The median waiting time on the kidney waiting list in the UK is just over 3 years. Highly sensitised patients (calculated PRA > 85%) tend to wait on average much longer. Strategies that have been used in the UK to increase the number of transplants in this group have included the prioritising of such patients for well matched kidneys based on the listing of all unacceptable mismatches, the increasing use of marginal donors to increase the number of transplants in all patients on the waiting list and more recently the use of paired exchange donations. Whilst these strategies have been useful, they do not always benefit the very highly sensitised patients. In addition, even patients who are not highly sensitised but who have HLA antibodies against or are ABO incompatible with their live donor can fail to benefit from these schemes depending on how rare their HLA types are and what their blood groups are. Such antibody incompatible transplants can be undertaken with suitable desensitisation protocols.
Antibody incompatible transplantation refers to the transplantation of patient across the previously insurmountable ABO and HLA barriers. Normally, blood group incompatible transplantation is precluded by the presence of blood group isohaemaglutinin in the blood of the recipient, leading to hyperacute rejection and allograft loss. From the HLA viewpoint, Patel and Terasaki demonstrated in 1969 that hyperacute rejection can result from allograft injury caused by preformed donor specific anti-HLA antibodies (DSA), making a positive cytotoxic HLA crossmatch a contraindication to transplant. It has however been shown as long ago as the 1980’s that it is possible to transplant across an ABO incompatibility by antibody removal prior to transplant with many such transplants now performed in Japan in particular but also in other countries. Similarly, removal of donor specific HLA antibodies to a point resulting in a negative crossmatch has been shown to avoid hyperacute rejection and permit HLA incompatible transplantation. Removal of ABO and HLA antibodies to permit antibody incompatible transplantation has come to be called desensitisation.
Careful patient selection for desensitisation is an important aspect of any antibody incompatible transplant program. Patients must be able to withstand whole volume plasma exchange, in some protocols multiple times. In additional, patients must be assessed for the likelihood of a successful desensitisation and for protocols involving plasmapheresis, an estimation of the number of treatments required to achieve a negative crossmatch. For HLA incompatible transplantation, this requires knowledge of the antibody specificity and strength. Antibodies to HLA DR51, 52 and 53 for instance often prove difficult to remove. Some transplant centres will for instance only include patients awaiting first transplant whilst other may accept re-transplant patients but avoid previous HLA mismatches. HLA antibody screening and identification has historically been by cell based techniques such as the Complement Dependent Cytotoxicity (CDC) assay or by cell based flowcytometric assays. Currently however, most HLA antibody testing in the UK is by the solid phase Luminex bead based assays. Use of Luminex assays has revolutionised HLA antibody investigation. The two main advantages are the speed with which tests can be turned around, and the sensitivity and specificity of the results obtained. When using single antigen bead assays, for each antibody detected, a Mean Fluorescence Intensity (MFI) value is obtained which provides a measure of the strength of the antibody. Studies have found good correlation between the MFI values obtained in Luminex and flowcytometric crossmatch results, however correlation with CDC results is not as good. Use of Luminex and other solid phase assays as part of the desensitisation protocols provides for precise characterisation of DSA and is one of the main reasons behind the success of antibody incompatible transplantation.
Early desensitisation protocols for HLA antibodies included the use of splenectomy. Typical protocols included plasmapheresis and IvIg with anti-CD20 and splenectomy. Most patents on this protocol did achieve a negative crossmatch. However patients on the same protocol without the splenectomy were later shown to achieve the same results. One protocol that is currently widely used for HLA desensitisation is high dose IvIg, typically given over the course of several months until a negative crossmatch is achieved. A common dose is 1g/kg patient weight. The exact mechanism of action of high dose IvIg is unknown. Proposed mechanisms include an anti-idiotypic effect of IvIg on HLA antibodies, saturation of the neonatal Fc receptor which would normally protect endogenous HLA IgG molecules and high dose IvIg is proposed to induce apoptosis in B cells. One disadvantage of high dose IvIg protocols is that they have been associated with thrombotic complications.
Another popular protocol is plasma exchange, with or without low doses of IvIg. Plasma exchange protocols aim to physically remove HLA antibodies to a negative crossmatch before transplantation. Some plasma exchange protocols also incorporate antithymocyte globulin (ATG) and anti-CD20 antibodies such as Rituximab. The use of Rituximab is controversial as it removes B cells but not plasma cells. For this reason, some transplant centres are evaluating the use of Bortezomib, which has anti plasma cell activity. Depending on the strength of the DSA present, plasma exchange protocol can involve several sessions of whole volume exchanges. HLA antibody removal is effective with the most commonly reported side effect being hypocalcaemia. Antibody removal by these protocols is however not specific to HLA IgG, removing other protective antibodies and clotting factor, potentially increasing the risk of infection or bleeding. This risk is reduced by the use of low dose IvIg and by transfusion if required.
A newer approach to desensitisation is the use of filtration or immunoadsorption. Immunoadsorption involves an apheresis procedure in which the patients’ blood is ‘filtered’ by passing it through a column containing beads coated with an IgG absorber such as Protein A which selectively filters out IgG molecules. This has few of the side effects of traditional plasma exchange. A single filtration session typically reduces HLA antibody MFI levels to half their pre filtration values. Several rounds of filtration are required to reduce antibody levels to achieve a negative crossmatch. HLA antibody levels rebound to some degree after each session as antibodies equilibrate between intravascular and extravascular spaces.
ABO desensitisation is by plasma exchange or by immunoadsorption. As in plasma exchange for HLA antibody removal, plasma exchange for ABO is also not specific to ABO antibodies, removing other protective antibodies and clotting factor and requires administration of IvIg in some protocols. Immunoadsorption for ABO desensitisation uses columns with a matrix of sepharose beads coated with blood group A or B carbohydrate antigens, typically in a double filtration configuration. As blood runs over the column, the blood type specific column removes isohaemaglutinin against the appropriate blood group. The column removes approximately 30% of anti-A or anti-B antibodies with each treatment. The columns are expensive but can be regenerated and reused for the same patient. Depending on the protocol in use, patients can proceed to transplant with a residual ABO antibody titre of 1:8 to 1:32.
H&I laboratories provide support for the HLA aspects of antibody incompatible transplant programs. Laboratory support for these programs can be grouped into support provided as part of the assessment and pre-treatment work up, support provided for treatment monitoring including the final pre-transplant crossmatch and support provided as part of the post transplant monitoring.
H&I laboratory support provided as part of the pre-treatment work up includes HLA typing and antibody screening and identification. Currently in the UK HLA typing is performed mainly by DNA based techniques. It is recommended that HLA typing is performed twice to confirm the type. Common HLA typing techniques include the polymerase chain reaction sequence specific primers (PCR-SSP) and polymerase chain reaction sequence specific oligonucleotide (PCR-SSO) techniques. HLA typing is carried out at HLA-A, B, C, DR (including DR51, 52 and 53) and DQ. HLA-DP and DQA typing are not generally undertaken unless the patient has HLA antibodies to DP and DQA. HLA antibody screening and identification is performed by solid phase assays such as Luminex single antigen beads (SAB) which give both the specificity and strength (MFI) for each antibody detected. H&I laboratories must be able to identify antibodies specific for HLA-A, B, C, DR, DQ and DP. In addition, the testing must be able to distinguish IgG allo and IgM allo and auto antibodies. It is particularly important to identify the MFI of DSA. Various studies have been conducted into the MFI value that correlates with a positive flowcytometric crossmatch with reports of positive CDC and flowcytometric crossmatch when MFI value are anywhere between 6000 and 10000. This inter laboratory variation makes it important for each laboratory to establish the MFI values that correlate with positive crossmatch in their hands.
The support provided by the laboratory as part of the treatment monitoring depends on the protocol in use. High dose IvIg protocols involves fewer, infrequent treatments whilst plasma exchange and filtration protocols may involve daily or alternate day treatment with HLA antibody testing before and after each treatment. This allows for an evaluation of the effectiveness of antibody reduction of each treatment, it allows an assessment of the antibody rebound between treatments and it helps to determine the number of additional or supplementary treatments (such as increased immunosuppression) that may be needed. The criteria for proceeding to transplant varies between transplant centres. Some will only proceed to transplant on the basis of a negative CDC and flowcytometric crossmatch, whilst others may proceed on the basis of a negative CDC, pos flowcytometric crossmatch provided the mean channel shift is below an acceptable level. The use of various immunosuppressive drugs such as Rituximab and alemtuzumab as part of the treatment protocol as well as the timing of the treatment and the availability of sample may make an actual crossmatch logistically difficult and some centres may proceed to transplant on the basis of a negative virtual crossmatch.
With high dose IvIg protocols, post transplant antibody monitoring is typically weekly for the first month, then monthly. For plasma exchange and filtration protocols, antibody monitoring is more frequent. The Johns Hopkins protocol involves weekly monitoring for the first month, then at 2 and 3 months, then quarterly thereafter. However, depending on the patients post transplant course, initial monitoring can be daily for the first few days and weeks post transplant. Antibody levels can sometime rebound significantly post transplant in many cases often higher than the pre transplant levels. This does not necessarily imply imminent graft failure as many patients appear to accommodate the graft even in the presence of DSA. Treatment should therefore be on the basis of actual proven rejection rather than on antibody levels alone. Potential treatment options include post transplant plasma exchange or plasma filtration and enhance immunosuppression. Some protocols also treat with IvIg and Rituximab though the use of Rituximab is not universal.
Patients are able to maintain grafts in the presence of DSA though Accommodation, the phenomenon in which a graft functions normally by acquiring resistance to immune-mediated injury despite the presence of anti-graft antibodies in the recipient. Proposed mechanisms for the Accommodation include the expression in the graft of several protective genes which block the activation of the transcription factor NF-KB, thereby suppressing induction of proinflammatory genes and inhibition of the membrane attack complex thereby disrupting the action of complement.
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