Indian Journal of Transplantation

: 2020  |  Volume : 14  |  Issue : 2  |  Page : 90--93

Testing for donor-specific antibodies in renal transplantation: Indian perspective

Praveen Kumar Etta 
 Department of Nephrology and Renal Transplantation, Virinchi Hospitals and Max Superspeciality Medical Centre, Hyderabad, Telangana, India

Correspondence Address:
Dr. Praveen Kumar Etta
Department of Nephrology and Renal Transplantation, Virinchi Hospitals and Max Superspeciality Medical Centre, Hyderabad - 500 034, Telangana


The evaluation of donor-specific antibodies by Luminex single-antigen bead assay (in addition to other crossmatch tests) to assess pretransplant immunological risk should be performed in recipients (especially with a history of prior sensitization) even in resource-constrained settings as this approach can help in better risk stratification, to decide on transplant eligibility, selection of immunologically favorable donor, to plan desensitization protocol and induction therapy that can lead to the reduction of posttransplant rejection rates and better graft survival. Despite the cost, it is justified to use these sensitive assays in selected cases even in a cost-limited setting as this enables earlier and better-matched transplant, and avoidance of morbidity and poor graft survival.

How to cite this article:
Etta PK. Testing for donor-specific antibodies in renal transplantation: Indian perspective.Indian J Transplant 2020;14:90-93

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Etta PK. Testing for donor-specific antibodies in renal transplantation: Indian perspective. Indian J Transplant [serial online] 2020 [cited 2020 Oct 29 ];14:90-93
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Renal transplantation (RT) is the best modality of renal replacement therapy for most patients with end-stage renal disease as it offers survival and quality of life benefit over dialysis.[1] The presence of preformed anti-donor antibodies (anti-human leukocyte antigen [HLA], anti-ABO, or non-HLA antibodies) is associated with greater risk of rejection (hyperacute and accelerated acute rejections) early after transplant, which may require graft nephrectomy.[2] These types of rejections are now rarely seen due to better pretransplant immunologic evaluation, which includes various crossmatch (XM) tests and testing for donor-specific antibodies (DSA). These also help in the risk stratification of recipients, to decide on transplant eligibility, desensitization, and induction therapy. The antibody-mediated rejection (ABMR) plays an important role in both short and long-term graft loss.[3] Almost a third of patients who are waitlisted for RT may have a degree of anti-HLA antibodies detected. The recipients with a history of blood transfusions, multiple pregnancies, and previous transplants are at higher risk for the development of DSA.

 Human Leukocyte Antigen and Epitope Matching

The degree of HLA mismatch influences long-term graft survival and risk of rejection. Among DSA, anti-HLA antibodies play a major role in graft rejection and survival. HLA Class 1 antigens (A, B, and C) are expressed on all nucleated cells; the epitopes reside only in the polymorphic α-chain. HLA class 2 antigens (DR, DQ, and DP) are normally restricted to antigen-presenting cells (dendritic cells, B-cells, and macrophages), but they can be upregulated and expressed after inflammatory insults, such as ischemia-reperfusion injury, infection, and rejection. Each Class 2 antigen consists of one α-chain and one β-chain, and both chains are polymorphic. HLA typing and matching in relation to transplant immunology conventionally consider only antigens in HLA-A, B, and DR loci. Recent evidence has shown that antibodies against other HLA loci (especially HLA-DP and DQ antibodies, which mostly arise de novo) also play a role in rejection.[4],[5]

The understanding of the reactivity of antibodies with HLA molecules is progressing currently. An antibody molecule has a paratope consisting of six complementary determining regions, three on the heavy chain, and three on the light chain, which interacts with the HLA antigen. The footprints of antibodies on their target antigens have been studied. In most cases, the presence of the immunogenic epitope, alone or in combination with one or two other polymorphic sites (crucial amino acid configurations), can explain the antibody reactivity pattern with the different HLA alleles. Recently, epitope- and eplet- based matching has been shown to be more predictive of XM results and subsequent graft outcome compared to HLA molecule matching.[6] While some of these epitopes are unique to a single HLA (private or foreign epitopes), others are shared among numerous antigens (public epitopes). Some HLA mismatches have many epitopes not shared by the HLA antigens of the patient. These mismatches are likely to be more immunogenic compared to an HLA mismatch, which shares most epitopes with the patient. Therefore, novel matching strategies aim at the limitation of the number of foreign epitopes rather than the number of HLA antigen mismatches. Hence “epitope” matching is superior over HLA antigen matching with respect to the prevention of de novo DSA formation and will enhance the prediction of acceptable HLA mismatches for sensitized patients. The degree HLA mismatch, list of acceptable and unacceptable (”taboo”) mismatch antigens using HLA Matchmaker is a common practice in western protocols. Individuals alloimmunized by a specific HLA type can make antibodies to several other HLA types due to the presence of cross-reactive groups on HLA molecules.

 Testing for Donor-Specific Antibody

Currently, we have both conventional cell-based assays and more advanced solid-phase assays to identify the presence of DSA, and each test has its own advantages and limitations.[7] Complement-dependent cytotoxicity XM (CDC-XM) was pioneered by Terasaki et al. in the 1960s. If complement-fixing DSA (immunoglobulin [Ig] G1 or IgG3) are present and bind to donor cells, the complement cascade will be activated through the classical pathway resulting in lysis of the lymphocytes. The addition of antihuman globulin enhances the sensitivity of the assay by cross-linking the antibodies. The proportion of lysed cells is assessed, and the XM is graded. An auto-XM or a repeat assay with dithiothreitol is useful to identify false-positive assays. DSA that is not complement-fixing (IgG2 or IgG4) can give a false-negative result. Flow cytometry XM (FC-XM) involves adding recipient serum to donor lymphocytes and then incubating them with fluorescein-labeled antibodies against human IgG. The strength of the fluorescence can be measured and expressed as “channel shifts” above the control sample. FC-XM is more sensitive for detecting DSA compared with CDC-XM. FC-XM detects DSA independent of complement fixation. Positive FC-XM with negative CDC-XM is likely to be caused by a noncomplement fixing antibody or a low-level antibody. Pronase treatment is used to improve the sensitivity and specificity of the B-cell FC-XM, but this may prone to false-positive reactions in the T-cell FC-XM test. If the patient was treated with rituximab or anti-thymocyte globulin, the B-cell FC-XM will likely be false positive and requires pronase XM.[8] The solid-phase assay, Luminex technology, consists of a series of polystyrene microspheres (beads) coated with HLA antigens and containing fluorochromes of differing intensity embedded within the bead. Recipient serum potentially containing anti-HLA antibodies is added to a mixture of synthetic beads. Beads may be coated with multiple HLA antigens (screening beads) or a single HLA antigen (single antigen bead [SAB] assay) for defining the specificity of antibodies more precisely. Positive results can then be graded semiquantitatively on the basis of the degree of fluorescence of the positive bead (mean fluorescence index [MFI]).[9] SAB detects only anti-HLA DSA and not the non-HLA antibodies. False-positive or high titers may be reported due to the presence of antibodies to denatured HLA molecules. A recent Indian study compared the MFI values of DSA detected by SAB assay to that of cell-based CDC and FC-XM results and concluded that a cut-off MFI value of 3000 for Luminex SAB-based assay was found to significantly correlate with the FC-XM positivity. In contrast, an MFI value of 7000 and above predicted a positive CDC-XM.[10] Recent evidence has shown that assessment of intra-graft DSA is a valuable tool for diagnosing ABMR, even if serum DSA is negative.[11] Virtual XM (VXM) is an assessment of immunological compatibility based on the profile of anti-HLA antibodies of the recipient by Luminex SAB assay compared with the HLA of the donor. It is not precisely an XM in the sense of mixing serum and lymphocytes. A patient will not be offered a kidney from the deceased donor who expresses an unacceptable HLA antigen (positive VXM). For VXM, we need HLA typing of donors, which is rarely feasible for deceased donors in the Indian setting. In the panel reactive antibody test, the recipient's serum is tested for antibodies against a panel of lymphocytes/HLA antigens from blood donors of the local population. It estimates the likelihood of positive XM to potential donors and is extremely useful in providing information about the sensitization of a recipient.

 Testing in Indian Setting

Although the SAB assay has a very high sensitivity for antibody detection, the CDC-XM still remains the most commonly used test in developing nations like India owing to its easy availability and financial restraints.[12] The pretransplant evaluation of DSA by only CDC-XM is likely to be associated with higher rejection rates and poor graft survival, especially in high-risk sensitized recipients.[13] The Luminex SAB may be useful even in cost-limited settings due to better risk stratification before transplantation.[14],[15] In a study published in this issue, the authors reported the clinical outcomes of ten sensitized patients (with positive anti-HLA antibodies) who were stratified and managed based on the Luminex SAB method for DSA detection in a cost-limited set-up from India.[16] In five patients, cadaveric transplantation was performed using VXM for kidney allocation; in four recipients with high DSA on SAB but XM compatible donors, desensitization was performed before living donor transplantation; and for one live donor-recipient pair with XM incompatibility (positive CDC-XM), combined kidney paired exchange with desensitization was performed (against a swapped donor with a lesser degree of incompatibility). The use of SAB assay has resulted in successful allograft outcomes in all the ten high-risk patients during follow-up. Hence wherever feasible, the evaluation of DSA by Luminex SAB (in addition to other XM tests) to assess pretransplant immunological risk should be performed in recipients (especially high-risk cases such as child to mother or husband to wife transplants; and those with history of prior sensitization) even in resource-constrained settings as this approach can help in better risk stratification, to decide on transplant eligibility, selection of immunologically favorable donor, to plan desensitization protocol and induction therapy that can lead to the reduction of posttransplant rejection rates and better graft survival. Despite their cost, it is justified to use these sensitive assays in selected cases even in a cost-limited setting as this enables earlier and better-matched transplant, and avoidance of morbidity and mortality while on the waitlist. We have proposed an algorithm for pretransplant immunological risk assessment in a cost-limited Indian setting for living-donor RT [Figure 1].{Figure 1}

 Pathogenicity of Donor-Specific Antibodies

The complex characteristics of DSA, like HLA class, specificity, strength, IgG subclass, and complement binding capacity, determine the pathogenicity of antibodies.[17] The modified solid-phase assays (C1q, C3d, and C4d-binding assays) can detect the complement-binding ability of antibodies more effectively. The presence of C3d-binding DSA at the time of ABMR is a strong independent predictor of allograft loss, as concluded in a recent study.[18] The authors have observed C3d-binding is a more sensitive marker than C1q-assay. C1q is the first component of the classic complement pathway, and it is therefore not surprising that a C1q-binding assay would exhibit a lower sensitivity than a C3d-binding assay. Another possible explanation could lie in the regulatory mechanisms preventing uncontrolled amplification of the complement cascade. By limiting C3 convertase formation even when substantial amounts of C1q bind to antibodies, they could reduce the specificity of a C1q-binding assay. In contrast, the presence of C3d on DSA proves the efficient cleavage of C3 and is, therefore, more closely related to the pathogenic processes damaging the graft. Recent evidence has also shown that C3d-binding (but not C1q-binding) antibodies are scarcely susceptible to extracorporeal removal (plasma exchange) and related to poor prognosis of ABMR.[19] In one study, low level preformed DSA able to bind C4d was reported to predict ABMR and graft loss, although the XM was negative before transplant.[20] The identification of the IgG subclass of DSA is of clinical importance. Serum IgG molecules can be divided into four subclasses (IgG1–IgG4) with varying capacity to activate complement and recruit effector cells through the Fc receptor (IgG3> IgG1> IgG2> IgG4). Recent evidence showed that complement binding IgG3 subclass of DSA was more pathogenic and was associated with active ABMR, shorter time to rejection, increased microcirculation injury, and C4d deposition. Noncomplement binding IgG4 DSA was associated with subclinical or chronic ABMR, late allograft injury with increased transplant glomerulopathy, and interstitial fibrosis/tubular atrophy lesions.[21]

 Nonhuman Leukocyte Antigen Antibodies

Non-HLA antibodies can also result in rejection. As recommended by Banff 2017 update of renal allograft pathology, testing for non-HLA antibodies is advised in cases where there are no detectable HLA-DSA but a biopsy specimen meeting criteria for ABMR.[22] These mainly include anti-angiotensin II type 1 receptor (anti-AT1R) antibodies, antiendothelin-1 type A receptor antibodies, and antiendothelial cell antibodies.[23] Four antigenic targets expressed on endothelial cells were identified in a recent study: endoglin, Fms-like tyrosine kinase-3 ligand, epidermal growth factor-like repeats and discoidin I-like domains 3, and intercellular adhesion molecule 4.[24] MHC1-related chains A and B (MICA and MICB) are minor histocompatibility molecules expressed on endothelial cells, and their antibodies can trigger ABMR.[25] The significance of anti-MICA antibodies was identified in a recent Indian study.[26] The authors have identified MICA alloantibodies in 14.6% of patients undergoing live-related donor RT; the presence of anti-MICA antibodies without any HLA antibodies was associated with poor outcomes in those patients. They have concluded that preformed MICA antibodies independently increase the risk of rejection. H-Y antigens encoded by Y chromosome in males and may cause rejection in male to female transplants. Other non-HLA antibodies identified in few studies include antibodies against agrin, vimentin, type IV collagen, fibronectin, perlecan, Kα-tubulin, protein kinase Cζ, and glutathione S-transferase T1.

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