The transplantation of ABO-identical or ABO-nonidentical ABO-compatible grafts has for decades been the mainstay of organ transplantation. As post-transplant survival improves and the demand for organ transplants grows, several attempts have historically been made to transplant liver grafts from ABO-incompatible (ABOi) donors, particularly in emergency situations, both in children and adults.
Historically, the transplantation of the kidney first broke the ABO blood group barrier, and since then, various therapeutic modalities have been introduced. In the 1980s and early 1990s, several trials of ABOi liver transplantation showed that the post-operative course was very poor, mainly because of severe rejection crises, thrombosis of the hepatic artery, and/or intrahepatic bile duct injury. Continuous improvement in liver transplantation and improved immunosuppression methods during the last decades brought back interest in more common use of ABOi grafts. A small number of cases reported by single centers showed good early results of ABOi transplantations. Since then, more ABOi liver transplantations have been performed, particularly in pediatric but also in adult recipients of the grafts from living donors under various protocols aimed at preventing typical complications resulting from blood group incompatibility. Improved immunosuppression has markedly decreased antibody-mediated rejection (AMR) and biliary complications but has increased infectious complications, which are now the major cause of morbidity and mortality after ABOi liver transplantation, but the outcome of ABOi liver transplantation has improved dramatically and now is similar to that of ABO blood type–matched transplantation.
To understand, however, the complex problems of ABOi organ, and particularly liver transplantation, one must take into consideration the basic immunological response for the ABOi organ developing within the liver graft and the differences between adults and children, as well as between infants and older children.
There are several differences between adults and children concerning the response to ABOi graft transplantation. Many reports show better outcomes in ABOi liver transplantation in infants. In early childhood, anti-A and anti-B antibody titers remain low because infants have limited ability to produce isohemagglutinins. Additionally, the activation of their complement system is suppressed. That is why AMR is not observed in children younger than 1 year of age and even up to 2 years.
In the clinical situation, there is a difference between emergency or elective transplantation and between the transplantation from a living donor or a deceased donor, particularly concerning the possibility of pretransplant treatment of the recipient. Therefore there is no single and best treatment protocol to propose as a standard.
Basic Pathophysiology and Pathological Findings
There are several responses triggered by antigen-antibody reactions between donor blood antigens on the graft and antibodies in the recipient’s serum. AMR initiates damage to ABOi liver graft by preformed isoagglutinins later triggered by antibodies produced by proliferating B cells activated by ABO antigens in the donor graft. ABO blood group antigens are expressed on all hepatic endothelial cells (vessels and bile ducts). The expression of human leukocyte antigens (HLA) class I (HLA-A or B) is weaker on hepatocytes, and HLA-DR class II expression is strongest on dendric cells (portal, perivenular, subcapsular) and Kupffer cells. The damage to the endothelial cells is caused by the production of cytokines, chemotactic factors, free radicals, and, later on, platelet and complement activation. It is followed by thrombus formation, migration of granulocytes and macrophages, and phagocytosis.
Complications that may develop with increased risk in patients after ABOi liver graft transplantation are multiple. The most rapid is the development of superacute or early acute AMR with a clinical course of intragraft disseminated intravascular coagulation (DIC), massive intrahepatic microcirculation thrombosis leading to hemorrhagic graft necrosis within a few days or weeks. The same mechanisms may be responsible for an increased incidence of hepatic artery thrombosis among recipients of ABOi liver transplants, which occur in up to 24% of these patients ( Figs. 9.1, 9.2, and 9.3 ).
This phenomenon is age dependent because production of anti-A/B isoagglutinins begins 3 to 4 months after birth and achieves the adult level between 4 and 7 years of age, whereas expression of A/B antigens in young children is lower. It explains, at least partially, the clinical observation that hyperacute or early AMR is almost nonexistent in infants and children younger than 2 years undergoing ABOi liver transplantation. The same was shown in heart transplantation in children.
There are also significant differences between the immunogenicity of grafts from A2 donors (about 20% of the population with blood group A) and from A1 or B donors because expression of A2 antigen is much weaker, and non-A group recipients possess a much lower titer of anti-A2 isoagglutinins. Therefore, differentiation of A donors to subgroups may be important in the elective living donor ABOi transplantations and the choice of perioperative immunosuppression.
Kishida et al. described the increased risk of development of thrombotic microangiopathy (TMA) in ABOi liver transplantation (LT) recipients. TMA is caused by the destruction of the microvascular endothelium and primary aggregation of platelets. It is demonstrated by thrombocytopenia and microangiopathic hemolytic anemia. Endothelial injury results in the release of large amounts of von Willebrand factor, which enhances adhesion of platelet formation of microthrombi.
The second common complication of ABOi liver transplantation is diffuse damage to the biliary tree, resulting in the development of multiple intrahepatic biliary stenoses. This complication usually develops within the first 3 months after transplantation and is also caused by immunological mechanisms related to humoral reactions because the donor blood group antigens are present on the epithelium of the graft bile ducts for about 3 to 6 months after transplantation ( Fig. 9.4 ). Some authors reported that high perioperative titers of specific anti-A/B antibodies are connected with acute hepatic necrosis (immunoglobulin G [IgG] isoagglutinins) and biliary complications (IgM isoagglutinins), as well as both immunoglobulins’ high post-operative titers.
All of the abovementioned complications usually lead to graft loss and the need for retransplantation or the patient’s death. In children, the incidence is very much age dependent; the lowest risk is in infants under 1 year of age, and the larger is in children older than 8 years of age.
The main characteristic pathological finding includes the deposition of antibody to sinusoidal and arteriolar endotheliums and hemorrhagic necrosis of the liver parenchyma. Periportal edema and necrosis seem to be the histological indication of an early phase of severe humoral rejection, causing massive parenchymal or biliary necrosis. The resulting vascular thrombosis causes graft ischemia, whereas bile duct strictures result in severe cholestasis. C4d staining facilitates an AMR diagnosis and should always be carried out when acute or chronic AMR is suspected. The Banff Working Group recommends C4d staining of frozen tissue immunofluorescence as well as of formalin-fixed, paraffin-embedded tissue using rabbit polyclonal or monoclonal antigen in several compartments (portal veins, portal capillaries, portal stroma, sinusoidal, and central vein endothelium). The result may be negative, minimal (< 10%), focal (10%–50%), and diffuse if more than 50% of structures are involved. Although C4d-positive staining is not a pathognomonic feature of AMR, detection of C4d has both a diagnostic and prognostic value and may be a hallmark of AMR in liver biopsies ( Fig. 9.5 ).
Strategies to Overcome ABO Incompatibility in Liver Transplantation
According to Warner’s statement, there are three preconditions for long-term survival of ABO-incompatible solid organ allografts:
low expression of antigen on the graft (as in the case of A2-positive organs, or organs from deceased young pediatric donors)
low titer of antidonor A/B antibodies in the recipient before transplantation
ability to maintain low titers of antidonor A/B antibodies in the recipient for at least 3 to 6 weeks after transplantation until a state of accommodation develops.
All these preconditions are necessary to prevent the development of early AMR in recipients of ABOi liver grafts. The safe recipient’s titers of anti-A/B isoagglutinins are considered to be 1:16 or less in the majority of publications, whereas titers 1:64 or above should be considered as a contraindication for ABOi transplantation. Because the anti-A/B antibody titer may increase early after transplantation, it should be monitored and adequately treated for the critical period of at least 6 weeks after transplantation. Several measures have been proposed and introduced historically to fulfill Warner’s conditions, and then various strategies combining these measures were proposed depending on the urgency of the transplant and type of donor. In an elective transplantation from a living related donor, treatment of the recipient may be started several days or even weeks before the transplant takes place, whereas in emergency transplantation or even elective transplantation with a graft from a deceased donor, pre-transplant treatment is limited to a few hours at most.
The measures used in the prevention of AMR of ABOi graft can be categorized into the following according to their effects:
Procedures aiming at B-cell depletion
Procedures resulting in inhibition of antibody production
intravenous immunoglobulins (IVIG)
Procedures eliminating anti-A/B antibodies
nonselective or semiselective plasmapheresis
selective apheresis: immunoadsorption (IA)
Graft local treatment to prevent single-organ DIC
portal vein infusion therapy
hepatic artery infusion therapy
Procedures Aimed at B-Cell Depletion
Procedures of B-cell depletion should be introduced early enough before transplantation to effectively suppress antibody production and prevent the development of AMR, which, once started, may be very difficult to reverse, leading to graft loss.
There is no general agreement on the role of splenectomy in ABOi transplantation. Logically, the removal of the spleen as a reservoir of the lymphoid tissue, particularly B cells and plasma cells producing large amounts of antibodies, should be very effective in depleting B cells and significantly lowering titers of anti-A/B antibodies in both classes IgG and IgM, as well as its rebound after transplantation. Clinical observations, however, do not confirm these expectations; no real immunological advantage has been observed by several authors with respect to anti-A/B isoagglutinin titers, the incidence of AMR, or late complications of ABOi transplantation in the biliary tract. Some reports, surprisingly, show even a higher rate of AMR in splenectomized recipients; other reports show, however, improved results after combining splenectomy with other regimens (local infusion therapy, rituximab).
In adult transplantation, particularly from living donors, splenectomy may be indicated to diminish portal flow and prevent overperfusion in the “small-for-size” graft (graft-to-recipient mass ratio < 1%). In children, this situation is rather uncommon; more often the graft is “large for size” (graft-to-recipient mass ratio > 3%) with the relative portal hypoperfusion. In both situations, splenectomy induces a high risk for portal vein thrombosis, particularly in children with biliary atresia with anyway hypoplastic and fibrotic portal veins.
In view of such observations, suggesting no or only minimal clinical efficacy of splenectomy in improving results of ABOi liver transplantation and relative contraindications because of the risk of portal vein thrombosis after transplantation, splenectomy does not seem to be a procedure to be recommended in pediatric ABOi liver graft recipients.
Rituximab is a monoclonal anti-CD20 antibody showing cytotoxic activity against both pre-B cells and mature B cells. This activity is complement dependent and antibody mediated and results in B-cell depletion, which cannot transform further into the antibody-producing plasma cells when the antigen is presented. Rituximab does not act against plasma cells directly because they do not express the CD20 receptor. Rituximab does affect mostly B cells present in the peripheral blood but does not suppress B cells present in the lymph nodes. After ABOi liver transplantation, they are activated by graft antigens, and some production of anti-A/B isoagglutinins is present for some weeks after transplantation, but usually not enough to induce severe AMR. Rituximab is most effective in living donor transplantations when given about 7 to 14 days before surgery to achieve the highest immunological effects.
The clinical efficacy of rituximab in preventing ABOi graft rejection was demonstrated initially by Japanese authors in the early 2000s in both ABOi kidney and liver transplantations from the living donors. Since then, rituximab has been widely used, with a high success rate in preventing early severe hemorrhagic AMR as well as in the treatment of AMR.
This anti-CD25 (anti-interleukin-2 receptor monoclonal antibody) IL2 receptor (present on activated T cells) monoclonal antibody is used commonly in sequential post–liver transplant immunosuppression to prevent cellular rejection by inhibiting T-cell proliferation. It has also been used successfully in some cases of severe steroid-resistant and anti-thymocyte globulin (ATG)-resistant cell-mediated rejection as a rescue therapy. The possible role of basiliximab in ABOi transplantation is based on inhibition of B-cell activation and, in this way, blocking the production of antibodies. Basiliximab has been used together with rituximab or as a single antibody in ABOi liver transplant recipients, but its efficacy is not proven in any prospective study and remains controversial.
Procedures Resulting in the Inhibition of Antibody Production: Intravenous Immunoglobulins
High-dose IVIGs have been used with significant success in a number of autoimmunologic diseases, as well as in the desensitization protocols for highly immunized against HLA patients awaiting kidney transplantation. It is also well recognized as supportive therapy for developed AMR, used in combination with steroid pulses. To achieve the expected effects of neutralization and inhibition of antibody responses, high doses of IVIGs are necessary, about 2 g/kg of body mass divided usually into two doses, given every other day. To eliminate positive donor-specific crossmatches and permit transplantation of broadly sensitized patients, they suggest a means to successfully treat AMR. The most important mechanism of action of IVIG in transplant recipients is that of IgG binding via their fragment crystallizable (Fc) region to Fc receptors on various cells including macrophages, natural killer cells, and B cells. Thus IgG interacts with signaling on antigen-presenting cells and their response, including antibody production, complement activation, and production of various inflammatory mediators. IVIG administration is connected with the passive transmission of some amounts of anti-A/B isoagglutinins; therefore, it is not recommended to administer them before ABOi transplantation, as this may increase transiently the anti-A/B immunoglobulins titer. Therefore, IVIG should be given after transplantation to these patients.
Procedures Eliminating Anti-A/B Antibodies
Plasmapheresis, or therapeutic plasma exchange (TPE), is widely used in various indications as well as in ABOi transplantations. It eliminates, in a nonselective manner, plasma immunoglobulins, including anti-A/B antibodies. Standard procedure clears about 60% to 70% of original plasma ingredients, but several procedures may be necessary to achieve safe titers of anti-A/B immunoglobulins (≤ 1:16) for effective prevention of AMR in ABOi liver transplant recipients. Some centers perform double-volume TPE, which eliminates up to 90% of the antibodies, thus allowing a much quicker drop of anti-A/B immunoglobulins titer to the prophylactic value. TPE may be used several days before living donor transplantation electively or immediately before and after transplantation performed urgently or from a deceased donor. Daily or every-other-day monitoring of anti-A/B titers allows keeping anti-A/B antibodies titers within therapeutic levels with repeated plasmaphereses during the initial 3 to 6 weeks after transplantation, when the titer usually naturally drops. In Japan, some centers perform so-called double-filtration plasmapheresis in ABOi liver transplantation from living donors, which extremely effectively eliminates both IgM and IgG, but the procedure is hardly used outside of Japan.
Other procedures have been developed for more specific antibody removal. The procedure of IA is similar to conventional dialysis, during which separated plasma is perfused (up to 3–6 plasma volumes per procedure) via special columns with specific affinity to anti-A or anti-B isoagglutinins (Glycosorb ABO; Glycorex Transplantation, Lund, Sweden). With IA, a large number of circulating anti-A/B immunoglobulins (both IgG and IgM) are depleted without measurable loss of other essential antibodies. Usually one to three sessions are necessary to achieve anti-A/B titers of 1:16 or less.
Less selective procedures dedicated to the removal of mainly IgG independently of their specificity are also available (Immunosorba, Globaffin; Fresenius Medical Care, Bad Homburg, Germany; TheraSorb; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
Graft Local Infusion Therapy
Portal Vein Infusion Therapy or Hepatic Artery Infusion Therapy, Local Graft Infusion Therapy
The idea of local graft infusion therapy via the portal vein or hepatic artery was based on the administration, directly to the liver, of agents preventing “single-organ DIC” resulting from a sharp immunoinflammatory reaction, which develops after transplantation of an ABOi organ. Several strong agents are used for portal vein infusion therapy (PVIT) or hepatic artery infusion therapy (HAIT): methylprednisolone as an immunosuppressant and antiinflammatory agent; prostaglandin E1 to prevent platelet aggregation and also to improve intrahepatic microcirculation; and protease inhibitor gabexate mesylate, which is a strong inhibitor of platelet aggregation, thrombin, and other coagulation factor inhibitors. Some procedures have been introduced and are mostly used in living donor liver transplantation (LDLT) in adults in Japan and are reported as significantly improving recipient and graft survival after ABOi liver transplantation. There is, however, a major risk of developing portal vein thrombosis after PVIT, which may be even much greater in pediatric recipients. Although the risk of HAT in adults with HAIT seems to be small, it could probably be larger in children; therefore, these procedures are not recommended for use in children in any publication.
Perioperative Strategies in ABOi Liver Transplantation
Several protocols have been proposed for immunosuppression in ABOi liver transplantation. Most of them were established for LDLT for adult patients, but some reports apply to the pediatric population as well.
Protocols Used in Adults
Elective Living Donor Liver Transplantation
Tanabe et al. performed in adults ABOi LDLT perioperative plasmapheresis and rituximab, splenectomy, and PVIT, in addition to standard immunosuppression with calcineurin inhibitors, antimetabolites, and steroids.
In another report, Ikegami et al. published good results with perioperative rituximab and plasma exchange, splenectomy, and post-operative IVIG treatment.
Rummler et al. compared data from several publications on adult ABOi LDLT and summarizing actual practices; most authors recommend:
Preoperative plasmaphereses or IA to achieve anti-A/B antibodies titers of 1:8 to 1:32 (most commonly ≤ 1:16)
Rituximab 300–375 mg/sq m given twice on days 21 and 14 or on days 14 and −1 + 1 day around transplantation; however, some centers use only one dose of rituximab between the 14th and 10th day before transplantation.
IVIGs are recommended by about 50% of centers and are given in a dose of 0.8 to 1.0 g/kg body mass 2 to 3 times on days 1, 3, and 5 after transplantation.
Nowadays, most centers do not recommend splenectomy.
PVIT or HAIT is used mainly in Japan and is not recommended by most other centers, particularly in children.
Post-operative immunosuppression consists of triple-drug therapy, but in a significant number of centers, basiliximab is also used in quadruple-drug protocol.
ABOi Deceased Donor Liver Transplantation or Emergency Living Donor Liver Transplantation in Adults
Thorsen et al. described a series of adult 48 ABOi liver transplantation from deceased donors performed over the last 20 years in which most patients were treated with preoperative induction with rituximab, or basiliximab or both monoclonal antibodies (> 70% of patients), post-operative standard triple-drug therapy, and post-operative IVIG (46%), but only 9% received pre- and post-operative plasmaphereses or IA procedures under anti-A/B isoagglutinin titer monitoring. All of the patients in the ABO-I LT group received a single dose of rituximab (375 mg/m 2 ) and IVIG (0.4 g/kg per day) at the beginning of the operation. IVIG (0.4 g/kg per day) for induction therapy was administered for 10 days after LT. A quadruple regimen was adopted to strengthen the immunosuppression in the two groups. Basiliximab with a dose of 20 mg was used twice for day 1 and day 4 after LT. Maintenance immunosuppressive therapy consisted of corticosteroids, tacrolimus, and mycophenolate mofetil (MMF). Corticosteroids were withdrawn 1 month after transplantation.
Tacrolimus was administered from the first post-operative day to achieve a trough plasma level of 8 to 12 ng/mL for the first post-LT month and was titrated down to 5 to 8 ng/mL for the next month. MMF was started and kept at 500 mg twice daily after LT. To confirm the suppressive effect of rituximab, the peripheral blood CD20 + B-cell counts were analyzed by flow cytometry for a period of 3 months post-transplantation.
Protocols Used in Pediatric Patients
There have been various approaches and contradictive results reported from the centers performing ABOi LT in children; however, all were based on a relatively small sample of patients. Most of the authors agree that children below 2 years do not need modification of standard immunosuppressive protocols used in ABOc transplantations, but in older children, who have a growing risk for complications of ABOi LT, perioperative immunosuppression should be modified.
Heffron et al. published a series of ABOi transplants in pediatric recipients using a standard protocol of anti-CD25 combined with tacrolimus (started on post-LT day 7), mycophenolate mofetil (30 mg/kg/d), and steroids tapered to 0.3 mg/kg/d on day 7 and stopped 12 months after LT (if not indicated by other factors). No splenectomy or plasmaphereses were used in the peritransplant period. With this regimen, long-term patient and graft survival were 100% and 83%, respectively. Almost the same protocol is used in the authors’ center, with tacrolimus administered from day 1 after transplantation as the only difference.
Honda et al. reported a series of 29 pediatric ABOi LDLTs. The basic immunosuppression consisted of tacrolimus, mycophenolate mofetil (20 mg/kg/d), and steroids, which were tapered off two times slower than in ABO-compatible transplants. Historically, they used plasmaphereses, infusion therapy, and splenectomy, but since 2010, they do not use any additional prophylactic procedures in children aged below 2 years. In older children, a single dose of rituximab is given 2 weeks before transplantation (310–500 mg/m 2 ).
Okada et al. tried various doses of rituximab in children older than 2 years receiving ABOi LDLT and concluded that a single dose of 200 mg/m 2 3 weeks before LT is optimal. In children with lower dosages, severe AMR developed, whereas after a dose of 375 mg/m 2 , high mortality from severe infectious complications was noted.
Markiewicz et al. described emergency ABOi LT in two children older than 10 years under immunosuppression with basiliximab, tacrolimus, mycophenolate mofetil, and corticosteroids. Additionally, in both patients, three selective IA sessions with the Glycosorb ABO system (Glycorex AB, Sweden) were performed with good results.
Shukfeh et al. reported their initial experience with ABOi LT in six infants. Immunosuppression consisted of tacrolimus, mycophenolate mofetil, and IVIG on days 1 and 3 after LT. Patient and graft survival in ABOi patients was 83%, whereas among ABOc patients, patient survival was 100%, and graft survival was 83% ( Table 9.1 ).