Master of Science

thesisAccurate genotyping of the human leukocyte antigens (HLA) are crucial for the success of hematopoietic stem cell and solid organ transplantation. Over the past 50 years, numerous methodologies have been used for HLA typing but technological limitations have prevented full interrogation of the HLA gene thus resulting in allelic ambiguity. In order to resolve these ambiguities, additional testing is often required, leading to increased expense and delay in reporting the genotyping results. Recently, advances in nucleic acid sequencing technologies, generally referred to as next-generation sequencing (NGS), have become available. HLA genotyping by NGS is poised to become the new gold standard for several reasons. First, the entire gene can be interrogated, as opposed to a few exons, thus enabling greater resolution of known polymorphisms outside of the T cell recognition site on the HLA molecule. Secondly, missing genetic sequences in the curated databases will become more complete thus improving the algorithms used for alignment to a reference sequence. Lastly, NGS will become the new gold standard because high-resolution genotyping can be achieved without the need for additional time and laboratory resources in order to resolve allelic ambiguities and meet typing requirements required by regulatory agencies. In this thesis I discuss my efforts to employ this new technology by developing an assay for genotyping of the HLA-A, B, C, DRB1, and DQB1 genes. In the new assay, 98% of all alleles typed correctly and unambiguously without the need for any secondary testing. This in-house method is later compared with two recently released commercial kits and 100% concordance was found between the three methods. Differences in workflow are compared and contrasted. In conclusion, we show that HLA genotyping by NGS produces more correct and unambiguous results than traditional Sanger sequencing without the need for reflex testing

Class I and class II anti-hla antibodies after implantation of cryopreserved allograft material in pediatric patients

AbstractObjectives: Very little is known regarding the immune response to cryopreserved allograft valves and patch material used in the surgical repair of congenital heart defects. Methods: We prospectively measured the frequency of panel reactive antibodies directed against HLA class I (HLA-A, B, and C) and class II (HLA-DR/DQ) alloantigens in 24 children receiving cryopreserved allografts. We compared them with results in 11 previously reported control patients. Sixteen of the study patients underwent placement of a valved conduit (11 pulmonic, 5 aortic) between the right ventricle and pulmonary arteries, 6 underwent patch angioplasty of stenotic vessels with cryopreserved pulmonary artery, and 2 underwent placement of a pulmonary monocusp patch. Study patients had panel reactive antibodies measured before, 1 month, 3 months, and 1 year after the operation. Results: With allograft implantation, panel reactive antibodies increased from 1.9% ± 5% before the operation to 62% ± 33% at 31 ± 8 days after the operation, 92% ± 15% at 3.3 ± 0.6 months after the operation, and 85% ± 18% at 1.1 ± 0.2 years after the operation. The control group showed no change in panel reactive antibodies, with a level of 1.6% ± 1% before the operation, 3.2% ± 1% 28 ± 5 days after the operation, and 1.7% ± 1% 2.7 ± 0.3 months after the operation. Class II antibodies (anti-HLA-DR/DQ) rose to 49% ± 35% at 30 ± 8 days and 70% ± 26% at 3.3 ± 0.6 months after the operation. Conclusions: Cryopreserved allograft material induces a marked response that involves both class I and class II anti-HLA antibodies within 3 months after operation in children. This alloantibody response may represent a form of “rejection,” may have implications for those who require subsequent cardiac transplantation, and may play a role in early allograft failure. (J Thorac Cardiovasc Surg 2000;119:324-30