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Keywords:

  • ABO-incompatible;
  • Humoral rejection;
  • Immunoadsorption;
  • Liver transplantation;
  • Plasma exchange;
  • Rituximab

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. DISCUSSION
  5. CONCLUSION
  6. REFERENCES

ABO-incompatible (ABO-I) liver transplantation has been performed essentially in patients with acute liver failure awaiting an urgent liver transplantation. Early results with ABO-I liver transplantation were disappointing with a very low graft survival rate (20–50%). The main risk is the occurrence of severe humoral and cellular rejection, vascular thrombosis, and biliary complications. In order to avoid humoral rejection and improve graft survival, total plasma exchange in combination with an intense immunosuppressive regimen has been proposed to decrease hemagglutinin titers in ABO-I liver grafts. In some centers, this regimen was associated with splenectomy, phototherapy, and portal or arterial intrahepatic infusion therapy; however, as these patients are at high risk of sepsis, a selective approach using antigen-specific immunoadsorption with immunoadsorbent columns has been successfully proposed for ABO-I living donor kidney transplantation. Few cases have been reported following liver transplantation. We report our recent experience with three adult patients (two patients with acute liver failure, and one with severe cirrhosis and hepatic encephalopathy) transplanted in an emergency situation with an ABO-I liver graft and managed with the use of GlycoSorb ABO immunoadsorbent columns and a quadruple immunosuppressive regimen with preservation of the spleen. Eight sessions were performed in the three patients. Antigen-specific immunoadsorption greatly lowered the anti-A hemagglutinin titers. None of the three patients developed acute humoral or cellular rejection. Two patients are alive at 1.5 and 3 years follow-up with a normally functioning graft. The third patient died with a functioning graft, one month after the transplantation, from septic complications.

During the last decade, the number of patients awaiting liver transplantation (LTx) has been dramatically increasing. To overcome the critical shortage of deceased organ donation, the use of marginal or extended criteria organ donors, non-heart beating and living donors, split and domino livers have been widely developed. ABO-incompatible (ABO-I) liver transplantation was first performed in patients with acute liver failure awaiting an urgent liver transplantation. In Eastern countries, where living donor livers provide the only source of organs, ABO-I transplantation has been also proposed for critically-ill patients and for those recipients with hepatocellular carcinoma (1). Viewing the reported literature and the current situation of organ donation, the problem of an ABO blood type incompatibility between voluntary donors and recipients in critical situations seems to be unavoidable.

Early results with ABO-I liver transplantation were disappointing because of the enhanced risk of severe humoral and cellular rejection of the graft, vascular thrombosis, and biliary tract complications. Angiocholitis related to ischemic cholangitis was common, often leading to liver abscesses and loss of the graft (2–4). Less frequently, signs of hyperacute rejection with acute periportal oedema have been described. Hyperacute rejection usually occurs during the first week after transplantation as a consequence of severe immunological reactions and leads to massive hepatocyte necrosis and graft loss (5,6).

In a report of our initial experience with 17/234 patients transplanted with an ABO-I graft in the late 1980s, the one-year patient survival rate was 66%, but the two-year graft survival rate was 30% as compared to 76% for compatible ABO transplants (2). Later, the analysis of our major cohort included 43 ABO-I liver transplantations; the five-year patient and graft survival rates were, respectively, 50% and 20%. The most common observed complications were vascular thrombosis (41%), biliary injury (34%), and hyperacute rejection (20%) (3).

Demetris et al. showed, in patients transplanted with an ABO-I graft, that liver allografts are susceptible to antibody-mediated rejection from preformed complement-fixing hemagglutinin leading to a hemorrhagic infiltration of portal tracts and deposition of fibrinogen and immunoglobulin (Ig)M on sinusoidal wall and vascular endothelium (4). The high rate of biliary complications leading to graft loss might be explained by the fact that the bile ducts may also express ABO antigens and serve as a target for antibody-mediated injury (7).

During the last two decades, several therapeutic regimens have been proposed to decrease hemagglutinin titers in ABO-I liver transplantations in order to avoid humoral rejection. The most common regimen included total plasma exchange in combination with immunosuppressive induction therapy (anti-thymocyte globulin, the monoclonal antibody OKT3, or cyclophosphamide). In some centers this regimen was associated with splenectomy, phototherapy, portal or arterial intrahepatic infusion therapy (6–12). The main risk of these intensive regimens is sepsis, commonly enhanced by the severity of the acute illness. More recently, a selective approach using antigen-specific immunoadsorption with immunoadsorbent columns (GlycoSorb ABO; Glycorex Transplantation AB, Lund, Sweden) (13,14), combined or not to anti-CD20 humanized monoclonal antibodies (rituximab [Mabthera; Roche Pharmaceuticals, Neuilly-Sur-Seine, France]), has been successfully proposed for ABO-I living donor kidney and liver transplantations (14–16). We report here our recent experience with three adult patients who received an ABO-I liver graft and managed with the use of GlycoSorb ABO immunoadsorbent columns and a quadruple immunosuppressive regimen with preservation of the spleen.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. DISCUSSION
  5. CONCLUSION
  6. REFERENCES

ABO-alloantibodies and antigen-specific immunoadsorption

Immunoglobulin G and IgM hemagglutinin titer measurements anti-A and anti-B were performed daily post-transplantation during the first 3–4 weeks, and also after immunoadsorption sessions. In addition, routine surveillance of liver enzymes and standard biological tests were performed twice daily. Immunoadsorption sessions were performed post-operatively according to the levels of hemagglutinin titers and whenever clinical, biological, or histological signs of rejection were suspected. The aim was to maintain hemagglutinin titers as low as possible below 1:16.

A conventional plasmapheresis device, supporting selective GlycoSorb ABO immunoadsorbent columns having a low molecular carbohydrate column with A or B blood-group antigen linked to a sepharose matrix specifically depleting anti-A or anti-B antibodies, was used to filter the patient's venous blood through a double-lumen central venous catheter. The duration of a session ranged from 3 to 4 h, based on estimated plasma volume.

Immunosuppressive regimen and antimicrobial prophylaxis

The three patients were treated with a quadruple immunosuppressive regimen. The immunosuppressive protocol consisted of:

  • • 
    Tacrolimus (Prograf; Astellas Pharma, Levallois Perret, France) given orally at a dose of 0.075 mg/kg between day 1 and day 4 when renal function was adequate, and progressively increased according to tacrolimus trough levels to a target of 11–15 ng/mL
  • • 
    Mycophenolate mofetil (CellCept; Roche Pharmaceuticals) administered at the daily dose of 2 gm intravenously, then switched to oral administration with the resumption of oral intake
  • • 
    Rabbit anti-human thymocyte immunoglobulin (Thymoglobuline; Genzyme Therapeutics, Saint-Germain-en-Laye, France) at a dose of 1.5 mg/kg/day, or an interleukin-2 receptor monoclonal anti-CD25 antibody, basiliximab (Simulect; Novartis Pharma, Rueil-Malmaison, France) at a dose of 20 mg intravenously at postoperative day 0 and day 4
  • • 
    Methylprednisolone 5 mg/kg intravenously at day 0 at the first incision and after skin closure, then tapered to 1 mg/kg/day over a week, with a maintenance dose of 0.1 mg/kg/day progressively decreased to 5 mg/day throughout the first year.

Antimicrobial prophylaxis consisted of 4.5 gm piperacillin-tazobactam (Tazocillin; Wyeth Pharmaceutical, Paris La Défense, France) every 8 h for 48 h. For cytomegalovirus antiviral prophylaxis the patients received i.v. ganciclovir at 5 mg/kg/day, and then they were switched to oral valganciclovir (900 mg/day) when oral food intake was allowed, for a period of three months. Antifungal prophylaxis consisted of the administration of 1 mg/kg/day of amphotericin B lipid complex (Abelcet; Cephalon, Maisons-Alfort, France) for the first week, then 2.5 mg/kg twice a week for an additional three weeks.

Liver biopsy and rejection treatment

Liver biopsies were performed whenever clinical or biological signs of rejection were suspected. Immunohistochemical analysis was done to investigate humoral rejection by determining the deposition of hemagglutinins and their complements in sinusoids (cytotoxic antibodies). Acute cellular rejection was defined according to the Banff criteria.

Humoral rejection was prevented or treated with an infusion of chimeric anti-CD20 monoclonal antibody, rituximab, at a dose of 375 mg/m2 over 8 h, with or without the administration of intravenous polyvalent immunoglobulin. Acute cellular rejection therapy consisted of 1–2 boluses of 1 g of methylprednisolone, according to the current practice in our center.

Patient 1

A 49-year-old woman was transferred to our intensive care unit (ICU) with the diagnosis of acute fulminant hepatitis of unknown etiology. At admission, she was mechanically ventilated in an advanced hepatic coma (grade 2) and with severe liver failure (prothrombin time level 8%, factor V 7%, and total bilirubin 565 µmol/L). She developed acute renal failure with a creatinine increase to 178 µmol/L. Her neurological status showed signs of cerebral oedema with dilated but reactive pupils. An intravenous infusion of mannitol (Macoflex®; Macopharma, Mouveaux, France) was then given as a first-line treatment of cerebral edema. She was registered on the national emergency transplant waiting list. She became hemodynamically unstable, necessitating the administration of noradrenaline at a dose of 0.5 mg/h. The first available cadaveric donor was a 56-year-old man of an incompatible blood group (type A+). Due to the severity of her condition, the graft was accepted and the patient underwent a total cadaveric A+/O+ incompatible liver transplantation. The main characteristics of the donor and recipient, and the main complications are summarized in Table 1. Five days after transplantation the inotropic support was stopped, the patient recovered consciousness, but remained on mechanical ventilation for mild confusion and bronchopneumonia for one month.

Table 1. Characteristics of recipients and donors, and the main post-operative complications
 Patient 1Patient 2Patient 3
  1. ALT, alanine transaminase; ARDS, acute respiratory distress syndrome; CVVHDF, continuous veno–venous hemodiafiltration; INR, international normalized ratio; LTx, liver transplantation.

Characteristics at admission   
 Age (years)492955
 SexFemaleFemaleMale
 Clinical presentationFulminant hepatitis of undetermined originToxic fulminant hepatitis (paracetamol)Alcohol cirrhosis
 Neurological status at admissionHepatic encephalopathy grade 3Hepatic encephalopathy grade 4Hepatic encephalopathy grade 2
 Liver tests at admission   
  Total bilirubin (µmol/L)14426390
  ALT (IU/L)154330034
  Prothrombin time (%)10919
INR3.66.34.7
Characteristics at LTx   
 Date of LTx6 June 200628 October 20061 January 2008
 Blood group of donorA+A−A−
 Blood group of recipientO+O+O+
 Donor age (years)565185
 Donor cause of deathCerebrovascular accidentRuptured cerebral aneurysmCerebrovascular accident
 Duration of cold ischemia5 h 22 min5 h 31 min6 h 48 min
 Blood transfusions (no. of units)81119
Main complications after LTx   
 Acute humoral rejectionNoneNoneNone
 Acute cellular rejectionNoneNoneNone
 Number of liver biopsies (timing)2 (days 6, 16)2 (days 10, 26)3 (days 15, 30; month 15)
 Renal failureYes, severeYes, severe (CVVHDF)Yes, moderate
 Infection present pre- and post-LTxBronchopneumoniaBronchopneumonia, ARDSBronchopneumonia, septicemia
Outcome   
 Length of hospital stay3.5 months26 days3 months
 Outcome status (follow-up)Alive (3 years)Died at 1 monthAlive (1.5 years)

The immunosuppressive prophylactic regimen included corticosteroids with regressive tapering, rabbit anti-human thymocyte immunoglobulin for five days, mycophenolate mofetil, and delayed introduction of tacrolimus at day 3 post-transplant due to renal failure. The hemagglutinin titers IgG and IgM anti-A ranged from 1:8 to 1:32. Antigen-specific immunoadsorption, using GlycoSorb anti-A immunoadsorbent columns, was performed on days 2, 8, and 20, when the initial isoagglutinin titers were raised; IgG and IgM anti-B hemagglutinin titers were measured concomitantly and served as controls (Fig. 1a).

image

Figure 1. Daily hemagglutinin titers for (a) patient 1, (b) patient 2, and (c) patient 3: immunoglobulin (Ig)M and IgG anti-A and the response to antigen specific immunoadsorption GlycoSorb anti-A. IgM and IgG anti-B served as the controls. The arrows indicate on which days antigen-specific immunoadsorption was performed.

Download figure to PowerPoint

Six days after transplantation the patient needed a laparotomy for intra-abdominal bleeding. A liver biopsy was then performed intra-operatively, which showed mild signs of heterogeneous sinusoidal congestion in the centrilobular area, but did not reveal any sign of humoral or cellular rejection. At post-operative day 16 the patient was found to have a marked increase in liver enzymes, highly indicative of an acute rejection episode. She was treated with an infusion of 375 mg/m2 rituximab, followed by an additional session of immunoadsorption while waiting for the results of a newly performed liver biopsy. The treatment led to a progressive decline of liver enzymes (mainly total bilirubin and γ-glutamyltransferase [GGT]) (Table 2). The latter biopsy showed mild inflammatory infiltrates with a marked cholangiolar proliferation, but failed to show frank signs of rejection. The patient was discharged from the ICU on day 43 after the operation, and from the ward on day 55, without any neurological sequelae and with a well-functioning graft and normal liver enzymes.

Table 2. Evolution of liver enzymes in the three patients after ABO-incompatible liver transplantation
 Day 1Day 5Day 10Day 151 month3 months1 year
  1. ALT, alanine transaminase; AST, aspartate transaminase; GGT, γ-glutamyltransferase.

Total bilirubin (µmol/L)       
 Patient 1293734180491310
 Patient 250192529315154
 Patient 3246169191235481514
AST (IU/L)       
 Patient 142433344127732
 Patient 2191224319421047
 Patient 3299475741662741
ALT (IU/L)       
 Patient 1213433538311224
 Patient 21556130230236663
 Patient 32291026431473839
GGT (IU/L)       
 Patient 1341051682072057269
 Patient 2224258383577165
 Patient 34470209253969167105
Prothrombin time (%)       
 Patient 158505762638198
 Patient 23337587354
 Patient 344517182839193

Patient 2

A 29-year-old woman was admitted to the ICU for acute fulminant hepatic failure due to a suicidal attempt 24 h after ingestion of 24 gm of paracetamol + codeine, 50 capsules of nitrazepam (Mogadon; Valeant Pharmaceuticals, Coumon, France), 45 capsules of bromazepam (Lexomil; Roche Pharmaceuticals), and alcohol. She had previously made four suicide attempts between 1995 and 2006. On admission her Glasgow coma score was 11 and she had a grade IV hepatic encephalopathy. She rapidly developed severe acute liver failure (prothrombin time level 9%, factor V 12%) within 24 h, leading to a grade II hepatic coma. She was then put on respiratory assistance and listed for transplantation.

The patient was transplanted 48 h after admission with an incompatible cadaveric liver graft (blood group A− to O+). The donor and recipient characteristics are summarized in Table 1. In the post-operative period the patient remained hemodynamically unstable and needed inotropic support, mainly due to septic complications (septicemia and pneumonia) associated to persistent mild intra-abdominal bleeding. She also developed an acute renal failure necessitating three sessions of continuous veno–venous hemodiafiltration.

Her immunosuppressive prophylactic regimen consisted of corticosteroids with regressive tapering, monoclonal anti-CD25 basiliximab (20 mg on days 0 and 4), mycophenolate mofetil, and the delayed introduction of tacrolimus on day 4 post-transplant due to renal failure. Hepatic cholestasis and cytolysis increased progressively from day 5 to day 10 (Table 2) and led to the performance of a transjugular liver biopsy. The latter revealed the presence of cholestatic liver, biliary clusters, hepatocyte ballooning evocative of functional cholestasis related to sepsis, and the absence of signs of rejection. Her IgG and IgM hemagglutinin titers remained very low between 1:2 and 1:4. A mild increase in IgG anti-A to 1:16 at day 15, in the context of severe cholestasis, led to the performance of one session of selective GlycoSorb anti-A immunoadsorption, which decreased IgG and IgM anti-A titers in the serum to undetectable levels (Fig. 1b).

After a period of a few days of stabilization, the patient's condition worsened progressively and she developed acute respiratory distress syndrome with septic shock leading to a multiple organ failure and death 26 days after transplantation. Liver biopsy performed at autopsy showed a cholestatic liver and cholangiolar proliferation related to the septic shock, as described in the previous biopsy, but didn't show any signs of acute cellular or humoral rejection.

Patient 3

A 55-year-old man, on the transplant waiting list for alcoholic liver cirrhosis (Child–Pugh stage C) and with a Model for End-Stage Liver Disease score of 34, was admitted to the hospital for spontaneous bacterial peritonitis that was successfully treated with a fluoroquinolone. Nevertheless, his liver function worsened rapidly (prothrombin level 22%, total bilirubin 460 µmol/L) and he developed an hepatic encephalopathy. Due to the severity of his illness, an ABO-incompatible cadaveric liver graft was accepted (blood group A− to O+) for transplantation. Characteristics of the donor and recipient and the main complications are shown in Table 1. The prophylactic immunosuppressive regimen used consisted of quadruple therapy, including corticosteroids with regressive tapering, basiliximab, mycophenolate mofetil, and tacrolimus.

On day 1 the IgG and IgM anti-A hemagglutinin titers were high, respectively, 1:512 and 1:256. An intravenous infusion of 375 mg/m2 of rituximab was administered. Antigen-specific immunoadsorption sessions using GlycoSorb anti-A were then performed on days 2, 3, 6, and 10, and allowed a significant decrease of hemagglutinin titers. Thereafter, his liver function tests stabilized without a further increase in the isoagglutinin titer (Fig. 1c). A major reduction of hemagglutinin anti-A titers was observed after the immunoadsorption sessions.

In the post-operative period the patient presented with persistent weakness, confusion, and tremor that necessitated prolonged respiratory assistance. Two weeks after transplantation, he developed biological signs of cholestasis with a moderate hepatic cytolysis. A liver biopsy performed on day 15, revealed an intense cholestasis, mild peliotic lesions, but no signs of rejection. His bilirubin and alanine transaminase levels decreased progressively and spontaneously. Another liver biopsy was performed at one month for elevated GGT levels and it showed the same features as the previous biopsy without signs of fibrosis or rejection. The patient was discharged from the ICU 27 days after the transplantation. Cholangiography was performed one month later via the T-tube, which showed a normal cholangiogram. By the end of the third month, the patient had recovered, his liver enzymes and graft function became normal and he was discharged from the ward. A routine liver biopsy performed one year after was within normal limits.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. DISCUSSION
  5. CONCLUSION
  6. REFERENCES

Early experience, in the late 1980s, in ABO incompatible liver transplantation showed serious complications of the graft in more than half of the patients with a major impact on morbidity and mortality. Despite that the liver appears to be less sensitive to antibody-mediated rejection, as compared to other organs, humoral rejection of the graft, ischemic cholangitis and vascular complications were common (2–4). Demetris et al. reported a 46% graft failure rate during the first 30 days for primary ABO-I grafts compared with an 11% graft failure rate for primary ABO compatible, cross-match negative, age, sex and priority-matched control patients (P < 0.02) (4). Recently, the two-year adult recipient survival rate after ABO-I living donor liver transplantation (LDLTx) in Japan was reported to be approximately 70%, while that for ABO-I LDLTx before 2002 was about 40% (17). The risk of graft dysfunction is mainly present during the first month, and tends to vanish later; this is probably related to at least a partial replacement of graft vasculature by endothelial cells that no longer carry ABO antigens (4).

Currently, ABO-I liver transplantation is performed in very rare situations and remains a subject of major debate. However, ABO incompatible liver transplantation could be a life saving procedure in particular situations such as acute liver failure, hepatocellular carcinoma that are at risk of dropout from the waiting list, living related donor transplantation, and in some other critical situations. Liver transplant surgery, performed in an emergency situation, has been considered as a salvage transplantation. Our three patients presented with a severe critical illness, as manifested by the neurological status of coma, hemodynamic instability, and rapid progression of the disease to a fatal outcome in the absence of transplantation. The short- and long-term outcomes of these patients transplanted with an ABO-I liver graft has been very encouraging with the recent improvement in immunosuppressive regimens and apheresis techniques.

It has been reported that the main complications of ABO-I LTx, such as hepatic necrosis and intrahepatic biliary complications, were closely related to the titer of perioperative anti-donor ABO antibodies (18). As a result, most of the efforts to improve the outcome of ABO-I LTx have been directed toward the lowering of anti-blood type antibodies. The impact of preformed anti-donor ABO antibodies and the strategy to reduce their titers play key roles in the success of ABO-I transplantation (8,9,16,19). In elective cases, plasmapheresis has been even initiated before transplantation in order to reduce the titer of IgM or IgG; this is more difficult to realize in emergency cases, such as the three patients described here.

Selective antigen-specific hemagglutinin immunoadsorption has been widely used in ABO-I kidney transplantation, but only in a few reported liver transplant recipients. It has been proposed in order to minimize side-effects and the morbidity related to total plasma exchange (20). Troisi et al. had previously reported in five recipients transplanted with an ABO-I graft, that antigen specific immunoadsorption using GlycoSorb ABO and quadruple immunosuppression, including daclizumab and mycophenolate mofetil, provide high efficiency to lower hemagglutinin titers over time, avoiding the need for splenectomy (13). Daclizumab and mycophenolate mofetil have been successfully used in ABO-I transplantation, therefore also minimizing the risk of infection (21). In the eight sessions performed in our three patients using GlycoSorb A, antigen-specific immunoadsorption showed to be extremely safe and provided efficient capacities to lower significant hemagglutinin titers to very low levels. We did not observe any side-effects related to the specific GlycoSorb immunoadsorption, nor any rituximab-related adverse effects in any of the three recipients.

In addition to the monitoring of hemagglutinin titers and liver enzymes, careful histological surveillance is also mandatory. Acute cellular rejection rates reported in the literature ranged from 20 to 90%, but have been less frequent in recent times (2–4). The three patients each had two liver biopsies performed during the first post-operative month that did not show signs of humoral or cellular rejection, highlighting the efficacy of antigen-specific immunoadsorption and the immunosuppressive regimen.

Toso et al. reported a comparable patient cohort of ABO-I liver transplants (N = 14) with acute liver failure or severely decompensate end-stage disease, intubated, and/or in the ICU. Using a quadruple immunosuppressive protocol without splenectomy, the authors did not observe a significant difference in regard to patient and graft survival when comparing one- and five-year data of ABO-incompatible, -compatible, and -identical liver graft recipients: 64 and 56% of ABO incompatible liver transplants remained functioning after one and five years, respectively. Three ABO-I grafts were lost (one from hyperacute rejection and two from hepatic artery thrombosis) (22). Plasmapheresis was used as adjuvant treatment for rejections in the first 12 patients, and as prophylaxis for high or rising isoagglutinin titers in the subsequent two.

CONCLUSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. DISCUSSION
  5. CONCLUSION
  6. REFERENCES

ABO-I transplantation remains an important and unavoidable therapeutic option in adult patients with acute liver failure awaiting an emergency procedure and in the context of a living donor liver transplantation. The use of a selective antigen-specific immunoadsorption GlycoSorb ABO combined with either basiliximab or a short course of antithymocyte globulin, tacrolimus, mycophenolate mofetil, and steroids appear to be safe and able to prevent acute humoral and cellular rejection. Careful daily monitoring of hemagglutinin titers during the first three weeks is essential, and early treatment with anti-CD20 antibody (rituximab) plus antigen-specific immunoadsorption has improved graft survival. The recent therapeutic approaches reported in the literature, as in our three patients, have provided encouraging long-term outcome results. Future studies are still needed to establish optimal management in this type of transplantation.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. DISCUSSION
  5. CONCLUSION
  6. REFERENCES