Utility of HLA Antibody Testing in Kidney Transplantation

Abstract

HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor-specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes.

Keywords: acute allograft rejection; chronic allograft failure; kidney transplantation.

Figure 1.

MFI of single antigen bead assays has analytic limitations and cannot be used as a quantitative metric of antibody amount. (A) An ideal test should always be able to distinguish antibody binding (green fluorescent signal) from negative control (white) with a clear threshold and no overlap between the MFI distributions. (B) Decreased density of antigen (Ag) on the surface of the bead will result in MFI measurement that underestimates the amount of the antibody present. (C) In contrast, nonspecific binding to the bead can result in artificially high background and signal MFI, with overestimation of antibody. (D) Interfering substances may prevent the detection of the antibody of interest with lower MFI. (E) Epitopes shared between different beads can dilute the amount of antibody bound to any single bead, with an erroneously low MFI on the given bead of interest.

Figure 2.

Individual single antigen beads within a single-assay reaction can have significant differences in the density of target antigen affecting maximum MFI. A saturating amount of W6/32 antibody that binds ubiquitously to class I HLAs was mixed with single antigen beads. The unadjusted results are shown, with the fluorescence representative of the density of the target antigen on each bead. Wide variation in density is seen.

Figure 3.

Removal of interfering factors with serum treatment can significantly increase MFI on beads of interest. (A) Neat serum (no treatment) SAB results of a sensitized renal transplant recipient. MFI appears on the y axis. Each bar represents a single bead. The bar graph illustrates MFI measurement for each HLA allele, with self-antigens (expected to be negative) identified with purple arrows and donor antigens indicated with red arrows. The A2 beads representing donor antigens are clearly negative. An XM would be predicted to be negative, because no antibodies to donor antigens are identified; however, the FCXM was strongly positive. (B) After treatment of the serum with dithiothreitol (DTT), antibody to donor HLA-A2 is detected at MFI>20,000.

Figure 4.

Dilution of serum in SAB assays does not result in a predictable or quantitative reduction in bead MFI. Serial dilutions were performed on dithiothreitol-treated serum tested on SAB in 10 consecutive patients with DSA. The expected change in MFI from baseline over dilution if neat serum MFI quantitatively represented antibody amount is shown with the dashed lines. MFIs of the highest bead(s) contributing to DSA in each of the sera are shown at each dilution as a percentage of the neat serum MFI (by definition, 100%). The MFIs vary widely through dilution; although antibody concentration is decreasing by a known amount through serial dilution, MFI changes are not proportional. Neat serum MFI does not, therefore, reliably represent antibody amount.

Figure 5.

Epitopes targeted by HLA antibodies may be shared across multiple beads in a single assay, lowering the MFI detected on any individual bead. Multiple beads share the Bw6 epitope. In this case, the mismatched donor antigen of interest is B35, with a normalized bead MFI of 1216 (weak). Many centers would predict a negative FCXM with this donor; however, it was strongly positive. Closer examination reveals that the majority of antigens sharing the Bw6 epitope are clustered together in this MFI range. Neither a B7 nor a B5 cross–reactive epitope group explains this reactivity pattern entirely; rather, it is likely that an antibody to Bw6 is diluted across multiple beads sharing this epitope. During in vitro XM, where only the B35 target was present, the antibody can bind without epitope dilution, yielding a positive result. CREG, cross reactive group.