|Year : 2018 | Volume
| Issue : 1 | Page : 1-6
Immunology in transplantation: Basics for beginners
Department of Nephrology, Osmania Medical College and General Hospital, Hyderabad, Telangana, India
|Date of Web Publication||29-Mar-2018|
Dr. Manisha Sahay
6-3-852/A, Ameerpet, Hyderabad - 500 016, Telangana
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Sahay M. Immunology in transplantation: Basics for beginners. Indian J Transplant 2018;12:1-6
| Introduction|| |
The immunological aspects of transplantation include testing for ABO compatibility, human leukocyte antigens (HLA) typing, testing for anti-HLA antibodies in the recipient serum against common antigens in population (panel reactive antibody [PRA]), and testing for antibodies in recipient serum against the specific donor, i.e., donor-specific antibodies (DSAs).
| Blood Group Matching|| |
ABO blood group matching is the first step in kidney transplantation. Rh matching is not required as Rh proteins are expressed only on red blood cells.
ABO system consists of four common blood groups (A, B, AB, and O). Types B and O most frequently found in the Indian population. Antigen is expressed on red blood cells, lymphocytes, platelets, and epithelial and endothelial cells. O blood group has no antigen while antibodies to both A and B are found in an individual with blood type O. An individual with blood type AB has A and B antigens but no antibodies. Blood group A has antigen A and antibodies to B and blood group B has antigen B and antibodies to A. Blood group A consists of two subtypes, A1 and A2. A2 is less antigenic than A1. Thus, kidneys from A2 blood group donor can be successfully transplanted into recipients with low pretransplant anti-A titers without desensitization.
The two methods used to reduce circulating ABO antibody titers are plasmapheresis and immunoabsorption. The goal is to achieve titers ≤1:8–1:32 depending on the center. Rituximab is used as adjunctive therapy to reduce antibody production and has replaced splenectomy. Long-term graft and patient survival of ABO-incompatible (ABOi) transplantation are comparable with those of ABO-compatible transplantation.
An important point to remember is that in ABOi recipients, the peritubular staining of C4d is usually positive in renal biopsy and reflects accommodation. Hence, only C4d is not sufficient to diagnose rejection in ABOi; in addition, the presence of histological criteria of rejection is essential.
| Methods of Antibody Detection|| |
The classic tube dilution method is most commonly used to report an immunoglobulin M (IgM) isoagglutinin titer and a total isoagglutinin titer. Improved assays, such as enzyme-linked immunosorbent assay (ELISA)-based technology, help standardize acceptable IgM and IgG isoagglutinin (antibody) titers at baseline and in the peritransplant period. If fresh frozen plasma (FFP) replacement is required as a result of plasmapheresis treatment, donor blood type or AB donor FFP should be given. This avoids administration of antiallograft isoagglutinin antibodies.
ABOi is no longer a barrier to transplantation as desensitization protocols have been developed that include use of rituximab, splenectomy, or plasmapheresis to successfully reduce antibody titers.
| Testing for Human Leukocyte Antigens|| |
The degree of HLA mismatch influences long-term graft survival rather than early rejection. As per the annual report of the Scientific Registry of Transplant Recipients, the 5-year allograft survival for deceased donor kidney transplants was 77% with zero HLA mismatch and 67% with six HLA mismatch., Two haplotype-matched living transplants are estimated to have a half-life of approximately 30 years while one haplotype-matched living transplants have a half-life of 18 years., Thus, HLA matching is important.
Genes coding for HLA is located on short arm of chromosome 6. These are classified as HLA I which includes A, B, and C. HLA II includes genes for DP, DQ, and DR antigens.,
HLA antigens can be tested by serology-based methods or by DNA typing methods. The cell-based (serology) assays are not specific for HLA antibodies, are time-consuming, require a sufficient supply of viable lymphocytes, and lead to unexpected positive crossmatches. In the 1990s, solid-phase assays were developed. These included ELISA-based and subsequently Luminex single bead assays. Luminex-based assays, using microbeads coated with soluble HLA protein as targets, are currently the most widely used.
The major techniques in DNA typing by solid-phase assays include sequence-specific primers (SSPs), sequence-specific oligonucleotide probes (SSOPs), sequencing-based typing (SBT), and next-generation sequencing (NGT).
SSP method uses polymerase chain reaction (PCR) and specificity of primers. A PCR undergoes amplification only if the sequences of primers are nearly perfect in binding. Primer sets have been designed to detect the variation in sequences for HLA typing. SSP method can thus detect the HLA typing of an individual. With reduction in cost for PCR, the technique is inexpensive and can provide HLA typing in few hours.
SSOP method utilizes 6–19 length nucleotide probes that bind to DNA sequences specific to HLA alleles. Both the SSP and SSOP methods can only identify known HLA alleles. The sample needs not be tested on the day of collection and does not require viable cells. [Figure 1]a,[Figure 1]b,[Figure 1]c shows the nomenclature of the reports.
|Figure 1: (a) Nomenclature human leukocyte antigens. (b) Human leukocyte antigens nomenclature. (c) Nomenclature human leukocyte antigens|
Click here to view
SBT method does not require prior knowledge of the HLA alleles, can reveal new alleles, and provides the most comprehensive understanding of HLA typing, but is time-consuming and expensive.,
Next-generation sequencing is the most advanced method but is currently not practiced widely due to cost issues.
Example of DNA-based report A * 33-Cw*0302-B* 5801- DRB*0301-DQB1 * 02
Epitopes and eplets
HLA antibodies recognize structural motifs (epitopes) that contain a set of amino acids present in the donor. Each HLA molecule contains multiple epitopes, and many epitopes are shared among different HLA antigens. These are called complement reactive eptiofpe groups (CREGS). Hence, identifying only HLA is not sufficient. These epitopes need to be identified. For antibodies against DQ or DP molecules, in which genes coding for both alpha (HLA-DQA1, DPA1) and beta (DQB1, DQB1) chains are polymorphic, epitopes specific for the alpha/beta chain, and for alpha-beta chain, combinations may be determined.
One of the approaches being currently used to identify epitopes is called Match Maker application that uses amino acid sequence comparison and molecular modeling to infer putative epitopes called eplets. Use of epitopes and epilets may help in determining permissible mismatches. How best to apply this knowledge in the clinical practice to improve transplant outcomes is under debate.
Besides major HLA, minor antigens play a significant role in survival of graft. An important antigen is MHC class I polypeptide-related sequence A encoded by the MICA gene. The MICA gene is located on human chromosome 6. It does not associate with beta-2-microglobulin and thus does not present antigens such as HLA class I molecules. It is polymorphic and about 50 antigens have been described. MICA is expressed on many cells such as endothelial cells, dendritic cells, fibroblasts, and epithelial cells but not on lymphocytes. Transplant recipients who have antibodies to MICA have worse graft survival.
Minor histocompatibility antigens (MiHA) are small endogenous peptides in antigen binding sites in MHC region which are recognised by CD8 cells and can cause rejection. H-Y MiHA is encoded by Y chromosome in male and may cause rejection in male to female transplant.
There are other antigens like angiotensin receptor, endothelial cell antigen etc which may cause rejections.
| Principles of Immunological Tests|| |
The important tests to detect the anti-HLA and other preformed antibodies in patient's serum include cell-based assays and solid-phase assays [Figure 2].,,,
| Cell Based Tests|| |
| i. Complement-Dependent Cytotoxicity Crossmatch Test|| |
The complement-dependent cytotoxicity (CDC) crossmatch test screens for preformed antibodies in the recipient that may immediately react against the donor. It can be a T-cell or a B-cell crossmatch.
T-cell crossmatch-T-lymphocytes are isolated from the donor and mixed with serum from the recipient. If preformed antibodies are present in the recipient, these recognize the HLA class I molecule and bind to them. When the complement is added, the cells undergo lysis. A dye that penetrates lysed cells is utilized to detect number of dead cells. In contrast, if no antibodies in the recipient's serum bind to the T-lymphocytes of the donor, complement will not be activated and there will be no cell death. No dye will be taken up by the cells and the test is considered negative [Figure 2]. The result of the crossmatch test is reported as the percentage of dead cells relative to live cells as determined by microscopy. The reading is on a semi-quantitative scale with 0 representing no dead cells and 8 representing complete lysis of cells. When there is >20% cell lysis, the test is reported as positive. At present, a positive CDC crossmatch test utilizing T-lymphocytes of the donor is considered an absolute contraindication for kidney transplantation. T-cells do not constitutively express HLA class II molecules. Hence, the result of a positive T-lymphocyte crossmatch test generally reflects antibodies to HLA class I only.
B-cell crossmatch-B-lymphocytes express both HLA class I and HLA class II molecules. A CDC crossmatch test that is positive against donor B-lymphocytes but negative against donor T-lymphocytes can be interpreted to represent a HLA class II antibody. HLA class II molecules are expressed on macrophages, dendritic cells, and B-lymphocytes. A positive B-lymphocyte CDC crossmatch test is not an absolute contraindication to transplantation. However, it has been associated with reduced long-term graft survival. A positive T-lymphocyte CDC crossmatch in the presence of a negative B-lymphocyte CDC crossmatch could be a technical error.
| Limitations of Complement-Dependent Cytotoxicity Crossmatch|| |
- False-negative lymphocyte CDC crossmatch test may occur if the concentration of antibodies binding to the lymphocytes is below the threshold for complement activation. Addition of antihuman globulin enhances the concentration of antibodies and thus increases the sensitivity of the CDC crossmatch test
- CDC crossmatch test cannot distinguish IgG from IgM antibodies as both IgG and IgM can fix complement. IgM antibodies are usually autoantibodies. IgM antibody can be removed by the use of the reducing agents such as 2-mercaptoethanol or dithiothreitol or by heating the serum to 63°C for 10 min. Thus, a CDC crossmatch test that is positive against B-lymphocytes but negative when the same serum is treated with heat at 63°C indicates an IgM antibody. The significance of the presence of IgM antibodies is not well understood
- This test requires the presence of live cells
- Although lymphocytes express class I HLA and class II HLA molecules, they do not provide the full representation of all antigens against which antibodies from a recipient can react. Thus, it does not detect DSAs against MICA or against donor endothelial cells. A CDC test with endothelial cells has recently been developed and employed.,
| ii. Flow Cytometry Crossmatch Test|| |
The flow cytometer utilizes laser to evaluate the status of single cells one at a time. The cells from the potential donor are isolated and are labeled using a fluorescent marker. A fluorescent-labeled antibody against CD3 or CD19 is used as a marker to distinguish T- from B-lymphocytes. The donor cells are incubated with the recipient's serum to allow for potential antibodies to bind. If there are donor-specific anti-HLA antibodies, the Fab portion of the antibody binds to the HLA antigens on the cell surface. Fluorescein-labeled goat antihuman antibody is then used as the reporter fluorescent dye to detect the binding of this alloantibody. This secondary antibody can detect either IgG or IgM antibodies (binding to IgM needs special technique). Thus, if there is a positive reaction between the recipient's serum and donor lymphocytes, the flow cytometer will be able to detect this interaction as it will recognize the fluorescent-labeled anti-CD3 or -CD19 antibody and the fluorescent-labeled antibody against the Fc portion of the DSA. If there is a negative reaction between the recipient's serum and donor lymphocyte, the flow cytometer will recognize the fluorescent-labeled anti-CD3 or -CD19 antibody but not detect any fluorescent-labeled antibody against the Fc portion of DSA. Flow cytometry results are reported as positive or negative based upon the median channel shift caused by the binding of a specific antibody.
| Advantages|| |
The flow crossmatch test is more sensitive than the CDC crossmatch test. It detects low titer antibodies. Unlike the CDC crossmatch test, the flow crossmatch test is also not dependent on complement. If antibodies that are binding to the donor T-lymphocytes are noncomplement fixing antibodies such as IgG2, CDC crossmatch test is negative against T-lymphocytes, but the flow crossmatch test is positive against T-lymphocytes.
| Limitations|| |
- The number of channel shifts required to call a test positive or negative has not been standardized
- Standard flow crossmatch test only detects IgG that is bound to donor cells. The flow crossmatch test will be negative if recipient has only donor-specific IgM antibody. Mostly, this is an advantage as IgM are nonpathogenic. However, role of IgM needs to be better defined and modifications can be made in flow crossmatch if needed to detect IgM as well
- The significance of a positive result in the presence of a negative CDC crossmatch is not entirely clear. In the absence of prior sensitization, a positive T- or B-lymphocyte flow crossmatch is not associated with increased risk of acute rejection. In patients who are sensitized before transplantation, the graft survival is inferior. The outcome of a positive B-lymphocyte flow crossmatch is less clear.
| Solid-Phase Assays|| |
The HLA antigen is immobilized on the surface in the solid-phase assays., Following methods are used:
- ELISA METHOD – Specific purified HLA molecules are immobilized on a plastic surface. The serum of the patient is then incubated on the plastic surface. If there are antibodies directed against a specific HLA, these antibodies bind to the antigen. A second antihuman IgG directed against the Fc portion of antibody is now added to detect the serum antibodies that have bound to the HLA. An enzyme is attached to this second antibody. The addition of a substrate for the attached enzyme will generate a colored product that is measured. If there is no anti-HLA antibody in patient's serum, then the antihuman IgG cannot attach and is washed away
- IN FLOW BEAD-dependent crossmatch (different from flow cytometry discussed above where lymphocytes are used), the antigens are placed on the latex beads (solid phase) which are fluorescent instead of using the lymphocytes. Multiple HLA antigens can be located on a bead. The antibodies bind to these antigens. These bound antibodies are tagged with FITC which is fluorescent and this shift is called transchannel shift. It can detect both HLA I and II antibodies and both IgM and IgG and complement fixing and noncomplement fixing antibodies are detected
- LUMINEX SINGLE ANTIGEN BEAD ASSAY – In Luminex, specific synthetic HLA molecules are immobilized on 100 polystyrene microspheres with fluorescent dyes. When excited with laser, each microsphere generates a unique spectral signature allowing for powerful multiplexing. The serum of the patient is incubated with the microspheres coated with HLA molecules. A second fluorescent-labeled antihuman IgG-directed against the Fc portion of the antibodies is then added to the system. A flow cytometer detects the amount of fluorescent-labeled antihuman IgG (phycoerythrin bound IgG) that is bound to a particular HLA molecule. The strength of the antibody titer is quantified as the mean fluorescence intensity (MFI). MFI values of >5000 are contraindication to transplant.
| Limitations With the Solid-Phase Assays|| |
- The panel of HLA in solid-phase assay represents the most prevalent HLA in the population and may miss the less common HLA
- The solid-phase assay is an “in vitro” test where HLA antigen is on microspheres and not the cells. Thus, the solid-phase assays will detect HLA in secondary structure but may miss detection of antibodies to HLA in quaternary structure unlike CDC crossmatch test which can detect them in quarternary structure also
- Commonly used solid-phase assays detect IgG antibodies and so an IgM antibody can be missed
- Solid-phase assays do not distinguish complement fixing from noncomplement fixing antibodies
- Blocking antibodies may lead to false negative test (prozone phenomenon)
- HLA antibodies levels may change over time. For example, an apparent sudden increase in antibody strength may occur due to proinflammatory events such as surgery or infection or of sensitizing events such as blood transfusions. The patient's immunological history should also be taken. Previous immunizing events (pregnancies, transfusions, and transplants) may recognize increased risk even in the absence of antibody. HLA typing of previous organ and blood donors and in the case of known pregnancies, HLA typing of offspring, when available, should be taken
- These methods do not detect memory B-cells which, although ideal for assessment of humoral immunological memory, are still being used mainly in the research setting because of cost.
Despite these limitations, solid-phase assays are the most sensitive tests that are currently available. Some antibodies may be present in high titre ie >5000 MFI eg HLA-DP antibodies but they do not react with the donor cells. Hence not only titre but nature of antibody is important. Cell binding is demonstrated by cell based assay. Solid-phase immunoassay (SPI) results must be combined with additional data from cell-based assays and from the patient's immunologic history.
| What Tests Are Done in Practice?|| |
Panel reactive antibody test
In the PRA test, the recipient's serum is tested for antibodies against a panel of lymphocytes from approximately 100 blood donors from local population. If the serum of a recipient causes lysis of cells in 80 out of 100 samples, it is PRA positive with value of the PRA being 80%. If a donor is available from that donor pool, the recipient will have acute rejection 80% of the time. PRA test has been extremely useful in providing information about the sensitization of a recipient. However, the panel does not represent all HLA class I and class II molecules. Further, the antigen specificity is not known.
PRA can be done by cell-based methods such as CDC method and flow cytometry.
Calculated PRA (cPRA) – Nowadays, it is possible to determine the antigen specificity against which an individual produces antibody. These antigens are called unacceptable antigens. The ELISA, flow cytometry, or Luminex assay is run against a fixed panel of HLA antigens from the past organ donors in a large computerized database and the percentage of specific unsuitable antigens reported. This “virtual crossmatch” is called as cPRA. Several centers do not perform PRA and instead calculate the PRA (cPRA). The PRA is calculated separately for class I HLA and class II HLA antigens. Patients with cPRA that is >80% receive additional points for the allocation of a kidney.
Donor-specific antibody test
DSAs are the antibodies in the recipient's serum which are specific to the donor and these in high titer are an absolute contraindication to transplant with that donor.
Potential transplant recipients may have preformed DSA against HLA.
- Pregnancies: About 30%–50% of women with three or more pregnancies will develop HLA antibodies. In some women, the antibodies could be present for just a short time (weeks to months), while in others they may persist for many years
- Blood transfusions: About 50% of patients who receive multiple transfusions will develop antibodies. However, blood transfusions using filters decrease the chances of sensitization
- Previous transplant: About 90% of patients develop HLA antibodies within 2 weeks of a failed graft. Over time, some of these patients will lose their antibodies
- Viral/bacterial infections: There are some reports that patients with virus infections develop HLA antibodies, although this is relatively rare.
DSAs can again be tested using cellular methods such as CDC or flow cytometry or by solid-phase assays such as flow bead assay, ELISA, or Luminex. T-cell CDC positivity is an absolute contraindication to transplant. CDC positive for B-cells is a relative contraindication and may impact long-term outcomes.
Luminex positive, flow negative, and CDC negative is low immunological risk as Luminex may detect antibodies in very low titer as well and these may not be complement fixing antibodies. Luminex and flow positive but CDC negative is intermediate risk, while all three positive is very high immunological risk for transplant.
Several studies have found association between preformed antibodies and worse graft survival., The strength of the DSA as measured by MFI also play a role in antibody-mediated rejection. DSAs of both classes HLA I and II are pathogenic. However, not all anti-HLA DSA may be pathogenic. Further research is needed to identify pathogenic DSA. Pathogenic DSA should be subjected to desensitization using plasmapheresis, rituximab, and high-dose intravenous Ig. DSA more than 5000 MFI is a contraindication to transplant.
De novo DSA-transplant recipients can develop DSA after transplantation. These may develop due to noncompliance with medications, viral infections, etc. De novo anti-HLA DSA has been found to be a risk factor for graft failure. DSA should be monitored 2, 4, and 8 weeks, 6 months, and 12 months and then less frequently thereafter.
| Newer Advances|| |
The complement cascade contributes to allograft damage; there are SPI assays that have been modified to detect complement-binding antibodies. Currently, the assays most commonly used detect: C1q (a fragment generated at the beginning of the classical pathway), C3d (a split product of C3 that is amplified midway in the complement cascade), and IgG subclasses, IgG1 and 3 being the strongest complement binders. The presence of these components portends a poor outcome. Although these new assays are valuable in the research setting, it is not yet clear whether they will be of utility in the clinical setting.
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[Figure 1], [Figure 2]