Application of H&I Laboratory Techniques – Histocompatibility & Immunogenetics

Pre-Transplant

Complex Unacceptable Antibody Listing

DQA antibodies cannot be listed as unacceptable with ODT but do cause a positive crossmatch and can lead to rejection. They therefore need to be taken into account when listing unacceptable mismatches and when accepting a kidney for a patient.

If an antibody can be confidently assigned to DQA i.e. all the DQA beads in the Luminex assay are positive and one or more of the non-masked DQB beads are Negative, then the DQB is not listed as unacceptable but would be reviewed at the time of a donor offer. For some patients, it may be possible to list a particular DQB as unacceptable in order to avoid donor offers with a particular DQA type e.g. It is possible to list DQB*05 as unacceptable to avoid offers from DQA*01 donors. It partly depends on the MFI and the risk of a positive crossmatch. A risk assessment is carried out at the time of a donor offer to assess whether or not the crossmatch is likely to be positive. For this, the donor DQA type would need to be known.

Managing Highly Sensitised Patients

Highly sensitised patients should be maintained on the waiting list as even a small chance of a deceased donor offer is better than no chance. Options to increase the chances of the patient getting a deceased kidney donor offer include:

Repeating the HLA antibody in case there have been any changes
Consider de-listing any unacceptable mismatches that have not been detected in the last 2 years and that are not repeat transplant mismatches
Review the Luminex MFI cut-off for listing unacceptable mismatches for this patient
If CDC screening has not been undertaken, do CDC to identify Luminex Pos., CDC Neg. specificities which could potentially be removed from the list of unacceptable mismatches
HLA antibody screening titres may be undertaken to determine which specificities titre out
Third party crossmatches may be undertaken to help predict crossmatch outcome

In addition to keeping the patient on the deceased donor waiting list, if the patient has a live donor, consider a live donor workup. If any potential live donors are HLA and or ABO incompatible but otherwise willing to donate, enter the patient and their donors into the paired/pooled exchange for at least two rounds.

If no offer is made through the paired/pool exchange then depending on the expertise in the local unit, the patient can be considered for antibody removal as part of an ABOi or HLAi transplant. Options for antibody removal include:

High dose IVIg. This will require regular, perhaps monthly HLA antibody removal
Plasma-exchange/Apheresis filtration. This will require HLA antibody screening titre to predict the number of rounds of antibody removal that may be required, together with pre and post antibody removal cycle tests

At Time of Transplant

Factors that can Affect the Crossmatch Results

Antibodies to Denatured Antigens on Beads

Although it is possible to have single Luminex beads reacting with very high MFI, these can also be denatured antibodies or non-specific binding to the beads, especially if the FCXM is negative.

In a live donor situation there is often time to investigate denatured antibodies either in your own lab or by sending the sample away to a lab with validated denatured antibody testing protocols. Acid treatment to strip the Luminex beads of bound antigen should result in a negative Luminex test if the antibodies are real HLA antibodies. If however the antibodies are binding non-specifically to the beads then acid treatment will not significantly reduce the MFI of the supposed DSA bead.

Where non-specific binding is confirmed and a repeat of the FCXM remains negative then it is safe to proceed to transplant.

Pan Reactive Luminex Beads

Occasionally, the Luminex antibody test can be pan reactive. Possible steps that can be taken in these situations include:

Repeat the Luminex screen to see if there are been an error
If Luminex ID beads available, test with those as well
I would also test with the alternate Luminex kit to see if there is a kit specific issue
If denatured bead protocol available would test to see if these antibodies are real
If C3d/C1q available to lab would test with those or
I would do CDC screening if Luminex Class I to see if these antibodies are cytotoxic
I would do high res typing of the patient to see if they have null or low expression at this locus to explain the antibody formation pattern
For completeness would do auto-crossmatch to see if negative
Would titre the antibody screening to see if any specificities titre out
Would do third party crossmatch to determine any specificities are crossmatch compatible
Ultimately would only transplant as part of a high risk immunosuppression protocol with post-transplant antibody monitoring

Prospective Pre-Transplant Test Decision Making

Antibody Screening Results: No detectable antibodies

Transplant can proceed without further pre-transplant testing provided this result has been seen on at least two samples bled at different times, the last sample tested was bled within the last three months and it is confirmed that patient has not had any sensitising events since the last sample was bled. Samples must be collected pre-transplant for retrospective crossmatching though these may potentially be stored and tested only if the patient is having issues

Antibody Screening Results: CDC IgM low titre Pos., IgG Neg, Luminex Neg.

Transplant can proceed without further pre-transplant testing provided the last sample tested was bled within the last three months and it is confirmed that patient has not had any sensitising events since the last sample was bled. Samples must be collected pre-transplant for retrospective crossmatching though these may potentially be stored and tested only if the patient is having issues

Antibody Screening Results: CDC Neg, Luminex Non DSA weak Pos. with peak MFI below 2000

Antibody Screening Results: CDC Neg, Luminex Non DSA Pos. with peak MFI above 2000 and less than 5000 but Luminex specificity cRF <75%

Minimum of pre-transplant SAB testing required assuming there have been no sensitising events since the last sample was bled. If DSA’s remain negative and MFI levels of non DSA have not changed significantly transplant can proceed without a wet pre-transplant crossmatch. Samples must be collected pre-transplant for retrospective wet crossmatching though these may potentially be stored and tested only if the patient is having issues

Antibody Screening Results: CDC high titre IgM Pos., IgG Neg, Luminex Neg.

Minimum of pre-transplant SAB testing required. If patient remains Luminex SAB Neg. then transplant can proceed without a wet pre-transplant crossmatch. Samples must be collected pre-transplant for retrospective crossmatching though these may potentially be stored and tested only if the patient is having issues

Antibody Screening Results: CDC Neg/NT, Luminex DSA weak Pos. (below 2000)

Minimum of pre-transplant SAB testing required. If patient remains Luminex SAB Weak Pos. then transplant can proceed without a wet pre-transplant crossmatch. Samples must be collected pre-transplant for retrospective crossmatching

Antibody Screening Results: CDC Neg/NT, Luminex DSA Pos. (above 2000)

Minimum of pre-transplant Flowcytometric crossmatch required. A CDC crossmatch may also be undertaken if Luminex SAB peak MFI > 5000 provided Renal Unit are happy to proceed to transplant with a CDC Neg. Flow Pos. crossmatch

Antibody Screening Results: Re-transplant patient who is CDC and Luminex Neg.

If full antibody history is available and patient has remained Neg throughout then minimum of pre-transplant SAB testing required. If patient remains Luminex SAB Neg. then transplant can proceed without a wet pre-transplant crossmatch. If there are repeat mismatches these must be highlighted to the renal unit. Samples must be collected pre-transplant for retrospective crossmatching though these may potentially be stored and tested only if the patient is having issues

Antibody Screening Results: Re-transplant patient who is CDC Neg and Luminex Pos.

Minimum of pre-transplant Flowcytometric crossmatch required. A CDC crossmatch may also be undertaken if Luminex SAB peak MFI > 5000. If there are repeat mismatches these must be highlighted to the renal unit. If the repeat mismatches are DSA this raises the risk level of the transplant. Interpretation of the crossmatch results must take into account any current immunosuppression and whether or not the previous graft is still in-situ

Antibody Screening Results: CDC Neg, Luminex Non DSA Pos. with peak MFI above 5000 and Luminex specificity cRF >75%

Minimum of pre-transplant Flowcytometric crossmatch required. A CDC crossmatch may also be undertaken if Luminex SAB peak MFI > 5000.

Antibody Screening Results: CDC Pos.

All patients who are CDC screen Pos. require a CDC crossmatch. A Flowcytometric crossmatch may also be carried out at the same time to help inform the immunosuppression strategy. If the CDC is negative but the Flow is positive, the transplant may still be able to proceed depending on the MCS/RMF provided the patient is able to tolerate enhanced immunosuppression. In such circumstance, if possible, a pre-transplant plasma exchange may be undertaken followed by close post-transplant antibody monitoring. If DSA antibody levels are rising post-transplant plasma exchange can be undertaken

Crossmatch Serum Selection

One possible policy is to select all samples from the last 12 months, the peak cRF% sample if older than the last 12 months and any samples collected post potential sensitising events if prior to the last 12 months.

Crossmatch results and immunological risk

Patel and Terasaki showed in 1969 that a positive cytotoxic crossmatch between a patient and donor due to pre-formed donor specific HLA antibodies (DSA) was associated with hyperacute rejection in kidney transplantation. This lead to a positive cytotoxic crossmatch becoming an absolute contraindication to transplantation in the absence of antibody removal. More recently however, improvements in immunosuppression and antibody definition have lead to a shift from a simplistic Pos/Neg. veto on transplantation to one of an assessment of the immunological risk posed.

HLA antibodies are known to contribute to all categories of rejection, including the Hyperacute, Accelerated, Acute and Chronic phases. Hyperacute rejection occurs within minutes and is due to pre-formed DSA present at the time of transplantation. Accelerated rejection takes place within days and is usually due to a memory or anamnestic response to pre-formed DSA which are absent at the time of transplantation. Acute rejection occurs within days or weeks and is usually due to de-novo DSA. Terasaki and others have also demonstrated a role for DSA in chronic rejection, though the timing of antibody formation and chronic rejection do not always coincide.

Consideration of the immunological risk associated with kidney transplantation needs to take into account the timing, duration, priming source, titre and specificity of any DSA. Antibodies formed as a result of transfusion are often IgM and IgG and may not be long lasting. Antibodies formed after transplantation or pregnancy are often IgG and involve immunological memory.

Gebel et al have proposed three levels of immunological risk associated with kidney transplantation, High, Intermediate and Low. In the UK, these categories have been incorporated into the BHSI/BTS guidelines on clinically relevant alloantibodies. Though the guidelines deal mainly with anti-AHLA antibodies, other antibodies such as anti-ABO and anti-endothelial cell antibodies are also relevant.

High immunological risk is indicated when the patient has circulating antibodies specific for mismatched donors HLA antigens. An example is a T + B cell Complement Dependent Cytotoxicity (CDC) current sample positive crossmatch in the presence of HLA class I DSA specific for the mismatched donor antigen. This carries a high risk of Hyperacute rejection in the absence of desensitisation. Another example of a High risk transplant is a B cell CDC current sample positive crossmatch in the presence of HLA class II DSA specific for the mismatched donor antigen. Transplants can also be high risk if only the historical sample is positive. For example a T + B cell CDC historical sample positive, current sample negative crossmatch in the presence of HLA class I DSA specific for the mismatched donor antigen or a B cell only CDC historical sample positive, current sample negative crossmatch in the presence of HLA class II DSA specific for the mismatched donor antigen are both High risk. Both have the potential to trigger an anamnestic or memory T and/or B cell response resulting in Accelerated rejection. In most cases, High risk transplants would be avoided unless as part of a validated desensitisation program.

Intermediate immunological risk is indicated by a flowcytometry positive, CDC negative crossmatch result with both current and historical samples in the presence of HLA class I DSA. Examples include T + B cell flow Pos, CDC Neg. current and historical samples in the presence of HLA class I DSA specific for the mismatched donor antigen.

Another example of an Intermediate risk transplant is a B cell only positive CDC current and historical sample in the presence of weak IgG HLA class I DSA specific for the mismatched donor antigen.

With Intermediate risk transplants, a clinical assessment is needed on the relative risk of proceeding to transplant with augmented immunosuppression and close post transplant monitoring versus remaining on the waiting list in the hope of obtaining a better match or taking part in a paired exchange if a live donor is available. Patients do die on the waiting list. This decision will be influenced by the experience of the transplant team and the H&I laboratory when it comes to monitoring and managing such patients, as well as the length of time the patient has already been on the waiting list, the CRF and the matchability of the patient.

Low immunological risk is indicated when the crossmatch result is negative and the patient is unsensitised. Low immunological risk is also indicated when the crossmatch result is negative, even if the patient is sensitised, provided the sensitisation is clearly shown to IgM only, is clearly shown to be Auto only or is otherwise considered to be non HLA.

Low immunological risk is also indicated when the crossmatch result is negative and any HLA antibodies present in the current and historical samples of the patient are not donor specific. In addition, Low immunological risk is also indicated when the crossmatch result is negative and HLA class I or II IgG DSA are detected in either the current or historical samples of the patient, provided these are detectable by Luminex SABs only.

Finally Low immunological risk is indicated when the crossmatch result is positive but the patient is either unsensitised or any HLA antibodies present in the current and historical samples are not donor specific.

Assessment of the immonological risks associated with a transplant requires close co-operation between the H&I laboratory and the transplant team. The lab should be informed of all potential sensitising event, including previous transplants, skin grafts, transfusion, pregnancies, miscarriages if known and recent infections or vaccinations. The laboratory crossmatch report to the transplant team should include appropriate advice on the clinical relevance of the results.

Complex Crossmatch Interpretation

Crossmatch results, especially FCXM results, do not always correlate linearly with MFI, especially when reviewed across different patients. However in general, presence of high MFI DSA should be predictive of a positive crossmatch and absence of DSA should be predictive of a Negative crossmatch.

If a crossmatch is Positive in the absence of DSA, potential reasons for the positive reaction would need to be investigated. Potential laboratory issues such as sample mix up and technical problems would need to be ruled out. If in doubt, the crossmatch would need to be repeated.

Clinical reasons such as patient underlying condition and treatment should also be investigated. Patients with autoimmune conditions often have a B Cell Positive crossmatch due to autoantibodies. If an auto crossmatch has not been set up this can be done to rule out auto antibodies. If the patient is on rituximab this can cause a B cell positive crossmatch. ATG can cause a positive T cell crossmatch.

Upon full investigation, if the crossmatch is still positive in the absence of DSA and auto antibodies and patient treatment have been ruled out then the positive reaction can be attributed to non-HLA and is not a contraindication to transplant. Post-transplant monitoring would be required.

If a crossmatch is unexpectedly Negative in the presence of high titre DSA, then like the situation where an unexpected positive reaction is seen in the absence of DSA, the potential reasons for the Negative reaction would need to be investigated. The patient antibody profile and donor type would need to be carefully reviewed to determine if the antibody is truly DSA. For instance are all the Luminex beads for that specificity actually positive? Could this be an antibody to an allele not expressed in the donor? Or has an antibody been assigned to DQB that is actually a DQA antibody and is therefore not actually DSA? Similarly, is an antibody actually to DPA but has been assigned to DPB?

Potential laboratory issues such as sample mix up and technical problems would need to be ruled out. If in doubt, the crossmatch would need to be repeated.

Upon full investigation, if the crossmatch is still negative, especially by CDC then it is safe to proceed with the transplant. Close post-transplant monitoring will be required.

Relevance of High MFI levels for DP antibodies

The decision to proceed to transplant or not would always be made in consultation with the renal unit, taking into account factors such as first or second transplant, is the DP DSA a repeat mismatch, the level of matching of the kidney at all other loci, the likelihood of the patient getting another offer that was otherwise as well matched, the CDC and Flowcytometric crossmatch results, the MFI level of the DP DSA and whether or not the patient was able to tolerate enhanced immunosuppression.

If this patient is going for first transplant or the DP DSA is not a repeat mismatch, the transplant can proceed if the CDC crossmatch is negative. If the CDC is negative but the Flow is positive, the transplant may still be able to proceed depending on the MCS/RMF provided the patient is able to tolerate enhanced immunosuppression. If possible, a pre-transplant plasma exchange may be undertaken followed by close post-transplant antibody monitoring. If DSA antibody levels are rising post-transplant plasma exchange can be undertaken.

Crossmatch Result Interpretation

CDC current and historic T Cell Pos B Cell Pos, FXCM current and historic T Cell Pos B Cell Pos, Luminex class I DSA in current and historic samples

Very High Risk – Transplant veto

CDC current and historic T Cell Neg B Cell Pos, FXCM current and historic T Cell Pos B Cell Pos, Luminex class II DSA in current and historic samples

High Risk

CDC current and historic T Cell Neg B Cell Neg, FXCM T Cell Pos B Cell Pos, Luminex class I DSA in current and historic samples

Intermediate Risk

CDC current and historic T Cell Neg B Cell Neg, FXCM T Cell Neg B Cell Pos, Luminex class II DSA in current and historic samples

intermediate Risk

CDC T Cell Neg B Cell Pos, FXCM NT, Luminex weak class I DSA in current and historic samples

High Risk

CDC Neg, FCXM T Cell Neg B Cell Neg, Luminex class I and/or II DSA Pos in current and historic samples, CDC screen Neg

Standard Risk – None complement fixing antibody

FCXM T Cell Neg B Cell Pos, CDC NT, Luminex class I and II DSA Neg

Standard Risk if results confirmed. Repeat to confirm. Test auto if not tested already

Factors which may Confound the Crossmatch Results

Goodpasture, Lupus and patients with some other autoimmune conditions can have autoantibodies which can confound HLA crossmatch tests. In addition, some of these patients are treated with plasmapheresis, cyclophosphamide or Azathioprine, Rituximab. There are a number of other clinical conditions and treatment which may also confound the crossmatch results.

Interpretation of crossmatch results in such patients can be complex. It may be necessary to repeat some unexpected positive crossmatch results to confirm and to review the HLA antibodies and potentially test with an alternative Luminex kit. Each H&I laboratory needs to evaluate the DSA levels which correspond with a positive crossmatch in their hands but typically, depending on the kit used, the flow would not be expected to be positive due to HLA DSA of below 2000 MFI.

A flow Pos. crossmatch in the absence of HLA DSA is considered to be standard risk according to BSHI/BTS guidelines and is not in of itself a contraindication to transplant. However, the positive reaction would ideally need to be investigated before proceeding with a transplant.

To investigate the positive flow result further, the flow crossmatch may need to be repeated and an auto crossmatch set up to determine if the reactivity is due to autoantibodies.

If autoantibodies and treatment are ruled out as potential causes of the positive flow the HLA DSA should be investigated further by for instance testing with an alternative Luminex antibody kit if not tested already and the patient should also be screened for non-HLA antibodies. Non-HLA antibodies, such as anti-angiotensin, anti-epithelial and anti HNA antibodies can cause a positive flow crossmatch and do contribute to rejection episodes post-transplant.

A CDC crossmatch may also be useful in determining the level of immunological risk. The ultimate decision to proceed or not will be made in consultation with the renal unit and as part of a high-risk strategy, taking into account clinical urgency.

ATG

Anti-Thymocyte Globulin (ATG) is an anti-human T cell antibody immunosuppressive used for transplant induction in highly sensitised patients and in the prevention and treatment of acute rejection in kidney and cardiac transplantation. Use of ATG in a patient pre-transplant will cause a Positive T Cell Crossmatch.

Rituximab

Rituximab is an anti-CD20 recombinant chimeric murine-human monoclonal antibody. It binds to CD-20 which is expressed on precursors and mature B cells but not on plasma cells. Binding triggers a series of cytotoxic immune response resulting in the elimination of B cells and the reduction in antibody formation. Use of Rituximab means that crossmatches need to be interpreted with care. The anti-CD20 means that the B cell CDC and flowcytometric crossmatch will likely be positive. It can also potentially reduce the number of B cells available in the crossmatch.

IVIg

The exact mechanism of action of IVIg is unknown but the 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 induction of apoptosis in B cells. IVIg is derived from pooled human plasma and can therefore cause a positive crossmatch if administered before crossmatch sample is taken.

Cyclophosphamide

Cyclophosphamide, an alkylating agent, is one of the most efficacious immunosuppressive drugs available. Cyclophosphamide is used for treatment of autoimmune disorders such as SLE and multiple sclerosis. Treatment with Cyclophosphamide can cause a loss of B cells which makes B cell auto crossmatching impossible.

Transplanting across a Positive Flowcytometric Crossmatch

A B Cell Pos. Flow crossmatch in the presence of HLA class II DSA in the current or historic sera would be considered an intermediate risk. Further testing such as auto-crossmatch may be required if not done already to eliminate auto antibodies and if not done yet, a Luminex SAB test would be carried out in the B cell weak Pos. sample to determine if the flow reactivity was due to HLA.

Enhanced immunosuppression and post-transplant antibody monitoring would be recommended to identify and potential changes in DSA. Frequency of testing could be 7, 14 and 28 days post-transplant, followed by 3, 6, 9 and 12 monthly testing if stable and also testing following any rejection episodes.

Marginal Deceased Donor offer for Patient with Live Donor

Patients being worked up for a live donor transplant are suspended from the ODT waiting list 1 month before the transplant to avoid this scenario. Ultimately the decision is made by the patient after full discussion with the clinician. It partly depends on an assessment of the offered deceased kidney in terms of DBD/DCD, donor age, level of match etc. Use of the deceased donor avoids an operation on the live donor and that live donor can still be kept in reserve for a future date.

Problems on Call

The main role of the consultant clinical scientist on call is to provide support to the state registered scientists who man the on-call rota, providing advice with interpretation of any complex results. They may also be required to discuss options and provide advice to the transplant team depending on the results.

Staff on the on call rota would normally be expected to be able to correctly select the sera to crossmatch based on written policies and to be able to interpret and report straightforward results but they know they have the assurance of a consultant clinical scientist on the other end of the phone should they need them.

Post-Transplant

Passenger Lymphocytes and kidney transplantation

Passenger lymphocytes are donor derived lymphocytes that are transferred into the patient during solid organ transplant. Passenger lymphocytes can cause conditions such as severe haemolytic anaemia in minor ABO mismatched transplantation in a condition known as Passenger lymphocytes syndrome(PLS) and Solid Organ associated GvHD.

In PLS, transplant of an O+ kidney into an AB+ recipient for example represents a minor ABO mismatch. Donor derived Passenger lymphocytes by be present and may produce anti-ABO antibodies directed at the patient’s AB+ red blood cells and can lead to sever haemolytic anaemia.

The condition is often self limiting after a few week but can be supported with blood transfusion if required. In severe cases, use of Ritiximab may be indicated.

In SO-GvHD, passenger lymphocytes from the transplanted organ, mismatched with the patient in the GvH direction but matched in the HvG direction may engraft and lead to GvHD which is often difficult to control as there is bone marrow involvement.

Biological Processes Involved in Cold Ischaemia and Reperfusion Injury

An extended period of cold ischaemia time (over 21hrs) is associated with delayed graft function (DGF). This is as a result of varying degrees of cold ischaemia and reperfusion injury despite improvements in hypothermic preservation solutions that have been developed to maintain tissue viability and reduce the accumulation of toxic substances.

A prolonged cold ischaemia time followed by reperfusion leads to the production of reactive oxygen species which induce adhesion molecule expression and increased production of cytokines, resulting in infiltration of neutrophils, followed by mononuclear cell infiltration and the upregulation of HLA antigens, all contributing to an inflammatory response which causes acute tubular necrosis.

Steps that can be taken to reduce cold ischaemia time include detailed and regular three monthly solid phase HLA antibody testing such as by Luminex to provide a detailed antibody history that would allow some carefully selected patients to proceed to transplant on the basis of a virtual pre-transplant crossmatch, with a retrospective crossmatch. For patients where a wet pre-transplant crossmatch is indicated, it is possible to request peripheral blood from the donor ahead of organ retrieval to allow a crossmatch can be performed and the cold ischaemia time kept to a minimum.

Post Solid Organ Transplant Monitoring

Solid organ transplantation has become an established therapy for the treatment of organ failure. Early allograft survival has improved dramatically over the last 3 decades with improvements in surgical and laboratory techniques as well as improvements in immunosuppressive regimes such as the use of the calcineurin inhibitors (CNI) Cyclosporin A and later Tacrolimus and the introduction of Sirolimus. Late graft loss however remains fairly static with 10 year graft survival at around 50% for kidneys and even lower for other organs.

Various techniques exist for monitoring graft function post transplant for signs non function and rejection, some of which do not typically involve the H&I laboratory. In the case of kidney transplantation, these include monitoring of serum creatinine levels for signs of a rise. This may be accompanied by monitoring of other signs of kidney dysfunction such as haematuria and proteinuria levels. The results of these may indicate a need for a biopsy. Biopsy signs of rejection include amongst other things, interstitial lymphocytic infiltration, interstitial fibrosis, tubulitis, tubular atrophy, vascular occlusion, thickening of glomerular capillaries, expansion and duplication of the glomerular basement membrane and C4d deposition. Other assays undertaken to assess renal function post-transplant include monitoring levels of cytotoxic T lymphocyte transcripts such as granzyme B and perforin.

The role of the H&I laboratory in post kidney transplant monitoring mainly involves testing for HLA antibodies post transplant but may also include testing for other antibodies such as anti-MICA or anti-endothelial cell antibodies. HLA antibodies have been implicated in all stages of allograft rejection from the hyperacute stage, to the acute and chronic phases. Hyperacute rejection occurs within minutes of transplant and is usually due to the presence of pre-formed donor specific antibodies. Acute rejection occurs within days to weeks, while chronic antibody mediated rejection occurs months to years post transplant. Recent data from the 14th international histocompatibility workshop demonstrated that four year deceased donor kidney allograft survival was 20% less in patients with donor specific antibodies compared to donors with no antibodies. Care is however needed as the time between detection of circulating donor specific antibodies and allograft failure does vary considerable in different studies, with some grafts functioning normally many years post the detection of DSA. Patients with non donor specific HLA antibodies also had a significant reduction in graft survival compared to non sensitised patients. In other studies, patients with HLA and MICA antibodies alone were shown to have significantly reduced allograft survival compared antibody negative patients. Patients with MICA antibodies alone also had reduced allograft survival though the effect was less than in patients with HLA antibodies alone.

The BSHI/BTS 2010 guidelines recommend that post-transplant antibody monitoring should be performed at agreed regular intervals, at the time of biopsy and in cases of suspected rejection. Antibody testing should also be undertaken at times of declining graft function when there is no other clinical cause. The guidelines do not however recommend a testing frequency.

The testing frequency is agreed between the H&I laboratory and the transplant centre. One example is to test at 1 week post transplant, then again at 1, 3, 6 and 12 months post transplant, then annually. Some labs collect samples at three monthly intervals as in the pre-transplant setting but store these rather than routinely test them unless the patient experiences declining renal function. This strategy allows a retrospective review of the patients post transplant antibody history should rejection be suspected without the costs of routine and regular screening of all post transplant samples. For patients who undergo HLA antibody incompatible transplantation, the BTS/BSHI guidelines recommend a post transplant testing frequency that matches the risk of adverse immunological events, especially for those patients who have antibody removal prior to transplant. The testing frequency for antibody incompatible transplantation with antibody removal in some protocols is daily for the first week, followed by weekly for the first month and monthly thereafter.

Post-transplant antibody testing in the UK is mainly carried out by Luminex solid phase assays. Any of the three types of Luminex assays may be used depending on the clinical scenario. Routine screening in suspected cases of rejection may use the mixed bead screen method or the phenotype panel system, with specificity confirmed by single antigen bead assays as required. Desensitisation programs will likely use single antigen bead methods. The Luminex solid phase assays have the advantage of also giving results for anti MIC antibodies.

Circulating donor specific antibodies together with C4d+ staining in biopsy in the presence of declining graft function indicate antibody mediated rejection. Early diagnosis is required if the graft is to be salvaged. Treatment options include removal of antibody by plasmapheresis, use of IvIg, use of anti-proliferative agents and Rituximab.

The H&I laboratory may also be involved in testing for antibodies to organ specific markers in other solid organ transplantation. Antibodies directed against the endothelial cell antigen vimentin for instance have been shown to be associated with the development of transplant associated vasculopathy in cardiac transplantation.