RHD-negative red cells may be avoided for patients with ambiguous serologic typing for the RHD antigen.

BACKGROUND AND OBJECTIVES: Serologic typing with monoclonal anti-D is mandatory for RHD antigen determination before transfusion, but due to aberrant (weak or partial) variants of RHD, results may be ambiguous and molecular RHD-typing is required. Before that, RHD-negative (RHD -) red blood cells concentrates (RBCs) shall be transfused to avoid anti-D formation, which probably leads to wastage of RHD - RBCs. STUDY DESIGN AND METHODS: All patients with ambiguous results in serologic RHD-typing and molecular RHD-typing were assessed retrospectively. The proportions of patients at risk for anti-D formation and the proportion of RHD - RBCs transfused unnecessarily were evaluated for the following transfusion strategies: (1) RHD-positive (RHD + )RBCs for all patients, (2) RHD + RBCs for patients with at least 2+ reaction with anti-D, (3) RHD + RBCs for patients with C and/or E in their RHCE-phenotype, (4) RHD + RBCs for patients with C and/or E and at least 2+ reaction, and (5) RHD - RBCs for all patients. RESULTS: A total of 112 patients were included. Most had weak D type 1-3 and a minority had other, rare RHD variants. The risk of anti-D formation was 4.5%, 2.9%, 1.8%, 1.0%, and 0% for strategies 1-5, respectively. The proportion of RHD - RBCs transfused unnecessarily was 0%, 49.5%, 0.9%, 50.5%, and 95.5%. CONCLUSION: Transfusing patients with a C and/or E in their RHCE-phenotype with RHD + RBCs resulted in a very low risk of immunization while avoiding wastage of RHD - RBCs. Therefore, this strategy should be used for some patients with ambiguous results in serologic RHD-typing and pending results of molecular RHD-typing.

GWAS meta-analysis of 16 790 patients with Barrett's oesophagus and oesophageal adenocarcinoma identifies 16 novel genetic risk loci and provides insights into disease aetiology beyond the single marker level.

OBJECTIVE: Oesophageal cancer (EC) is the sixth leading cause of cancer-related deaths. Oesophageal adenocarcinoma (EA), with Barrett's oesophagus (BE) as a precursor lesion, is the most prevalent EC subtype in the Western world. This study aims to contribute to better understand the genetic causes of BE/EA by leveraging genome wide association studies (GWAS), genetic correlation analyses and polygenic risk modelling. DESIGN: We combined data from previous GWAS with new cohorts, increasing the sample size to 16 790 BE/EA cases and 32 476 controls. We also carried out a transcriptome wide association study (TWAS) using expression data from disease-relevant tissues to identify BE/EA candidate genes. To investigate the relationship with reported BE/EA risk factors, a linkage disequilibrium score regression (LDSR) analysis was performed. BE/EA risk models were developed combining clinical/lifestyle risk factors with polygenic risk scores (PRS) derived from the GWAS meta-analysis. RESULTS: The GWAS meta-analysis identified 27 BE and/or EA risk loci, 11 of which were novel. The TWAS identified promising BE/EA candidate genes at seven GWAS loci and at five additional risk loci. The LDSR analysis led to the identification of novel genetic correlations and pointed to differences in BE and EA aetiology. Gastro-oesophageal reflux disease appeared to contribute stronger to the metaplastic BE transformation than to EA development. Finally, combining PRS with BE/EA risk factors improved the performance of the risk models. CONCLUSION: Our findings provide further insights into BE/EA aetiology and its relationship to risk factors. The results lay the foundation for future follow-up studies to identify underlying disease mechanisms and improving risk prediction.

Completing the Donor History Questionnaire before the Donation Visit Can Improve Blood Safety.

Background and Objectives: In Germany, the donor history questionnaire (DHQ) is traditionally filled in at the donation center to avoid any influence of others. Since March 2020, it has been suggested to donors to answer the DHQ already at home and to call if they have any concerns to reduce the number of ineligible donors on-site during the COVID-19 pandemic. Materials and Methods: We evaluated the rate of ineligible donors before and after March 2020. Additionally, an anonymous online survey asking for the donors' attitude towards the DHQ was performed. It included questions on whether and for what reason the DHQ had been answered incorrectly in the past. Results: The rate of ineligible donors decreased by 27% (from 7.1% to 5.2%). In total, 5,556 of 10,252 invited donors completed the survey (54.2%). 88.6% reported either going through the DHQ at home or knowing all questions from their previous donations. 444 donors (8.0%) had at least once postponed a donation after reading the DHQ at home. 68 donors (1.2%) admitted having intentionally provided false answers in the past (9 at home, 43 on-site, 14 both, 2 unknown). Not wanting to be rejected once arriving at the donation center was an important motivation for 42% of donors answering incorrectly on-site. Details on 46 incorrect answers were provided: only 17 had no influence on donor eligibility or product quality. In 5 cases, some blood products might have had impaired quality. Truthful answers to 17 questions would have led to deferral, mostly due to increased risk for unrecognized viral infections transmitted by sexual contacts. For a further 7 questions, there was insufficient information available to determine possible consequences. Asked about their general opinion, 753 (13.6%) of all donors estimated the risk of incorrect answers being greater on-site, while 239 (4.3%) presumed an increased risk at home. Conclusion: Answering the DHQ prior to a donation visit prevented ineligible donors from visiting the donation center. Furthermore, it might improve honesty, as the discomfort of being deferred after arriving at the donation center was an important reason to answer incorrectly. Overall, there was no increased risk of donor or product safety, and potentially even a benefit.

Long-Lived Individuals Show a Lower Burden of Variants Predisposing to Age-Related Diseases and a Higher Polygenic Longevity Score.

Longevity is a complex phenotype influenced by both environmental and genetic factors. The genetic contribution is estimated at about 25%. Despite extensive research efforts, only a few longevity genes have been validated across populations. Long-lived individuals (LLI) reach extreme ages with a relative low prevalence of chronic disability and major age-related diseases (ARDs). We tested whether the protection from ARDs in LLI can partly be attributed to genetic factors by calculating polygenic risk scores (PRSs) for seven common late-life diseases (Alzheimer's disease (AD), atrial fibrillation (AF), coronary artery disease (CAD), colorectal cancer (CRC), ischemic stroke (ISS), Parkinson's disease (PD) and type 2 diabetes (T2D)). The examined sample comprised 1351 German LLI (≥94 years, including 643 centenarians) and 4680 German younger controls. For all ARD-PRSs tested, the LLI had significantly lower scores than the younger control individuals (areas under the curve (AUCs): ISS = 0.59, p = 2.84 × 10-35; AD = 0.59, p = 3.16 × 10-25; AF = 0.57, p = 1.07 × 10-16; CAD = 0.56, p = 1.88 × 10-12; CRC = 0.52, p = 5.85 × 10-3; PD = 0.52, p = 1.91 × 10-3; T2D = 0.51, p = 2.61 × 10-3). We combined the individual ARD-PRSs into a meta-PRS (AUC = 0.64, p = 6.45 × 10-15). We also generated two genome-wide polygenic scores for longevity, one with and one without the TOMM40/APOE/APOC1 gene region (AUC (incl. TOMM40/APOE/APOC1) = 0.56, p = 1.45 × 10-5, seven variants; AUC (excl. TOMM40/APOE/APOC1) = 0.55, p = 9.85 × 10-3, 10,361 variants). Furthermore, the inclusion of nine markers from the excluded region (not in LD with each other) plus the APOE haplotype into the model raised the AUC from 0.55 to 0.61. Thus, our results highlight the importance of TOMM40/APOE/APOC1 as a longevity hub.

Prevalence of antibodies against Hepatitis D virus (HDV) in blood donors in Northern Germany.

OBJECTIVE: To assess the prevalence of HDV infections in German blood donors. METHOD: 167 donors with acute/chronic or resolved HBV infection and detectable antibodies against Hepatitis B core antigen (anti-HBc) were tested for antibodies against HDV (anti-HDV) by competitive ELISA. Samples with detectable anti-HDV or with HBsAg and/or HBV DNA were additionally investigated for HDV RNA. RESULTS: In nine (5.4 %) of the 167 donors, also HBsAg and HBV DNA were detectable. Anti-HDV was detectable in two of the 167 donors (1.2 %), additional four donors (2.4 %) had a borderline result. All of these donors tested negative for HBsAg and HBV DNA. Neither in samples with anti-HDV nor in HBsAg-/HBV DNA-positive samples, HDV RNA was detectable. CONCLUSIONS: At least 1.2 % of anti-HBc-positive blood donors have had an HDV infection. Although there is some evidence for a somewhat higher prevalence of HDV, the overall prevalence of HDV in Northern Germany is low.

Infectivity of blood products containing cytomegalovirus DNA: results of a lookback study in nonimmunocompromised patients.

BACKGROUND: DNA of human cytomegalovirus (CMV) is frequently detected in plasma of donors with primary CMV infection. It is unknown, however, whether leukoreduced blood products from these donors contain sufficient amounts of infectious virus to cause transfusion-transmitted CMV infections (TT-CMV). STUDY DESIGN AND METHODS: During a 14-year period, CMV DNA-positive donations were identified as part of several previously published studies. Additionally, further donors with seroconversion were tested for CMV DNA. The serostatus of patients who had received a CMV DNA-positive blood product was determined out of pretransfusion samples. Later samples were examined for development of CMV antibodies. Patients with a follow-up of less than 140 days were also tested for CMV DNA. RESULTS: A total of 221 blood products from CMV DNA-positive donations were transfused to 219 recipients. Pretransfusion samples were available for 179 patients, of whom 62 (34.6%) were seronegative. For 39 seronegative recipients of 40 blood products follow-up samples drawn at least 30 days after transfusion were available. The median duration of follow-up was 287 days (range, 38-3784 days). Thirty-six patients were still CMV seronegative in their last sample. Three patients were CMV seropositive due to passive antibody transfer by plasma rich products from seropositive donors, but CMV DNA negative in all tested samples. CONCLUSION: TT-CMV was excluded in all recipients of 40 blood products from CMV DNA-positive donations. This corresponds to a 95% interval of confidence for the risk of TT-CMV of less than 7.4%. Because no patient belonged to a typical at-risk population, the results are only valid for immunocompetent subjects.

Unacceptable human leucocyte antigens for organ offers in the era of organ shortage: influence on waiting time before kidney transplantation.

BACKGROUND: The assignment of human leucocyte antigens (HLAs) against which antibodies are detected as unacceptable antigens (UAGs) avoids allocation of HLA- incompatible allografts. There is uncertainty as to what extent UAGs decrease the probability of receiving a kidney offer. METHODS: Kidney transplantations in 3264 patients on the waiting lists of six German transplant centres were evaluated for a period of at least 2 years. The proportion of excluded offers due to UAGs was calculated as virtual panel-reactive antibodies (vPRAs). RESULTS: In the common Eurotransplant Kidney Allocation Scheme, the transplant probability was unaffected by vPRAs in exploratory univariate analyses. In the multivariable model, a 1% increase in vPRA values was outweighed by an additional waiting time of 2.5 weeks. The model was confirmed using an external validation cohort of 1521 patients from seven centres. If only patients with standard risk were considered (e.g. no simultaneous transplantation of other organs), only 1.3 weeks additional waiting time was needed. In the Eurotransplant Senior Program, patients with vPRA values >50% had a strongly reduced transplant probability in the unadjusted analyses. In the multivariable model, a 1% increase in vPRA values was outweighed by an additional waiting time of 5 weeks. CONCLUSIONS: This study demonstrates that the assignment of UAGs decreases the transplant probability in both main Eurotransplant allocation programs because of insufficient compensatory mechanisms. At present, for immunized patients, a prolonged waiting time has to be weighed against the increased immunologic risk due to donor-specific antibodies not assigned as UAGs.

Development of a high-resolution NGS-based HLA-typing and analysis pipeline.

The human leukocyte antigen (HLA) complex contains the most polymorphic genes in the human genome. The classical HLA class I and II genes define the specificity of adaptive immune responses. Genetic variation at the HLA genes is associated with susceptibility to autoimmune and infectious diseases and plays a major role in transplantation medicine and immunology. Currently, the HLA genes are characterized using Sanger- or next-generation sequencing (NGS) of a limited amplicon repertoire or labeled oligonucleotides for allele-specific sequences. High-quality NGS-based methods are in proprietary use and not publicly available. Here, we introduce the first highly automated open-kit/open-source HLA-typing method for NGS. The method employs in-solution targeted capturing of the classical class I (HLA-A, HLA-B, HLA-C) and class II HLA genes (HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1). The calling algorithm allows for highly confident allele-calling to three-field resolution (cDNA nucleotide variants). The method was validated on 357 commercially available DNA samples with known HLA alleles obtained by classical typing. Our results showed on average an accurate allele call rate of 0.99 in a fully automated manner, identifying also errors in the reference data. Finally, our method provides the flexibility to add further enrichment target regions.

Comparison of Two Test Strategies for Clarification of Reactive Results for Anti-HBc in Blood Donors.

OBJECTIVE: Testing for antibodies against hepatitis B core antigen (anti-HBc) was introduced to detect blood donors suffering from occult hepatitis B infection. Confirmation of specification of reactive results in the anti-HBc screening assay is still a challenge for blood donation services. METHODS: Two different test strategies for confirmation of specification of reactive anti-HBc tests, one performed in our institute and one suggested by the German authority (Paul-Ehrlich-Institut (PEI)), were compared. The first strategy is based on one supplemental anti-HBc test, the other requires two supplemental anti-HBc tests. RESULTS: 389 samples from 242 donors were considered. Both test strategies yielded concordant results in 117 reactive samples termed 'true-positive' or 'specificity confirmed', in 156 reactive samples termed 'false-positive' or 'specificity not confirmed', and in 99 negative samples. In 17 samples obtained from 11 donors, both test strategies gave discrepant results ('false-positive' but 'specificity confirmed'). In 10 of 11 donors, a real HBV infection was very unlikely, one remained unclear. 30 donors considered 'false-positive' became negative in all anti-HBc tests after follow-up testing and thus eligible for donor re-entry. CONCLUSIONS: The test strategy suggested by the PEI yielded no additional information but induced an overestimation of HBV infections and unnecessary look-back procedures. Many anti-HBc-reactive donors can be regained after follow-up testing.

Allelic and copy-number variations of FcγRs affect granulocyte function and susceptibility for autoimmune blistering diseases.

Low-affinity Fcγ receptors (FcγR) bridge innate and adaptive immune responses. In many autoimmune diseases, these receptors act as key mediators of the pathogenic effects of autoantibodies. Genes encoding FcγR exhibit frequent variations in sequence and gene copy number that influence their functional properties. FcγR variations also affect the susceptibility to systemic autoimmunity, e.g. systemic lupus erythematosus and rheumatoid arthritis. This raises the question whether FcγR variations are also associated with organ-specific autoimmunity, particularly autoantibody-mediated diseases, such as subepidermal autoimmune blistering diseases (AIBD). A multitude of evidence suggests a pathogenic role of neutrophil granulocyte interaction with autoantibodies via FcγR. In a two-stage study, we analyzed whether the FcγR genotype affects neutrophil function and mRNA expression, and consequently, bullous pemphigoid (BP) disease risk. We compared this to findings in pemphigus vulgaris/foliaceus (PV/PF), two Fc-independent AIBDs. Our results indicate that both allele and copy number variation of FcγR genes affect FcγR mRNA expression and reactive oxygen species (ROS) release by granulocytes. Susceptibility of BP was associated with FcγR genotypes that led to a decreased ROS release by neutrophils, indicating an unexpected protective role for these cells. BP and PV/PF differed substantially regarding the FcγR genotype association patterns, pointing towards different disease etiologies.

Co-occurrence of autoantibodies in healthy blood donors.

Autoimmune diseases are rare, but their incidence has increased over the past decades. Interestingly, the co-occurrence of autoimmune diseases is well documented; however, data on the presence of more than one specific autoantibody in healthy individuals are not available. Here, we investigated the prevalence of several autoantibodies in a cohort of over 6000 healthy persons. While individual autoantibodies were rarely detected (i.e. ranging from 0.3% for ANCA to 4.6% for anti-TPO), the cumulative prevalence of the tested autoantibodies was as high as 10%. Furthermore, our results demonstrate co-occurrence of ANA with specific autoantibodies that target TPO, CCP and Dsg1/3, while ANCA and autoantibodies to PCA and BP180/BP230 were not more frequent in ANA-positive compared to ANA-negative samples. This indicates that shared and independent mechanisms influence loss of tolerance to distinct sets of self-antigens.

Seroprevalence and incidence of hepatitis E virus infection in German blood donors.

BACKGROUND: Hepatitis E virus (HEV) is transmissible by transfusion. More data are needed about seroprevalence, incidence, and viremia in blood donors for the assessment of risk of transfusion-transmitted (TT)-HEV infections. STUDY DESIGN AND METHODS: Samples from 1019 whole blood donors were tested for anti-HEV immunoglobulin (Ig)G by enzyme-linked immunosorbent assay and Western blot. The incidence of HEV and presence of HEV RNA in donors who seroconverted were determined by testing archive samples and recipients of viremic donations were traced. Anti-HEV IgM and alanine transaminase (ALT) testing were also performed to assess the value of such measures in the prevention of TT-HEV infections. RESULTS: A total of 69 of 1019 donors tested positive for anti-HEV IgG (6.8% seroprevalence), and seroconversion for anti-HEV IgG occurred in seven of 69 donors within 2 years (incidence, 0.35%/year). Three of seven (42.8%) seroconverting donors provided an archive sample in which HEV RNA was detectable. One recipient of these donations was traceable; anti-HEV IgG, IgM, and HEV RNA testing were negative 41 days after transfusion. Neither ALT levels nor anti-HEV IgM detection correlated with the presence of HEV RNA. CONCLUSIONS: The seroprevalence of HEV was 6.8%, and the annual incidence 0.35%. HEV RNA was detectable in several seroconverting donors, without evidence for HEV transmission in the only traceable recipient. Since neither ALT nor anti-HEV IgM testing correlate with the presence of HEV RNA, HEV nucleic acid testing currently provides the only method for the prevention of TT-HEV infection. However, before implementation, more data about clinical relevance of TT-HEV infections and infectious dose of HEV are required.

Parvovirus b19 infections and blood counts in blood donors.

OBJECTIVES: Parvovirus B19 (B19V) is a transfusion-transmissible virus. To obtain data about the prevalence, incidence, the course of B19V infection in blood donors and whether B19V might impair their blood counts, samples from blood donors with B19V infection were investigated. METHODS: Blood donations were screened for B19V DNA using the Cobas TaqScreen DPX Test® in mini-pools. B19V DNA concentration, anti-B19V IgG antibody titer and blood counts were determined in positive donors. RESULTS: 157/23,889 (0.66%) donors provided 347 B19V DNA-positive samples. Prevalence of B19V infection was 0.45%, incidence 0.20%. B19V DNA concentrations were predominantly low; only in 8 samples were viral loads of ≥10(5) IU B19V DNA/ml plasma detectable. Besides a slight decrease in hemoglobin, hematocrit, mean corpuscular volume, mean cellular hemoglobin and mean hemoglobin concentration, no major differences in blood counts occurred in B19V DNA-positive samples. In samples with a low B19V DNA concentration, anti-B19V IgG titers were rather high. 98 donors provided at least 1 B19V DNA-positive follow-up sample, indicating a prolonged viremia. CONCLUSIONS: B19V infection induced no major impairment in the blood counts. In donors with low-level viremia, infectivity through their donations is probably reduced by high antibody titers. Low-level viremia is prolonged, probably exceeding 1 year in many cases.

The impact of donor cytomegalovirus DNA on transfusion strategies for at-risk patients.

BACKGROUND: Cytomegalovirus (CMV) DNA is frequently detected in plasma of newly seropositive donors. Selection of leukoreduced blood products from donors with remote CMV infection could avoid transfusion-transmitted CMV infections (TT-CMV) due to primarily infected donors. However, there are no data about the prevalence of reactivations in long-term seropositive donors compared to the incidence of window period donations in seronegative donors. Therefore, the optimal transfusion strategy for at-risk patients is unclear. STUDY DESIGN AND METHODS: Whole blood samples from 22,904 donations were tested for CMV DNA, and CMV DNA-positive donations were categorized as donations from 1) seronegative donors, 2) newly seropositive donors, and 3) long-term seropositive donors. RESULTS: Twenty-one donors were reproducibly CMV DNA-positive (0.09%). Frequency of detection and concentration of CMV DNA in whole blood were comparable for seronegative and long-term seropositive donors. Nonreproducibly positive results for CMV DNA in whole blood were more frequent in long-term seropositive donors (0.16% vs. 0.01%, p<0.01). Only low concentrations of CMV DNA in plasma were detectable in two seronegative donors and one long-term seropositive donor. Highest concentrations of CMV DNA in both whole blood and plasma, however, were found in newly seropositive donors. CONCLUSION: Prevalences of window period donations among seronegative donors and reactivations among long-term seropositive donors, as well as the CMV DNA concentration in whole blood and plasma samples from these donors, are comparable. Therefore, blood products from both groups could be used for patients at risk for TT-CMV, while those of newly seropositive donors seem to bear an increased risk.

Window period donations during primary cytomegalovirus infection and risk of transfusion-transmitted infections.

BACKGROUND: Donors with short interdonation intervals (e.g., apheresis donors) have an increased risk of window period donations. The frequency of cytomegalovirus (CMV) window period donations is important information to decide whether selection of seronegative donors might be advantageous for patients at risk for transfusion-transmitted CMV infections (TT-CMV). STUDY DESIGN AND METHODS: CMV seroconversion in 93 donors with positive results in routine CMV antibody testing within at most 35 days after the last seronegative sample was evaluated by Western blot and/or a second antibody test. In donors with unconfirmed seroconversion, an additional later sample was tested. Concentration of CMV DNA was determined in pre- and postseroconversion samples. RESULTS: CMV seroconversion was confirmed in 12 donors (13%). Among these, the last seronegative sample was CMV DNA positive in three donors (25%, below 30 IU/mL). The first seropositive sample was CMV DNA positive in 10 donors (83%, maximum 1600 IU/mL). Both prevalence and median concentration of CMV DNA were higher in the first seropositive sample (p = 0.004 and p = 0.02), with maximum concentrations being reached about 2 weeks after seroconversion. No CMV DNA was detected in samples from donors with unconfirmed seroconversion. CONCLUSION: At least in donors with short interdonation intervals, most suspected CMV seroconversions are due to false-positive results of the screening test. As window period donations are rare and contain less CMV DNA than the first seropositive donation, avoidance of blood products from primarily seropositive donors is especially helpful to avoid TT-CMV if donors with short interdonation intervals are concerned.

Monitoring the SARS-CoV-2 Pandemic: Prevalence of Antibodies in a Large, Repetitive Cross-Sectional Study of Blood Donors in Germany-Results from the SeBluCo Study 2020-2022.

SARS-CoV-2 serosurveillance is important to adapt infection control measures and estimate the degree of underreporting. Blood donor samples can be used as a proxy for the healthy adult population. In a repeated cross-sectional study from April 2020 to April 2021, September 2021, and April/May 2022, 13 blood establishments collected 134,510 anonymised specimens from blood donors in 28 study regions across Germany. These were tested for antibodies against the SARS-CoV-2 spike protein and nucleocapsid, including neutralising capacity. Seroprevalence was adjusted for test performance and sampling and weighted for demographic differences between the sample and the general population. Seroprevalence estimates were compared to notified COVID-19 cases. The overall adjusted SARS-CoV-2 seroprevalence remained below 2% until December 2020 and increased to 18.1% in April 2021, 89.4% in September 2021, and to 100% in April/May 2022. Neutralising capacity was found in 74% of all positive specimens until April 2021 and in 98% in April/May 2022. Our serosurveillance allowed for repeated estimations of underreporting from the early stage of the pandemic onwards. Underreporting ranged between factors 5.1 and 1.1 in the first two waves of the pandemic and remained well below 2 afterwards, indicating an adequate test strategy and notification system in Germany.

Prognostic value and cost-effectiveness of different screening strategies for HLA antibodies prior to kidney transplantation.

HLA antibody screening is conducted routinely prior to kidney transplantation, but the comparative prognostic value and cost-effectiveness of different methods are unclear. Pre-transplant sera of 141 patients transplanted between 1998 and 2000 were screened by ELISA and Luminex assays, and antibody specificities of reactive sera determined using bead array techniques. ELISA screening detected donor-specific antibodies (DSA) in 19 patients, who had a higher incidence of impaired graft function (60% vs. 20%, p = 0.04) and antibody-mediated rejection (AMR) within 90 d after transplantation (AMR, 35% vs. 5%, p = 0.02). Luminex screening detected eight additional patients with DSA, among those one with AMR. Six of eight patients with Luminex-only-DSA reported no prior immunizing events. Death-censored graft survival was shorter only in patients with DSA and AMR (median, 1.7 yr instead of between 9.5 and 11.0 yr for patients without DSA or patients with DSA but no AMR, p < 0.001). Material costs per detected clinically relevant DSA were about 57% higher for Luminex screening, but this increase could be avoided by modifying the cut-off recommended by the manufacturer. Conclusively, specification of antibodies only in sera reactive in screening tests was cost-effective to prevent shortened graft survival. Preformed DSA were only harmful if AMR was diagnosed within 90 d after transplantation.

High-throughput method for the hybridisation-based targeted enrichment of long genomic fragments for PacBio third-generation sequencing.

Hybridisation-based targeted enrichment is a widely used and well-established technique in high-throughput second-generation short-read sequencing. Despite the high potential to genetically resolve highly repetitive and variable genomic sequences by, for example PacBio third-generation sequencing, targeted enrichment for long fragments has not yet established the same high-throughput due to currently existing complex workflows and technological dependencies. We here describe a scalable targeted enrichment protocol for fragment sizes of >7 kb. For demonstration purposes we developed a custom blood group panel of challenging loci. Test results achieved > 65% on-target rate, good coverage (142.7×) and sufficient coverage evenness for both non-paralogous and paralogous targets, and sufficient non-duplicate read counts (83.5%) per sample for a highly multiplexed enrichment pool of 16 samples. We genotyped the blood groups of nine patients employing highly accurate phased assemblies at an allelic resolution that match reference blood group allele calls determined by SNP array and NGS genotyping. Seven Genome-in-a-Bottle reference samples achieved high recall (96%) and precision (99%) rates. Mendelian error rates were 0.04% and 0.13% for the included Ashkenazim and Han Chinese trios, respectively. In summary, we provide a protocol and first example for accurate targeted long-read sequencing that can be used in a high-throughput fashion.

B-cell responses to vaccination with BNT162b2 and mRNA-1273 6 months after second dose.

OBJECTIVES: To examine the state of B-cell immunity 6 months after the second vaccination against SARS-CoV-2 in comparison to the state observed 2 weeks after vaccination. METHODS: Sera of 439 participants, whose immune responses to two doses of an mRNA-based vaccine (BNT162b2 or mRNA-1273) were previously characterized, was examined for anti-S1 IgG and IgA, anti-NCP IgG and neutralizing antibodies (nAb), and antinuclear antibodies (ANA). RESULTS: Levels of all examined markers decreased significantly from 2 weeks to 6 months after second vaccination (anti-S1 IgG: 3744 ± 2571.4 vs. 253 ± 144 binding antibody units (BAU)/mL; anti-S1 IgA: 12 ± 0 vs. 1.98 ± 1.75 optical density (OD) ratio; nAb: 100% ± 0% vs. 82% ± 19.3%), the vast majority of participants retaining reactive levels of anti-S1 IgG (436/439) and anti-S1 IgA (334/439) at 6 months. Immune responses were stronger for mRNA-1273 compared with BNT162b2 (anti-S1 IgG: 429 ± 289 vs. 243 ± 143 BAU/mL; anti-S1 IgA: 5.38 ± 3.91 vs. 1.89 ± 1.53 OD ratio; nAb: 90.5% ± 12.6% vs. 81% ± 19.3%). There was no meaningful influence of sex and age on the examined markers. There was a strong correlation between anti-S1 IgG and the surrogate neutralization assay (rho = 0.91, p <0.0001), but not for for IgA and the surrogate neutralization assay (rho = 0.52, p <0.0001). There was a ceiling effect for the association between anti-S1 IgG titres and the inhibition of binding between S1 and ACE2. ANA prevalence was unchanged from 2 weeks to 6 months after the second vaccination (87/498 vs. 77/435), as were the median ANA titres (1:160 vs. 1:160). DISCUSSION: Although the clinical consequences of decreasing anti-SARS-CoV-2 antibody titres cannot be estimated with certainty, a lowered degree of clinical protection against SARS-CoV-2 is possible. Persistently stronger responses to mRNA-1273 suggest that it might confer greater protection than BNT162b2, even 6 months after the second vaccination. Neither examined vaccinations induced ANA within the examined time frame.

The temporal course of T- and B-cell responses to vaccination with BNT162b2 and mRNA-1273.

OBJECTIVES: To investigate the response of the immune system (and its influencing factors) to vaccination with BNT162b2 or mRNA-1273. METHODS: 531 vaccinees, recruited from healthcare professionals, donated samples before, in between, and after the administration of the two doses of the vaccine. T- and B-cell responses were examined via interferon-γ (IFN-γ) release assay, and antibodies against different epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (S1 and NCP) were detected via ELISA and surrogate neutralization assay. Results were correlated with influencing factors such as age, sex, prior infection, vaccine received (BNT162b2 or mRNA-1273), and immunosuppression. Furthermore, antinuclear antibodies (ANAs) were measured to screen for autoimmune responses following vaccination with an mRNA vaccine. RESULTS: No markers of immunity against SARS-CoV-2 were found before the first vaccination. Two weeks after it, specific responses against SARS-CoV-2 were already measurable (median ± median absolute deviation (MAD): anti-S1 IgG 195.5 ± 172.7 BAU/mL; IgA 6.7 ± 4.9 OD; surrogate neutralization 39 ± 23.7%), and were significantly increased two weeks after the second dose (anti-S1 IgG 3744 ± 2571.4 BAU/mL; IgA 12 ± 0 OD; surrogate neutralization 100 ± 0%, IFN-γ 1897.2 ± 886.7 mIU/mL). Responses were stronger for younger participants (this difference decreasing after the second dose). Further influences were previous infection with SARS-CoV-2 (causing significantly stronger responses after the first dose compared to unexposed individuals (p ≤ 0.0001)) and the vaccine received (significantly stronger reactions for recipients of mRNA-1273 after both doses, p < 0.05-0.0001). Some forms of immunosuppression significantly impeded the immune response to the vaccination (with no observable immune response in three immunosuppressed participants). There was no significant induction of ANAs by the vaccination (no change in qualitative ANA results (p 0.2592) nor ANA titres (p 0.08) from pre-to post-vaccination. CONCLUSIONS: Both vaccines elicit strong and specific immune responses against SARS-CoV-2 which become detectable one week (T-cell response) or two weeks (B-cell response) after the first dose.