Determining the specificity of HLA antibodies is arguably one of the most important contributions by the HLA laboratory today. For patients that have low-to-moderate levels of antibody, using a specificity assay that is based on a phenotypic panel is practical and cost effective. Individual microparticles are coated with a full HLA class I (HLA-A, -B and -C antigens) or HLA class II type (HLA-DR, -DQB, -DQA and -DP antigens). The specificity assay based on flow cytometry contains a panel of 55 HLA class I beads and 32 HLA class II beads. The majority of HLA antigens are represented on the beads, which allow a PRA percentage and HLA specificity to be determined by software analysis. The drawback of this approach arises from the panel employed to detect the HLA antibodies. Certain antibody specificities may not be detected due to the masking effect of linkage disequilibrium mentioned previously.
The HLA system is the most polymorphic system in the human body and not all antigens are represented with the same frequency in all ethnic groups. Some antigens only occur in certain ethnic groups and have very low frequency within a given population. To address this issue, multiplexing platforms have been developed for specificity testing that contain either HLA class I and class II proteins from platelets, or antigens from transformed cell lines that represent all major HLA antigens. This method allows for volume batching and a rapid turnaround time.
However, for patients with multiple antibody specificities the best approach is single-antigen technology. Single-antigen methods can define every antibody specificity for which there is a single-antigen bead designed. In the assays that are being used today, this includes the majority of antigens in HLA-A, -B and -Cw, and HLA-DR, -DRw, -DQA, -DQB and -DP loci. In the flow cytometry method, individual HLA molecules are coupled to microparticles, resulting in the ability to detect a single HLA antibody. Within each group of beads, there are eight different beads coated with various levels of fluorescent dye, allowing for individual identification (Figure 3A). Figure 3A depicts a serum that contains no anti-HLA antibodies and, thus, there is no shift of the beads outside of the boxes established by the negative-control sera. Binding of antibody to the beads causes a shift in fluorescence, which is detectable by flow cytometry, as seen in Figure 3B. Figure 3C shows a patient serum that is positive for multiple anti-HLA class I antibodies. While Figure 3 shows a representative snapshot of single-antigen beads, there is a total of ten groups of eight beads that detect HLA class I loci antibodies, and four groups of beads for MHC class II antibody detection. The Luminex assay, illustrating single-antigen technology, is depicted in Figure 4. Each bead is represented individually in the diagram and marked as positive or negative, according to the mean fluorescent intensity (MFI) value. High levels of antigen-antibody binding result in a shift of fluorescence intensity, culminating in increased MFI values. The significance of being able to detect both MHC class I and class II antibodies lies in the fact that patients develop antibodies to HLA class II antigens with the same frequency that they develop anti-HLA class I antibodies. However, because of the aforementioned difficulties in detecting and defining these anti-MHC class II antibodies, their role in graft rejection was unknown. Since these new technologies have become mainstream, the forthcoming data clearly illustrates that anti-MHC class II antibodies are also responsible for graft rejection and detrimental for long-term graft survival.[28,29,30,31,32]
Dot plot analysis of flow cytometry single-antigen beads. Each group of beads labeled R2 through to R10, represents individual beads expressing one single antigen. (A) HLA class I-negative control beads. (B) HLA class I single-antigen bead-positive control beads, showing fluorescent shift due to antibody binding. (C) A patient positive for HLA class I antigens A1, A2, A25 and A29, weakly positive for A26 and positive for B49. FITC: Fluoroisothyocyanate.
Luminex™ single-antigen beads of a patient serum showing the presence of HLA-A and -B antibodies. A positive reaction is determined by the level of mean fluorescent intensity and cut-offs are established by the manufacturer or, alternatively, are modified by individual laboratories through internal validation studies.
While single-antigen methods provide information that is critical for quality patient care, there are technical issues with the assays that can render in terpretation of the data problematic, and these issues need to be addressed. Patients that have antibodies to latex will react against the beads themselves, thus resulting in a positive shift of all beads, irrespective of the presence of HLA antibodies, which renders the data uninterpretable. Sometimes, adsorption of the patient sera with unbound beads can remove enough of the bead antibody to determine which HLA antibodies are present. Additionally, it has been noted that solid-phase technology can detect patient antibodies against denatured HLA molecules, as well as the native HLA molecules. In essence, the anti-HLA antibodies can bind to regions of bead-coupled HLA molecules that are normally not accessible if they are masked by ß2-microglobulin, for example. Some reports have indicated that these antibodies are not clinically relevant and, thus, are not a contraindication to transplantation [M Lopez-Cepeno, pers. comm.]. However, it is not currently possible to discriminate these irrelevant antibodies from antibodies that have developed against the native HLA molecules and which would constitute a risk for transplantation.
As mentioned previously, typing for HLA antigens was, until recently, performed using a cellular-based assay, the CDC test. All HLA molecules identified during this era were assigned a numeric designation based on cellular reaction patterns, and were referred to as HLA antigens. For example, HLA-A2,3;B7,8;DR4,7 was the common method of designating a HLA phenotype. With the development of molecular-based typing methods, HLA molecules can now be defined based on their DNA sequence, and these molecules are referred to as HLA alleles.[33,34] The correct nomenclature for allele level typing by molecular methods is HLA-A*0201, *0301;B*0702, *0801 DRB1*0404, *0701. Therefore, one can distinguish how the HLA typing was performed by the nomenclature used. Not surprisingly, the new solid-phase methods can detect antibodies at the allele level, causing a conundrum for the patients that are waiting for deceased donor transplantation. The issue is that, while it is now possible to define antibodies at the allele level, it is not possible to enter this information into the UNOS computer as an unacceptable antigen at the allele level. A patient can develop a HLA antibody against an allele that is similar to a self-antigen, but the antibody cannot be listed as an unacceptable antigen without manipulation of data entry. For example, a patient who types as a HLA-B*4402 can develop an antibody against a HLA-B*4403 allele. Normally, the patient's HLA type would be entered into the UNOS computer as a B44 antigen, however, that would preclude listing HLA-B*44 as an unacceptable antigen due to the restrictions of the listing a 'self-antigen'. This can be overcome by not listing the patient's type as HLA-B44 and listing the antigen as unacceptable. However, at the same time, this practice might prove disadvantageous for obtaining a zero-mismatch donor. In the future, it would be beneficial to have the UNOS computer program adapted to allow entry of unacceptable antigens at the allele level.
Finally, the level of sensitivity of antibody detection should match the level of sensitivity utilized to perform the final crossmatch, which would allow a 'predictive' response for compatibility. However, not all antigens are coated onto the surface of the beads at the same density level. Studies have shown that certain beads carry more antigen than other beads, thus rendering detection of some antibodies more sensitive than others. Furthermore, density differences between a coated bead and the antigen density on the cell surface can cause differences in the level of sensitivity between antibody detection and the crossmatch assay.
Expert Rev Clin Immunol. 2008;4(3):391-399. © 2008 Expert Reviews Ltd.
Cite this: Trends in HLA Antibody Screening and Identification and Their Role in Transplantation - Medscape - May 01, 2008.