P105 An accelerated method for reverse sequence-specific oligonucleotide probe-based HLA typing

Aim Turnaround time (TAT) for HLA typing is critical for transplantation. We sought to establish a faster protocol for low-resolution HLA typing using the rSSOP methodology. Methods The LABType™ rSSOP method (One Lambda) was altered to reduce hybridization time. Modifications included centrifugation and incubation duration reductions, and reagent volume adjustments. Single parameters were first changed and tested. Successful modifications were used in the next round of tests. All modifications were tested with a set of PCR amplicons prepared from three samples, which contained previously HLA-typed high quality DNA. Following a series of rSSOP typings for these samples at HLA-A, B, C, DRB1, and DQB1/DQA1 loci, an accelerated protocol was established. Discovery phase concluded with protocol validation at all loci including HLA-DRB3/4/5 and DPB1/DPA1. Full validation confirmed intra-operator, inter- and intra-assay, as well as multiple DNA source reproducibility. DNA was extracted from different samples including ACD blood (n = 4), EDTA blood (n = 4), cord segment (n = 5), buccal swab (n = 5) and mouth-wash samples (n = 4). Results The original centrifugation of 3000 rpm for 2 min (total 2 min 22 s) was changed to a quick spin (up to 3000 rpm; 33 s), reducing each centrifugation step by 1 min 50 s. Additionally, one centrifugation and one transfer of plate step were eliminated. The duration of incubation time was changed for denaturation (from 10 min to 2.5 min), hybridization (from 15 min to 5 min), and streptavidin-PE (SAPE, from 5 min to 2.5 min). The reagent volume modifications includes the reduction of washing buffer (from 100 μl to 50 μl per well) and increase in SAPE (from 25 μl to 50 μl per well). Compared to the original protocol, the accelerated protocol reduced the hybridization time approximately 30 min. This method allowed us to obtain consistent and accurate typing results for all tested HLA loci among different operators, intra- and inter-assays, and a variety of DNA samples. Conclusions Fulfilling requests for faster TAT may be achieved by adaptation of current methodologies, which may help improving patient management. The accelerated rSSOP protocol is an effective method for reducing testing time, while still giving robust results for HLA class I and class II typing. (RLU and JJX contributed equally).