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Aim Given evidence that donor specific antibodies (DSA) to Human Leukocyte Antigens (HLA) adversely affect kidney transplant outcome, sensitive techniques for detecting HLA antibodies have been developed. Single-antigen (SA) Luminex-based tests, in particular, are highly sensitive and are used to define ‘unacceptable’ donor antigens in a virtual crossmatch. Although it has largely revolutionized HLA antibody detection, the Luminex SA bead assay however does occasionally present with some problematic results such non-specific reactivity and interfering substances which can make interpretation challenging. One phenomenon that labs encounter is the inhibition or ‘prozone’ effect in cases when antibody is in excess. A consequence of this is that the antibody specificities with the highest concentration can appear to have weak or absent reactivity to specific SA beads. This is believed to be caused by complement components competitively displacing the anti-IgG detecting antibody because it has been shown to be reversed with complement inhibitors such as EDTA, heat inactivation, serum dilution and C1-inhibitors. However, this inhibition effect occurs only with some patient serums and not across all the antigens. We investigated under which circumstances this occurred and whether some antigens were more likely to be affected. Methods Post-transplant serum was tested with and without EDTA by Single Antigen Bead assay. Results In our center we found the inhibition effect predominantly in highly sensitized patients that had previously received grafts. Class II antigens, in particular HLA-DQ, was found to be inhibited by the presence of antibody excess, and reversed with EDTA treatment. Interestingly, the inhibitory effect was often seen with beads that specifically expressed donor specific alpha and beta chain antigen combinations, whereas beads carrying only the beta chain antigen of the donor but a different alpha chain were not affected. Conclusions This indirectly suggests that the number of epitopes targeted on each HLA molecule, rather than just antibody titre, may influence the ‘prozone’ phenomenon.

Next generation sequencing (NGS) offers a new paradigm in HLA genotyping that will fundamentally change the way clinical laboratories report patient genotypes. The technique reveals entire gene sequences with ultra-high base resolution and can resolve phase ambiguities for multiple samples in a single assay. While implementation of NGS is challenging in clinical diagnostics, the definite advantages and new diagnostic possibilities make the switch to the technology inevitable. Recently a number of commercially available NGS-based HLA kits have come on the market. On behalf of the Canadian HLA network, we present a comparative study of 4 of these. HLA genotyping requires three basic steps; PCR, sequencing of amplicon library, and allele assignment. The relative performance of each step was assessed. We evaluated three commercially available NGS HLA typing kits; Illumina’s TruSight, Omixon’s Holotype HLA and GenDx’s NGSgo kit. We also had early access to OneLambda’s kit. All amplicon libraries were sequenced using either the Illumina MiSeq or Ion Torrent PGM platform. Each of the commercial kits included the necessary proprietary allele assignment software. All methods provided targeted capturing of the classical class I (HLA-A, B, C) and class II HLA genes (HLA-DRB1, DQA1, DQB1, DPB1) as a minimum. All calling algorithms provided allele-calling to at least three-field resolution. A validation panel of 48 clinical and proficiency testing samples were analyzed using all 4 methods and performed by the same technician. All samples had known HLA alleles obtained by LABType SSO/SSP typing (4 digit). A total of 1680 loci were analyzed. An overview of each protocol with our experience on sequence performance efficiencies, read depth uniformity and ambiguity analysis will be presented. Briefly, targeted NGS HLA typing kits were found to be easy to use, flexible to the need of the clinical laboratory and priced comparable to HD SSO. Results were highly congruous with standard SSO/SSP typing. Our studies show that NGS is fully feasible for routine use, and offers precise, ultra-high resolution, complete sequence, cost-efficient high-throughput HLA DNA typing which provides informative data and improved HLA matching for medical research, transplantation medicine, and HLA-related disease diagnosis.

Two polymorphic regions have been described within the IL-2 and IL-2 receptor β genes comprising 15 and 8 alleles, respectively. Whether these polymorphisms have biologic importance is unknown, although they have been variably identified in associated with certain chronic disease states. We report here the detection of four new alleles designated IL-2 A∗ (122 bp), IL-2R-2 (169 bp), IL-2R 0 (165 bp), and IL-2R 9 (147 bp) in patients with rheumatoid arthritis and normal controls from the Pacific Northwest. The number of alleles now recognized at these loci within the IL-2 and IL-2Rβ genes increases to 16 and 12, respectively.

This report describes a new MHC class II allele, HLA-DRβ1 ∗ 0306, discovered in a 31-year-old Norwegian male. The allele typed serologically as DRw52 (DR3) and amplified in PCR using DR52-associated group primers. This product could not be identified using established restriction digests, however. Use of Asp 700, Msp I, Hha I, Bse RI, Mnl I, Hph I, and Bsrb I gave banding patterns expected for a DRβ1 ∗ 03011 allele, but Rsa I had an additional site at codon 47. Sequencing showed a single base change at this position, with the substitution of tyrosine for phenylanine in this new allele. The biologic impact of this substitution remains to be determined.

Five polymorphic regions (a to e) have been recognized within the TNF gene region. These polymorphisms appear to be of biological importance as individual alleles have been associated with higher production of TNF and/or an increased risk of rheumatoid arthritis or diabetes mellitus. We report here the detection of four new alleles designated a14 (122 bp), b8 (131 bp), b9 (132 bp), and d0 (122 bp) in patients with rheumatoid arthritis and normal controls from the Pacific Northwest. This increases to 39 the number of alleles now recognized at these loci.