The success of transplantation is inextricably linked with the measures aimed to reduce rejection. Human leukocyte antigens (HLA) have a crucial role in defence via antigen presentation but, in the context of transplantation, are responsible for recognition of non-self thus initiating an immune response. Patients can form anti-HLA antibodies in response to pregnancy and transfusion, and in response to transplant-mismatched antigens; however, pro-inflammatory events such as surgery, trauma, infections, and vaccinations are also associated with an increase in HLA antibodies. Characterisation of patient anti-HLA antibodies forms an integral part of histocompatibility testing to support clinical transplantation.
Pre-transplant, a patient antibody profile is used to define Unacceptable Antigens (UA) against which they have existing HLA antibodies; during organ allocation, organs expressing UAs are censored and therefore not offered as an option for that patient. The patient antibody profile is also increasingly used by laboratories for virtual crossmatching (VXM) between specific patient-donor pairs; this is a risk assessment tool to predict the result of physical assays such as complement-dependent cytotoxicity and flow cytometry crossmatches.
While non-sensitised patients with no circulating HLA antibodies can be assumed to have a negative VXM with any prospective donor, VXM provides risk stratification for sensitised patients as the patient antibody profile is compared against the donor HLA type; this is especially important in time-critical scenarios such as cardiothoracic transplantation. In addition, the association of donor-specific antibodies (DSA) with subsequent graft loss also suggests that post-transplant screening for DSA leading to early intervention could improve graft outcomes.
While there are currently thousands of known HLA alleles with this number still increasing, only ~30% have been reported commonly in unrelated individuals; these are documented in the Common and Well-Documented (C/WD) alleles catalogue. When developed, the LABScreen Single Antigen Bead products were designed to give us breadth of antigen coverage over all serological specificities known at the time. However, our knowledge has moved beyond this to allele-specific antibodies and now to epitope-based analysis. Rather than serological specificities, we now know that anti-HLA antibodies are specific for epitopes; small configurations of amino acids which may be shared between several HLA alleles leading to cross-reactivity. The degree of eplet mismatching between donor-recipient pairs can be determined using bioinformatic programs such as HLAMatchmaker, which is freely available within the One Lambda HLA Fusion software. HLAMatchmaker can also be put to use to facilitate antibody or epitope specificity definition through epitope-based analysis.
Building on this knowledge, One Lambda have released the next step in HLA-specific antibody testing with two LABScreen products; the Single Antigen Supplement (for use on Luminex 200 instruments) and ExPlex panels (for use on LABScan3D/FlexMAP3D instruments). When combined with the classic SAB kit, the Supplement/ExPlex kits provide coverage of 151 class I and 119 class II alleles. While there are many examples of common (Caucasian) alleles in the extended panels, there are also alleles that are more prevalent in ethnically diverse populations; some examples include A*02:05, B*39:06, C*07:01, DRB1*13:02, DQB1*05:03, and DPB1*02:02 alleles. These products have been designed to expand antibody coverage to better complement molecular typing results and provide more data for the assessment of antibodies at the eplet level. The newest additions to the LABScreen portfolio follow the same protocol, use the same analysis software, and can be easily implemented into existing laboratory workflow.
One Lambda’s aim here is not to disadvantage patients by listing more unacceptable specificities, but to enable labs to define antibody reactivity more clearly. Epitope-based analysis helps identify windows of transplantation for highly sensitised patients by determining acceptable mismatches and expanding their donor pool. The additional allele information also gives laboratories a more thorough understanding of the pre-transplant antibody profile; this leads to risk-reduction for patients through a better informed VXM by confirming unacceptable epitopes which would likely cause reactivity. VH Bio feels these products provide significant value to histocompatibility labs by improving patient safety and risk assessment.
Several labs have already presented their clinical experience with using the extended LABScreen panels, such as the ASHI presentation given by Dr Kelley Hitchman (San Antonio, USA – https://bit.ly/3Rx83lY) or the EFI presentation by Dr Rob Liwski (Dalhousie, Canada). These included several case studies highlighting the benefits of ExPlex/Supplement panel usage for highly sensitised patients (cRF >95%):
- Unexpected positive flow crossmatch – analysis using ExPlex found this to be caused by DRB1*13:02 DSA (not present on classic panel) and antibodies against the 37N epitope.
- Patient denied transplant – classic panel showed C*16:01 positive so rejected donor with C*16:02 type due to DSA; analysis using ExPlex found they were C*16:02 negative as this has a different epitope therefore no DSA present.
- Undetected DSA – patient tested negative for C*16:01 on classic panel; analysis using ExPlex found DSA against C*16:02 and the 80k epitope.
- Transplant loss due to undetected DSA – patient tested negative for B*35:01, *35:03, and *35:08 antibodies using classic panel; analysis using ExPlex found DSA against B*35:02 and *35:12.
These examples and more show the efficacy of LABScreen ExPlex/Supplement panels through assaying cases of unexplained positive crossmatches, biopsy-proven rejection for DSA-negative patients, and unusual patterns of Single Antigen bead reactivity which may be explained by epitope analysis. This saved laboratories time and resources by reducing the need for additional testing.
If you want to know more, feel free to get in touch directly or join VH Bio’s Ben Adams for his symposium at the BSHI conference on 13th September 2022; he will be discussing the use of these extended panels alongside HLAMatchmaker as we shift as a community towards epitope-based analysis.