ABO Incompatible Liver Transplantation: Current Status

Introduction

Living donor liver transplantation (LDLT) is the primary method of liver transplant in countries where cadaveric donors are limited. More than 80% of liver transplants are living donor-related in East and South Asian countries. Breaching of ABO barrier has led to further expansion of donor pools in these countries and ABOi LDLT constitute 10-20% of living donor liver transplants.^1-3

Alexandre first performed the successful human ABO incompatible (ABOi) renal transplant in 1985.⁴ However, initial attempts at ABOi liver transplants yielded disappointing results. High rates of graft loss and mortality were seen due to hyperacute rejection, antibody-mediated rejection (AMR) related complications and increased rate of infections in these patients due to immunosuppression.^5,6 The use of plasmapheresis, splenectomy and local infusion therapy helped in improving the results of ABOi LDLT. The introduction of rituximab dramatically reduced the risk of graft rejection in these patients.^7-9 Further improvement in immunosuppression and pretransplant desensitization protocols has led to comparable outcomes of graft and patient survival in ABOi liver transplant and ABO compatible (ABOc) liver transplant.^10,11 ABOi liver transplant has the risk of AMR that can manifest as graft dysfunction, rapid graft loss due to hepatic necrosis, and non-anastomotic biliary strictures. Management of these patients in the pre- and post transplant stages requires a complex multi-disciplinary approach.

Management in pre-transplant period

One of the most important steps in ABOi LDLT is pretransplant desensitization to prevent ABO antibody-related rejection and graft dysfunction. Desensitization reduces the titre of pre existing ABO antibodies in recipients and decreases the B cell population, thereby decreasing the further formation of these antibodies. Effective removal of preformed anti-ABO antibodies from recipients’ blood before the transplant surgery determines the post-transplant outcomes. Past studies have shown that high perioperative titre of anti-A and anti-B antibodies increase the risk of biliary strictures, necrosis, and allograft rejection.^12,13

A. Rituximab

The use of rituximab for desensitization has revolutionized the management of ABOi LT. It is an anti-CD-20 chimeric murine monoclonal antibody. Rituximab plays an important role in the management of lymphomas, autoimmune and rheumatoid disorders. Tyden et al. first used rituximab in ABOi kidney transplant.¹⁴ Following the successful use in kidney transplant, Monterio et al. reported its first use in liver transplant.⁷ Mechanism of action include selective binding to CD-20 receptors present on most of the human B lymphocytes. Rituximab causes cell injury and death of CD-20 positive cells via direct signalling, complement-dependent cellular toxicity (CDCC) and antibody-dependent cellular toxicity (ADCC) resulting in marked depletion of the B cell population.^15,16

B cells play important roles like antibody production, cytokine production and antigen-presenting cells so their depletions adversely affect both humoral as well as T cells mediated immune response.^17,18

The standard dose of rituximab is 375 mg/m², given as a slow intravenous infusion. A single dose is sufficient most of the time and multiple administration can lead to infectious complications.^19,20 Doses as low as 200 mg/m2 have also been described in some studies. Egawa et al. showed that rituximab in the dose of 200 mg/m2 did not increase the risk of AMR and lower doses can be considered for selective patients, especially in those with higher risk of infections.²¹ Rituximab is given 2 weeks before the liver transplant as it has a long elimination half-life of about 3 weeks. It has been used even 7 days before a liver transplant in urgent situations. Studies have reported its use even within 72 hours of surgery in emergency liver transplants.^22-23 Care should be taken to avoid plasmapheresis soon after rituximab injection to prevent its elimination. Ideally, it should not be done within three days of rituximab injection. B cell depletion from the peripheral blood after rituximab administration usually happens within 48-72 hours, although complete depletion can take three weeks. Rituximab dose response is assessed by measuring the B cells population (CD 19+ cells) in peripheral blood. Samples are taken before rituximab dose and after 2 weeks. Desired results are defined as reduction in CD 19+ cells to less than 1 %. A second dose can be repeated in case of suboptimal effect after first dose. Effect of rituximab after a single dose can last for several months and the second dose administration is rarely needed and can increase infectious complications.^24,25

There is complete long-term elimination of B cells and memory B cells from peripheral blood, and spleen after a single dose of rituximab. However, the B cells can persist in lymph nodes, so the risk of AMR is markedly reduced but not eliminated.^22,26 Use of rituximab in ABOi LDLT is responsible for a marked improvement in graft survival due to decreased risk of AMR.²⁷ In a multicentre study conducted in Japan, the absence of rituximab prophylaxis was the only significant factor associated with AMR. The incidence of AMR in rituximab and non-rituximab group was 6% and 23% respectively. Local infusion therapy, intravenous immunoglobulins (IVIG) and splenectomy had no impact on AMR and overall survival.19,23

B. Plasma exchange therapy and immunoadsorption

Plasmapheresis involves exchange of patient plasma and the removal of various plasma proteins, and immunoglobulins including ABO antibodies. The presence of preformed ABO antibodies or isoagglutinins in recipients increases the risk of rejection and complications due to AMR. Risk of these complications has been shown to increase with rising titres of these antibodies.^6,13 Plasmapheresis and immunoadsorption can effectively remove ABO antibodies from the blood and bring down the titre. These methods work only on preformed antibodies present in the blood and more than one session may be required if titres rise again due to ongoing synthesis.²⁸

A safe pre-transplant level of ABO isoagglutinin titre is not defined and may vary from less than 1:8 to 1:64 in the immediate pre-transplant period according to the transplant centre protocol. Rituximab administration may result in sufficient reduction in B cells and low titres before the transplant plasmapheresis may not be needed in these patients.^29,30 About 50%-60% reduction in IgG and Ig M level can be achieved after one session of plasmapheresis.^12,31 Depending on the titre level, one or more sessions of plasmapheresis will be needed to achieve the target. Common side effects of plasmapheresis include hypersensitivity, hemodynamic stress and citrate toxicity.³² Plasmapheresis can also be used to reduce the post-liver transplant rebound of ABO isoagglutinins.

Immunoadsorption (IA) works in the same way as plasmapheresis by removing ABO isoagglutinins, however, it differs from plasmapheresis as it removes only preselected specific antibodies. Antigen-specific columns of immunoadsorption adsorb ABO antibodies and the rest of the immunoglobulins and plasma components are returned back to recipients. The adverse effects profile of IA is relatively better than nonselective plasmapheresis.³³ Many studies in ABOi kidney transplantation have shown the efficacy of IA, however, experience is limited in liver transplants.^27,34,35

Intraoperative period

Splenectomy

Spleen is a major lymphoid organ and a reservoir of plasma cells. It plays an important role in AMR because of antibody production by plasma cells. Splenectomy can decrease the risk of AMR by eliminating the plasma cell reservoir in the spleen. Before the use of rituximab in liver transplants, splenectomy during transplant surgery was the standard practice in ABOi LDLT.¹² With the use of rituximab, splenectomy is not necessary and can also increase the risk of infections in immunocompromised recipients.^36-38 Rout et al. reported similar ABO isoagglutinins titre, AMR rate and survival outcomes in patients with and without splenectomy, both groups received preoperative rituximab.³⁹

Post-transplant period

Target ABO isoagglutinins titres in the pre transplant and post-transplant period

Still, no consensus has been reached regarding the safe levels of anti-A and anti-B titres at which the risk of AMR and other related complications is minimal and the target level varies from centre to centre.²⁷ Target pre-transplant level antibodies vary from ≤1:64 to ≤1:8 in various studies.^40,41 In a study of 381 adult ABOi LDLT patients, Egawa et al. showed that the risk of AMR increased at the level of ≥1:16 and ≥1:64 in the pre-transplant and post-transplant period respectively. The use of rituximab significantly decreased the risk of AMR in this study.19 Although evidence suggests that pre-transplant ABO isoagglutinins titre directly correlates with the risk of AMR, some studies have shown contrary results. Some researchers have suggested that anti-A and B titres may not affect the incidence of AMR in liver transplant recipients.⁶ Sometimes, non-anastomotic intrahepatic biliary strictures, a complication of AMR can occur without obvious rebound in isoagglutinins titres.⁴⁴

Post-transplant immunosuppression

In ABOi LDLT, post-transplant immunosuppression protocol consists of a standard triple drug regime similar to the ABOc LDLT protocol. Three immunosuppressive drugs are steroids, calcineurin inhibitors (CNIs) and mycophenolate. Tacrolimus is a more commonly used CNI compared to cyclosporin. An initial 500 mg-1000 mg dose of IV methylprednisolone is given intraoperatively before the perfusion. Intravenous methylprednisolone 100 mg is given on postoperative day 1 followed by slow tapering over a week. Oral prednisolone is started at the end of first week and continued usually for the next three months and then stopped after slow tapering. Tacrolimus and mycophenolate are started on post-operative day 1 and tacrolimus trough levels are maintained between 10-12 ng/ml for the initial few weeks. Mycophenolate mofetil (MMF) is given in a dose of 500-1000 mg/day and a higher dose can be used in patients with renal dysfunction to allow a lower dose of tacrolimus.^23,45 Some authors have recommended to start tacrolimus and MMF in pre-transplant period to decrease the risk of future allograft rejection although the practice is not common and can increase the post-transplant infectious complications.⁴⁶ Use of Basiliximab as a post-transplant induction agent in patients with renal dysfunction helps in delaying the use of CNIs but it’s use has been associated with increasing risk of infection and experience in ABOi LDLT settings is currently limited.^47,48

Intravenous Immunoglobulins (IVIG)

IVIG primarily consists of polyspecific IgG and is prepared by using the plasma of multiple healthy blood donors. Common uses of IVIG have been in immunodeficiency disorders and systemic inflammatory syndromes like Guillain barre syndrome.²³ High dose IVIG have been used for the treatment of steroid-resistant severe acute rejection in kidney and liver transplants.49 IVIG has also been used in ABOi LDLT for post-transplant rebound of ABO antibodies titres.⁵⁰ IVIG acts as an immunomodulator and has a multifactorial action in solid organ transplant recipients that includes inhibition of alloreactive T cells, blocking of CD19 expression on activated B cells, blockade and neutralization of allo-antibodies. It also blocks Fc (crystallizable fragment) receptors on mononuclear phagocytes and causes complement inhibition.^12,51 Use of IVIG as a part of desensitization protocol in ABOi LDLT has been reported in a few studies. Kim et al. used a simplified protocol without plasmapheresis in 43 patients. After receiving pre-transplant rituximab, patients were given IVIG (0.8 gm/kg) on post operative day 1 and 4 along with three-drugs immunosuppression. No patients had AMR, but 30% of patients had biliary strictures. ABO antibody titres were below 1:16 at the time of transplant in all patients. Limited experience, absence of long-term outcomes data, high treatment cost and adverse reactions are the main limiting factors for routine use of IVIG for desensitization ABOi LT.

Management algorithm

The desensitization protocol is started with the administration of rituximab two weeks before the liver transplant surgery. The commonly used dose is usually 350 mg and lower (300 mg) can be given in patients with acute or chronic liver failure or who have been in ICU for a prolonged period as the risk of infection is increased in these patients. CD 19+ and CD 20+ mononuclear cells are used to assess the response to rituximab and the presence of less than 1% of these cells is considered a satisfactory response. Pre-transplant plasmapheresis or IA is done to achieve the target ABO antibody titre of ≤1:16 immediately before the transplant surgery. Splenectomy, Lymphocyte Immunoabsorption Therapy (LIT) or IVIG are not part of the routine protocol at most centres. Anti-IL-2 agents like Basiliximab are not used routinely for induction but can be considered in selected cases with renal dysfunction. The recipients receive 500-1000 mg dose of IV methyl prednisolone intraoperatively during the anhepatic phase. Post-transplant immunosuppression consists of standard triple drugs including CNIs, MMF and steroids. Tacrolimus trough levels are maintained between 10-12 ng/ml for first 2-3 weeks. MMF is usually started at dose of 500 mg twice a day and can be increased to 750 1000 mg twice a day. ABO antibody titres (Both IgG and IgM) should be measured twice a day for first two weeks and then on daily basis for next two weeks. Indication of plasmapheresis in post-transplant period includes two log rises in titres or rise in titre to 1:64 or more and an unexplained rise in bilirubin and liver transaminases. Threshold for using plasmapheresis can be lower or higher at liver transplant centres. Protocol liver biopsy is avoided at most centres. Liver biopsy can be done in suspected cases of allograft rejection and unexplained derangements in liver biochemistry. Acute rejection episodes are managed with plasmapheresis, steroid pulse therapy and modulation of doses of CNIs and MMF. As these patients have a high risk of post-transplant infections, broad-spectrum antibiotics are routinely used in the early post- transplant period. In addition, these patients should receive prophylactic antifungal therapy and pre-emptive treatment for cytomegalovirus (CMV) infection (Figure 1).

Figure 1: ABO incompatible liver transplantation²⁰

Abbreviation: CMV, Cytomegalovirus

Diagnosis and management of acute antibody mediated rejection

Although ABOi LDLTs are being performed routinely and outcomes have improved significantly, many areas need improvement and warrant further studies. Early diagnosis and management of AMR is one of the critical factors to improve graft survival rate and outcomes in ABOi LDLT. Validation of potential biomarkers and laboratory tests for early diagnosis of AMR before pathological changes in liver biopsy is one potential area for future research. Reported incidence rate of AMR in ABOi liver transplant is 5%-7 % in post-rituximab era.46 Diagnosis of AMR is based on deranged liver biochemistry in the absence of vascular, biliary and other cause of deranged liver functions. A rise in Anti A and Anti B antibody titres support the diagnosis of allograft rejection. Radiological features in early stage include portal oedema and areas of necrosis while biliary changes in form of intrahepatic strictures appear in late stage.52 Histology helps make the diagnosis although features are not entirely specific. Main histological features of AMR include endothelial cells injury and hypertrophy, endotheliitis with inflammatory cells infiltrates. Diffuse complement component C4d deposition along endothelial lining is one of the specific features of AMR. Diagnosis is made by the presence of consistent histology, C4d depositions, donor specific antibodies in recipient blood and exclusion of other causes of graft insult.⁵³

Treatment of AMR is still not standardized and prevention remains the most clinically effective and cost-effective method for AMR. Unnecessary blood transfusions in pre-transplant period should be avoided to decrease the risk of sensitization. Post-operative rise in isoagglutinins titre is managed by plasmapheresis. Early plasmapheresis may prevent severe clinical allograft rejection. Mild to moderate cases of AMR can be treated with methylprednisolone pulse therapy and up titration of tacrolimus and MMF. Treatment options for those refractory to steroid pulse therapy include anti-thymocyte globulin (ATG), IVIG and rituximab.54,55 Use of proteasome inhibitor Bortezomib and complement inhibitor Eculizumab in refractory AMR have been used in limited studies only and need more evidence for their regular use.56-58 Other emerging therapies for AMR include IL-6 receptor antagonist Tocilizumab and T cell co-stimulating agent Belatacept. Experience with these newer agents is very limited but preliminary data seems promising.23,59,60 Newer anti-CD20 drug Obinutuzumab has been used in rituximab refractory cases of B cell lymphomas. Obinutuzumab has a more potent B cell depletion effect compared to rituximab. It can be used in ABOi transplant as an alternative to rituximab and a small study showed its safety and tolerability in kidney transplant patients.61-63 Table 1 includes the emerging therapies for prevention and treatment of AMR.

Table 1: Newer drugs in ABO incompatible liver transplantation

Abbreviations: AMR: Antimicrobial resistance, IL: Interleukin, BAFF: B-cell activating factor, IgG: Immunoglobulin, GVHD: Graft versus host disease

ABOi LDLT in paediatric population

Studies have reported better outcomes in ABOi LT in children as compared to adults. In a retrospective analysis of national registry database by Stewart et al., paediatric (<17years) ABOi DDLT graft survival was similar to ABOc DDLT.⁶⁴ Children under 2 years have the best outcomes in paediatric ABOi LDLT and 11 months is the minimum age at which antibody-mediated complications have been reported. The immature immune system and the resulting decreased risk of AMR are likely reasons for better outcomes in children.^65-68 Mysore et al. devised a new strategy for ABOi DDLT based on pre-transplant ABO antibody titres. Children with pre-transplant titre of ≥1:32 received rituximab and plasmapheresis before transplant followed by standard triple drug immunosuppression in post transplant period. Children with pre-transplant isoagglutinins titre of <1:16 received only steroids and tacrolimus. Authors reported 100% graft survival rate over a 3.3 year follow up. However, in this study only ten patients underwent ABOi liver transplant.⁶⁹ Given the absence of AMR and related complications in children below 1 year, use of rituximab and plasmapheresis is avoided in this group of patients. Anti AB isoagglutinins titres in small children can be below the pre-transplant target level without any desensitization method because development of these antibodies is age-dependent. Post-transplant immunosuppression protocol usually consists of three drug regimes as in adult patients including CNIs, MMF and steroids. Use of IVIG has been reported by Markiewicz Kijewska et al. in children during post-transplant period for desensitization and in treatment of severe AMR.⁷⁰

Conflicts of interest:

All authors declare no conflicts of interest.

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None of the authors have any financial, professional or personal conflicts that are relevant to the manuscript.