The major histocompatibility complex is comprised of integral membrane glycoproteins that function in the recognition of self vs. nonself antigens and presentation of nonself antigens to the immune system. It was discovered on leukocytes and hence is referred to as the human leukocyte antigen (HLA) complex. HLA antigens are encoded on chromosome 6p, and this is the most polymorphic system in the human body.
Several scientists contributed to the discovery of the HLA complex. The discovery of the first HLA antigen is credited to French immunologist Jean Dausset (1916-2009). Dausset studied sera of patients who had received multiple blood transfusions and observed that these sera agglutinated leukocytes of other individuals but not self. He hypothesized that these leukocyte antigens would become important in tissue and bone marrow transplantation. The first alloantigen on leukocytes was called MAC (later known as HLA-A2).1
HLA antigens are classified into HLA Class I, Class II, and Class III. Class I antigens are present on the surface of all nucleated cells and platelets. They present intracellular derived foreign peptides to the cells of the immune system. MHC Class I antigens play a key role in alerting the immune system of viral infection by presenting internal viral peptides to cytotoxic T lymphocytes. Class II antigens are found primarily on antigen-presenting cells such as macrophages, dendritic cells, monocytes, and B-lymphocytes and present extracellular-derived foreign peptides. During an immune response against a bacterial infection, macrophages that have ingested the bacteria utilize HLA Class II antigens to present the extracellular bacterial peptides to T-helper lymphocytes to expedite the immune response. Class III antigens play a role in the inflammation and complement system.
HLA Class I and II antigens are important in solid organ and hematopoietic stem cell transplantation. Each class has three loci. For Class I, these are HLA-A, HLA-B and HLA-C. For Class II, the loci are HLA-DR, HLA-DQ and HLA-DP.
HLA typing
HLA typing consists of identifying the unique combination of antigens present in an individual’s tissue. This is done by either low- or high-resolution molecular techniques. In low-resolution typing, the patient is typed to the point where the HLA antigen is identified by the allelic group. A patient can be typed and identified as HLA-A*02, which states that this individual presents HLA-A2 antigen but does not identify the specific allele of A2. High-resolution typing, on the other hand, does identify the specific allele the individual possesses. For example, typing by high resolution methods could be identified as HLA-A*02:01, which states that this person possesses HLA A2 antigen coded by gene A allele 01.2
Antibody testing
In the same manner that an individual develops antibodies against bacteria, viruses, and parasites via natural exposure or vaccinations, an individual can develop antibodies against non-self HLA antigens found in the population through blood transfusions, previous transplantations, and pregnancy. Patients who become sensitized to allogeneic HLA antigens run the risk of antibody-mediated rejection (AMR) of the transplanted organ. Anti-HLA antibodies are either IgG or IgM. The IgM antibodies are usually non-complement-binding and therefore usually non-consequential. IgG antibodies, on the other hand, depending on their sub-type (IgG1, IgG2, IgG3, and IgG4), can be complement-binding, and can contribute to AMR of the transplanted organ, thrombi formation, and possible loss of the graft if not treated successfully and promptly. IgG3 binds complement the most, followed by IgG1, IgG2, and IgG4.
In order to determine whether the recipient is sensitized to allogeneic HLA antigens, the clinical HLA laboratory performs a panel reactive antibody test (PRA), a screening assay to detect anti-HLA antibodies. If the PRA is positive, a single antigen bead assay (SAB) is performed to detect the specificity of the anti HLA Class I and/or Class II antibodies. The basic principle for both tests (PRA and SAB) is the same. Antibody screening is performed using a solid phase bead assay platform. HLA Class I and II antigens are extracted and purified from cell lines, usually an EBV cell line, and bound onto microparticles. Patient serum is incubated with these microparticles. If the patient possesses antibodies against allogeneic HLA antigens, they will bind to the complementary antigens on the microparticles. This binding is detected by adding an anti-human monoclonal IgG antibody that is tagged with a fluorescent tag. When the specimen is examined on the analyzer, the laser detects this fluorescent tag as a positive signal and the results from this procedure are reported out as the percent PRA. The PRA is essentially the transplantability index of the patient. If there are no anti-HLA antibodies, the PRA is 0 percent. This means the patient’s transplantability index is 100 percent minus 0 percent—that is, 100 percent.
The optimal result of a PRA would be 0 percent, which means that the patient does not have detectable preformed HLA antibodies. A high PRA percentage does not necessarily indicate an abundance of antibodies. The patient could have low titers of an antibody that is cross-reactive with shared HLA epitopes. Likewise, a low PRA does not necessarily indicate that a recipient is suitable for transplantation. Even though the PRA is low, if the antibody that the recipient possesses is donor-specific, it could cause rejection. The PRA establishes the probability of a positive crossmatch with donor antigens, therefore indirectly giving an estimate of how appropriate a donor is for a recipient. The most important aspect of antibody testing is the specificity of the antibodies identified and, in the case of solid organ transplantation, whether the anti-HLA antibodies detected in the recipient’s serum are against donor HLA antigens (also called donor-specific antibodies or DSA).
Crossmatch and virtual crossmatch
A crossmatch is a procedure in which serum of the recipient is incubated with lymphocytes of the donor, to determine whether the recipient has DSA. Even if the PRA/antibody identification tests determine that the recipient has HLA antibodies, this does not mean that the immune system of the recipient will attack the donor kidney. If the donor does not possess the HLA antigens that the recipient has antibodies against, the recipient’s immune system won’t readily attack the transplanted organ. However, if the donor does possess this antigen, then the crossmatch would show that the recipient has a DSA.
HLA crossmatch is performed via flow cytometry, utilizing donor lymphocytes and recipient serum. T cells possess HLA Class I antigens, whereas B cells possess both Class I and II HLA antigens. Donor lymphocytes are allowed to react with recipient serum. Positive crossmatches indicate that the recipient possesses DSA that can potentially cause rejection, and therefore a more suitable donor must be found. If both the T and B cell crossmatch are positive, then the recipient has anti Class I DSA and/or both Class I and Class II DSA. If only the B cell crossmatch is positive, then the recipient probably has anti Class II DSA or weak anti Class I DSA.
A virtual crossmatch is an assessment of the immunological compatibility between recipient and donor based on the recipient’s HLA antibody profile and the HLA antigens of the donor. In order for a virtual crossmatch to be acceptable, there needs to be sufficient, current data on the recipient’s antibody profile.
Developing a new testing algorithm
Tambur et al studied the effects of altering the testing procedure on the transplantability of sensitized recipients and the success of transplantation. Two groups were established by the researchers. Group I consisted of individuals whose percent PRA was determined via solid phase-based testing with limited antibody identification. Group II consisted of individuals who had gone through more complete testing, including antibody identification, strength assessment, and use of pronase for crossmatch.3 The researchers hypothesized that more in-depth and complete analysis of antibody makeup would increase the transplantability of sensitized patients. The more complete antibody testing allowed the center to “define the specificity of HLA directed antibodies for patients in Group II,” which led to an increase in the number of sensitized patients who were transplanted, in comparison to patients in Group I. Although the percent PRA was determined in the same manner for both groups, they differed in the methods used to identify the specificity of the antibodies. The transplantability of sensitized patients in Group II was higher than that of Group I (49 percent vs. 40 percent), though the viability of the kidney at one year after transplantation was similar for both groups.
Bostock et al wanted to determine the probability of receiving a kidney transplant from a deceased donor waiting list based on the percent PRA. The group conducted a retrospective study, looking at the deceased donor waiting list of their institution, specifically looking at ABO type, lymphocyte crossmatch results, percent PRA, and time on the wait list. Potential recipients were classified into four groups based on the percentages of their PRA: 0 percent, one percent to 19 percent, 20 percent to 79 percent, and 80 percent to 100 percent.4 The research group found that the probability of receiving a kidney decreases as the percent PRA of the potential recipient increases. The group also discovered that a higher risk of no transplantation is apparent once the PRA increases above 20 percent.
Mazuecos et al conducted a study in which kidney transplantations were constructed on a protocol based on virtual crossmatch analysis with a final crossmatch before transplantation for highly sensitized patients (PRA > 80 percent). Out of the 52 patients who were transplanted, five patients experienced AMR, and DSAs developed in ten patients. These results were compared to 35 patients who were not classified as highly sensitized. In terms of acute rejection, no significant difference was observed, but highly sensitized recipients were more prone to develop DSA. Virtual crossmatch assessment with a final crossmatch before procedure increased access to kidney grafts in highly sensitized patients with a low probability of AMR and high survival rate.5
A highly sensitive solid-phase bead assay for HLA antibody detection has recently become available and should be appropriately used to improve laboratory resource consumption by avoiding unnecessary testing and associated costs. Currently, all donors are HLA typed by low-resolution molecular methods and crossmatched with potential recipients. We propose a change in this testing algorithm for living kidney donation. For a recipient with 0 percent PRA detected by assay (and hence who is virtual cross match-negative), HLA typing and crossmatch should be performed on the donor only if he or she is approved for donation, since the process of finding a suitable donor includes several clinical and laboratory parameters other than PRA and crossmatch results. As laboratory professionals, we must emphasize appropriate test utilization. Testing should be performed only when necessary. High-complexity testing is expensive, and HLA laboratories should continuously look for ways to improve efficiency and reduce cost without compromising patient safety.
References
- Thorsby E. A short history of HLA.Tissue antigens. 2009;74(2):101-116.
- Choo SY. The HLA system: genetics, immunology, clinical testing, and clinical implications.Yonsei Medical Journal. 2007;48(1);11-23.
- Tambur AR, Leventhal J, Kaufman DB, Friedewald J, Miller J, Abecassis M. Tailoring antibody testing and how to use it in the calculated panel reactive antibody era: the Northwestern University experience.Transplantation. 2008;86(8):1052-1059.
- Bostock IC, Alberú J, Arvizu A, et al. Probability of deceased donor kidney transplantation based on % PRA. Transplant Immunology. 2013;28(4):154-158.
- Mazuecos A, Alvarez A, Nieto A, et al. Kidney Transplantation Results in very highly sensitized patients included in a virtual crossmatch program: analysis of kidney pairs. Transplantation Proceedings. 2016;48(9): 2899-2902.
