Antibody Incompatible Transplantation (AIT)

Def: transplantation of an organ into a recipient who is either ABO-incompatible or current or pre-conditioning DSA.
Hyperacute rejection (HAR) is a key risk in AIT and its preventable by avoiding transplantation against a strongly positive flow cytometry (FC) crossmatch or a positive complement-dependent cytotoxic (CDC) crossmatch in the immediate pre-operative period.
accelerated or acute AMR in the early period after transplantation is considered as the second key risk in AIT, usually occurring in the first 2 weeks. pre-treatment FC crossmatch positive is the most important risk factor for this type of rejection due to pre-existing AB but some patients with detectable donor-specific antibodies (DSA) and negative FC crossmatch do experience early acute AMR so we need to improve our risk assessment by adding new other parameters such as
·       the source of sensitization.
·       detailed characteristics of DSA.
·       novel serum or urine biomarkers measured either pre-conditioning or in the very early post-transplant period for early diagnosis of rejection.
Chronic AMR is an important cause of graft failure after AIT and is considered a third key risk in AIT, and also occurs in many patients who develop de novo HLA antibodies after standard transplantation. There are no graded recommendations for the effective prevention or treatment of chronic AMR.
In ABO-incompatible transplantation, the donor to recipient blood group combinations of A to O, B to O, AB to O, B to A, AB to A, A to B, and AB to B are incompatible.
There is insufficient evidence to incorporate recommendations about antibodies against non-HLA antigens like (angiotensin receptors, non-ABO blood groups, and endothelial antigens).

Histocompatibility and Immunogenetics Laboratories

the degree of immunological incompatibility between donor and recipient depends on the level of preformed anti-donor reactive antibodies using both solid-phase assays and traditional serological assays after taking the sensitization history of the recipient.
The three commonly used methods for assessing HLA-specific antibody levels are
·       the complement-dependent cytotoxic crossmatch (CDC)
·       the flow cytometry crossmatch (FC)
·       HLA microbead analysis
although it should be noted that the CDC and FC techniques detect antibodies other than HLA. antibody compatible transplant is defined as a donor/recipient with negative CDC and FC cross match and low levels of detectable donor-specific antibodies (DSA) by microbead analysis.
We recommend that pre-transplant assessment includes the use of microbead analysis plus a cellular crossmatch method.
A positive cellular crossmatch in the absence of detectable donor-specific HLA antibodies by microbead may be a false positive and should be investigated further.
A positive microbead test without a positive cellular crossmatch should also be investigated. In general, a transplant will not fulfill the requirements to be defined as an antibody incompatible with HLA-specific antibodies unless an appropriate cellular crossmatch is used.
Unlike CDC and FC crossmatching, microbead analysis does not rely upon a constant supply of fresh donor lymphocytes to allow for daily monitoring. Instead, target HLA proteins are purified and attached to polystyrene beads which are incubated with patient sera and any HLA-specific antibody present will bind to the HLA protein coupled to the microbead and allows the laboratory to detect it.
Although the development of Luminex-based technology has led to substantial progress in HLA antibody detection compared with cell-based techniques, technical issues can confound assay interpretation. Samples with high levels of antibodies exhibit prozone and high-dose hook effects resulting in a falsely low mean fluorescence intensity (MFI) level, or in some cases high levels of antibodies not being detectable at all. The prozone concept also applies to cellular assays. the solution to overcome the prozone effect is to add a small amount of ethylenediaminetetraacetic acid (EDTA) to the serum prior to testing or sera dilution. Also, the addition of DTT to the sera or heat inactivation of the sera prior to testing are effective alternatives to the addition of EDTA. We recommend that all laboratories involved in HLA transplantation, in cases where a prozone or hook effect is considered to have a protocol for treating the sera and DTT controls should always be applied and reported for the CDC assay. if treating the sera, it should be mentioned to be used in interpretation and translating guidance of MFI threshold into the local practice.
Another technical issue surrounding the use of HLA antigen-coated beads has recently emerged which is the presence of denatured antigen on the bead surface can lead to the presentation of a number of non-native HLA epitopes due to altered conformation of the protein. A simple and effective way of avoiding the detection of ‘naturally’ occurring antibody artifacts is to use two manufacturer’s kits. Antibody to denatured antigen should always be considered in cases of negative crossmatch with positive microbead assay results. Alternative screening methods should be considered in these cases to confirm, or deny, the presence of true donor HLA-specific antibodies before classification as potential HLAi.
DSA C1Q is a modification of the Luminex assay have also been developed to be able to detect antibody’s ability to fix complement from those that do not However, the clinical utility of these assays in renal transplantation is not clear cut and further research is therefore needed before a full recommendation.
In ABOi transplantation, measurement of blood group antibody levels is required. At present this is performed by hemagglutination testing and it’s better to be done by gel-card-based assays because there is a degree of standardization.

SELECTION, RISK ASSESSMENT, AND MAKING CHOICES

Statements of Recommendation We recommend that:
• Any patient considering AIT must be fully counseled regarding the procedures, risks, and potential outcomes, and must also be informed of the alternative routes to standard transplantation including exchange transplantation and the option of deceased donor transplantation. (1D)
• Patient counseling must include a risk assessment of the likelihood of accelerated acute, acute, and chronic antibody-mediated rejection, graft loss, and death using appropriate local and national/international data. (1D)
• In HLAi transplantation, patients must be risk assessed according to the principal risk factors for adverse outcomes. These include a positive CDC crossmatch or a high FC crossmatch and may include high levels of cumulative DSA beyond MFI 10000, multiple donor-specific antibodies, transplantation of a kidney from a deceased donor, and repeat mismatches including those related to pregnancy. (1C)
• In ABOi transplantation, patients must be risk assessed for acute AMR. Risk factors include ABO antibody titers above 1/256 and additional HLA antibody incompatibility. (1C)
Informed Choices and Kidney Sharing Current data indicate that the results of AIT are not as good as those of standard transplantation as alternatives include finding an alternative living donor, entering into a kidney sharing scheme, waiting for a deceased donor organ, or dialysis.
We recommend that patients with living donors should be encouraged to enter a kidney sharing scheme in order to have the chance of being offered a standard transplant. A few patients are not suitable for the sharing scheme due to medical urgency or any other reasons also paired or pooled donation is a potential opportunity to receive an immunologically low-risk transplant. Since January 2012 it has been possible for patients to have a modified profile of unacceptable antigens listed for the NLDKSS compared to the deceased donor waiting list, with the removal of those antigens against which the recipient has only low-level antibodies or removal antibodies that were felt to be amenable to desensitization according to local policy.

Outcomes and Risk factors for HLAi

The outcomes of AIT are not well defined, partly because of short-term follow-up and partly because of likely publication bias. The outcomes of HLAi transplants have also been reported in large collaborative studies from the USA and showed
Five-year unadjusted graft loss was
·       16.6% in transplants with no known antibody incompatibility
·       20.1% in those with DSA detectable by microbead but crossmatch negative
·       28.8% in those who were FC crossmatch positive and CDC crossmatch negative
·       39.9% in those who were CDC crossmatch positive.
Five-year mortality in these groups was 9.3%, 9.6%, 12.9%, and 19.1% respectively. Mortality is an important additional risk factor in HLAi transplantation, partly due to the more intense immunosuppression needed, and partly because many of the recipients have experienced long periods of previous dialysis and transplantation and may have acquired significant comorbidities. Over time, however, the risk of death is statistically higher in those patients that remain on dialysis.
the most important of these risk factors is a positive CDC crossmatch, ABOi, or a single or cumulative DSA measured by microbead with MFI >10000. There are also other emergentHLAi transplantation from a deceased donor that has few good results in the current NHS BT Registry outcomes. Two approaches are being considered to address this. First a modified profile of unacceptable antigens Second, desensitization.

Outcomes and Risk Factors for ABOi

The outcomes of ABOi transplants have been described in large studies from the USA, Japan, Europe, and the UK. The USA study, and to a lesser extent the European study, show
an early graft loss with a relative risk of around 2 compared to other transplants.
There is also an increased risk of complications, consequent from the more intense treatment given.
Acute AMR in ABOi is far less frequent than in HLAi, but may be of rapid onset and be hard to treat.
 The antibody level in ABOi is measured using a hemagglutination technique. AMR is more likely when the ABO titer is greater than 1/256. Most reports have shown no significant association between maximum anti-A/B IgM titers and graft survival.
Grafts from blood group A2 donors appear to be at lower risk than those from A1 donors, as the antigen is expressed at lower levels on tissue, but despite this rejection and graft loss have been reported in this donor group. The major increased risk of graft loss occurs very early post-transplantation and ABOi transplants that last greater than 2 weeks have the same survival as standard transplants.

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CONDITIONING TREATMENT BEFORE TRANSPLANTATION

Statements of Recommendation We recommend that:
• Extracorporeal therapies must be used to remove HLA or ABO antibodies to the level which gives a negative cytotoxic crossmatch or microbead measurement of MFI <5000, but this level may be flexible depending on an overall risk assessment. In ABOi, a haemagglutination titer of <1/8 is considered to be acceptable. (1C)
• In HLAi, immunosuppression protocol and induction agent should be specified in the unit’s guidelines. Tacrolimus and mycophenolate may be started before the transplant. Combinations of IVIg and rituximab may also be used. (1C)
• In ABOi, Combinations of IVIg, rituximab, and mycophenolate may be used. (1C)
We suggest that:
• There are several methods available for extracorporeal antibody removal (plasma exchange, cascade plasmapheresis, immunoglobulin immunoabsorption, and specific antigen adsorption (ABOi only)). At present, there is no evidence that one particular method produces superior clinical outcomes. (2C)
Strategies for Conditioning Therapy
There are three conditioning strategies that may be used before AIT is performed.
First, (long duration desensitization) treatment may be given to reduce HLA antibody sensitization over a period of weeks or months, which may enable a more compatible organ to be sourced if there is no living donor and may reduce the risks of transplantation in that HLA antibody production is reduced.
Second, (short duration therapy) conditioning may be performed over a period of days or hours before a transplant. This strategy creates a safer window of opportunity for transplantation. The majority of AIT is currently performed in this setting of short-term conditioning.
Third, there may be no conditioning, although the immunosuppression given at the time of transplantation may be more intense than for a standard transplant. It may be possible to achieve good outcomes with this approach even with high DSA at the time of transplantation, as reported in France.

Extracorporeal Antibody Removal Therapy

For whom:
·       patients are CDC crossmatch negative with their donor’s kidney but may have microbead measured DSA of MFI >10000. Conditioning may be needed to reduce antibody levels until achieve CDC crossmatch negative, FC crossmatch negative and microbead MFI <5000. However, it is not an absolute requirement to achieve these levels.
·       In ABOi, antibody removal is used to achieve a haemagglutination titer of <1/8 at the time of transplantation.
In HLAi the available extracorporeal techniques: include plasma exchange, double filtration plasmapheresis, and immunoadsorption (Protein A or immunoglobulin-specific adsorption). Any of these techniques may be used with careful monitoring of coagulation.
In ABOi, the available extracorporeal techniques include plasma exchange, double filtration plasmapheresis, and immunoadsorption (Protein A, immunoglobulin specific adsorption, or ABO specific adsorption). Any of these techniques may be used.
Pre-transplant Drug Therapy
There is no good evidence that any particular drug therapy given alongside extracorporeal antibody removal will effectively suppress donor-specific antibody production but IVIG, rituximab, MMF, and interleukin 6 receptors blockers are mostly used with no strong evidence yet.
Many series of ABOi have reported the use of rituximab and IVIg.
Splenectomy was used prior to ABOi transplantation after Alexandre reported its use was associated with successful outcomes in the 1980s. However, splenectomy is no longer recommended and many centers with excellent outcomes use rituximab.

INITIAL THERAPY AND MONITORING IN THE EARLY POST-TRANSPLANT PHASE

Statements of Recommendation
We recommend that:
• The highest risk period for acute AMR is the first 2 weeks after transplantation. Patients must be monitored carefully in a hospital or in the clinic during this period. (1C)
• In HLAi, drug therapy during the first two weeks post-transplant should include tacrolimus and mycophenolate. Prednisolone, basiliximab, alemtuzumab, IVIg, ATG, and bortezomib may be used according to local guidelines and risk assessment. (1C)
• In ABOi, drug therapy during the first two weeks post-transplant should include tacrolimus and mycophenolate. Prednisolone, basiliximab, alemtuzumab, IVIg, and ATG may be used according to local guidelines. (1C)
We suggest that:
• Daily measurement of HLA or ABO antibody levels is not mandatory, but daily samples should be taken when in hospital and at each clinical visit and be available for urgent analysis if required. (2D)

Occurrence of Acute AMR and Monitoring

It is clear from many reports that the highest risk period for acute AMR in AIT is in the first 2 weeks after transplantation. It is not possible to recommend exact follow-up regimens for all patients and they should be individualized depending on the intensity of anti-rejection therapy and any other complications, as well as the risk of AMR. However, hospitalization is likely to be longer and outpatient monitoring more frequent than for ‘standard’ transplants. There is not a direct correlation between HLA or ABO antibody levels measured post-transplant and the onset of AMR and antibody levels cannot be interpreted in the absence of clinical information.
Choice of Immunosuppression in HLAi
Optimal post-transplant therapy for the prevention of acute AMR has not been defined in HLAi transplantation and there are few randomized trials to provide guidance. Tacrolimus and mycophenolate and steroids are still the standard of care as maintenance immunosuppression. Good outcomes have been reported with using basiliximab as non-depleting induction therapy. However, most units report using more intense routine induction immunosuppression such as ATG and alemtuzumab. IVIg also may be included in a unit’s guidelines.
rituximab, and bortezomib their evidence for induction remains debatable, and their use is not recommended in lower-risk transplants. Eculizumab is not recommended as a routine preventative therapy for AMR in HLA AIT transplants.
Choice of Immunosuppression in ABOi
The peak risk period for acute AMR is also in the first 14 days after ABOi transplantation. There are no randomized trials that indicate the optimal therapy after ABOi transplantation to prevent AMR. However, unlike HLAi, ABO antibodies do not seem to cause late AMR or chronic AMR, so the focus is on the prevention of early AMR. tacrolimus, mycophenolate, and steroids are still the standard of care for maintenance immunosuppression. Prednisolone-free immunosuppression has been reported with the use of alemtuzumab. We recommend that IVIG, rituximab, ATG, or alemtuzumab may be included in a unit’s guidelines for ABOi. We do not recommend the routine use of bortezomib or eculizumab.

DIAGNOSIS AND TREATMENT OF ACUTE ANTIBODY-MEDIATED REJECTION

Statements of Recommendation We recommend that:
• The diagnosis of acute antibody-mediated rejection (AMR) is made following allograft biopsy and according to diagnostic criteria of the last Banff updates.
• Patients with histologically proven acute AMR must be screened for the presence of donor-specific antibodies (DSA) at the time of diagnosis. (1C)
• Patients with acute AMR receive (or are switched to) baseline immunosuppression including tacrolimus, mycophenolate mofetil, and corticosteroids, and are treated with high-dose steroids. (1C)
• Patients with early acute AMR in the presence of a detectable DSA receive extracorporeal antibody removal with five cycles of treatment or until the DSA is no longer detectable according to unit policy. (1C)
• In ABOi renal transplantation, AMR may occur rapidly so multiple therapies may need to be used. (1C)
We suggest that:
• IVIg, ATG, rituximab, or bortezomib may be used in combination with other agents until evidence emerges to the contrary. (2D)
• Eculizumab may be considered for rescue therapy in resistant acute AMR in cases that are C4d positive or the DSA have complement-fixing properties. (2D)
• Splenectomy may be considered (with or without additional eculizumab) to rescue acute AMR presenting with acute onset oligo/anuria in the early period after AIT. Where possible, the diagnosis should be confirmed pre-splenectomy by biopsy. (2D)
Diagnosis of AMR
Kidney biopsy, immunofluorescence staining to distinguish the predominant cell type involved in rejection (monocytes vs T cells) and analysis of gene transcripts, Detection of circulating DSA at the time of histological evidence of AMR is required to meet the full criteria of acute AMR.
Treatment of Acute AMR
Given the lack of randomized control trials, the optimal treatment for AMR remains uncertain. The current KDIGO guidelines suggest that acute AMR should be treated with one or more of the following, with or without steroids: plasma exchange, intravenous immunoglobulin, anti CD20 antibody, and lymphocyte-depleting antibody; however, this advice was based upon level 2C evidence or less. It is recommended that AIT recipients with acute AMR receive baseline maintenance treatment with tacrolimus, mycophenolate mofetil, and steroids at the usual maintenance doses. This is based on evidence drawn from studies assessing maintenance immunosuppression protocols associated with lower rates of acute rejection. The more specific treatment options used in practice are described below
Extracorporeal Antibody Removal
Plasmapheresis, plasma exchange, and immunoadsorption remove circulating DSA and are used in the treatment of AMR. one of the few randomized controlled trials had to be terminated due to the significant benefit seen in the interventional arm receiving immunoadsorption.
There is recent debate regarding its efficacy in the treatment of late acute AMR because of the nature of antibodies and its mechanism of graft injury but most centers still using it.
IVIg Intravenous
immunoglobulin (IVIg) has numerous potential effector mechanisms which could attenuate the treatment of AMR. These include the ability to neutralize circulating DSA, inhibition of complement activation, and blocking immune activation by competing for FcγRs.
IVIG can be used alone or with plasma exchange therapy.
ATG
ATG consists of polyclonal antibodies which predominantly act against T-cells; however, it also contains antibodies against activated B-cell and plasma cell surface antigens.
Observational studies indicate that ATG on its own or in conjunction with plasmapheresis is effective at reversing histologically proven AMR. ATG may also be effective in treating concurrent acute T cell-mediated rejection.
Rituximab
Rituximab is a B-cell depleting monoclonal antibody directed against the cell surface molecule CD20. Following administration, rituximab depletes both immature and mature B-cells, but not plasma cells or memory B cells. Rituximab is now the commonest add-on agent used in the treatment of acute severe AMR. However, Ritux ERAH study (NCT01066689) is a randomized controlled trial, which has published one-year outcomes in abstract form. This double-blinded randomized controlled trial analyzed the effectiveness of rituximab versus placebo in patients with acute AMR receiving plasmapheresis, IVIg, and corticosteroids. At one year, there was no benefit of adjuvant rituximab in terms of allograft survival or improvement in function. The long-term outcomes have yet to be reported.
Bortezomib
Bortezomib is a proteasome inhibitor that induces apoptosis in metabolically active plasma cells. BORJECT Study showed no benefits in the treatment of AMR.
Eculizumab
Eculizumab is a humanized monoclonal antibody directed against the complement component C5. Its use to treat severe, refractory AMR is limited to case reports and small case series where there is likely to be a strong element of publication bias. a recent randomized controlled trial showed no difference between treatment and control groups in relation to the composite primary endpoint. There have also been reports of the failure of eculizumab to rescue allografts with acute AMR maybe because the antibody-mediated injury may occur in a non-complement-dependent manner and eculizumab may not be of benefit in this setting.
Splenectomy
Splenectomy has been used for rescue therapy in patients with severe AMR in HLAi. One study from Johns Hopkins University reported on five patients, all of who were failing to respond to plasmapheresis and IVIg (± rituximab and ATG).
AMR in ABOi
Diagnosis: Based on Graft dysfunction + antibody level increasing one or more dilutions based on the same hemagglutination method with fragmented RBCs+pathology evidence.
Antibodies monitoring:
Minor changes (for example titers changing by one dilution (e.g. 1/4 to 1/8)) may prompt a plan of plasmapheresis or immunoadsorption for the following day but If the change is two dilutions, e.g. 1/4 to 1/16 from the previous day, then treatment may be initiated that day. Rising titers despite immunoadsorption combined with graft dysfunction mean that the graft may thrombose as the antibody is being synthesized at a faster rate than it can be removed. Daily antibody level measurement may indicate an early sign of immune activity. if the following day’s antibody titer is worse. Vascular patency needs to be confirmed by ultrasound and then the recipient is treated with complement blockade (eculizumab), which may need to be administered within 24-48 hours of graft dysfunction if antibody titers are rapidly increasing.
Graft dysfunction with low levels of AB antibody and without cell fragmentation should prompt the normal approach of ultrasound scan and biopsy and the more normal steroid-based treatment, as T cell-mediated rejection may be present.
Regarding treatment
In contrast to HLAi transplantation, acute AMR is uncommon following ABOi transplantation but it can be catastrophic when it occurs, leading to allograft infarction in a matter of hours. Whilst most acute AMR after HLAi transplantation may respond, at least initially, to treatment so it’s better to use of multiple therapies from the start even before kidney biopsy usually Combining extracorporeal antibody removal with eculizumab is the most common to be used with optimization of standard immunosuppressive medications.

DIAGNOSIS AND TREATMENT OF CHRONIC ANTIBODY-MEDIATED REJECTION

Statements of Recommendation We recommend that:
• The diagnosis of chronic antibody-mediated rejection (cAMR) is made on renal allograft biopsy. (1C)
• Patients with histological changes consistent with cAMR are screened for the presence of DSA. (1C)
• In ABOi transplantation, the risks of late AMR related to blood group antibodies are very low. If there is a suspicion of AMR, the patient’s current HLA antibody status should be checked. (1C)
• Other causes of ‘glomerular double contours’ are excluded. (1C) We suggest that
• In order to prevent cAMR, there is no evidence that maintenance immunosuppression need be more intense than for standard transplants, but there should be careful attention to advising and supervising adherence to care. (2C)
• Immunosuppressive agents used for the treatment of acute AMR may be considered for the treatment of cAMR in the presence of coexisting acute features of AMR. (Not graded)
A number of studies are being undertaken in this field as outlined previously.
The RituxiCAN-C4 study aims to assess if rituximab can improve allograft function and reduce proteinuria in renal transplant recipients with chronic AMR.
The TRIBUTE study is an efficacy study that aims to assess the benefit of bortezomib in conjunction with plasmapheresis and IVIg in preventing the progression of the histological features of chronic AMR. There is an ongoing study assessing the use of eculizumab in the treatment of chronic complement-mediated injury of renal allografts. In the absence of strong evidence, we suggest that patients be maintained on triple immunosuppression including tacrolimus and mycophenolate. Additional measures to reduce proteinuria, including blockade of the rennin-angiotensin system, should also be taken.