Automated High Resolution HLA Typing of Class I & II Alleles Using the SBT Method. W. Dong1, C. Carozza1, E. Pimpinella1, J. Barone1, S. Hsu1, N. Flaherty2, L. Tack2 1) Histocompat/Molecular Genetics, American Red Cross Penn-Jersey Region, Philadelphia , PA; 2) PerkinElmer, Inc, 2200 Warrenville Rd, Downers Grove, IL.
Human Leukocyte Antigen (HLA) matching is a critical element for solid organ or bone marrow transplantation success. The HLA Lab of the PJ Region of the American Red Cross provides allelic level HLA typing supporting bone marrow transplantation to hospitals and also carries out DNA-based typing of unrelated donors and cord blood units as a contract lab for the National Marrow Donor Program. We screen for HLA polymorphisms using molecular methods such as Sequence Specific Oligonucleotide Probing (SSOP) and Sequencing-based Typing (SBT) methods. Our strategy is to use the SSOP method as an intermediate resolution testing process along with SBT for higher resolution typing. High resolution typing using the more informative Gold Standard SBT method is common for successful bone marrow and stem cell transplantation. SBT also allows the detection and identification of new alleles, not possible with most other techniques. The AlleleSEQR SBT kits from Celera are used for testing both Class I (HLA-A, B, C) and Class II (HLA-DRB1, DQB1 and DPB1) loci and requires setting up hundreds of PCR reactions, making this procedure an ideal candidate for automation. Our lab processes more than 1000 samples/week for HLA typing. To meet the turn around time of 3 to 9 days for clinical purposes, we used a JANUS Liquid Handling Workstation from PerkinElmer to automate the SBT typing process. The JANUS workstation sets up (1) primary PCR amplification plates, and (2) SBT cycle sequencing reactions using forward and reverse sequencing primers to detect polymorphic sequences in both Class I and Class II regions. Amplified dye-terminator labeled reaction products are analyzed using an ABI Prism 3730XL DNA Analyzer. Sequence files are imported into the Assign-SBT data analysis software and electropherograms aligned. Data for each sample template analyzed is compared to known alleles in the library for identification. Automation, by increasing SBT throughput, shortens turnaround time, reduces the potential for manual procedural errors, and maintains consistency between runs.