Pre-formed antibodies to non-self Human Leucocyte Antigens (HLA) represent a major barrier to kidney transplantation. HLA antibodies capable of causing a positive flow cytometric crossmatch (FCXM) against donor lymphocytes are associated with an increased risk of antibody-mediated rejection (AMR). The registration of incompatible (i.e. antibody positive) HLA with the UK kidney allocation system reduces the risk of a positive FCXM and early AMR. HLA antibodies in patient sera are routinely defined using sensitive high-throughput LuminexÂ® single antigen bead (SAB) assays, but these antibodies are not always capable of causing a positive FCXM. As such, the âclinical relevanceâ of HLA antibodies identified solely by SAB assays is debated. UK guidelines state that a negative pre-transplant FCXM equates to standard immunological risk to transplant. Therefore, the use of SAB assays to define incompatible HLA potentially restricts opportunities for patients to receive a kidney transplant offer, particularly for those who have HLA antibodies directed against â¥85% of HLA in the donor population. The aim of this project was to establish whether innovative application of alternative commercial methods for HLA antibody characterisation could improve prediction of clinical relevance for individual antibodies. Over a four-year period, 8% (58/730) of crossmatches were performed against patient sera containing donor-specific HLA antibodies (DSA) identified using the One Lambda LABScreenâ¢ Single Antigen Bead (LABScreen) assay. However, an unexpected negative FCXM was seen for 66% (38/58) of sera containing LABScreen DSA. Retrospective analysis demonstrated that FCXM outcome could not be reliably predicted by LABScreen based on the number of DSA, DSA specificity, or assay output value. Additional HLA antibody testing was performed using Immucor LIFECODESÂ® LSA (LIFECODES), Immucor LIFECODESÂ® C3d Detection (LIFECODES-C3d) and BAG Healthcare HISTO SPOTÂ® HLA AB Screen/ID (HISTO SPOT) assays. These three assays improved accurate prediction of a negative FCXM from 0% to 89%, although positive FCXM prediction decreased from 100% to 68%. Consideration of HISTO SPOT and LABScreen results alone improved prediction of a negative FCXM from 0% to 68% and positive FCXM prediction to 88%. LIFECODES-C3d positivity demonstrated an association with a positive FCXM independent of LABScreen output value, contrary to previous reports. Antibody-epitope analysis using HLAMatchmaker did not offer further FCXM predictive value but enhanced understanding of the validity of putative antibody targets identified by each assay, particularly for sera with complex antibody reaction patterns. The HISTO SPOT assay demonstrated high concordance with FCXM outcome for this cohort. This represents the first report supporting application of the HISTO SPOT assay in assessment of clinical relevance for pre-transplant HLA antibodies. A clinical case study for a patient with high panel HLA antibody reactivity illustrated that a small decrease in the number of registered incompatible HLA increased the proportion of compatible UK donors. The use of HISTO SPOT, LIFECODES-C3d and HLAMatchmaker analysis, alongside the routine LABScreen assay, offers a novel strategy for improved clinical risk stratification of pre-transplant HLA antibody. Implementation of this testing strategy could improve access to HLA compatible transplantation, by reducing the number of incompatible HLA registered with the UK allocation system, and thereby decreasing waiting time, particularly for patients who are currently considered HLA incompatible with more than 85% of the UK donor population.
|Date of Award||1 Aug 2021|
- The University of Manchester
|Supervisor||Judith Worthington (Supervisor) & Anne White (Supervisor)|
- HISTO SPOT