|Year : 2021 | Volume
| Issue : 1 | Page : 4-13
Pretransplant histocompatibility testing algorithm: Laboratory and clinical approach in the Indian context
Feroz Aziz1, Aseem K Tiwari2, Himanshu V Patel3, Rajni Chauhan2
1 Department of Nephrology and Transplantation, IQRAA International Hospital and Research Center, Laboratory of Transplant Immunology, Aster MIMS Hospital, Kozhikode, Kerala, India
2 Department of Transfusion Medicine, Molecular and Transplant Immunology Laboratory, Medanta - The Medicity Hospital, Gurugram, Haryana, India
3 Department of Nephrology and Transplantation, Institute of Kidney Diseases and Research Center, Dr. H L Trivedi Institute of Transplantation Sciences, Ahmedabad, Gujarat, India
|Date of Submission||19-Jul-2020|
|Date of Acceptance||08-Dec-2020|
|Date of Web Publication||31-Mar-2021|
Dr. Feroz Aziz
Department of Nephrology and Transplantation, Laboratory of Transplant Immunology, Aster MIMS Hospital, IQRAA International Hospital and Research Center, Kozhikode - 673 016, Kerala
Source of Support: None, Conflict of Interest: None
Remarkable advances in histocompatibility testing have immensely improved the safety of transplantation and have decreased the incidence of rejections. Human leukocyte antigen (HLA) typing and crossmatches by complement-dependent cytotoxicity or flow cytometer-based methods are the vital tests involved in pretransplant histocompatibility testing. Continuous development in these testing technologies since the 1960s has refined these methodologies that can be used to predict graft rejection. Advancements from polymerase chain reaction-based methods to sequence based in HLA typing, and from cell-based cross-matches to virtual cross-matches using advanced solid-phase platforms, have enhanced our understanding about the donor-specific antibodies (DSAs) and have challenged the concept that the presence of DSA is an absolute contraindication to transplantation. Despite various developments, it is very difficult to perform a plethora of tests for pretransplant workup due to cost constraints in a developing country like India. In this review, we would discuss the advantages, limitations, and cost involved in the pretransplant immunologic workup along with an algorithmic approach for physicians that may help in decision-making amidst multiple information from different platforms.
Keywords: Crossmatches, donor-specific antibody, histocompatibility, sensitized
|How to cite this article:|
Aziz F, Tiwari AK, Patel HV, Chauhan R. Pretransplant histocompatibility testing algorithm: Laboratory and clinical approach in the Indian context. Indian J Transplant 2021;15:4-13
|How to cite this URL:|
Aziz F, Tiwari AK, Patel HV, Chauhan R. Pretransplant histocompatibility testing algorithm: Laboratory and clinical approach in the Indian context. Indian J Transplant [serial online] 2021 [cited 2021 Jun 16];15:4-13. Available from: https://www.ijtonline.in/text.asp?2021/15/1/4/312760
| Introduction|| |
Improved allograft outcome and decreased incidence of rejections in the current era of transplantation owes a lot to advancements in histocompatibility testing along with that of immunosuppression and understanding of posttransplant infections. Human leukocyte antigen (HLA) typing and crossmatches are the two huge components of histocompatibility testing for solid organ transplantation. With the advent of polymerase chain reaction (PCR) in the 1980s, the molecular-based HLA typing methods have virtually replaced serology-based typing methods. Our understanding of crossmatches has evolved over the last five decades from first-generation, tray-based cytotoxic crossmatch to the latest platform of solid-phase bead assays. As crossmatches and antibody detection methods have become more and more sensitive, clinicians are fed with a lot of information from a histocompatibility test report standpoint, which makes the decision-making process difficult. For a developing country like India, the financial burden and logistics of transplantation make the challenge even more formidable. Our aim is to provide a practical approach to histocompatibility by utilizing information from newer platforms in solid organ transplantation, “keeping-in-mind” the aforementioned limitations of the developing world.
| Human Leukocyte Antigen Typing|| |
Molecular-based HLA typing has become the “standard of care” across the globe. There are three methods of molecular typing namely (1) sequence-specific primer (SSP), (2) sequence-specific oligonucleotide (SSO), and (3) sequence-based typing (SBT). HLA typing for solid organ transplantation is primarily done by SSP and SSO techniques which are based on primers and probes made of well-documented alleles. Both can give low- to intermediate-resolution typing based on primer/probe that is being used. SSO platform is more advanced compared to SSP; is automated; and has higher throughput that is, it can type multiple samples simultaneously, making it less labor intensive, provided there are enough numbers to make it financially viable. SBT, though the standard of care for hematopoietic progenitor cell pretransplant testing, is not routinely used for solid organ transplantation. In solid organ transplants, SBT is needed only if one needs a high-resolution typing of a donor locus (or loci) to allele level to characterize an antibody as “donor specific” or “not donor specific,” which might not be possible with low-resolution typing.
In India, HLA typing is mandatory from a legal standpoint for living-related donor transplantations as per the Transplantation of Human Organ Act 1994, subsequently amended several times, with the latest amendment being in 2014. Living-related donor transplant program dominates the transplant scenario in India and in the developing world, and options within the family are restricted. The clinical utility of doing HLA typing in living donor transplants is a complex issue. Parents who constitute almost 35% of the living donors would be haplo-identical and do not add any further information from immunological standpoint. No HLA compatibility is expected in spousal donation, leaving sibling donation as the real “must-need” scenario for HLA compatibility. Siblings have a 25% chance to be HLA identical (two haplo-identical), 50% chance to be one haplo-identical, and 25% chance to be completely nonidentical. Being a two haplo-identical sibling has a phenomenal immunological advantage with an estimated graft half-life of over 30 years.
If the recipient has an anti-HLA antibody detected by new-generation testing methods, the HLA typing of the donor is needed irrespective of the donor's relationship to the patient, to characterize it as donor specific or not. Donor-specific antibodies (DSAs) are identified prior to transplantation, if antibody screen or crossmatch is positive. DSAs are to be identified after transplantation, only when humoral immune injury is suspected. In living-related donor scenario, if a recipient has no option of choosing from multiple potential donors, the need of HLA typing has clinical significance only in sibling donation or to characterize DSA.
Solid organ transplantation has been traditionally done with low-resolution (two-digit antigen level) typing with primary focus on A, B, and DR loci. Recent advancements and understanding have warranted the need of increasing the depth and breadth of HLA typing. Certain antibodies in single antigen bead (SAB) analysis readout can be identified as DSA against non-self HLA antigens only if we have high-resolution typing. Antibodies against HLA-C, HLA-DQ, and HLA-DP are being found to be important and warrant typing for these loci, if potential antibodies exist., High-resolution typing to four-digit allele level is needed for epitope matching using tools such as HLA-Matchmaker or PIRCHE., HLA typing for a single locus costs around 3000 Indian National Rupee (INR); HLA-A, -B, and -DR loci of an individual cost around 9000 INR and more loci means bigger expenditure. Futuristically, we are heading toward sequence-based typing using “next-generation sequencing” (NGS) as it is becoming more affordable with a tradeoff of slightly longer turnaround time (TAT). With increasing loci and resolution, the increment in the cost of NGS is much less compared to that in SSP or SSO methods. Living donor kidney transplantation being the primary type of transplantation in India, the high TAT of NGS should not be an impediment, especially considering that NGS provides higher resolution at a lower cost.
Futuristically, the histocompatibility community is exploring the option of matching at epitope level rather than HLA antigen level. Each HLA antigen is constituted by many regions of antigenic determinants called epitopes which are specific sequences of amino acids on the antigen recognized by the immune system. The part of the antibody that recognizes the epitope is called the paratope. Specialized software-based programs such as HLA-Matchmaker and PIRCHE are available which can tease out and compare the epitope information between patient and donor when the patient's antibody/antibodies identified by SAB and high-resolution HLA typing information of donor are available. There are accruing data which show certain nature and number of epitope mismatches that are associated with the development of DSA and inferior graft survival. We need more data from across the globe that could identify the immunogenicity of epitope mismatch combinations before it can be incorporated into routine clinical practice. Once in place, the epitope matching is predicted to be simpler than HLA matching as the number of crucial epitopes might be only 100 or 200 instead of the ever-expanding HLA loci which are in thousands and still counting. Further discussion on this evolving concept is beyond the scope of the current review.,,
| Detection Of Anti-Human Leukocyte Antigen Antibodies – Contexts And Tools|| |
After HLA typing, testing for anti-HLA antibodies in a recipient constitutes the most important work done in the transplant immunology laboratory. The ultimate testing in the area of histocompatibility evolves around the detection of donor-specific anti-HLA antibodies which are detrimental to allograft outcome. The selection of antibody testing tool is highly contextual.
Methods to identify antibodies against donor HLA antigens have evolved over three phases – complement-dependent cytotoxicity cross-match (CDC-XM) test, flow-cytometer cross match (FCXM) test, and solid-phase immunoassays (SPI), of which the Luminex platform is the dominant player. CDC-XM and FCXM are used in the context of pretransplant crossmatch which is needed before any transplant with an identified donor. “Screening for the presence of anti-HLA antibodies” as part of broad sensitization and to characterize an antibody as “DSA” against a particular HLA antigen can be done by new-generation, solid-phase, bead-based assays performed on “Luminex platform.” The utilization of the aforementioned tools should fit into an algorithmic approach in decision-making.
From a clinician's perspective, the important logical questions that come up sequentially with regard to testing for anti-HLA antibodies can be as follows:
- Does my patient have an anti-HLA antibody?
- If my patient has an anti-HLA antibody, what are the chances of getting a donor against whom the recipient does not have an antibody?
- If a donor is available – deceased or living, is the antibody directed against the donor?
- Even if there is an antibody against the donor, could it be so harmful that I should preclude transplantation or can I circumvent it?
Histocompatibility testing methods have evolved by addressing the last question, first. The final gatekeeper who decides whether an individual is transplantable with an identified donor from an immunological standpoint is the “pretransplant crossmatch.” A negative crossmatch is imperative for any transplantation. The immunological profiling done by undertaking newer platforms helps in risk stratification, selection of compatible donor, and deciding on the need of pretransplant immunomodulation therapies.
| Pretransplant Crossmatches - The Final Gatekeeper|| |
Development of cytotoxic crossmatch by Professor Terasaki in 1969 was a landmark in the journey of transplantation, and all ensuing advancements in histocompatibility owe a lot to this fundamental accomplishment.
Complement-dependent cytotoxicity cross-match
CDC-XM has been the sole and main tool of histocompatibility for over five decades. It still continues to be used by a good proportion of centers in the developing world as the sole crossmatch technique. In the seminal study by Terasaki et al. 15% of the recipients developed hyper-acute rejection despite a negative CDC-XM, and still the concerns regarding the sensitivity of the test remain unresolved. Albeit CDC-XM enhanced with secondary antibody (anti-human globulin) has increased the sensitivity by 10%–30% compared to that of the standard CDC-XM, it falls short compared to newer platforms. The transplant community is unequivocal on agreeing that a positive CDC-XM after excluding technical errors and inbuilt variations is a certain “no” for transplantation. Studies by multiple centers have shown that we may be missing 20% of recipients with DSAs, if we rely only on CDC-XM.,, Early antibody-mediated rejection (ABMR) occurs in 30%–50% of recipients with DSA which can subsequently cause allograft loss in around 15% of recipients. In brief, CDC crossmatch alone is incapable of identifying around 15% of recipients with DSA, which is detrimental to allograft outcome. This is attributable to the fact that CDC-XM is dependent on cell viability and the need of higher titers of antibodies to make the cytotoxicity appreciable. Lower titers of antibodies which are potentially harmful are not identified by CDC-XM. The cost of CDC-XM is around 5000 INR [Figure 1].
|Figure 1: Comparison of sensitivity between CDC-XM and FCXM. CDC-XM: Complement-dependent cytotoxicity crossmatch; FCXM: Flowcytometry crossmatch|
Click here to view
Flow cytometer cross-match
FCXM is the most sensitive crossmatch technique; it is a cell-based test but not dependent on cell viability and is more objective. In developed world, FCXM has almost replaced CDC-XM as the primary crossmatch technique. Many studies have proved the increased sensitivity of FCXM in identifying donor-specific reactivity compared to that of CDC-XM; and recipients with positive FCXM results in increased rejection and increased graft loss.,, A negative FCXM is certainly reassuring and provides approval for transplantation from histocompatibility standpoint in certain terms, especially for sensitized patients. In the course of this review, we shall discuss how a well-standardized FCXM report can help in the decision-making for or against offering transplantation amidst the myriad of information that pours on to clinician's desk from more sensitive SPI assays. Accessible FCXM facility is not available for a good proportion of transplant centers in India. To develop an in-house FCXM facility, the transplant program should be of high volume or the laboratory should be one that caters to multiple services other than transplantation, for example, hematological malignancy workup. The estimated cost for doing an FCXM is around 7000 INR [Figure 1].
Interpretation of crossmatches
CDCXM and FCXM vary in sensitivity and specificity and provide an opportunity to profile the immunological response characteristics of a recipient. CDCXM of the donor lymphocytes is performed using serial doubling dilutions of the recipient's serum. Controls are run with each assay, and the positive and negative controls should give cytotoxicity results of >80% and <15%, respectively. During microscopic analysis, the percentage of dead cells (lymphocytotoxicity) is observed and numerical score should be used as recommended by the ASHI laboratory. False-positive results are sometimes caused by IgM autoantibody. However, this can be overcome by the addition of dithiothreitol which helps prevent IgM autoantibody-mediated complement activation and allows only IgG (DSAs) to act. False-negative reactions may occur when DSA levels are too low to result in the activation of the complement cascade or if the antibodies are of the type that do not cause complement activation. One of the advantages of using CDCXM is the resolution of prozone phenomenon as the serial dilutions are already included in the assay. In FCXM, T and B cells are interpreted distinctly using respective fluorochrome conjugated antibodies. T lymphocytes have only HLA Class I antigens, whereas B lymphocytes have both HLA Class I and II antigens. The density of HLA class I antigens is in fact more in B lymphocytes compared to that in T lymphocytes., If a patient has only antibodies against class II, only Flow B cell crossmatch would be positive and if the patient has antibodies against class I, both Flow B and T cell crossmatch should be positive. FCXM positivity due to anti-HLA antibodies involves Flow B cell positivity either alone or along with Flow T positivity depending on the class and strength of antibodies. FCXM with B cells could be obscurely positive due to auto-antibodies, nonspecific antibodies binding to Fc receptor on B cells, or non-HLA antibodies binding on to B cells, raising the concern for specificity of the donor-directed antibodies. Of importance is that a negative FCXM reassures the absence of any concerning antibody population, especially in sensitized patients, and helps in decision-making to facilitate transplantation [Figure 1].
| Profiling of Anti-Human Leukocyte Antigen Antibodies: Screening to Characterization of Donor-Specific Antibody|| |
Exposure of foreign HLA antigens through sensitizing events (such as blood transfusion, pregnancy, or transplant) can, sometimes, trigger an allo-immune humoral response by generating anti-HLA antibodies. Development of humoral allo-immune response varies from individuals and also depends on the nature of immunologic trigger. Many HLA antigens share similar epitopes and result in cross-reacting antibodies, resulting in broader sensitization. Irrespective of the immunological trigger, a proportion of subjects who are identified as responders develop a humoral response, whereas the remaining majority are labeled as nonresponders who do not mount a humoral re-sponse.
Assessing the baseline status of sensitization has become the first part of immunological assessment for prospective living and deceased donor recipients. All potential recipients could be qualitatively “screened” for the presence of anti-HLA antibodies or their degree of sensitization could be semi-quantified by testing against a representative panel of HLA antigens to give a percentage value called “panel-reactive antibody” (PRA). Newly evolved testing platforms based on solid-phase beads have made the aforementioned primary profiling more objective with good reproducibility.
Screening for anti-human leukocyte antigen antibodies and panel-reactive antibody assessment
Testing the sera against a panel of antigens by different methods to assign a value in percentage (PRA) has been used traditionally as the index of sensitization for potential recipients. First-generation PRA testing with CDC trays with a representative panel of live cells had to give way for “SPI” based on ELISA and flow-cytometer because of limitations of CDC such as lower sensitivity, poor representation of potential donor pool, subjectivity in interpretation, and cumbersome nature to maintain the viability of cells. Initially, ELISA-based PRA estimation using recombinant HLA antigen replaced CDC-PRA with better representation and sensitivity. Subsequently, PRA testing was taken over by flow-cytometer-based PRA (flow-PRA) which was based on latex beads coated with denatured HLA from representative donor pool. (3) Flow-PRA is a very sensitive test with better representation and is still being used by many centers for initial screen for the presence of anti-HLA antibodies [Table 1] and [Table 2].
|Table 1: Comparison of the four methods of testing for panel-reactive antibody-target antigen representation: Advantages and disadvantages|
Click here to view
|Table 2: Luminex platform tests - three distinct types of target antigens can give three different types of information|
Click here to view
The advent of “bead-based solid-phase” platform has revolutionized antibody testing for over a decade, and the working principle is that of a flow-cytometer. It utilizes polystyrene beads coated with recombinant HLA as antigen targets, avoiding the need of real cells. It has a dual laser system in which one laser identifies the antigen target to which antibodies bind and other laser gives a quantitative assessment for the “amount” of antibodies that bind to the target. It provides the most sensitive platform in the current era.
The result from the “bead-based solid-phase” platform depends on the type of antigenic bead used. We have the following three types of beads for antibody profiling – (1) multiple antigen screening bead which has the most common well-documented HLA antigens to give a qualitative report for the presence of antibodies, (2) phenotypic beads – where each bead represents the phenotype of an individual and gives a PRA report in percentage depending on the proportion of beads that react with the given serum and finally, (3) SAB where each bead stands for a particular antigen and reports the specific antigen against which the patient has an antibody; if the donor has the HLA antigen corresponding to the antibody, it is called DSA [Table 3].
|Table 3: Characterization of donor-specific antibody based on single-antigen bead readout and donor human leukocyte antigen type|
Click here to view
Limitations of panel-reactive antibody in the Indian context
The PRA beads have not been validated in the Indian population, and there is no estimate as to how much representative it is for Indian population? The clinical utility of a PRA value in the Indian context is unclear. In countries where organ allocation is from deceased donor program through organ-sharing networks, PRA score helps in the prioritization of recipients in the waitlist based on the sensitization status.
The flow-PRA and “phenotype bead-” based PRA have been replaced with calculated PRA in the Western world. The calculated PRA is a computed score which gives the probability of getting a donor from the representative population calculated by factoring in the HLA statistics of nearly 12,000 donors from yesteryears and the SAB profile of the recipient.
In India, because we have neither a phenotypic bead representative of our population nor a data bank of HLA details of our donor profile, the PRA testing does not have any bonafide role in the decision-making process from a histocompatibility perspective.
Antibody screen with multiple antigen bead: A reasonable option in the Indian context
Despite exposure to sensitizing events, only a minority gets sensitized. The primary assessment from a histocompatibility standpoint of a potential renal recipient is to assess whether he/she has any anti-HLA antibody at all. HLA antibody screen with the primary mixed antigen bead is a good option despite some limitation in sensitivity. The presence of any antibody in screening warrants further scrutiny to define the nature and vehemence of the antibody, and is detailed in the ensuing discussion. As mentioned earlier, the utility of testing for PRA with phenotypic beads will not offer much in the way of decision-making with a related donor in the frame.
Steps in patients with anti-human leukocyte antigen antibodies: Characterization of donor-specific antibodies to feasibility of transplantation
If a recipient has been detected to have anti-HLA antibodies by any of the screening methods, subsequent histocompatibility evaluation becomes complex and is being discussed in the ensuing sections of this article.
When one patient is identified to have anti-HLA antibodies by flow PRA, multiple antigen bead screen, or phenotypic PRA bead, the next step is to characterize the antibody and see whether it is against the specific donor or not. SAB testing involves polystyrene beads, in which each bead is being coated with a single recombinant HLA antigen. Analysis of serum with SAB can list out the HLA antigens against which the patient has antibodies. [Figure 2] and [Table 3] show the comparison of the antibody profile with donor HLA typing and the identification of DSA. This is called a virtual cross-match.
The characterization of DSA need not be straightforward many a times. The SAB analysis output in terms of mean fluorescent intensity values denoting the presence of anti-HLA antibodies is not approved as a quantitative assay as the bead's relative fluorescence is without reference to a standard. More importantly, the antibodies may be produced against discrete amino acid sequences called public epitopes shared between many HLA antigens. The group of HLA antigens sharing same public epitope and constituting a pattern of reactivity is called a cross-reactive group (CREG). The antibody profile of recipient's reactive serum will have to be analyzed for a pattern with respect to the already-identified CREGS for meaningful interpretation as DSAs [Table 4]. Quite often, antibodies might be against DP loci or DQ loci which may not be typed routinely, and hence HLA typing may have to broaden as per the SAB profile. In brief, SAB interpretation to characterize a DSA involves identifying and interpreting reactive patterns of the antibody profile.
|Table 4: Cross-reactive groups associated with different human leukocyte antigen specificities|
Click here to view
A potential limitation which is yet to be studied in depth is the underrepresentation of SAB antigens pertinent to non-Caucasian population. Single-center data from India have shown that there is underrepresentation of HLA antigens in the currently available SAB beads where they compared the coverage of the SAB in the context of HLA alleles identified by “NGS.”
Once screen is positive, although the next logical step is to characterize the antibody by the aforementioned steps, in a primarily living donor program, the physician has the liberty to assess the feasibility of transplantation in certain terms with the identified donor by doing a crossmatch upfront without pursuing the antibody whose detection would be far more expensive. Of the two crossmatches, flow crossmatch is the more sensitive and should be the cross-match of choice in sensitized individuals with anti-HLA antibodies. Solid-phase bead-based platform is a very sensitive platform and to decipher the immunological risk in conformity with a CDC result which is least sensitive of tools, is challenging. With low-level antibodies, the correlation becomes very difficult and indeterminate.
| Algorithm for the Clinician Who Deals Primarily with Living Donor Program|| |
The basic clinical evaluation starts from a proper history taking of a sensitizing event, if any. In a developing country like ours, where the dialysis care is heterogeneous and poorly regulated with suboptimal data collection, retrieving a reliable history of sensitization is challenging. At one end, more and more triggers are being identified as potential sensitizing events and on the other end, nonsensitized individuals are being shown to have antibodies., The aforementioned reasons have made the need for an objective testing to identify the presence of anti-HLA antibody indispensable.
- Step I: Does my patient have any anti-HLA antibody? The initial test in the algorithm should be an HLA antibody screen using the “multiple-antigen” bead test. Phenotypic PRA bead or flow PRA may be used but is more expensive with little additional advantage in our scenario
- Step II: The subsequent step is based on the presence or absence of the anti-HLA antibody
- Step II-A: If no anti-HLA antibody is present – absence of anti-HLA antibody places the patient immunologically toward the benign end of the spectrum, with low risk of ABMR. Absence of any sensitizing event asserts the attribution, and we can proceed with a crossmatch, CDC or FCXM [Figure 3]a. A genuine crossmatch positivity due to anti-HLA antibodies would be an unlikely scenario. Any noise in the crossmatch in such scenario warrants us to evaluate for situations such as auto-antibodies, IgM antibodies, and non-HLA antibodies as the culprit rather than rushing to SAB testing as the next step. If the patient has sensitizing events, especially in scenarios like husband to wife donation, re-transplant candidates, blood transfusion, or historical crossmatch positivity, we may do SAB analysis as a priority even if the antibody screen is negative due to the concern of lack of sensitivity of screening beads reported in some previous studies and the binding need of identifying possible DSA in such high-risk cases. Such sensitization events call to attention the need of more sensitive FCXM rather than CDC-XM. FCXM positivity due to rituximab may be resolved with the addition of Pronase in FCXM.
- Step II-B: If anti-HLA antibody is present – presence of anti-HLA antibodies with or without the presence of an identifiable sensitizing event drifts the candidate toward the less benign end of the immunological spectrum, with higher risk of ABMR. If financial resources are not a constraint, the next logical step would be to do an SAB analysis and to do donor HLA typing for assessing the presence of DSA. The cumulative cost of SAB and HLA typing of donor with high resolution test would amount to 40,000 INR. Both the clinician and patient would be more interested to know the feasibility of transplantation with the proposed donor as a definitive end point which can be provided only by a crossmatch. Rather than spending a huge amount on SAB with many inherent discrepancies, it is prudent from an immunological and financial standpoint to do a crossmatch preferably an FCXM as the next step following detection of antibodies in screen. A positive crossmatch makes the transplant prospects evasive and challenging, and a negative crossmatch makes transplant option feasible [Figure 4].
- Presence of anti-HLA antibodies by screening methods is just qualitative information highlighting that the recipient harbors antibodies which might or might not be of significance. Sensitive crossmatch by FCXM can provide an insight into the feasibility of transplantation. A detailed analysis of antibody profile with SAB is essential and donor specificity is assessed by looking at the donor HLA profile. The SAB profile is based on high resolution, and HLA typing of higher resolution might be needed in certain cases. As mentioned in [Figure 3]b, in recipients with anti-HLA antibodies, based on their profile and behavior of the antibodies, they can be divided into the following three sub-populations [Figure 3]b.
|Figure 3: (a and b) Practical histocompatibility approach in patients with antibody negativity and antibody positivity|
Click here to view
|Figure 4: Practical histocompatibility approach in patients with anti-human leukocyte antigen antibodies|
Click here to view
Antibody screen positive, flow-cytometer cross match negative, and no donor-specific antibodies
Recipients of this subgroup belong to the benign end of the spectrum, near the most benign group of “screen and crossmatch negative,” as depicted in [Figure 3]a. This sub-group could be considered as immunologically low risk from a histocompatibility standpoint.
Antibody screen positive, flow-cytometer cross match positive, and donor-specific antibodies present
It is the group with the highest immunological risk carrying DSAs that results in positive crossmatch. Transplantation has to be avoided for recipients in this sub-group with corresponding donors. Recipients have to resort to alternative donors or kidney exchange programs for a suitable donor, preferably without DSA and negative crossmatch.
Antibody screen positive, flow-cytometer cross match negative, and donor-specific antibodies present
This subgroup provides the most challenge in clinical decision-making for transplant physicians. The transplant community is unambiguous in withholding transplantation in the context of CDC-XM positivity. FCXM- and CDC-XM-negative patients in the presence of DSA is a heterogeneous population influenced by the limitations of solid-phase bead assays. Each transplant immunology laboratory standardizes their FCXM crossmatch with the corresponding SAB-DSA data to provide lab-centric results to formulate histocompatibility protocols., Studies have also been done correlating the posttransplant DSA profile and posttransplant flow crossmatches, and its association with early humoral rejections. This group definitely falls in an intermediate position in the immunological spectrum with no strong evidence-based strategies of immunomodulation to prevent ABMR. Studies have shown that presence of SAB-DSA with negative-flow crossmatch is associated with increased rejection and graft loss. Different transplant programs subject this group to their center-specific immunomodulation strategies and proceed with transplantation. Plasma exchange, intravenous immunoglobulin, and induction with depleting agents are the most utilized desensitization protocols., Many centers resort to alternate donors or paired kidney exchange programs to facilitate transplantation for this sub-group based on their historical immunological behavior.,
Occasionally, instances arise where the crossmatch, especially flow-crossmatch (mainly B cell FCXM), is positive with no identified antibody in screen or SAB analysis, which are usually due to auto-antibodies, medications such as rituximab, nonspecific antibodies binding to Fc receptor on B cells, or non-HLA antibodies binding on to B cells. Many laboratory interventions such as auto-crossmatch, pronase treatment, and repeat testing in serial dilutions or pretreatment of sera can help to identify the reasons for incongruity.,
| Relevance of Profiling|| |
The three generations of antibody-based histocompatibility testing that have evolved over five decades, from CDC-XM (1969) through FCXM (1980s) to bead-based solid-phase assay (2000s), help in profiling the transplant recipients based on their immune reactivity pattern [Figure 5]. At the one end of the spectrum, we have immunologically benign recipients where all platforms are negative and on the other end, we have patients who are immunologically high risk, showing the presence of DSAs in all platforms. Each platform has its advantages and disadvantages which the transplant physician should be literate of. Histocompatibility testing has to be done on more than one platform as per the risk profile of the recipient.
| Conclusion|| |
Advancements in histocompatibility methods have contributed to improved outcomes in solid organ and stem cell transplantation. The advent of PCR has revolutionized HLA typing methods, and a paradigm shift has occurred by identifying newer alleles on a continuous basis. Looking forward, high-resolution NGS-based HLA typing is becoming accessible and cost-effective. In a living donor program, from a histocompatibility standpoint, HLA typing has a distinct role as per the donor relation and the antibody profile of the recipient. Crossmatch is the final gatekeeper in histocompatibility testing and has evolved over the last five decades in the pursuit of increased sensitivity and specificity. Each technique has inherent limitations, and needs evaluation with multiple platforms to proceed with certainty.
In India, a good proportion of transplant centers are dependent solely on CDC-XM which is deficient in sensitivity and objectivity and falls short of being a comparable platform utilizing solid-phase bead assays. Interpretation of profiles from solid-phase bead assays is best done when analyzed along with FCXM results in majority of the centers, worldwide. The crossmatch protocol of Indian transplant programs has to imbibe FCXM as the primary crossmatch test which can provide better clinical decision-making with increased objectivity and accuracy. Active interaction with the transplant immunology laboratory has to be done on a routine basis by the transplant physician in teasing out the intricacies of different testing methods.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Schaffer M, Olerup O. HLA-AB typing by polymerase-chain reaction with sequence-specific primers: More accurate, less errors, and increased resolution compared to serological typing. Tissue Antigens 2001;58:299-307.
Gebel HM, Bray RA. Laboratory assessment of HLA antibodies circa 2006: Making sense of sensitivity. Transplant Rev 2006;20:189-94.
Bray RA, Nickerson PW, Kerman RH, Gebel HM. Evolution of HLA antibody detection: Technology emulating biology. Immunol Res 2004;29:41-54.
Sheldon S, Poulton K. HLA typing and its influence on organ transplantation. Methods Mol Biol 2006;333:157-74.
Ministry of Law, Justice and Company Affairs (Legislative Department) New Delhi, the 11. 1994. p. 1-13.
Mittal T, Ramachandran R, Kumar V, Rathi M, Kohli HS, Jha V, et al
. Outcomes of spousal versus related donor kidney transplants: A comparative study. Indian J Nephrol 2014;24:3-8.
] [Full text]
Cecka JM. The UNOS renal transplant registry. Clin Transpl. 2001:1-18. PMID: 12211771.
Mulley WR, Hudson F, Lee D, Holdsworth RF. Tissue typing for kidney transplantation for the general nephrologist. Nephrology (Carlton) 2019;24:997-1000.
Jolly EC, Key T, Rasheed H, Morgan H, Butler A, Pritchard N, et al
. Preformed donor HLA-DP-specific antibodies mediate acute and chronic antibody-mediated rejection following renal transplantation. Am J Transplant 2012;12:2845-8.
Cross AR, Lion J, Loiseau P, Charron D, Taupin JL, Glotz D, et al
. Donor specific antibodies are not only directed against HLA-DR: Minding your Ps and Qs. Hum Immunol 2016;77:1092-100.
Duquesnoy RJ. Epitope-based human leukocyte antigen matching for transplantation: A personal perspective of its future. Curr Opin Organ Transplant 2018;23:486-92.
Geneugelijk K, Spierings E. PIRCHE-II: An algorithm to predict indirectly recognizable HLA epitopes in solid organ transplantation. Immunogenetics 2020;72:119-29.
Kosmoliaptsis V, Mallon DH, Chen Y, Bolton EM, Bradley JA, Taylor CJ. Alloantibody responses after renal transplant failure can be better predicted by donor-recipient HLA amino acid sequence and physicochemical disparities than conventional HLA matching. Am J Transplant 2016;16:2139-47.
Larkins NG, Wong G, Taverniti A, Lim WH. Epitope matching in kidney transplantation: Recent advances and current limitations. Curr Opin Organ Transplant 2019;24:370-7.
Sypek M, Kausman J, Holt S, Hughes P. HLA epitope matching in kidney transplantation: An overview for the general nephrologist. Am J Kidney Dis 2018;71:720-31.
Phanish MK. Immunological risk assessment and human leukocyte antigen antibody testing in kidney transplantation. Indian J Nephrol 2016;26:80-5.
] [Full text]
Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. Engl J Med 1969;280:735-9.
Kerman RH, Kimball PM, Van Buren CT, Lewis RM, DeVera V, Baghdahsarian V, et al
. AHG and DTE/AHG procedure identification of crossmatch-appropriate donor-recipient pairings that result in improved graft survival. Transplantation 1991;51:316-20.
Lefaucheur C, Suberbielle-Boissel C, Hill GS, Nochy D, Andrade J, Antoine C, et al
. Clinical relevance of preformed HLA donor-specific antibodies in kidney transplantation. Am J Transplant 2008;8:324-31.
Lefaucheur C, Loupy A, Hill GS, Andrade J, Nochy D, Antoine C, et al
. Preexisting donor-specific HLA antibodies predict outcome in kidney transplantation. J Am Soc Nephrol 2010;21:1398-407.
Amico P, Hönger G, Mayr M, Steiger J, Hopfer H, Schaub S. Clinical relevance of pretransplant donor-specific HLA antibodies detected by single-antigen flow-beads. Transplantation 2009;87:1681-8.
Gloor JM, Winters JL, Cornell LD, Fix LA, DeGoey SR, Knauer RM, et al
. Baseline donor-specific antibody levels and outcomes in positive crossmatch kidney transplantation. Am J Transplant 2010;10:582-9.
Morris PJ, Ting A. The crossmatch in renal transplantation. Tissue Antigens 1981;17:75-82.
Karpinski M, Rush D, Jeffery J, Exner M, Regele H, Dancea S, et al
. Flow cytometric crossmatching in primary renal transplant recipients with a negative anti-human globulin enhanced cytotoxicity crossmatch. J Am Soc Nephrol 2001;12:2807-14.
Pelletier RP, Adams PW, Hennessy PK, Orosz CG. Comparison of crossmatch results obtained by ELISA, flow cytometry, and conventional methodologies. Hum Immunol 1999;60:855-61.
Pidwell DJ. Interpretation of crossmatch results. ASHI La-boratory Manual 2000;1(IC13):1.
Khodadadi L, Adib M, Pourazar A. Immunoglobulin class (IgG, IgM) determination by dithiothreitol in sensitized kidney transplant candidates. Transplant Proc 2006;38:2813-5.
Mulley WR, Kanellis J. Understanding crossmatch testing in organ transplantation: A case-based guide for the general nephrologist. Nephrology (Carlton) 2011;16:125-33.
Bishara A, Nelken D, Brautbar C. Differential expression of HLA Class-I antigens on B and T lymphocytes obtained from human lymphoid tissues. Immunobiology 1988;177:76-81.
Pellegrino M, Belvedere M, Pellegrino AG and Ferrore S, B peripheral lym-phocytes express more HLA antigens than I peripheral lymphocytes. Transplantation 1978;25:93.
Gebel HM, Bray RA, Nickerson P. Pre-transplant assessment of donor-reactive, HLA-specific antibodies in renal transplantation: Contraindication vs. risk. Am J Transplant 2003;3:1488-500.
Kaufman A, de Souza Pontes LF, Queiroz Marques MT, Sampaio JC, de Moraes Sobrino Porto LC, de Moraes Souza ER. Analysis of AHG-PRA and ELISA-PRA in kidney transplant patients with acute rejection episodes. Transpl Immunol 2003;11:175-8.
Chandraker A, Sayegh MH, Singh AK. Core concepts in renal transplantation. Core Concepts in Renal Transplantation US: Springer; 2012. p. 1-242. https://doi.org/10.1007/978-1-4614-0008-0.
Gebel HM, Bray RA. HLA antibody detection with solid phase assays: Great expectations or expectations too great? Am J Transplant 2014;14:1964-75.
Cecka JM. Calculated PRA (CPRA): The new measure of sensitization for transplant candidates. Am J Transplant 2010;10:26-9.
Colombo MB, Haworth SE, Poli F, Nocco A, Puglisi G, Innocente A, et al
. Luminex technology for anti-HLA antibody screening: Evaluation of performance and of impact on laboratory routine. Cytometry B Clin Cytom 2007;72:465-71.
Sullivan HC, Gebel HM, Bray RA. Understanding solid-phase HLA antibody assays and the value of MFI. Hum Immunol 2017;78:471-80.
Pratheeba M, Doss SA. The Impact of Next Generation Sequence Based HLA Typing of donor-Recipient Pairs on Interpreting Virtual Crossmatch – A Single Centre Study; TRANS 163; Abstracts _ TRANSMEDCON 2019.
Couzi L, Araujo C, Guidicelli G, Bachelet T, Moreau K, Morel D, et al
. Interpretation of positive flow cytometric crossmatch in the era of the single-antigen bead assay. Transplantation 2011;91:527-35.
Reinsmoen NL, Lai CH, Vo A, Cao K, Ong G, Naim M, et al
. Acceptable donor-specific antibody levels allowing for successful deceased and living donor kidney transplantation after desensitization therapy. Transplantation 2008;86:820-5.
Morales-Buenrostro LE, Terasaki PI, Marino-Vázquez LA, Lee JH, El-Awar N, Alberú J. “Natural” human leukocyte antigen antibodies found in nonalloimmunized healthy males. Transplantation 2008;86:1111-5.
Shankar N, Daly R, Geske J, Kushwaha SK, Timmons M, Joyce L, et al
. LVAD implant as a bridge to heart transplantation is associated with allosensitization as measured by single antigen bead assay. Transplantation 2013;96:324-30.
Burns JM, Cornell LD, Perry DK, Pollinger HS, Gloor JM, Kremers WK, et al
. Alloantibody levels and acute humoral rejection early after positive crossmatch kidney transplantation. Am J Transplant 2008;8:2684-94.
Mohan S, Palanisamy A, Tsapepas D, Tanriover B, Crew RJ, Dube G, et al
. Donor-specific antibodies adversely affect kidney allograft outcomes. J Am Soc Nephrol 2012;23:2061-71.
Montgomery RA, Lonze BE, King KE, Kraus ES, Kucirka LM, Locke JE, et al
. Desensitization in HLA-incompatible kidney recipients and survival. N Engl J Med 2011;365:318-26.
Hachem RR, Yusen RD, Meyers BF, Aloush AA, Mohanakumar T, Patterson GA, et al
. Anti-human leukocyte antigen antibodies and preemptive antibody-directed therapy after lung transplantation. J Heart Lung Transplant 2010;29:973-80.
Segev DL, Gentry SE, Warren DS, Reeb B, Montgomery RA. Kidney paired donation and optimizing the use of live donor organs. JAMA 2005;293:1883-90.
Kute VB, Agarwal SK, Sahay M, Kumar A, Rathi M, Prasad N, et al
. Kidney-paired donation to increase living donor kidney transplantation in India: Guidelines of Indian Society of Organ Transplantation-2017. Indian J Nephrol 2018;12:67.
Weinstock C, Schnaidt M. The complement-mediated prozone effect in the Luminex single-antigen bead assay and its impact on HLA antibody determination in patient sera. Int J Immunogenet 2013;40:171-7.
Bearden CM, Agarwal A, Book BK, Sidner RA, Gebel HM, Bray RA, et al
. Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab. Hum Immunol 2004;65:803-9.
Tait BD, Süsal C, Gebel HM, Nickerson PW, Zachary AA, Claas FH, et al
. Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation. Transplantation 2013;95:19-47.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]