Molecular HLA typing techniques are currently undergoing a rapid evolution. While real‐time PCR is established as the standard method in tissue typing laboratories regarding allocation of solid organs, next generation sequencing (NGS) for high‐resolution HLA typing is becoming indispensable but is not yet suitable for deceased donors. By contrast, high‐resolution typing is essential for stem cell transplantation and is increasingly required for questions relating to various disease associations. In this multicentre clinical study, the TGS technique using nanopore sequencing is investigated applying NanoTYPE™ kit and NanoTYPER™ software (Omixon Biocomputing Ltd., Budapest, Hungary) regarding the concordance of the results with NGS and its practicability in diagnostic laboratories. The results of 381 samples show a concordance of 99.58% for 11 HLA loci, HLA‐A, ‐B, ‐C, ‐DRB1, ‐DRB3, ‐DRB4, ‐DRB5, ‐DQA1, ‐DQB1, ‐DPA1 and ‐DPB1. The quality control (QC) data shows a very high quality of the sequencing performed in each laboratory, 34,926 (97.15%) QC values were returned as ‘passed’, 862 (2.4%) as ‘inspect’ and 162 (0.45%) as ‘failed’. We show that an ‘inspect’ or ‘failed’ QC warning does not automatically lead to incorrect HLA typing. The advantages of nanopore sequencing are speed, flexibility, reusability of the flow cells and easy implementation in the laboratory. There are challenges, such as exon coverage and the handling of large amounts of data. Finally, nanopore sequencing presents potential for applications in basic research within the field of epigenetics and genomics and holds significance for clinical concerns.

Background: Autoimmune neutropenia of early childhood (AIN) is caused by autoantibodies directed against antigens on the neutrophil membrane. The ABO, secretor, and Lewis histo‐blood group systems control the expression of carbohydrate antigens and have previously been linked to autoimmune diseases. We aimed to investigate the association between genotypes and the risk of AIN in Danish patients.Study Design and Methods: One hundred fifty‐four antibody‐positive AIN patients were included. Controls (n = 400) were healthy unrelated Danish blood donors. Molecular determination of ABO, secretor (FUT2), and Lewis (FUT3) genotypes were determined using real‐time polymerase chain reaction (qPCR) or Sanger sequencing to infer the prevalence of Lewis antigens (Le^a and Le^b) and secretor (SeSe or Sese) or nonsecretor (sese) phenotypes.Results: Blood type O was more common in controls (46.8%) than in AIN patients (36.4%) (OR = 0.65; p = 0.028). Secretors of H Le^b antigens were less frequent among AIN patients (25.2%) than controls (35.0%) (OR = 0.62; p = 0.037).Discussion: ABO blood group antigens and the secretion of these antigens are associated with a diagnosis of AIN. The mechanism underlying the association between autoimmunity and interaction among ABO, secretor, and Lewis genotypes has not yet been elucidated, but several studies indicate a connection to the gut microbiota.

The importance of structural genetic variants, such as copy number variations (CNVs), in modulating human disease is being increasingly recognized. Several clinical conditions require investigation of human neutrophil antigen (HNA-1), which is encoded by the Fc gamma receptor IIIb gene (FCGR3B), including suspicion of neutropenia, infections, and proactive testing of blood component donors to reduce the potential risk in transfusion. In this study, we compared real-time quantitative polymerase chain reaction (qPCR) with two digital PCR (dPCR) platforms, namely droplet digital PCR and an array-based platform, to determine CNV in FCGR3B. We initially tested 400 anonymous blood donors with qPCR using a commercially available TaqMan probe assay (Applied Biosystems) on a Quant Studio 12 Flex. CNV was determined for all 400 tested individuals with copy numbers ranging from zero to four. Zero copies were detected in 0.2% (1/400), one copy was detected in 3.8% (15/400), two copies were detected in 87.8% (351/400), three copies were detected in 8.0% (32/400), and four copies were detected in 0.2% (1/400) of tested individuals. From this cohort, we selected 32 donors with copy numbers from zero to four for analyses with Digital Real-Time PCR (dPCR) using Lab on an array (LOAA) on an On-Point analyzer from Optolane Technologies Inc. and the Droplet Digital PCR (ddPCR) platform from Bio-Rad Laboratories. We compared the obtained copy numbers of FCGR3B on the three platforms and found full concordance between the copy numbers obtained. We therefore conclude that all three platforms can be used for quantification of copy numbers for FCGR3B, and although dPCR has some advantages over qPCR, it was not necessary for reliably estimating CNV of the FCGR3B gene.

The objective of this study was to evaluate the diagnostic and prognostic potential of soluble CD163 (sCD163) in patients with pancreatic ductal adenocarcinoma (PDAC). Preoperative serum samples from 255 patients with PDAC were analyzed for sCD163 using a commercially available enzyme-linked immunosorbent assay. The diagnostic value of sCD163 was evaluated using receiver operating characteristic (ROC) curves. The prognostic significance of sCD163 was evaluated by Cox regression analysis and Kaplan–Meier survival curves. sCD163 was significantly increased in patients with PDAC, across all stages, compared to healthy subjects (stage 1: p value = 0.033; stage 2–4: p value ≤ 0.0001). ROC curves showed that sCD163 combined with CA 19-9 had the highest diagnostic potential compared to sCD163 and CA 19-9 alone both in patients with local PDAC and patients with advanced PDAC. Univariate and multivariate analysis showed no association between sCD163 and overall survival. This study found elevated levels of circulating sCD163 in patients with PDAC, regardless of stage, compared to healthy subjects. This suggests that sCD163 may have a clinical value as a novel diagnostic biomarker in PDAC.

Autoimmune neutropenia (AIN) in early childhood is caused by autoantibodies directed against antigens on the neutrophil membrane and is a frequent cause of neutropenia in children. Association of AIN with Fcγ receptor (FCGR) 3B variants is well described. In this study, we investigate genetic variations in the FCGR locus and copy number variation of FCGR3B. A total of 130 antibody‐positive AIN patients, 64 with specific anti‐HNA‐1a antibodies and 66 with broad‐reacting anti‐FcγRIIIb antibodies, were genotyped with a multiplex ligation probe assay and compared with healthy controls. Positive findings were confirmed with real‐time q‐PCR. We determined copy numbers of the FCGR2 and FCGR3 genes and the following SNPs: FCGR2A Q62W (rs201218628), FCGR2A H166R (rs1801274), FCGR2B I232T (rs1050501), FCGR3A V176F (rs396991), haplotypes for FCGR2B/C promoters (rs3219018/rs780467580), FCGR2C STOP/ORF and HNA‐1 genotypes in FCGR3B (rs447536, rs448740, rs52820103, rs428888 and rs2290834). Generally, associations were antibody specific, with all associations being representative of the anti‐HNA‐1a‐positive group, while the only association found in the anti‐FcγRIIIb group was with the HNA‐1 genotype. An increased risk of AIN was observed for patients with one copy of FCGR3B; the HNA genotypes HNA‐1a, HNA‐1aa or HNA‐1aac; the FCGR2A 166H and FCGR2B 232I variations; and no copies of FCGR2B 2B.4. A decreased risk was observed for HNA genotype HNA‐1bb; FCGR2A 166R; FCGR2B 232T; and one copy of FCGR2B promoter 2B.4. We conclude that in our Danish cohort, there was a strong association between variation in the FCGR locus and AIN. The findings of different genetic associations between autoantibody groups could indicate the presence of two different disease entities and disease heterogeneity.