LAUSR

Aim A young pediatric small bowel recipient was transplanted with a donor who was typed only at low resolution by an outside center. For 6 years, the patient received multiple blood transfusions and was monitored by IgG and C1q using LabScreen SAB (One Lambda). High MFI DSA to DQ2 was treated by IVIG desensitization; however, based on the low resolution typing, DQ2 DSA remained. To determine whether this was real DSA or whether the donor had a different DQ2 heterodimer, a fresh frozen biopsy sample was obtained to try to identify the donor HLA alleles by Next Generation Sequencing (NGS). Methods Full length genomic DNA from the patient and biopsy was sequenced for 11 loci (A,B,C,DRB1/3/4/5,DQA1/B1,DPA1/B1) using TruSight HLA v2 Sequencing Panel (Illumina), and analyzed by Assign v.2.1 (Illumina). Two to 3 field resolution was obtained. Results At each locus on the biopsy DNA, the sequence showed discontinuous alignments and much higher numbers of mismatched nucleotide positions than usual due to the mixture of both patient and donor. Individual sequenced reads containing mismatched nucleotides compared with the two predominant alleles were applied to BLAST using both the NCBI and IMGT websites. Once multiple alleles of the first field were obtained, polymorphic positions among exons were compared with other mismatched positions of the sequenced reads to determine the second and third allelic fields. Among all loci, more than two alleles were observed. By eliminating the recipient HLA alleles from the output, the donor’s HLA typing became clear. Typing of the donor by NGS from a biopsy sample revealed that the donor did not have the target heterodimer that had been giving high MFI values by both IgG and C1q. The DQ2 that had been followed was, in fact, not a DSA. Since a prior weak DR53 DSA had already resolved, continued treatment for AMR (Antibody-Medicated Rejection) was not indicated and augmented immunosuppression was discontinued. Conclusions This case decisively demonstrates the power of NGS technology to identify 2–3 field allele level HLA typing from a chimeric specimen using a frozen biopsy specimen. It also highlights the power of the technology to inform clinical decision making.