Autoimmune neutropaenia (AIN) in early childhood is characterized by chronic neutropaenia and positivity for human neutrophil antibodies (HNA), resulting in the excessive destruction of neutrophils. The association between regulatory T cells (Tregs) and AIN has been described, and in this study, we investigated three Treg-associated genes, IL-2, IL-10 and FOXP3. The frequencies of three single nucleotide polymorphisms (SNPs) in IL-2 -330T>G (rs2069762), +114G>T (rs2069763) and IVS3-116 A>G (rs2069772), four SNPs in IL-10 -3575T>A (rs1800890), -1082G>A (rs1800896), -819 C>T (rs1800871) and -592 C>A (rs1800872) and three SNPs in FOXP3 -3499 A>G (rs3761547), -3279 C>A (rs3761548) and -924 A>G (rs2232365) were compared between 166 Danish AIN patients and 358 healthy controls. Disease association was observed for IL-2 IVS3-116 GG (p = 0.0081, OR = 0.35 [0.15-0.80]), IL-10 -3575 TT (p = 0.0078, OR = 1.71 [1.16-2.54]) and IL-10 -1082 AA (p = 0.014, OR = 1.76 [1.14-2.72]) in all patients and FOXP3 -924 (p = 0.0005, A OR = 0.41 [0.25-0.68] and G OR = 2.42 [1.46-4.01]) in male patients. None of the associations were linked to antibody specificity. Disease-associated haplotypes were observed in IL-2 and FOXP3. IL-2 -330T/+114 T/IVS3-116A was associated with anti-Fc gamma RIIIb-positive patients (p = 0.012, OR = 2.07 [1.18-3.62]). FOXP3 -3499A/-3279C/-924A was associated with anti-HNA-1a-positive male patients (p = 0.016, OR = 0.41 [0.20-0.83]), and ACG was associated with female patients, both in the combined group (p = 0.006, OR = NA) and the anti-Fc gamma RIIIb-positive group (p = 0.002, OR = NA). We conclude that our findings reveal a correlation between SNP in Treg-associated genes and AIN, indicating that AIN could be driven by dysfunction of immune homeostatic-evolving Tregs.

BACKGROUND:In 2010, Denmark was the first country to implement a targeted routine antenatal anti-D prophylaxis (tRAADP) program, offering fetal RHD genotyping to all nonimmunized D negative pregnant women. The program represented a shift from only postnatal prophylaxis to a combined antenatal and postnatal prophylaxis. This study aimed to evaluate the clinical effect of tRAADP in Denmark. STUDY DESIGN AND METHODS:This nationwide registry-based cohort study included all D negative women who gave birth between 2004-2020, identified through the National Medical Birth Register and the Departments of Clinical Immunology in Denmark. The clinical effect of tRAADP was assessed by comparing the incidence of new D immunization between 2004-2009 (non-tRAADP-cohort) and 2011-2018 (tRAADP-cohort). RESULTS:A total of 282 women were D immunized during pregnancy between 2004-2009 (non-tRAADP-cohort), and 167 between 2011-2018 (tRAADP-cohort). The incidence of new D immunization decreased from 0.46% (95% CI 0.41-0.52) in the non-tRAADP-cohort to 0.22% (95% CI 0.19-0.25) in the tRAADP-cohort. The risk reduction was statistically significant p < 0.001. Notably, in the tRAADP cohort 0.1% (95% CI 0.08-0.12) of new D immunizations occurred before the time of antenatal prophylaxis. DISCUSSION:tRAADP significantly reduced the incidence of new D immunization by more than half, thus demonstrating the expected effect. However, even with full adherence to the current program, some women with early fetomaternal hemorrhage (FMH) were still at risk. Future studies may evaluate the impact of administering an additional tRAADP dose earlier in the second trimester to prevent this.

The clinical presentation of CD21 deficiency in 2 siblings caused by a novel mutation in the CD21 gene is reported, and the frequency of this mutation in the Danish population is explored. Successful treatment with IgG replacement in both patients with CD21 deficiency is also reported.

HLA studies in patients with autoimmune neutropenia (AIN) have shown very consistent results for the association with HLA class II alleles at low resolution. This study aimed to examine the association of both HLA class I and class II at high resolution to clarify the contribution of risk alleles to the disease. A total of 107 AIN patients were genotyped for six loci of HLA class I (HLA‐A, ‐B and ‐C) and class II (HLA‐DRB1, ‐DQB1, and ‐DPB1) genes by a high‐resolution (3‐field, 6‐digit) analysis and compared with HLA typing of 1000 healthy controls. Compared with the controls, the allele frequencies were significantly higher in AIN patients for A*02:17:01G, C*01:02:01G, DRB1*10:01:01G, DRB1*14:01:01G, DRB1*16:01:01G, DQB1*05:02:01G, and DQB1*05:03:01G but lower significant for C*03:04:01G, DRB1*04:01:01G, DRB1*13:02:01G, DQB1*03:02:01G, and DQB1*06:04:01G. Frequently associated two‐locus haplotypes were found to be DRB1*10:01:01G‐DQB1*05:01:01G and DRB1*16:01:01G‐DQB1*05:02:01G, while the S2 (Q‐ or D‐KRAA) shared epitope (SE) was associated with lower risk. A unique association with HLA alleles was observed between patients with specific anti‐HNA‐1a antibodies and broad‐reacting anti‐FcγRIIIb. Anti‐HNA‐1a antibody‐positive patients were associated with C*01:02:01G, DRB1*01:01:01G, DRB1*16:01:01G, DQB1*05:01:01G, DQB1*05:02:01G, DQB1*06:04:01G, and DPB1*10:01:01G; the two‐locus haplotypes DRB1*01:01:01G‐DQB1*05:01:01G and DRB1*16:01:01G‐DQB1*05:02:01G; and the S3P (Q‐ or R‐RRAA) SE. Anti‐FcγRIIIb antibody‐positive patients were associated with the alleles A*02:17:01G, DRB1*10:01:01G, and DQB1*05:02:01G; the haplotypes DRB1*10:01:01G‐DQB1*05:01:01G and DRB1*11:01:02G‐DQB1*05:02:01G; and the S3D (DRRAA) SE. The different associations regarding FcγRIIIb antibody specificities could indicate disease heterogeneity.

Genetic variation in the FCGR3B gene is responsible for different variants of human neutrophil antigen 1 (HNA-1). Laboratory techniques currently utilized for routine HNA-1 genotyping, predominantly PCR-sequence-specific primer (PCR-SSP) and PCR-sequence-based typing (PCR-SBT), lack specificity for FCGR3B. This study compares the capabilities and limitations of existing technologies including an in-house TaqMan PCR, a commercial PCR-SSP test, PCR-SBT and multiplex ligation-dependent probe amplification (MLPA) with those of a long-read nanopore sequencing assay. Testing was performed with both related and unrelated Danish samples with different copy numbers and/or rare alleles. Long-read nanopore sequencing was validated by blind testing of ten English samples. The results showed that FCGR3B copy numbers correlate with a dose-dependent distribution of alleles that complicates genotyping by TaqMan PCR, PCR-SSP and PCR-SBT, due to co-amplification of the homologous FCGR3A gene. MLPA can correctly quantify the dose-dependent distribution but not detect novel variants. Long-read nanopore sequencing showed high specificity for FCGR3B and was able to detect dosage-dependent distribution, and rare and novel variants that were previously not described. Current HNA-1 genotyping methods cannot produce unambiguous allele-level results, whereas long-read nanopore sequencing has shown the potential to resolve observed ambiguities, identify new HNA-1 variants and allow definitive allele assignment.

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.

Autoimmune neutropenia (AIN) of early childhood is caused by autoantibodies against antigens on the neutrophil membrane. Human leukocyte antigens (HLA) have previously been associated with AIN. This study investigated HLA-DRB1 and HLA-DQB1 alleles in 160 antibody positive patients and compared with 1000 controls. Increased risk was observed for DRB1*10, DRB1*14, DRB1*16 and DQB1*05, and lower risk for DRB1*04, DRB1*13 and DQB1*03. Haplotypes with higher risk included: DRB1*10/DQB1*05, DRB1*14/DQB1*05 and DRB1*16/DQB1*05, while DRB1*04/DQB1*03, DRB1*07/DQB1*02, and DRB1*13/DQB1*06 were associated with lower risk. Associated HLA-DRB1 and –DQB1 differed between patients positive for anti-HNA-1a-specific antibodies and patients positive for broad reactive anti-FcγRIIIb antibodies. DRB1*01, DRB1*04 and DQB1*03 was only associated for anti-HNA-1a positive, and DRB1*10 was restricted to broad reactive anti-FcγRIIIb positive. Strong association between AIN and HLA-DRB1 and -DQB1 alleles and haplotypes suggested that they play a role in susceptibility or protection. Different associations regarding FcγRIIIb antibody specificities could indicate disease heterogeneity.

Background Copy number variation (CNV) in the genome is difficult to analyze with standard genotyping methods. CNV in the Fcγ receptor region (FCGR) is associated with risk of autoimmune disease. Genetic variation in Fc-gamma coding genes is also abundant, and several functional polymorphisms are described. Autoimmune neutropenia (AIN) primarily affects children under the age of five but can also affect adults. The disease is caused by autoantibodies reactive to neutrophil surface antigens, primarily the FCGR3B receptor. We investigated the association between common SNPs and CNV in Fcγ receptors and AIN. Aims Establish the association between CNV and genetic variation in Fc-gamma receptor coding genes in Danish patients. Methods 127 Danish AIN cases were compared with 610 healthy and unrelated Danish blood donors for FCGR2A-H166R, and with 919 Caucasians (1) for FCGR3A-V158F, FCGR2B-B2.4, FCGR2B-I232T and CNV. Molecular determination of copy number and genotypes was determined using Multiplex Ligation Probe Assay (MLPA) with the following SALSA MLPA probemixes: P110-C1 FCGR mix 1 and P111-C1 FCGR mix 2. Statistical analysis was performed with Fisher Exact test. Results The polymorphisms FCGR2A-166H, FCGR3A-158F, FCGR2B-232I were statistically significantly associated with a higher risk of AIN (Table 1). The presence of a FCGR2B-2B.4 promoter haplotype was associated with a significantly lower risk of AIN (OR=0.5). There was not observed a difference in the distribution of CNV for the Fc-gamma receptor genes (data not shown). Summary/Conclusion In our Danish cohort, we report an association between AIN and polymorphisms in Fc-gamma receptor 2A, 3A and 2B, indicating a significance of other Fc-gamma receptors than FCGR3B. We do not observe a difference in the distribution of CNV of the Fc-gamma receptor genes. 1) Nagelkerke SQ, Tacke CE, Breunis WB, Tanck MWT, Geissler J, Png E, Hoang LT, van der Heijden J, Naim ANM, Yeung RSM, Levin ML, Wright VJ, Burgner DP, Ponsonby A-L, Ellis JA, Cimaz R, Shimizu C, Burns JC, Fijnvandraat K, van der Schoot CE, van den Berg TK, de Boer M, Davila S, Hibberd ML,Kuijpers TW and the International Kawasaki Disease Genetics Consortium (2019) Extensive Ethnic Variation and Linkage Disequilibrium at the CGR2/3 Locus: Different Genetic Associations Revealed in Kawasaki Disease. Front. Immunol. 10:185. doi: 10.3389/fimmu.2019.00185 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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 (Lea and Leb ) 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 Leb 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.

Transfusion-Related Acute Lung Injury (TRALI) has been associated with neutrophil reacting antibodies in transfused blood products. We report a case of life-threatening TRALI in an obstetric patient triggered by transfusion from a non-transfused male blood donor. A residual risk of TRALI exist, even in a male-only plasma setting.

Autoimmune neutropenia of infancy (AIN) is a frequent cause of neutropenia in children. The disease is caused by antibodies against epitopes on the immunoglobulin G (IgG) Fc receptor type 3b (FcγIIIb). We investigated the possible association of human neutrophil antigens (HNA), human leukocyte antigen (HLA)‐DR, and HLA‐DQ alleles with AIN and the association of these genotypes with the presence of autoantibodies.

Amniotic fluid embolism (AFE) is a rare obstetric emergency with high maternal morbidity and mortality. Despite ongoing research, the pathogenesis of AFE remains unresolved, and AFE is a diagnostic and therapeutic challenge. We present a case report of suspected AFE and disseminated intravascular coagulation occurring during caesarean delivery in a pregnancy with oocyte donation and intracytoplasmic sperm injection. Awareness of the diagnosis, rapid initiation of treatment and the use of rapid tests of haemostasis in targeted coagulopathy treatment may improve clinical outcome.

Background The pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has tremendous consequences for our societies. Knowledge of the seroprevalence of SARS-CoV-2 is needed to accurately monitor the spread of the epidemic and also to calculate the infection fatality rate (IFR). These measures may help the authorities to make informed decisions and adjust the current societal interventions. Blood donors comprise approximately 4.7% of the similarly aged population of Denmark and blood is donated in all areas of the country. The objective of this study was to perform real-time seroprevalence surveying among blood donors as a tool to estimate previous SARS-CoV-2 infections and the population based IFR. Methods All Danish blood donors aged 17-69 years giving blood April 6 to 17 were tested for SARS-CoV-2 immunoglobulin M and G antibodies using a commercial lateral flow test. Antibody status was compared between areas and an estimate of the IFR was calculated. The seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (CI). Results The first 9,496 blood donors were tested and a combined adjusted seroprevalence of 1.7% (CI: 0.9-2.3) was calculated. The seroprevalence differed across areas. Using available data on fatalities and population numbers a combined IFR in patients younger than 70 is estimated at 82 per 100,000 (CI: 59-154) infections. Conclusions The IFR was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. The IFR, including only individuals with no comorbidity, is likely several fold lower than the current estimate. This may have implications for risk mitigation. We have initiated real-time nationwide anti-SARS-CoV-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic.

Abstract Identification of risk factors for contracting and developing serious illness following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of paramount interest. Here, we performed a retrospective cohort analysis of all Danish individuals tested for SARS-CoV-2 between 27 February 2020 and 30 July 2020, with a known ABO and RhD blood group, to determine the influence of common blood groups on virus susceptibility. Distribution of blood groups was compared with data from nontested individuals. Participants (29% of whom were male) included 473 654 individuals tested for SARS-CoV-2 using real-time polymerase chain reaction (7422 positive and 466 232 negative) and 2 204 742 nontested individuals, accounting for ∼38% of the total Danish population. Hospitalization and death from COVID-19, age, cardiovascular comorbidities, and job status were also collected for confirmed infected cases. ABO blood groups varied significantly between patients and the reference group, with only 38.41% (95% confidence interval [CI], 37.30-39.50) of the patients belonging to blood group O compared with 41.70% (95% CI, 41.60-41.80) in the controls, corresponding to a relative risk of 0.87 (95% CI, 0.83-0.91) for acquiring COVID-19. This study identifies ABO blood group as a risk factor for SARS-CoV-2 infection but not for hospitalization or death from COVID-19.