P239 More with less: multiplexed real-time PCR HLA typing using SYBR® and multi-peak TM analysis

Aim HLA typing plays a critical role in solid organ transplantation. The degree of HLA matching and especially the avoidance of immunogenic antigens in pre-sensitized patients is directly correlated with improved outcomes. As of October 2016, the IMGT/HLA database has grown to contain over 15,600 alleles and will certainly continue to expand. Current technologies used to evaluate immune status will also need to expand and broaden their ability to detect allele specific differences that code for immunogenic antigens. Epitope mapping will likely play an important role in this effort and provide a framework to group alleles in a practical way. HLA typing will also need to evolve to split these differences with improved resolution. This study validates a new multiplexing HLA typing method that allows better resolution and flexibility to add additional resolution over time. Methods The LinkSēq™ typing kit from Linkage Biosciences uses real-time PCR combined with melt curve analysis to define HLA specificity. An evolution of this approach consists of multiplexing several targets into a single reaction and identifying the products by way of distinct melt curve signatures, thereby drastically reducing the number of required reactions. Results Among the new multiplex assays that were validated, the example presented in this study combines 3 separate reactions targeting HLA-B*15, HLA-DPB1*03 and HLA-DQB1*02 alleles into a single reaction without requiring any protocol changes. Software algorithms were updated to define these new multiplexed results while keeping the same analysis workflow and thus a transparent modification to end-users. Conclusions This study demonstrates that multiplexing several LinkSēq assays into the same reaction is a practical and reproducible approach and should be applicable across the full spectrum of reactions. In addition to cost and labor advantages, multiplexing represents a leap forward for high-throughput HLA applications where the need for increased resolution is growing.