Despite improvements in outcomes of solid organ transplantation (SOT), chronic deterioration of allografts remains an important problem and is associated with increased morbidity and mortality 1. With the implementation of sensitive and specific solid phase immunoassays (SPI) for identification of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA), and with the improved understanding of the pathogenic effect of alloantibodies, it is well accepted that chronic graft loss is part of the antibody-mediated rejection (AMR) spectrum 1,2. Circulating DSAs, either present prior to transplant or developed de novo, may bind to graft endothelial cells, promoting the classical pathway of complement activation, and the inactive product C4d bound covalently to tissue, provides the ‘footprint’ of antibody-mediated allograft damage 3. Correlation between the level of DSA in the serum, C4d positivity in the biopsy and severity of microvascular damage has been shown by several investigators 4,5. However, C4d-negative biopsies with circulating DSA also showed microcirculation damage and increased expression of endothelial genes 6. A recent publication summarizes the revised Banff criteria for AMR in renal and non-renal allografts, including C4d-negative AMR 7.
DSAs have been shown to promote various types of AMR, from the acute to the chronic form of rejection also termed as transplant glomerulopathy in renal, cardiac allograft vasculopathy in heart and bronchiolitis obliterans in lung transplants. Although the HLA antibodies may appear before loss of function and are highly predictive of poor outcome, there is still controversy concerning: (i) whether antibodies detected solely by highly sensitive techniques are clinically relevant, (ii) how often to monitor post-transplant and (iii) when to implement antibody removal therapies in the absence of clinical dysfunction.
Detection and characterization of HLA-antibodies using new diagnostic tools
A variety of assays are available for HLA antibody identification that differ in the type of technique, target, sensitivity and specificity. These include cell-based assays, where the target can be tested in a cytotoxicity or flow cytometry assay, and solid phase assays (SPI) where soluble antigens are tested 2. SPI use solubilized HLA molecules that are bound to a solid matrix that is either a microtitre plate or polystyrene beads. In the bead-based array, the level of antibody binding is expressed as the mean fluorescence intensity (MFI) 2.
The single-antigen bead (SAB) assay enables precise identification of all antibody specificities in complex sera and the broad categorization of antibody levels based on mean fluorescent intensity (MFI) into low, intermediate and high. This assay was developed and licensed as a qualitative assay 2. While the MFI does not represent the titre of HLA antibody, the MFI result is used universally to gauge antibody strength. However, one limitation of this method is that strong antibodies may be inhibited by immunoglobulin (Ig)M or the C1 component of complement in undiluted sera 2. Nevertheless, the SAB assay, with additional modifications to also detect antibody titre, is a valuable tool to support a diagnosis of humoral rejection in routine monitoring both pre- and post-transplantation, providing information as to the type of intervention and how aggressively a patient should be treated, prior to transplantation.
Furthermore, modification of the SAB assay to detect C1q binding has provided a new tool, the C1q assay, for risk stratification of transplant recipients who exhibit DSA 2. The C1q assay detects antibodies capable of binding and fixing the first complement component, C1q 8,9. The C1q-binding potential differs between the IgG subclasses: IgG3 and IgG1 bind complement more strongly than IgG2 and IgG4. Damage to the allograft is caused not only by the binding of complement, but through inflammation which is dependent upon the specific cells that infiltrate the allograft. All IgG subclasses recruit monocytes to the graft and cause mild inflammation and some cell damage. However, the IgG3 and IgG1 antibodies also recruit natural killer (NK) cells, leading to the release of interferon gamma, which activates monocytes and leads to a more aggressive inflammation and increased pathology in the graft 10.
In cardiac transplantation, correlations have been demonstrated between C1q positive antibodies and early AMR 11. Furthermore, in renal transplants, the presence of complement binding DSA was associated with a more severe graft injury phenotype and significant risk for graft failure 12. Therefore, this assay should identify all C1q-binding DSAs that could prove problematic in the future, even if they are not currently activating the complement cascade 8.
In summary, improved tools are available for determining DSA specificity, level and function. Early detection of DSA and risk stratification for intervention based on DSA characteristics may impact long-term allograft survival.
Acknowledgments
The author would like to thank Meridian HealthComms Ltd for providing medical writing services.
Disclosures
A. Z. is a recipient of a research grant from CSL Behring.
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