Trends in HLA Antibody Screening and Identification and Their Role in Transplantation

Cathi L Murphey; Thomas G Forsthuber


Expert Rev Clin Immunol. 2008;4(3):391-399. 

In This Article

Abstract and Introduction

HLA testing has been a staple in transplantation since the recognition that antibodies, directed against lymphocytes, were associated with allograft failure. This seminal finding led to the discovery of the MHC and the appreciation of the importance of HLA testing in transplantation. Early approaches focused on the importance of HLA matching, and were an important aspect of deceased organ donor allocation. More recently, and as a direct result of improvements in immunosuppression, there has been a movement away from 'matching' as the driving force in organ allocation. By contrast, we are now challenged with selecting donor-recipient pairs based on acceptable mismatches. For patients devoid of HLA antibodies, this is not an issue. However, for patients with HLA alloantibodies, that is, the sensitized patient, we face significant challenges in assessing the repertoire of the HLA antibody reactivity they possess. Over the past several years, significant advances in HLA antibody detection have occurred. Solid-phase, multiplex testing platforms have replaced traditional cell-based assays, and have provided better sensitivity and specificity in antibody detection. As a direct result of improved antibody identification, many programs are moving into the realm of the 'virtual crossmatch'. The virtual crossmatch has proven to be successful in renal, cardiac and lung transplantation, and has resulted in a greater percentage of sensitized patients gaining access to transplantation. This review will be devoted to highlighting the latest developments in antibody assessments and discussing their utilization in transplant testing.

HLAs are glycoproteins that reside on the surface of almost every cell in the body. The primary function of these antigens is to serve as recognition molecules in the initiation of an immune response. HLA antigens on specialized immune cells present peptides from foreign substances (e.g., viruses and bacteria) to effector cells of the immune system. Effector cells are then responsible for driving both the cellular and humoral arms of the response. There are two major classes of HLA antigens, HLA class I (HLA-A, -B and -C) and HLA class II (HLA-DR, -DQ and -DP). With regard to transplantation, HLA antigens play a major role in the rejection of foreign tissue. While not insurmountable, overcoming the MHC barrier has made the field of transplantation quite challenging. Before the recent era of new immunosuppressive drugs, patients who were well matched for HLA loci had better graft survival than recipients who were not well matched.[1,2,3] Although allocation based on HLA matching provided superior graft survival for well-matched patients,[1,4] matching proved to be a distinct disadvantage for patients who had rare or unusual HLA phenotypes.[5,6] The recent addition of new and more powerful immunosuppressive regimens has increased the survival for poorly matched recipients, and driven organ allocation in the USA away from 'matching' and more towards considering the overall survival benefit and equitable allocation.[6,7] With this in mind, two groups of patients have emerged: those with HLA antibodies and those without. For patients without HLA antibodies, allocation can be based on wait time and overall survival benefit. However, for patients with HLA antibodies, allocation must be driven by the probability of finding 'acceptably mismatched' organs.

HLA antibodies are not naturally occurring and arise following pregnancy, transfusion or previous transplantation. Transfused females that have had multiple pregnancies frequently find themselves highly sensitized. In essence, they have developed antibodies against many HLA targets. Finding an acceptable mismatch for patients that are highly sensitized is dependent on the ability to correctly identify all HLA antibodies present in patient sera. In the past, identification of relevant HLA antibodies has been hindered by:

  • The inability to distinguish IgM from IgG antibodies

  • Accurate identification of anti-HLA class II antibodies

  • The inability to detect antibodies masked by linkage disequilibrium

For example, if all cells in a particular panel that have HLA-B8 also have HLA-A1 present owing to linkage disequilibrium, then it is impossible to determine if the patient has a HLA-B8 antibody only, a HLA-A1 antibody only, or both.

One of the assays used to determine the presence of preformed donor-specific antibody is the lymphocyte crossmatch test.[8] Typically, the crossmatch test and the antibody-determining assay should have the same level of sensitivity so that the outcome of the final crossmatch testing performed between the patient and the prospective donor is predictably positive or negative. However, previously utilized complement-dependent cytotoxicity-based (CDC) assays lack the level of sensitivity of the assay utilized for the final crossmatch, thus, sometimes resulting in positive crossmatches at the time of transplant.[9,10,11] Crossmatching is routinely performed today by flow cytometry and, since the new solid-phase antibody-screening and -identification assays are also based on flow-cytometry technology, the incidence of false-positive crossmatches due to mismatched sensitivity levels has been greatly reduced.[11]

Advancement in HLA antibody screening and identification by solid-phase multiplex platforms has overcome many of the issues stemming from previous methods. One of the most commonly used methods to screen patients for HLA antibodies before the development of the solid-phase assays was the CDC assay, with or without antihuman globulin (AHG) enhancement. The CDC assay is a complement-dependent method that detects antigen-antibody binding in vitro.[12] The assay is less sensitive than flow cytometry, which can detect antigen-antibody binding independent from the activation of complement. The addition of AHG increased the sensitivity of the CDC assays, as well as allowed for detection of cytotoxicity-negative, absorption-positive HLA alloantibody.[13,14] Since both IgG and IgM can bind complement, neither the CDC nor AHG-CDC assays can distinguish between the immunoglobulin (Ig) classes without the manipulation of sera. While data show that donor-specific anti-IgG HLA antibodies are deleterious to graft survival,[15,16,17,18,19] the role of anti-IgM HLA antibodies and non-HLA antibodies are less well established. Although controversial, data exist showing that IgM and non-HLA antibodies are not associated with poor graft outcomes.[20,21,22,23] Therefore, it is important to have a screening/identification method that can distinguish between Ig classes to provide clinically relevant information to clinicians.

Another problem with the previous antibody testing arose with the cell panels used for CDC or AHG-CDC testing. Since the HLA system is highly polymorphic, large cell panels were necessary to provide coverage for detecting the most common HLA antigens. Rare or unusual antigens were frequently not included in most clinical laboratory panels, owing to the unavailability of appropriate donor cells. Additionally, detection of both anti-MHC class I and class II antibodies was problematic. The predominant problem underlying class distinction arises from the distribution of HLA antigens. MHC class I molecules are found on all nucleated cells, whereas MHC class II molecules are expressed predominantly on antigen-presenting cells (APCs), B lymphocytes, activated T cells and microvascular endothelial cells. At the time, routine cellular-based antibody testing used lymphocytes as the target source, resulting in the inability to distinguish the existence of HLA class II antibodies in the presence of HLA class I molecules.

The new solid-phase technologies can easily distinguish between HLA IgM and IgG antibodies by using a secondary antibody that is specific for IgG or IgM. In addition, these methods can simultaneously discriminate HLA class I and II antibodies. Single-antigen methods, which carry only one antigen/allele per bead, allow for the unique identification of HLA specificities. Since there is no competition with other antigens on the same bead, the issue of antigen masking resulting from linkage disequilibrium has been eliminated.

The benefit of these new technologies is most apparent in the highly sensitized patient awaiting deceased donor transplantation. In the USA, patients are placed onto a national list managed by the United Network for Organ Sharing (UNOS), and allocation is currently based on a combination of factors, including wait time, ABO, HLA matching and HLA sensitization.[53] To avoid unnecessary testing in highly sensitized patients, the HLA antigens to which a patient has developed an antibody are entered into the UNOS computer as 'unacceptable' and, thus, are excluded as a candidate for a donor to whom they might be incompatible. Therefore, having the ability to assign the full repertoire of unacceptable antigens has changed the role of antibody testing in the HLA laboratory.


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