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Why HLA-DQ?
Part 4: Limitations of current HLA assays
Despite being one of the most important laboratory assays that revolutionized HLA antibody testing, it’s important to discuss some of the shortcomings of the single antigen bead (SAB) assay. There are three major limitations to consider when performing and interpreting the assay: saturation, inhibition, and shared epitopes. A short description of what they look like and why they can be problematic are below.
Example titration curves for “normal” serum, two different sera with inhibition, and one with saturation
1. Inhibition – Also known as the “prozone effect” or “hook effect” from other fields of medicine, inhibition is a phenomenon observed when SAB testing is performed on serially diluted serum, wherein mean fluorescence intensities (MFI) of the diluted samples increase as the serum is diluted. It is most commonly observed in highly sensitized patient sera that contain high-titer antibodies. Inhibition is thought to be caused by an overabundance of complement split products that interfere with binding of the fluorescent reporter antibody, resulting in underestimated or false negative results. We suspect that other factors may contribute to this phenomenon. Dilution of the serum removes enough of the inhibitory factors and restores the ability of the reporter antibody to bind to the bead-bound antigen-antibody complex.
Multiple interventions can reduce this type of inhibition. Ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and heat treatment have all been shown effective methods to mitigate inhibition and are routinely performed by HLA labs, although attention should be given to the concentration used. Serial dilution can also detect inhibition, and even provide patterns of inhibition, although it is an expensive and time consuming approach to use if the end result required is determination whether the sample is affected by inhibition or not
2. Saturation – The SAB assay is a solid-phase platform that semi-quantitatively measures antibody levels using about 100 microscopic beads coated in recombinant HLA antigens. There is a finite amount of antigens that can be attached to each bead. Saturation occurs when the amount of antibody exceeds the number of available binding sites (antigens) presented by the microbeads, effectively “maxing out” the MFI. Serum samples with antibody levels higher than the antigen level on the bead will show steadily high MFI values on multiple dilutions until the serum is diluted enough to have an antibody concentration that falls below that of the antigen. Although this can often occur hand in hand with inhibition, saturation and inhibition are driven by two different principles.
Saturation can be problematic when analyzing SAB results, especially when titration isn’t performed. Assuming no inhibition, low MFI values (<15,000) roughly correspond to antibody strength (titer). However, as MFI values approach saturation-level, an accurate assessment of antibody levels becomes near impossible without adding titration studies
Figure: (Top) Table showing the median neat MFI of HLA antibodies based on titer strength of the antibody. Number of antibodies for each locus is shown in the right-most column. Median MFI increases linearly for antibody titers between 20 and 26, however the relationship begins to level off once antibody strength exceeds a titer of 27 due to bead saturation. The orange box indicates the range of titers that were affected by saturation. (Bottom left) Graphical representation of the table. Each locus is represented by a different color line. (Bottom right) Variability in neat MFI values for 717 HLA-DQ antibodies
Adapted from: Tambur AR & Wiebe C. 2018. HLA diagnostics: Evaluating DSA strength by titration. Transplantation: 102
The figure above summarizes the results of a 2018 study that compared DSA strength by titration and neat (undiluted serum) MFI values. Serum was not treated for inhibition in order to get an appreciation of the extent of inhibition possible, if it was present. As shown in the table (graphically represented by the left plot), the correlation between neat MFI and antibody strength is lost after the titer exceeds 27 (1:128). Bead saturation masks the true strength of high-titer antibodies, making it difficult to interpret high MFI values. The figure also illustrates how inhibition can impact MFI values – the large valleys in the HLA-DQ and -DR345 curves indicate extremely high-titer antibodies that exhibited low MFI values in the neat serum.
Because HLA-DQ are often the strongest antibodies, they are also the most affected by both inhibition and saturation. The right graph in the figure shows the variability in neat MFI values based on antibody strength for 717 different HLA-DQ antibodies. Inhibition causes low MFI values to be seen in both low- and high-titer antibodies (bottom circle). Likewise, saturation causes MFIs to max-out around 20,000, making titers between 1:128 and 1:32,768 indistinguishable (top circle). Because of saturation, titration is still considered the gold standard for determining antibody strength.
3. Shared Epitopes – Even though HLA loci are the most genetically polymorphic gene system known in humans, with over 27,000 alleles currently described, most of the polymorphisms within each locus are isolated to specific sites on the protein. The overall structure of the HLA antigens is surprisingly conserved. Because of this, there can be considerable cross-reactivity between the different HLA alleles, and even between different HLA antigens. The phrase “shared epitope” refers to a conserved region found on multiple HLA antigens that can be recognized by the same antibody. A well-known example of this are the Bw4 and Bw6 public epitopes. Other examples include the well-defined CREGs (Cross REactive Groups)
In SAB testing, shared epitopes can lead to falsely low MFI values. Because the antibody recognizes an epitope present on multiple different HLA antigens (and beads in the assay), the antibody gets “spread-out” among the epitope-positive antigen beads, causing low fluorescent signals when the reporter antibody is added. Conversely, if the antibody were to recognize a single private epitope found only on HLA-B18, it would bind only to that bead in the assay, generating a much stronger fluorescent signal. HLA labs must be mindful of shared epitopes when interpreting SAB results, so that DSA containing shared epitopes are not missed.
Figure: Simplified illustration of SAB result differences when an antibody recognizes a private epitope vs. a shared epitope. Red triangles represent a shared Bw6 epitope, while other colored triangles represent private epitopes found only on that specific antigen. Assuming similar starting concentrations of antibody, those that recognize a shared epitope can cause artificially decreased MFI values due to additional available binding sites that are found on multiple different antigen beads.