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#38
ALLELE-LEVEL CLASS I HLA TYPING BY SBT AND TARGETTED SSP.
Mary A. Portas BS, CHT 1, Michael T. Aubrey MS, CHS 1 and Brian M. Freed PhD, DABHI 1. 1 Histocompatibility, ClinImmune Labs, Aurora, CO, USA .
The pace of HLA class I allele discovery requires innovative strategies to maintain allele-level typings. We have developed a procedure, based upon commercial reagents, that stages sequencing followed by targetted SSP. This strategy minimizes DNA consumption, reagent costs, and turn-around-time while maximizing allele resolution.
For this pilot study, DNA was extracted from N=17 bone marrow recipients with the GenoM6 robot and characterized by heterozygous sequence-based typing (SBT, Abbott Laboratories) on an ABI 3100 automated sequencer. If SBT alone achieved an allele-level typing we stopped further testing. For ambiguities outside the sequenced exons we applied sequence-specific AmbiSolv (Dynal Biotech) primer mixes. Routine class I serology sometimes ruled out null alleles. Samples with cis-trans ambiguities were resolved using subsets of Dynal Biotech high-resolution SSP Unitrays. Subsets were prepared by cutting the Unitrays into 8 to 12 individual 8-well strips. Although cutting the Unitrays increased handling and management, it mitigated DNA consumption and costs.
The resolution at each locus was evaluated after SBT and also after application of secondary technologies. Allele-level typings (e.g., B*0801/B*4402) were defined as having two possible alleles per locus. Only 59%, 59%, and 47% of HLA-A,B,Cw typings, respectively, were allele-level by SBT; after targetted SSP, 100% of HLA-A,B,Cw typings were allele-level (see below table). Thus, a combination of SBT, serology, and targetted SSP routinely achieves allele-level typing.
Allele-Level Resolution After SBT and Targetted SSP HLA-A HLA-B HLA-Cw SBT/Serology 59% 59% 47% SBT plus AmbiSolv 65% 71% 71% SBT plus 1 Strip 82% 82% 65% SBT plus 1 Strip plus Ambisolv 88% 94% 94% SBT plus >1 Strip plus Ambisolv 100% 100% 100%