Tucson, AZ). For antigen retrieval, deparaffinized and rehydrated sections were treated with protease I (Ventana; 0.5 units/mL) at 37°C for 20 minutes.
We used lymphoid tissue with follicular hyperplasia as a positive control for C4d staining.14 The reticular staining pattern in the germinal centers was confirmed in every C4d immunostaining. Ten wedge biopsies of the liver allografts taken during graft resection (time 0 biopsy) were used as negative controls. For comparison of ABO blood type-incompatible and non-ABO blood type-incompatible cases, 10 needle biopsies with typical acute cellular rejection from ABO-identical transplants were assessed for C4d immunostaining.
Evaluation of C4d Immunostaining
Three pathologists (H.H., T.S., and A.M.) independently evaluated the C4d immunostaining slides of the initial biopsies. The identification numbers were removed from the slides and the pathologists were not given any clinical information. C4d staining was semiquantitatively evaluated in terms of the percentage of portal tracts containing distinctly stained stroma and/or endothelium. Biopsies containing 50% or more stromal-positive portal tracts were evaluated as positive. Specimens with less than 50% stromal-positive portal tracts or positive staining only in the vascular endothelium or sinusoids were classified as indeterminate. Completely negative staining was evaluated as negative. Extraportal endothelial staining was recorded but not graded. Any staining in the liver capsule or extraliver tissue was omitted to evaluate.
A couple of discordant cases were classified as indeterminate, and we did not perform additional C4d immunostaining because of the small size of the specimens. Immunostaining of the follow-up biopsies of C4d-positive cases (n = 21) and the second biopsies of C4d-negative cases (n = 17) was evaluated with clinical data and previous biopsies.
Statistical significance of differences among the groups was assessed by Fisher's exact test. Laboratory data were analyzed using Mann-Whitney's U test. Patient survival was determined by Kaplan-Meier analysis, and differences in survival were analyzed with a log-rank test. For all analyses, P values of less than 0.05 were taken as significant.
C4d Immunostaining and Clinical Outcomes
Five of 10 time 0 allograft biopsies obtained at liver transplantation showed focal endothelial or stromal C4d staining. These positive stainings did not exceed 10% of the number of the portal tracts and were classified as indeterminate. The other 5 time 0 biopsies were negative for C4d. In ABO blood type-identical specimens with a diagnosis of acute rejection, 1 specimen showed strong and diffuse portal stromal staining (positive), 6 showed focal or diffuse endothelial staining (indeterminate), and 3 negative. Distinct C4d staining was seen in the perivenular areas in the C4d-positive case.
Among 34 initial biopsies from ABO blood type-incompatible transplants, 17 cases (50%) were positive for C4d in the portal stroma, 8 (24%) were indeterminate, and 9 (26%) were negative. Representative staining patterns are shown in Figure 1. Preoperative clinical features were not significantly different between the groups (Table 1). However, high-postoperative antidonor antibody titer (×64 or more) was more frequently seen in C4d-positive patients than in C4d-indeterminate and -negative ones (Table 1). When the C4d-indeterminate group was regarded as negative for C4d, C4d positivity was significantly associated with high titers (88 vs. 35%; P = 0.002). Hepatic necrosis demonstrated by computed tomography was seen only in the C4d-positive patients (24 vs. 0%; P = 0.052). Patients who underwent postoperative plasma exchange were statistically more frequently seen in C4d-positive patients than in C4d-indeterminate and -negative patients (53 vs. 6%; P = 0.003). The timing of postoperative plasma exchange was clinical humoral rejection, which occurred within 14 postoperative days. One to 6 (median, 3.5) courses of plasma exchange were performed, and 4 of 10 (40%) grafts recovered.
Table 1. C4d Immunostaining Pattern and Clinical Features
Overall, patient survival was significantly worse for C4d-positive than for C4d-indeterminate and -negative patients (41 vs. 88%; P = 0.007; Fig. 2A). The high-titer group (n = 21) also showed poorer overall survival than the low-titer group (n = 13), which was not significant in this study (56 vs. 83%; P = 0.072; Fig. 2B).
C4d Immunostaining and Histology
Findings of initial biopsies are summarized in Table 2. In C4d-positive patients, the most common histology at initial biopsy was PEN/portal hemorrhagic edema. Ten of 11 cases (91%) with PEN/portal hemorrhagic edema showed C4d stromal deposition. The median postoperative day of C4d-positive PEN cases was day 7 (Table 3).
Table 2. C4d Immunostaining and Initial Biopsy Within the First 3 Postoperative Weeks
The other histology in C4d-positive cases included purulent cholangitis (n = 4; median posttransplant day 18), coagulative hepatocyte necrosis (n = 1; posttransplant day 17; clinically hepatic artery thrombosis), moderate acute cellular rejection (n = 1; posttransplant day 15), and hepatocanalicular cholestasis (n = 1; posttransplant day 13).
In C4d-indeterminate and -negative patients, the most common histology was hepatocanalicular cholestasis (n = 5; median posttransplant day 13), followed by purulent cholangitis (n = 4; median posttransplant day 18), mild lobular inflammation (n = 4; median posttransplant day 16), mild to moderate acute cellular rejection (n = 3; median posttransplant day 12), and PEN (n = 1; posttransplant day 10; RAI = 6).
PEN/portal hemorrhagic edema was statistically more frequently observed in C4d-positive patients than in non-C4d-positive patients (59 vs. 6%; P = 0.0012). Levels of serum transaminases tended to be higher in C4d-positive patients but did not reach statistical significance. Total bilirubin level was significantly higher in C4d-positive patients (P = 0.009).
PEN/Portal Hemorrhagic Edema Cases
Among 11 PEN/portal hemorrhagic edema cases, the histology of 5 patients (cases 3, 5, 12, 15, and 1 C4d-negative case) was PEN (Table 3). PEN included portal hemorrhage, mild to moderate portal neutrophilic infiltration, perivenular endothelialitis without significant perivenular necrosis, and focal periportal necrosis (Fig. 3A). The portal stroma adjacent to the parenchyma and peribiliary stroma were positive for C4d (Fig. 3B). The terminal hepatic venules showed occasional faint positivity, but the sinusoids in zones 2 and 3 were negative for C4d. Cholangitis with mild to moderate neutrophilic infiltration was seen in all cases, but ductopenia or duct atrophy was not observed with CK7 immunostaining. The RAI score of PEN cases was 6 or 7, and all patients with PEN resulted in graft failure or severe graft damage.
The other 6 patients showed portal hemorrhagic edema (cases 8, 11, 13, 14, 16, and 17). Cellular infiltration was mild (Fig. 3C), but all revealed a C4d staining pattern identical to that of PEN (Fig. 3D). Some cases had perivenular C4d deposition, but perivenular inflammation was none or minimal. The RAI of portal hemorrhagic edema was 4 or less (Table 3). Only 1 patient resulted in graft failure (case 14; RAI = 2).
Among 17 C4d-positive patients, 5 patients (3 with PEN, 1 with periportal hemorrhagic edema, and 1 with cholangitis) resulted in rapid graft failure and follow-up biopsies were not available (cases 1, 3, 12, 14, and 15; Fig. 4).
In the other 12 C4d-positive patients, at least 1 follow-up biopsy was available at various times. Five patients (cases 2, 4, 10, 13, and 16) were negative for C4d at the last follow-up biopsy (range, 34-792 posttransplant days). The other 7 showed positive C4d immunostaining in the last follow-up biopsy (range, 52-508 posttransplant days). Six patients (case 4 with acute cellular rejection and cases 8, 11, 13, 16, and 17 with portal hemorrhagic edema) were clinically free of symptoms and achieved normalization of the levels of serum transaminases and bilirubin as of April 2005. Among them, 3 (cases 8, 11, and 17) were positive for C4d at the last biopsy (posttransplant days 77, 336, and 209, respectively).
Any second biopsy from patients whose initial biopsies were indeterminate or negative for C4d did not reveal C4d positivity in the portal stroma (median posttransplant day 31; range, 19 - 603).
Cases with Stromal C4d Immunostaining without Elevation of the Antidonor Antibody (IgM) Titers
Cases 6 and 9 showed C4d stromal positivity, but their postoperative titer of antidonor IgM antibody remained low. In case 6, the peak antidonor IgM titer was ×16, but the antidonor IgG titer was elevated up to ×256 and lasted for 4 days. Every biopsy of case 6 showed purulent cholangitis, and subsequent computed tomography revealed hepatic necrosis. The patient died of chronic cholangitis.
Case 9 was a patient with multivisceral transplantation from 2 different donors; the liver transplantation was ABO blood type incompatible (AB to B), while the small intestine graft was ABO blood type compatible (blood type O). The postoperative peak antidonor IgM titer was low (×8) and the antidonor IgG was not detected. However, preoperative cross-match tests were interpreted as weakly positive against both donors. Liver biopsies revealed purulent cholangitis with ductular cholestasis, which was attributed to sepsis (Fig. 5A). C4d staining was positive (Fig. 5B). The ABO blood type-compatible small intestine graft showed severe acute rejection with microthrombi, and C4d deposits were observed in the endothelium (Fig. 5C and D). The patient died on posttransplant day 58. Necropsy revealed severe mucosal hemorrhage in the small intestine and monoclonal posttransplant lymphoproliferative disorder. Preoperative blood exchange and induction therapy with basiliximab were used for this patient, but no postoperative cross-match test was performed.
In ABO blood type-incompatible liver transplantation, preoperative plasma exchange was performed to lower the titers of antidonor blood group A/B antigen antibodies. The onset of acute humoral rejection is thus suspected when liver dysfunction occurs along with the elevation of titers of those antibodies. Liver biopsies soon after titer elevation usually demonstrated characteristic features such as portal edema, hemorrhage, and periportal necrosis. In some cases, however, elevation of the antidonor antibody titers was not associated with graft dysfunction, or late graft dysfunction occurred even when patients did not show significant titer elevation. In such cases, histological demonstration of deposition of immunoglobulin or complements in the allografts is important to demonstrate humoral reactivity and to decide patients' treatment.12, 15, 16 Immunostaining of IgM or other complements such as C3c, however, was technically difficult in paraffin-embedded formalin-fixed liver tissue, and we were unable to make reproducible results.4, 5 Moreover, severe tissue damage other than humoral rejection can cause immunoglobulin and complement deposition.12, 17 Interpretation of the staining results can be difficult if you do not routinely perform immunofluorescent study of the allograft liver. On the other hand, C4d immunoperoxidase staining was relatively easy to perform, and the results were reliable since positive and negative controls were readily available.
Still, there have been few data about the significance of antibody-mediated rejection in clinical liver transplantation, and the criteria of histological diagnosis and therapeutic options remain unclear. Recently, a couple of studies demonstrated C4d deposition in liver allografts with acute rejection using paraffin-embedded sections, although the presence of alloreactive antibodies was not clearly stated.18, 19 Instead, they suggested that the local B-cell response might be specifically involved in C4d deposition in human liver allograft rejection. In their reports, C4d deposited in the pericapillary or periportal areas of the portal tracts, and the staining patterns were nearly identical to those of our cases. These staining patterns denoted that periportal areas may be the main targets of both humoral and acute cellular rejection, and that lobular deposition of immunoglobulin and complement may be a secondary change. In ABO blood type-incompatible transplantation, the findings may be compatible with the observation that capillaries in the portal tracts are the primary sites of ABH blood type antigen expression in the liver allografts.20 The staining pattern may also support that periportal necrosis seen in experimental and clinical rejection can be a diagnostic histological feature of humoral rejection.5, 21
On the other hand, the significance of C4d staining only in the endothelium was not clear in this study. Since the endothelial staining pattern was reproducible, and was common in acute cellular rejection of ABO blood type-identical cases, we speculated that it represented mild humoral reaction of the grafts. Neither patient survival nor severe liver damage, however, was associated with the endothelial staining pattern.
We were able to demonstrate in this study that C4d deposition in the portal stroma was correlated with poor patient survival and characteristic histology, as well as with postoperative titers of ABO blood type alloantibodies. Conversely, PEN/portal hemorrhagic edema showed a high percentage of C4d positivity and was probed as a useful histological feature in establishing the diagnosis of acute humoral rejection. The histology of PEN/portal hemorrhagic edema was transient, and there was a spontaneous decrease of ABO blood type alloantibody titers in the first postoperative month, but some follow-up biopsies more than a year after transplantation remained positive for C4d. This suggests that nonspecific histology such as cholangitis or coagulative necrosis can be regarded as a result of humoral rejection when C4d was detected in the allografts. For example, case 7, an 11-month-old boy, was the youngest patient with humoral rejection in this study, while hepatic artery thrombosis was a clinical cause of hepatocyte necrosis.
C4d staining was also correlated with radiological findings. It is known that there are 2 major complications associated with ABO blood type-incompatible liver transplantation.4, 15 One is massive hepatocyte necrosis occurring within the first 2 weeks, which is refractory to postoperative therapy and fatal. The other is intrahepatic biliary complication that is not often associated with early liver graft dysfunction, but becomes evident several months after liver transplantation. In this study, hepatocyte necrosis found by computed tomography was associated with only C4d positivity and 3 of 4 patients revealed PEN, while no hepatocyte necrosis was found in C4d-indeterminate and -negative patients. This suggests that hepatocyte necrosis is a result of severe humoral rejection. Intrahepatic biliary complications, on the other hand, occurred in both C4d-positive and -negative patients. Since the focus of this study was concentrated on the patients showing early graft dysfunction and receiving biopsy within the first 3 postoperative weeks, we think that a study based on protocol biopsy is necessary to elucidate late-onset biliary complications associated with ABO blood type-incompatible transplantation.
Once humoral rejection was defined by both morphology and C4d deposition, the RAI of the Banff schema with minor modification reliably predicted the outcome of humoral rejection. PEN corresponded to acute humoral rejection with a RAI of 6 or 7 and was associated with graft failure. On the other hand, portal hemorrhagic edema had minimal inflammatory cell infiltrate and an RAI score of 4 or less, and was related to better prognosis and complete recovery in some cases. The results suggested that the Banff schema could be useful for the evaluation of both acute cellular and acute humoral rejection. It must be kept in mind, however, that the prognosis of acute humoral rejection was much poorer than acute cellular rejection, and that even acute humoral rejection with low RAI required treatment, including repeated plasma exchange, to rescue the patients.
C4d stromal deposition in the liver allograft was demonstrated without elevation of the ABO blood type alloantibody titer in case 9. Since this patient showed a weakly positive T-cell cytotoxic cross-match and severe rejection in the ABO blood type-compatible small intestine graft, this humoral response might be mediated by lymphocytotoxic antibodies.22, 23
We conclude from this study that the immunohistochemical detection of C4d has both diagnostic and prognostic value and could be a hallmark of antibody-mediated rejection in liver biopsy. In combination with conventional histological criteria, the demonstration of C4d in the portal stroma may be useful to determine the indication of postoperative plasma exchange and to assess the current and future protocols for ABO blood type-incompatible liver transplantation. Further studies are needed to clarify the significance of C4d and other markers of humoral immunity in ABO blood type-matched liver transplantation.
We thank Dr. Daisuke Morioka (Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine) for his useful advice about the histology and treatment of the patients.