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Year : 2021  |  Volume : 15  |  Issue : 4  |  Page : 295-299

Tools for histocompatibility testing and significance of panel reactive antibodies - A narrative review

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

Date of Submission18-Oct-2020
Date of Decision02-Dec-2020
Date of Acceptance08-Dec-2020
Date of Web Publication30-Dec-2021

Correspondence Address:
Dr. Praveen Kumar Etta
Department of Nephrology and Renal Transplantation, Virinchi Hospitals and Max Superspeciality Medical Centre, Hyderabad - 500 034, Telangana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijot.ijot_120_21

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Immune response directed towards the allograft is a major barrier to the longterm graft survival in kidney transplantation. The importance of various tools for histocompatibility testing including the significance of panel reactive antibodies in transplant immunology is discussed here.

Keywords: Histocompatibility testing, human leukocyte antigen, kidney transplantation, panel reactive antibodies

How to cite this article:
Etta PK. Tools for histocompatibility testing and significance of panel reactive antibodies - A narrative review. Indian J Transplant 2021;15:295-9

How to cite this URL:
Etta PK. Tools for histocompatibility testing and significance of panel reactive antibodies - A narrative review. Indian J Transplant [serial online] 2021 [cited 2022 May 25];15:295-9. Available from: https://www.ijtonline.in/text.asp?2021/15/4/295/334298

  Introduction Top

Alloimmunity remains an important barrier to the long-term graft survival in kidney transplantation (KT).[1] Remarkable advances in histocompatibility testing has immensely improved the safety of KT and decreased the incidence of rejections. The availability of various tools such as human leukocyte antigen (HLA) typing of donor-recipient pair and assessing its matching, performing cross-match (XM) tests, identification of anti-HLA antibodies and donor-specific antibodies (DSAs), and testing for panel reactive antibodies (PRAs) has enabled our understanding of transplant immunology in managing KT recipients. These tools can help in better risk stratification, prognostication, to decide on transplant eligibility, selection of immunologically favorable donor, to plan desensitization and immunosuppression including induction therapy that can lead to better long-term graft survival.

  Methodology Top

We reviewed published literature from PubMed database with the following search terms crossmatch, histocompatibility, panel reactive antibodies, HLA typing, and donor specific antibodies. We included studies relevant to kidney transplantation and of English language literature. We reviewed 26 full text articles after excluding abstracts and articles which are not relevant.

  Human Leukocyte Antigen Typing and Matching Top

HLA typing and matching remains one of the standard immunological tests in KT.[2] In humans, the major histocompatibility complex (MHC) genes are encoded on the short arm of chromosome 6 and include the class I genes HLA-A,-B, and-C, and the class II genes HLA-DR,-DQ, and-DP. MHC molecules are highly polymorphic and each MHC locus has 1000-5000 allelic variants. HLA class I antigens are expressed on all nucleated cells; the epitopes reside only in the polymorphic α-chain. HLA class II antigens are normally restricted to antigen-presenting cells (dendritic cells, B cells, and macrophages), but they can be expressed under inflammatory conditions (ischemia-reperfusion injury, infection, and rejection) on a variety of cell types including endothelial cells and epithelial cells. HLA-DR,-DQ, and-DP molecules all have polymorphic beta chains. HLA-DR molecules have a conserved alpha chain, while HLA-DQ and HLA-DP have polymorphic alpha chains. HLA antigens may share a common (public) epitope and belong to a cross-reactive epitope group.

HLA typing and matching in relation to transplant immunology traditionally consider only antigens in HLA-A,-B and-DR loci. However, all HLA proteins including HLA-C,-DQ, and-DP are now recognized as antigenic targets.[3] HLA typing was initially performed using serologic-based assays, but this has been replaced by the use of DNA-based molecular techniques (sequence-specific primers and sequence-specific oligonucleotide probes [SSOP] typing), which allow for higher resolution and more accurate typing. Due to the need of rapid turnaround time, typing of the deceased donors is typically performed using real-time polymerase chain reaction or premade, reverse-SSOP trays. High-resolution typing methods (sequence-based typing [Sanger-based sequencing] and next generation sequencing) may be considered for use in typing of recipient or live donor evaluations.

In India, HLA typing is mandatory from legal standpoint for living related donor transplantations as per Transplantation of Human Organ Act 1994, subsequently amended several times, the latest amendment being 2014.[4] This information also aids the clinician in assessing the patient's likelihood of receiving a HLA-compatible transplant, identifying “unacceptable antigens” to avoid transplanting grafts from donors expressing these antigens, and classifying those at higher immunologic risk who may benefit from a more aggressive immunosuppressive regimen and/or more intensive posttransplant monitoring.

HLA mismatch refers to an HLA antigen found on the cells of the allograft but not in the recipient. Incremental HLA mismatches are associated with increased risk of rejection and allograft loss.[5] The HLA epitope matching may be superior to traditional 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.[6] High resolution typing to 4 digit allele level is needed for epitope matching using tools such as HLA-matchmaker or Predicted Indirectly ReCognizable HLA Epitopes.[7]

  Cross-Match Top

Sensitized transplant candidates with preformed antibodies against donor HLA antigens may experience positive XM before or rejection after transplant. In complement dependent cytotoxicity (CDC) XM, donor lymphocytes are first separated (usually by magnetic bead isolation) into a CD3+ T-cell fraction and a CD19 + B-cell fraction. Serum from the recipient is then added to the cells, followed by the addition of complement and a viability dye. If DSA is present, cell lysis is observed, and the XM is deemed positive. CDC assay can result in false-positive results due to the presence of immunoglobulin M (IgM) or non-HLA antibodies. The flow cytometry XM (FCXM) assay has a greater degree of sensitivity and can also detect non-complement binding DSA. Recipient serum is added to the donor lymphocytes, followed by the addition of a secondary fluorochrome-conjugated antibody that detects human IgG. In this assay, donor lymphocytes do not need to be separated into their T-and B-cell fractions. Rather, additional detection antibodies conjugated to different fluorochromes are added to distinguish between the two subsets of lymphocytes. Samples are analyzed on a flow cytometer and results are expressed as channel shifts of median fluorescence (median channel shifts) above the baseline. Common false-positive results encountered in FCXM testing include high background signal (particularly with B cell FCXM) and the presence of non-HLA antibody. Individuals who have only anti-HLA IgM antibody may have a negative flow cytometric XM since the fluorochrome-labeled detection antibody is selective for IgG.[8]

  Anti-Human Leukocyte Antigen Antibodies and Donor-Specific Antibodies Top

The presence of preformed anti-HLA antibodies in highly sensitized patients remains a major barrier for successful KT. The development of solid phase immunoassays (SPIs) that use solubilized HLA antigens as targets have greatly increased the ability to detect and identify these antibodies. SPIs are more sensitive than cytotoxic assays and are more specific for anti-HLA antibodies.[9] They do not detect IgM and non-HLA antibodies.

The initial enzyme-linked immunosorbent assay (ELISA) and the flow cytometric assays are replaced in most places by more advanced and sensitive Luminex assay. In flow cytometric assay, recipient serum is added to a platform of polystyrene beads, to which purified HLA antigens are attached. A fluorochrome-conjugated anti-IgG detection antibody is then added, and the presence of anti-HLA IgG antibody is identified by flow cytometric methods. In Luminex platform, the panel of beads can be screening beads, phenotypic beads or single antigen beads (SAB). In Luminex SAB assay, each of the distinct recombinant HLA molecule is uniquely expressed on a particular polystyrene bead impregnated with two fluorescent dyes. It is used to determine the precise specificity of the HLA antigen against which the antibody is directed. The degree of fluorescence exhibited by the presence of alloantibody is measured in terms of its mean fluorescence intensity (MFI) and can provide some clue as to the amount and strength of alloantibody present. Although results are provided as a numerical value, MFI values are not synonymous with concentration or titer of antibody.

The density of HLA antigen expressed on individual beads may not correspond to its density found on cell surfaces (HLA-C and-DP have low level cell surface expression); two different antibodies with the same MFI level may show disparate XM results. Binding of antibody to denatured antigens can result in a false positive detection of antibody that has no clinical significance. Inhibitors such as various complement components can bind to the anti-HLA antibody and stereotypically hinder the ability of the detection antibody to bind (the prozone effect) and can lead to under-recognition of antibodies. The presence of intravenous immune globulin or IgM antibody can also mask the recognition of IgG alloantibody. Pretreating the patient's serum with ethylene diamine tetraacetic acid or dithiothreitol or heat inactivation or titration studies ameliorate this effect. When the alloantibody reacts against a public/shared epitope, the binding of the antibody is distributed across all beads containing antigens expressing the common epitope, effectively “diluting out” the antibody. This leads to lower MFI values and underestimation of the true antibody burden.[10]

The term “unacceptable antigen” refers to a donor HLA antigen against which the patient has performed antibody, and should be avoided because of an increased risk of antibody mediated rejection (ABMR).[11] Candidates are eliminated from the match list for donors with unacceptable HLA antigens (positive virtual XM). Virtual XM compares recipient antibody specificities with donor antigens, permitting rapid and earlier identification of immunologically compatible donors in deceased donor and living donor paired exchange programs.[12] This method showed excellent correlation with predicted positive XM. It would increase the efficiency of deceased donor kidney allocation, reducing the number of final positive XM (and organ refusals) and avoids unnecessary organ shipments with time and cost savings to transplant centers. Complement fixing ability of antibodies can be detected by modified SPIs such as C1q, C3d, and C4d-binding assays. Identifying IgG subclass of antibodies also has prognostic significance. IgG3 antibodies have greater capacity to activate complement and recruit effector cells through the Fc receptor, and are more pathogenic and associated with active ABMR, shorter time to rejection, increased microcirculation injury, and C4d deposition.

  Panel Reactive Antibodies Top

The traditional way to measure the sensitization status is the PRA assay. PRA identifies several antibodies to a potential cluster of donors, whereas the XM test will identify if a recipient had antibodies to a specific donor of interest. PRA is calculated separately for class I and II HLA antigens. High level PRA itself is a risk factor for allograft rejection. PRA is the percentage reactivity of a patient's serum antibodies against of known panel of HLA antigens presented either on donor lymphocytes (cell based CDC assay), or in more recent years on SPI matrices. It is considered as a measure of the proportion of potential donors who would be found to be incompatible with a recipient. Patients with high PRA have to wait for a very long time until a compatible donor becomes available. Centers either use a peak or current PRA level for a given patient in wait list.

Nearly one-third of deceased donor wait listed candidates have a PRA of 10% or more. Primary sensitization results from exposure to foreign HLA antigens via transplantation, transfusion, or pregnancy, although infection (cross-reacting antibodies) and other conditions can also alter sensitization status. About 30%–50% of women with three or more pregnancies develop anti-HLA antibodies. About 50% of patients who receive multiple transfusions develop antibodies. Leukodepleted blood may reduce the risk of sensitization. About 90% of transplant patients develop HLA antibodies within 2 weeks of a failed graft.[13] In some cases, the antibodies could be transiently present for just a short time, while in others they may persist for several years.

Testing for panel reactive antibody

The conventional PRA testing is based on the original cell based (CDC) assays for detecting HLA antibodies. It involves exposing a panel of cells from different donors to a recipient's serum, to which complement and a vital dye are added. The donors are selected to represent the common HLA frequencies in a potential donor population. The percentage of the cells in the panel which are lysed is an estimate of the percentage of donors from that population against whom the recipient has cytotoxic antibodies. Only a sufficiently higher titer of antibodies is able to activate complement cascade resulting in cell lysis. The limitations of cell-based PRA assays include lack of sensitivity, limited specificity with high false-positive rates due to non-HLA antibodies, IgM, and autoantibodies. The antigen specificity of antibodies is not known. The heterogeneity is also due to cell panel phenotype variation regardless of recipient serum changes, subjectivity in interpretation and cumbersome procedure to maintain viability of cells. It detects only complement fixing antibodies (IgG1 or IgG3) and results also depend on the quality of lymphocytes and rabbit complement used.

Newly evolved SPI platform using denatured or recombinant HLA antigens has made PRA assay more objective with good reproducibility. It does not require complement activation for a positive reaction. It can be performed on ELISA platform (ELISA-PRA, using recombinant HLA antigens), flow cytometric (Flow-PRA, based on latex beads coated with denatured HLA) or luminex platform (Luminex-PRA, based on polystyrene beads coated with recombinant HLA using dual laser system). The bead-based assays are associated with increased sensitivity and capacity for high-volume testing. Luminex platform utilizing phenotypic beads, where each bead represents phenotype of an individual is the most sensitive PRA assay in the current era. After antibody binding to its respective HLA antigen bead, reaction is said to be positive when its fluorescence is above the threshold level validated by the laboratory and manufacturer. The PRA estimate is defined based on the percentage of total beads with positive fluorescence.

Limitations of panel reactive antibody

The value of PRA depends both on the panel composition and the technique used for antibody detection. The composition of the antigen panel varies considerably with the use of locally procured cell panels or different commercially available SPIs which often do not represent the potential donor population. Detection methods range from the relatively insensitive CDC assay to highly sensitive SPIs. SPIs using solubilized HLA antigens as targets by virtue of their greater sensitivity could detect low levels of antibodies and give higher PRA values, giving an unfair advantage over those who had PRAs by conventional cell panel methods. Because of these variables, PRA does not provide a realistic measure of sensitization and its value is highly variable and inconsistent. There was also heterogeneity in that transplant centers could choose to list either peak PRA or current value.

Panel reactive antibody in Indian scenario

In India, the KT program is constituted mainly by live related donors. There are little data about PRA and its utility in live donor transplant program. A prospective observational study from India, evaluated the role of PRA in immunological risk stratification and its correlation with antecedent events and posttransplant outcomes in a live donor KT program. Flow-PRA positivity (>10%) was observed in ≈15% end-stage kidney disease patients. Female sex, longer duration of dialysis and hepatitis C virus infection were significantly associated with PRA positivity. Luminex SAB test was done in patients with high PRA to identify DSAs before taking them for KT. Overall, sensitizing events were more commonly found in PRA-positive group, however, single blood transfusion alone was not a significant contributor to sensitization. Cross-reactivity with viral infections and vaccination could also contribute to sensitization. HCV infection was significantly more common in PRA positive group in this study. Less number of patients in PRA positive group received transplant as compared to PRA negative group (41.4% vs. 91.7%, P < 0.0001) due to immunological reasons (XM positivity or high levels of DSAs). There was no difference in short-term outcomes between PRA positive and negative groups after exclusion of high risk patients from transplantation, this could also be due to better HLA-matched donors in PRA-positive group. The study concluded that PRA testing even in live donor transplant program was useful in identifying high risk patients and evaluating them further with more specific tests including SAB assay in a cost-effective manner.[14]

  Calculated Panel Reactive Antibody Top

To circumvent the limitations associated with conventional or measured PRA, virtual or calculated PRA (cPRA) was introduced, and it has become the primary measure of sensitization in Western protocols. The cPRA is calculated using an algorithm that correlates recipient anti-HLA antibody profile as measured by SAB assay with the population frequency of unacceptable HLA antigens to generate a percent score. It is presently the best estimate of likelihood of a positive XM to a randomly selected donor.

In the West, transplant centers enter the details of unacceptable antigens for their candidates. Depending on the prevalence and frequency distribution of unacceptable HLA antigens in a donor pool, the likelihood that the recipient and donor would be incompatible can be calculated. The universal listing of unacceptable antigens for sensitized patients with validated HLA frequencies has enabled a consistent reporting of cPRA. In deceased donor kidney allocation programs, extra allocation points are being awarded to sensitized patients with high cPRA to increase their access to potentially compatible donors and to minimize waitlist time.[15]

The computer calculates the cPRA using the unacceptable values that have been entered for a candidate. With every addition or deletion to the patient's unacceptable list, the value will be automatically recalculated. Different algorithms are available in various organ allocation systems such as eurotransplant (EUTR), United Network for Organ Sharing (UNOS) and Canadian Transplant Registry depending on the population and ethnicity as the frequency distribution of antigens differ.[16] To avoid errors in the allocation, due to different allelic frequencies among geographical areas, every transplant program ideally should use its local donor HLA database to calculate the cPRA. Special allocation programs such as EUTR acceptable mismatch program and Organ Procurement and Transplantation Network (OPTN) kidney allocation scheme use cPRA value for sensitized candidates to increase their chances to get an organ. In the OPTN website, unacceptable antigens can be entered for the following HLA loci: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3/4/5, HLA-DQA, HLA-DQB, and HLA-DPB. Antibodies directed against HLA-DPA are not considered in the allocation schema. Implementation of the cPRA policy in OPTN/UNOS national kidney organ allocation in the US in year 2009 has increased efficiency in organ allocation and is helping to facilitate transplantation of highly sensitized candidates (cPRA >80%).[17] The median number of unacceptable antigens listed for highly sensitized patients with cPRA values of 80 or greater was 35.[18] Broadly, sensitized patients have antibodies against numerous HLA antigens, but, if these are rare in frequency in the donor population, they may not have a high cPRA.

Limitations of calculated panel reactive antibody

The listing of unacceptable antigens among transplant centers is also variable depending upon the level of risk of ABMR they are willing to accept. Thus, some centers may assign unacceptable antigens based on low antibody levels, whereas others may accept higher levels. The cPRA may be systematically underestimated when donor typing is not complete at loci where HLA antibodies to those loci are utilized in donor decision-making.

  Novel Assays Top

The evaluation for non-HLA antibodies is indicated in cases of histological ABMR (microvascular inflammation [MVI] score ≥1 and C4d-positive or MVI score ≥2 and C4d negative) with no identifiable anti-HLA antibodies (DSA) by SPIs. Some of these clinically significant antibodies include anti-angiotensin II type 1 receptor (anti-AT1R) antibodies, anti-endothelin-1 type A receptor antibodies, antibodies to MHC1-related chains A and B (MICA and MICB) and other types of anti-endothelial cell antibodies (AECAs). Specific novel assays including endothelial cell crossmatch test are useful in identifying them, however their role as pretransplant histocompatibility assay is still not well established. Alloreactive memory T cells are central mediators of renal allograft rejection, and monitoring the activity of these cells may help to identify patients who are at risk for rejection. The interferon (IFN)-gamma enzyme-linked immunospot assay (ELISPOT), which measures IFN-gamma secretion by recipient T cells in response to donor antigens, has been used to assess anti-donor T cell alloreactivity in vitro.

To conclude, genetic disparities between transplant candidate and donor may lead to alloimmune response. The availability of various tools for histocompatibility testing enabled better risk stratification and prognostication. Currently, no single assay is capable of capturing all aspects of alloreactive cellular and humoral immune responses. Therefore, combinations of different assays in addition to conventional tests should be used to have a complete picture of the alloimmune response. It is justified to use PRA assay in selected cases even in living donor transplant setting as this enables better risk stratification and further evaluation by more specific assays like SAB assay. This is especially relevant to resource poor countries like India, where these specific assays are costly and are not routinely done.

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Conflicts of interest

There are no conflicts of interest.

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Etta PK. Comprehensive management of the renal-transplant recipient. Indian J Transplant 2019;13:240-51.  Back to cited text no. 1
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William RM, John K. Understanding crossmatch testing in organ transplantation: A case based guide for general nephrologist. Nephrology 2011;16:125-33.  Back to cited text no. 8
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