HLA genotyping using the Illumina HLA TruSight next-generation sequencing kits: A comparison.

Illumina first introduced their TruSight human leucocyte antigen (HLA) next-generation sequencing (NGS) typing kit in 2015 and subsequently followed up with a new version in 2016. Here we report on our experience comparing the two versions of the Illumina HLA NGS kits.

HLA genotyping in the clinical laboratory: comparison of next-generation sequencing methods.

Implementation of human leukocyte antigen (HLA) genotyping by next-generation sequencing (NGS) in the clinical lab brings new challenges to the laboratories performing this testing. With the advent of commercially available HLA-NGS typing kits, labs must make numerous decisions concerning capital equipment and address labor considerations. Therefore, careful and unbiased evaluation of available methods is imperative. In this report, we compared our in-house developed HLA NGS typing with two commercially available kits from Illumina and Omixon using 10 International Histocompatibility Working Group (IHWG) and 36 clinical samples. Although all three methods employ long range polymerase chain reaction (PCR) and have been developed on the Illumina MiSeq platform, the methodologies for library preparation show significant variations. There was 100% typing concordance between all three methods at the first field when a HLA type could be assigned. Overall, HLA typing by NGS using in-house or commercially available methods is now feasible in clinical laboratories. However, technical variables such as hands-on time and indexing strategies are sufficiently different among these approaches to impact the workflow of the clinical laboratory.

Report on the effects of fragment size, indexing, and read length on HLA sequencing on the Illumina MiSeq.

Single-molecule sequencing should allow for unambiguous, accurate, and high-throughput HLA typing. In this proof of principle study, we investigated the effects of fragment size for library preparation, indexing strategy, and read length on HLA typing. Whole gene amplicons of HLA-A, B, C, DRB1, and DQB1 were obtained by long-range PCR. For library preparation, two fragment sizes were evaluated: 100-300bp and 300-600bp. For sample multiplexing, two indexing strategies were compared: indexing-by-amplicon, where each individual amplicon is barcoded, and indexing-by-patient, where each patient's five loci are equimolarly pooled after PCR and indexed with the same barcode. Sequencing was performed on an Illumina MiSeq instrument using paired-end 150bp and 250bp read lengths. Our results revealed that the 300-600bp fragments in the 2×250 MiSeq group gave the most accurate sequencing results. There was no difference in HLA typing results between the two indexing strategies, suggesting that indexing-by-patient, which is much simpler, is a viable option. In conclusion, enzymatic fragmentation of pooled whole gene amplicons is a suitable strategy for HLA typing by next-generation sequencing on the Illumina MiSeq.

HLA alleles and drug hypersensitivity reactions.

The human leucocyte antigen (HLA) system is well known for its association with certain diseases such as ankylosing spondylitis, celiac disease and many others. More recently, severe and even fatal drug hypersensitivity reactions linked to particular HLA alleles have been discovered. The significance of these discoveries has led the European Medicines Agency (EMA) and its member state agencies to recommend HLA gene testing before initiation of drug treatment. To date, the following drugs have been identified as causing significant drug hypersensitivity reactions in patients who have the following HLA alleles: abacavir and HLA-B*57:01, carbamazepine and HLA-B*15:02/A*31:01 and finally allopurinol and HLA-B*58:01. This review will outline and discuss these three drugs and their associated HLA alleles as well as examine the pathogenesis of the drug hypersensitivity reactions.

Celiac disease and HLA typing using real-time PCR with melting curve analysis.

Celiac disease (CD) is an autoimmune disease characterized by chronic diarrhea, inflammatory lesions of small bowel and nutritional malabsorption. CD is strongly associated with the presence of HLA-DQB*02, DQB*03:02 and DQA*05. The absence of any one of these three human leukocyte antigen (HLA) alleles rules out the diagnosis of CD in suspected patients. Here, we describe a novel method to detect the presence of these specific HLA alleles using real-time polymerase chain reaction (PCR) with melting curve analysis. Compared with current HLA typing assays, the real-time PCR method is faster, requires fewer handling steps and provides 100% sensitivity and specificity for typing of HLA-DQB*02, DQB*03:02 and DQA*05 alleles.

Prospective randomized trial of azathioprine in cryopreserved valved allografts in children.

BACKGROUND: The purpose of this study was to prospectively assess the effects of azathioprine on the humoral immune response to HLA alloantigens and allograft function in children receiving cryopreserved valved allografts. METHODS: We randomized 13 children to receive azathioprine or not to receive azathioprine (controls) after receiving a cryopreserved valved allograft. Azathioprine patients received intraoperatively 4 mg/kg of azathioprine and 2.0 +/- 0.5 mg/kg once daily for 3 months after operation. Panel reactive antibodies against HLA class I and class II alloantigens were measured before, 1 month, and 3 months after operation. RESULTS: Panel reactive antibodies were not significantly different between the azathioprine and control groups before (0.0% +/- 0% versus 1.6% +/- 1%), 1 month (59% +/- 17% versus 71% +/- 12%), or 3 months (84% +/- 15% versus 96% +/- 1.3%) after operation. There were no differences in degree of allograft valve stenosis between azathioprine (31.5 +/- 26 mm Hg, 13.4 +/- 7 months postoperatively) and control groups (25.4 +/- 11 mm Hg, 17.2 +/- 10 months postoperatively) or allograft valve insufficiency. CONCLUSIONS: Azathioprine does not significantly decrease the immune response to HLA alloantigens or affect the function of cryopreserved valved allografts used in children to repair congenital heart defects.

A prospective analysis of the immunogenicity of cryopreserved nonvalved allografts used in pediatric heart surgery.

BACKGROUND: The purpose of this study was to prospectively determine the immunogenicity of nonvalved allograft tissue used to repair congenital heart defects. METHODS AND RESULTS: We prospectively analyzed the immune response of 11 children, 1.4 months to 10 years of age, who required nonvalved allografts to alleviate stenosis during repair of congenital heart defects. In 7 patients, pulmonary arterial grafts were used; in 3 patients, monocusp pulmonary artery grafts were used; and in 1 patient, a section of glutaraldehyde-preserved allograft pericardium was used. We measured the level of HLA panel-reactive antibody (PRA) before surgery, 1 week after, 1 month after, and 3 months after surgery. PRA was determined by the antiglobulin technique and flow cytometry. HLA class I and class II antibodies measured by either technique were negligible before and 1 week after surgery. Nine of 11 patients (82%) exhibited a significant immune response at 1 month after surgery that further increased at 3 months. The measured PRA for class I antibodies with the antiglobulin technique increased to 43+/-36% at 1 month and to 69+/-38% at 3 months after surgery. Flow cytometry class I PRA measurements were similar. Class II PRA increased to 26+/-34% at 1 month and to 41+/-36% at 3 months. Age negatively correlated with the degree of elevation of PRA, but neither allograft area nor the area indexed to patient body surface area correlated with PRA. CONCLUSIONS: Cryopreserved nonvalved allografts induce a strong HLA antibody response in the majority of children.

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

OBJECTIVES: 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.

Repeat donor HLA-DR mismatches in renal transplantation: is the increased failure rate caused by noncytotoxic HLA-DR alloantibodies?

INTRODUCTION: Data from the UCLA/UNOS and Collaborative Transplant Studies Registries indicate that mismatched HLA-DR alloantigens expressed on a former donor renal allograft should not be repeated because of significantly poorer long-term survival. METHODS: Retransplant candidates waiting for another renal allograft were screened for HLA class II alloantibodies (aAb) using direct complement-dependent cytotoxicity and several sensitive aAb binding assays. RESULTS: When screened by complement-dependent cytotoxicity, 46% of the patients were aAb negative. In contrast, using aAb binding assays, 90% of the patients had HLA-DR aAb specific for previous HLA-DR allograft mismatches. Most important, no directly cytotoxic HLA-DR antibody was detected in 9 of 27 patients. CONCLUSION: Our studies suggest that crossing the same HLA-DR mismatch in a subsequent transplant may result in poorer survival due to underlying donor-specific HLA-DR aAb. If confirmed in a retrospective study of retransplant patients, B cell donor cross-matches using antiglobulin complement-dependent cytotoxicity or flow cytometry would appear essential if this barrier were to be crossed.